Agent for cardiopathy

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns an agent for myocardial infraction containing an antibody which identifies IL-6 receptor as an active ingredient.

EFFECT: invention provides improved state of an affected zone in myocardial infraction and inhibited left ventricular remodelling following myocardial infraction.

23 cl, 5 ex

 

The technical field

The present invention relates to means for treating myocardial infarction, containing the inhibitor of IL-6 as an active ingredient, and to the use of such funds. In addition, the present invention relates to an overwhelming remodeling of the left ventricle after myocardial infarction, which contain the inhibitor of IL-6 as an active ingredient, and to the use of such funds.

The level of technology

Myocardial infarction is one of ischaemic heart disease. It is a disorder that causes necrosis of the myocardium, where spasm of the coronary artery of the heart occurs due to atherosclerosis and similar disorders, and blood flow in the coronary arteries is significantly reduced or stopped. Expansion and/or deterioration affected by the infarct region is the cause of the complications, such as heart failure and/or ischemia-induced severe arrhythmia, and becomes life-threatening.

As myocardial infarction progresses, the cells in the affected myocardium infarct areas of the die and/or rejected and replaced by fibrous tissue, such as collagen fiber. In an area of infarction is observed defect contractility, and this area does not stand intracardiac pressure, which increases p and the contraction of the heart, and then the fibrous wall is gradually expanding. As a result, for compensation hypofunction occurs hypertrophy endocardial cavity in areas not affected by the heart attack, and dilation of the whole left ventricle. This phenomenon is called remodeling of the left ventricle, and it further reduces the function of the heart and improves soreness and later mortality. Therefore, to improve prediction of myocardial infarction, it is important to suppress the progression of remodeling of the left ventricle as soon as possible, and the development of effective treatment is desirable.

IL-6 is a cytokine named stimulating B-cell factor 2 (BSF2), or interferon-β2. IL-6 was opened as a differentiation factor involved in the activation of b-lymphocytes (non-patent document 1), and was later found as a multifunctional cytokine that affects the function of various cells (non-patent document 2). It was shown that IL-6 induces the maturation of T-lymphocytes (non-patent document 3).

IL-6 transmits its biological activity by means of two types of proteins in the cell. One of the protein is the receptor for IL-6, which is legendbase.ui protein, which binds IL-6, and he has a molecular weight of approximately 80 KD (non-patent documents 4 and 5). In addition to membranous the authorized forms, which permeates the membrane and is expressed on the cell membrane, the receptor for IL-6 is present as a soluble receptor of IL-6, which mainly consists of the extracellular region, membrane-bound form.

Another protein is a membrane protein gp130, which has a molecular mass of approximately 130 KD and is involved in signal transmission, is not associated with the ligand. The biological activity of IL-6 is passed into the cell through the formation of complex IL-6/receptor, IL-6, and then binding complex with gp130 (non-patent document 6).

Inhibitors of IL-6 are substances that inhibit the transfer of the biological activity of IL-6. To date, known antibodies against IL-6 (anti-IL-6 antibodies), antibodies against receptors of IL-6 (antibodies against the receptor for IL-6), antibodies against gp130 (anti-gp130 antibodies), variants of IL-6, partial peptides of IL-6 or receptors of IL-6 and the like.

There are several reports regarding antibodies against the receptor for IL-6 (non-patent documents 7 and 8; patent documents 1-3). Known humanitariannet RM-1-antibody (patent document 4), which was obtained by transferring into a human antibody hypervariable segment (CDR), antibodies mouse RM-1 (non-patent document 9), which is one of the antibodies against the receptor for IL-6.

To date, researchers have suggested that IL-6 affects the function and structure of the heart based on these facts, IL-6 has a negative effect on the contractility of the heart muscle (non-patent document 10), cardiac hypertrophy develops in the mouse, which gp130 is activated due to high expression of IL-6 and receptor of IL-6 (non-patent document 11), and so on. After myocardial infarction IL-6 is expressed in the left ventricle, particularly in the border zone of myocardial infarction associated with reperfusion (non-patent document 12), and the expression level is associated with the size of the left ventricle (LV) after myocardial infarction (non-patent document 13). In addition, it was shown that the cells of the myocardium to produce IL-6 at low load, oxygen (non-patent document 14) and that the expression of cytokines in the second cells during post-infarction remodeling plays a regulatory role in the changes of the extracellular matrix (non-patent document 15). In addition, concerning the relationship between myocardial infarction and IL-6, it is shown that the system of JAK/STAT, activated IL-6 has a protective effect against myocardial infarction (non-patent document 16).

On the other hand, on the basis of the experiment on IL-6-knockout mice it was shown that the lack of IL-6 had no effect on the amount affected by the infarct zone, remodeling of the left ventricle or the like (non-patent document 17). As described above, the role of IL-6 in the development of heart attack miokar the a and the remodeling of the left ventricle after myocardial infarction is unknown.

Links of the prior art in this field related to the present invention, is presented below.

[Non-patent document 1] Harada, T. et al., Nature (1986) 324, 73-76

[Non-patent document 2] Akira, S. et al., Adv. in Immunology (1993) 54, 1-78

[Non-patent document 3] Lotz, M. et al., J. Exp. Med. (1988)167, 1253-1258

[Non-patent document 4] Taga, T. et al., J. Exp. Med. (1987) 166, 967-981

[Non-patent document 5] Yamasaki, K. et al., Science (1988) 241, 825-828

[Non-patent document 6] Taga, T. et al., Cell (1989) 58, 573-581

[Non-patent document 7] Novick, D. et al., Hybridoma (1991) 10, 137-146

[Non-patent document 8] Huang, Y. W. et al., Hybridoma (1993) 12, 621-630

[Non-patent document 9] Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906

[Non-patent document 10] Finkel, M. S. et al., Science (1992) 257, 387-389

[Non-patent document 11] Hirota, H. et al., Proc. Natl. Acad. Sci. USA (1995) 92, 4862-4866

[Non-patent document 12] Gwechenberger, M. et al., Circulation (1999) 99, 546-551

[Non-patent document 13] Ono, K. et al., Circulation (1998) 98, 149-156

[Non-patent document 14] Yamauchi-Takihara, K. et al., Circulation (1995) 91, 1520-1524

[Non-patent document 15] Yue, P. et al., Am. J. Physiol. (1998) 275,H250-H258

[Non-patent document 16] Negoro, S. et al., Cardiovasc. Res. (2000) 47, 797-805

[Non-patent document 17] M. Fuchs et al., FASEB J. (2003) 17, 2118-2120

[Patent document 1] international publication WO 95/09873

[Patent document 2] French patent publication No. FR 2694767

[Patent document 3] U.S. Patent No. 5216128

[Patent document 4] international publication WO 9219759

Description of the invention

[Problems that can be solved by the invention]

Up to the present time believed that IL-6 participates in the development of myocardial infarction and subsequent remodeling of the left ventricle. However, its specific role is not clear. In addition, not found, what type of effect the introduction of the inhibitor of IL-6 can be demonstrated in myocardial infarction and subsequent remodeling of the left ventricle.

The present invention was accomplished in a similar situation, and the purpose of the present invention was to develop tools for the treatment of myocardial infarction, containing the inhibitor of IL-6 as an active ingredient. In addition, the present invention relates to suppressing remodeling of the left ventricle after myocardial infarction, containing the inhibitor of IL-6 as an active ingredient. In addition to the other purposes of the present invention includes the development of methods of treatment of myocardial infarction and methods of suppressing remodeling of the left ventricle following myocardial infarction, and all methods include the stage of introduction of the inhibitor of IL-6 subjects who have suffered a myocardial infarction.

[Means of solving problems]

To solve the above problems, the authors of the present invention investigated the influence of antibodies against IL-6 to improve the condition of affected infarct zone after myocardial infarction and for the suppression of remodeling of the left ventricle after myocardial infarction.

First, the authors present invention has created a model of myocardial infarction by ligating the left anterior descending branch vessels in male Balb/c mice. Then 500 μg antibodies against the receptor for IL-6 (MR16-1) was administered intraperitoneally to mice models of myocardial infarction.

As a result, the increase in the activity of myeloperoxidase (MPO) in the area affected by the myocardial infarction was significantly suppressed. Was also suppressed the expression of chemotactic protein-1 monocyte infarction (MCP-1) as in the infarction area, and in areas not affected by the heart attack, in mice with the introduction of antibodies against the receptor for IL-6. In addition, echocardiographic and histological verification found that cardiac hypertrophy was suppressed in mice with the introduction of antibodies against the receptor for IL-6.

Thus, initially, the authors of the present invention discovered that by injecting antibodies against the receptor for IL-6 can improve the condition of the affected infarct zone of myocardial infarction and suppress remodeling of the left ventricle after myocardial infarction, and finally completed the present invention.

More specifically, the present invention relates:

[1] a means for treatment of myocardial infarction, containing the inhibitor of IL-6 as an active ingredient;

[2] to the product p. [1], where the inhibitor of IL-6 is an antibody that p will spausnet IL-6;

[3] to the product p. [1], where the inhibitor of IL-6 is an antibody that recognizes the receptor for IL-6;

[4] to the product p. [2] or [3], where the antibody is a monoclonal antibody;

[5] to the product p. [2] or [3], where the antibody is an antibody against IL-6 person or receptor of IL-6 person;

[6] to the product p. [2] or [3], where the antibody is a recombinant antibody;

[7] to the product p. [6], where the antibody is a chimeric antibody, humanitariannet antibody or human antibody;

[8] a means for suppressing remodeling of the left ventricle after myocardial infarction, containing the inhibitor of IL-6 as an active ingredient;

[9] to the product p. [8], where the inhibitor of IL-6 is an antibody that recognizes IL-6;

[10] to the product p. [8], where the inhibitor of IL-6 is an antibody that recognizes the receptor for IL-6;

[11] to the product p. [9] or [10], where the antibody is a monoclonal antibody;

[12] to the product p. [9] or [10], where the antibody is an antibody against IL-6 person or receptor of IL-6 person;

[13] to the product p. [9] or [10], where the antibody is a recombinant antibody;

[14] to the product p. [13], where the antibody is a chimeric antibody, humanitariannet antibody or human and tetelo;

[15] the agent according to any one of paragraphs. from [8] to [14], which is used to treat myocardial infarction;

[16] the method of treating myocardial infarction in a subject, comprising the stage of introduction of the inhibitor of IL-6 to a subject, who had myocardial infarction;

[17] the method of suppressing remodeling of the left ventricle after myocardial infarction in a subject, comprising the stage of introduction of the inhibitor of IL-6 to a subject, who had myocardial infarction;

[18] the method according to p. [16] or [17], where the inhibitor of IL-6 is an antibody that recognizes IL-6;

[19] the method according to p. [16] or [17], where the inhibitor of IL-6 is an antibody that recognizes the receptor for IL-6;

[20] the method according to p. [18] or [19], where the antibody is a monoclonal antibody;

[21] the method according to p. [18] or [19], where the antibody is an antibody against IL-6 person or receptor of IL-6 person;

[22] the method according to p. [18] or [19], where the antibody is recombinant antibody;

[23] the method according to p. [22], where the antibody is a chimeric antibody, humanitariannet antibody or human antibody;

[24] the use of an inhibitor of IL-6 to obtain an agent intended for the treatment of myocardial infarction;

[25] the use of an inhibitor of IL-6 to obtain funds intended to suppress remodeling of the left ventricle of th is myocardial infarction;

[26] for use on p. [24] or [25], where the inhibitor of IL-6 is an antibody that recognizes IL-6;

[27] for use on p. [24] or [25], where the inhibitor of IL-6 is an antibody that recognizes the receptor for IL-6;

[28] for use on p. [26] or [27], where the antibody is a monoclonal antibody;

[29] for use on p. [26] or [27], where the antibody is an antibody against IL-6 person or receptor of IL-6 person;

[30] for use on p. [26] or [27], where the antibody is a recombinant antibody; and

[31] for use on p. [30], where the antibody is a chimeric antibody, humanitariannet antibody or human antibody.

The best way of carrying out the invention

The inventors have found that the improvement of the affected area in myocardial infarction and suppression of remodeling of the left ventricle after myocardial infarction can be achieved by introducing antibodies against the receptor for IL-6. The invention is based on the received data.

The present invention relates to a means for treatment of myocardial infarction and means for suppressing remodeling of the left ventricle after myocardial infarction, containing the inhibitor of IL-6 as an active ingredient.

As used throughout this description, the term “inhibitor of IL-6” refers to a substance that Blo is range-mediated IL-6 signaling and inhibits the biological activity of IL-6. Preferably, the inhibitor of IL-6 is a substance which exhibits inhibitory activity against the binding of IL-6 receptor, IL-6 or gp130.

Inhibitors of IL-6 according to the invention include, but are not limited to, for example, anti-IL-6 antibodies, antibodies against the receptor for IL-6, anti-gp130 antibodies, variants of IL-6, soluble variants of the receptor for IL-6 and partial peptides of IL-6 or receptors of IL-6 and low molecular weight compounds that show similar activity. Preferred inhibitors of IL-6 according to the invention include antibodies that recognize the receptor of IL-6.

The source of antibodies is not very limited in the present invention; however, preferred if the antibody is produced by mammalian cells and, more preferably, human cells.

Anti-IL-6 antibody used in the present invention, can be obtained as polyclonal or monoclonal antibodies by known methods. In particular, monoclonal antibodies produced by mammalian cells include antibodies produced by hybridomas and antibodies produced by host cells transformed expressing vector that comprises a gene antibodies, genetic engineering methods. Binding to IL-6 antibody prevents the binding of IL-6 receptor, IL-6, and blocks the transmission of biological the activity of IL-6 in the cell.

Such antibodies include MN (Matsuda, T. et al., Eur. J. Immunol. (1988) 18, 951-956), SK2 antibody (Sato, K. et al., proceedings of the 21stAnnual Meeting of the Lapanese Society for Immunology (1991) 21, 166), etc.

Basically hybridoma producing anti-IL-6 antibody can be obtained by known methods as follows. More precisely, such hybridoma can be obtained by using IL-6 as a sensitizing antigen for the implementation of immunization with conventional methods of immunization, merge the obtained immune cells with known parent cells in the normal method merge cells, and screening for cells producing a monoclonal antibody, a conventional screening method.

More precisely, anti-IL-6 antibodies can be obtained in the following way. For example, IL-6 person used as a sensitizing antigen for obtaining antibodies, can be obtained by using the gene IL-6 and/or amino acid sequences described in the publications Eur. J. Biochem. (1987) 168, 543-550; J. Immunol. (1988) 140, 1534-1541; and/or Agr. Biol. Chem. (1990) 54, 2685-2688.

After transformation of an appropriate host cell known expressing vector, the sequence of the gene IL-6, the desired protein IL-6 clean-known method from the host cell or from the culture supernatant. A purified protein IL-6 can be used as sensitizing is ntigen. Alternatively, a fused protein composed of protein, IL-6 and another protein may be used as a sensitizing antigen.

Antibodies against the receptor for IL-6 used in the present invention, can be obtained as polyclonal or monoclonal antibodies with known methods. In particular, antibodies against the receptor for IL-6 used in the present invention are preferably monoclonal antibodies produced by mammalian cells. Monoclonal antibodies produced by mammalian cells, include antibodies derived from hybridomas, and such antibodies, which are produced from host cells transformed by expressing the vector containing the gene antibodies, genetic engineering methods. By binding to the receptor IL-6 antibody prevents the binding of IL-6 receptor, IL-6, and blocks the transmission of the biological activity of IL-6 in the cell.

Such antibodies include antibody MR16-1 (Tamura, T. et al., Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928); antibody PM-1 (Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906); antibody AUK12-20 antibody AUK64-7 and antibody AUK146-15 (WO 92/19759), etc. Among them the antibody PM-1 can serve as an example of a preferred monoclonal antibodies against the receptor for IL-6, and antibody MR16-1 - example of a preferred monoclonal antibodies against the receptor for IL-6 mouse.

More specifically, antibodies against the receptor for IL-6 can be obtained as follows. For example, the receptor for IL-6 of human rights or the receptor of IL-6 mouse, used as a sensitizing antigen for obtaining antibodies, can be obtained by using the genes of the receptor of IL-6 and/or amino acid sequences described in the published European patent application No. EP 325474 and in the publication of Japanese patent application (Kokai) No. (JP-A) Hei 3-155795, respectively.

There are two types of proteins-receptors IL-6, protein expressed on the cell membrane, and the protein is separated from cellular membrane (soluble receptor IL-6) (Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676). Soluble receptor of IL-6 is mainly composed of the extracellular region associated with the cell membrane receptor of IL-6 and differs from the membrane-bound receptor of IL-6 is eat, what it lacks the transmembrane region or the same as the transmembrane and intracellular region. Any receptor of IL-6 can be used as a protein-receptor, IL-6, since it can be used as a sensitizing antigen for obtaining antibodies against the receptor for IL-6 used in the present invention.

After transformation of an appropriate host cell known expressing vector system introduced with the gene sequence receptor, IL-6, the desired protein is the receptor for IL-6 clean-known method from the host cell or from the culture supernatant. Purified protein is the receptor for IL-6 can be used as a sensitizing antigen. Alternatively, the cell expressing the receptor of IL-6 or a fused protein composed of protein-receptor IL-6 and another protein may be used as a sensitizing antigen.

Anti-gp130 antibodies used in the present invention, can be obtained as polyclonal or monoclonal antibodies with known methods. In particular, anti-gp130 antibodies used in the present invention are preferably monoclonal antibodies produced by mammalian cells. Monoclonal antibodies produced by mammalian cells include antibodies, Paul is obtained from hybridomas and antibodies derived from host cells transformed by expressing vector that contains a gene antibodies, genetic engineering methods. By binding to gp130 antibody prevents the binding of gp130 to the IL-6/receptor of IL-6 and blocks the transmission of the biological activity of IL-6 in the cell.

Such antibodies include antibody AM (JP-A Hei 3-219894); 4B11 antibody and antibody N (U.S. patent 5571513); antibody B-S12 and antibody-R8 (JP-A Hei 8-291199) etc.

In essence, hybridoma producing monoclonal anti-gp130-antibody, can be obtained by known methods as follows. More precisely, such hybridoma can be obtained by using the gp130 as a sensitizing antigen for the implementation of immunization conventional method of immunization, merge the obtained immune cells with a known parent cell by usual method merge cells and screening of producing a monoclonal antibody of the cells of the conventional screening method.

More specifically, the monoclonal antibody can be obtained as follows. For example, gp130, is used as a sensitizing antigen for obtaining antibodies, can be obtained by using the gp130 gene and/or amino acid sequences described in the published European patent application No. EP 411946.

After transformation of an appropriate host cell Izv the STN expressing vector system, introduced with the gp130 gene sequence, the desired protein gp130 clean-known method from the host cell or from the culture supernatant. A purified protein gp130 can be used as a sensitizing antigen. Alternatively, a cell expressing gp130 or a fused protein composed of gp130 protein and another protein may be used as a sensitizing antigen.

Mammal species that are subjected to immunization sensitizing antigen is not very limited, but preferably they are chosen taking into account compatibility with the parent cell used to merge cells. As a rule, the use of rodents, such as mice, rats and hamsters.

Immunization of animals sensitizing antigen is carried out by the known methods. For example, as a conventional method, immunization is performed by injection mammals sensitizing antigen intraperitoneally or subcutaneously. In particular, sensitizing antigen is preferably diluted with or suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline or a similar solution, is mixed with an appropriate amount of a conventional adjuvant (such as complete adjuvant's adjuvant), emuleret and then administered to the mammal was carrying the only time every day 4-21. In addition, the appropriate media can be used for immunization sensitizing antigen.

After this immunization serum is achieved an increased level of the desired antibody, and then the immune cells are collected from the mammal to perform methods for cell fusion. Preferred immune cells that are destined to merge cells, include, in particular, cells of the spleen.

Regarding the myeloma cells of mammals, which can be used as parent cells, i.e. cells of the partner, which is subjected to fusion with the above immune cells, various known strains of cells, for example, P3X63Ag8.653 (Kearney, J.F. et al., J. Immunol. (1979) 123, 1548-1550), P3X63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8, 405-415), SP2/0 Shulman, M. et al., Nature (1978) 276, 269-270), F0 (de St. Groth, S.F. et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I.S., J. Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc. are used appropriately.

In essence, the merger described above immune cells and myeloma cells can be performed with known methods, for example, by the method of Milstein et al. (Kohler, G. and Milstein, C., Methods Emzymol. (1981) 73, 3-46), and a similar method.

More precisely, merge cells, described above, reach nutrient culture medium in the presence of increasing the existing merge cells means. For example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used as amplifier merge cells. To increase the effectiveness of the merger may be added as auxiliary tools, such as dimethyl sulfoxide.

The terms of immune cells and the myeloma cells is preferably, for example, from 1 to 10 immune cells for each cell myeloma. The culture medium used for the above-described merge cells, represents, for example, culture medium RPMI1640 or MEM, which is suitable for proliferation of the above myeloma cells. Normal culture medium used for culturing this cell type, can also be used. In addition, additives serum such as fetal calf serum (FCS)may be used in combination.

As for the merging of cells of interest fused cells (hybridoma) are formed by mixing predetermined amounts of the above immune cells and the myeloma cells in the above culture medium, and then by adding and mixing with a solution of PEG at a concentration of from 30% to 60% (wt./about.) (for example, a solution of PEG with an average molecular weight from about 1000 to 6000), pre-heated to approximately 37°C. Then, the means, the amplifying cell fusion, and so on is one, which is not desirable for the growth of hybridomas can be removed by repeating the stages of the successive addition of the appropriate culture medium and removing the supernatant by centrifugation.

The above hybridoma subjected to selection by culturing cells in a conventional selective culture medium, for example, culture medium NAT (cultural medium containing gipoksantin, aminopterin and thymidine). Culturing in a culture medium NAT continues for a sufficient period, usually within a few days to a few weeks to destroy cells other than interest hybrid (not merged cells). Then carry out the method of serial dilutions for screening and cloning of hybridomas producing an antibody.

In addition to the method of immunizing an animal other than human, antigen for obtaining the above-described hybrid, a desired human antibody that is capable of binding to the desired antigen or antigen-expressing cell, can be obtained by sensitization of human lymphocyte desired protein antigen or antigen-expressing cellin vitroand merge sensitized b-lymphocyte with a myeloma cell (for example, U266) (see publication of Japanese patent bid, medium, small is (Kokoku) No. (JP-B) Hei 1-59878 (considered approved Japanese patent application published for opposition). In addition, a desired human antibody can be obtained by injection of antigen or antigen-expressing cells of the transgenic animal that has a set of genes of human antibodies, and then by implementing the above-described method (see international patent application No. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735).

Thus obtained hybridoma that produce monoclonal antibodies can be subjected to subculturing in normal culture medium and can be stored in liquid nitrogen for a long period.

To obtain monoclonal antibodies from the above-described hybrid, you can use the following methods: (1) the way in which hybridoma cultivated by conventional methods, and the antibodies they receive in the culture supernatant; (2) the way in which hybridoma quickly multiply by introducing them compatible to the mammal, and the antibodies are obtained as ascites; and so on. The first method is preferred for obtaining antibodies of high purity, while the latter method is preferred for the production of antibodies on a large scale.

For example, development of hybridomas producing antibody against the receptor of IL-6 may be implemented is prohibited method described in Japanese patent application JP-A Hei 3-139293. Obtaining can be carried out in a manner that involves the injection of hybridoma producing antibody RM-1, in the abdominal cavity of mice of BALB/c, obtaining ascites, and then purification of antibodies RM-1 from ascites, or in any manner involving the cultivation of hybridoma in an appropriate environment (e.g., RPMI1640 medium containing 10% fetal bovine serum and 5% BM-Condimed H1 (Boehringer Mannheim); environment for SFM hybrid (GIBCO-BRL); environment PFHM-II (GIBCO-BRL), and so on) and then obtaining antibodies RM-1 from the culture supernatant.

Recombinant antibody can be used as the monoclonal antibodies according to the invention, where the antibody produced by genetic recombination by gene cloning antibodies from hybridoma, introducing the gene into an appropriate vector, and then introducing the vector into a host organism (see, for example, Borrebaeck, C.A.K. and Larrick, J.W., THERAPEUTIC MONOCLONAL ANTIBODIES, published in the UK by MACMILLAN PUBLISHERS LTD., 1990).

More precisely, mRNA (mRNA)encoding the variable (V) region of an antibody, isolated from a cell that produces an antibody, such as hybridoma. The selection of mRNA can be performed by obtaining the total RNA of known methods such as a method of ultracentrifugation using guanidine (Chirgwin,J.M. et al., Biochemistry (1979) 18, 5294-5299) and AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159), and obtain mRNA using the kit for purification of mRNA (Pharmacia) and similar methods. Alternatively, mRNA can be directly obtained by using the cleaning kit QuickPrep mRNA (Pharmacia).

cDNA V region antibodies are synthesized from the obtained mRNA using reverse transcriptase. Synthesis of cDNA can be achieved by using a set of synthesis of the first cDNA strands containing AMV reverse transcriptase (AMV Reverse Transcriptase First-strand cDNA Synthesis Kit), and so on. In addition, for synthesis and amplification of cDNA can be used for 5'-RACE method (Frohman, M.A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using a set of 5'-Ampli FINDER RACE (Clontech) and PCR (PCR). Of interest, the DNA fragment is purified from the obtained PCR products and then “sew” with the vector DNA. Then the recombinant vector obtained using the above-described DNA and contribute toEscherichia colior similar bacteria, and colonies are selected to obtain the desired recombinant vector. The nucleotide sequence of the desired DNA can be confirmed, for example, dideoxy-method.

After obtaining the DNA encoding the V region of interest antibodies, sew it with the DNA that encodes the constant region (C-region) of the desired antibodies, and the lead in expressing vector. Alternatively, DNA encoding the V region of the antibody can be introduced in expressing a vector containing a DNA encoding the C-region of the antibody.

To obtain antibodies that wish to use in the present invention, as described below, the gene of the antibody is administered in expressing the vector in such a way that it is expressed under the control of regulating the expression of the area, such as enhancer and promoter. Then the antibody can be expressed by transforming the host cell data expressing vector.

In the present invention to reduce heterologous antigenicity against humans and the like can be used artificially modified recombinant antibodies, such as chimeric antibodies, humanized antibodies or human antibodies. These modified antibodies can be obtained by known methods.

A chimeric antibody can be obtained by ligating the DNA encoding the V region of the antibody obtained as described above, DNA encoding the C-region of human antibodies, DNA introduction in expressing vector and introducing the vector host for the production of antibody (see European patent application No. EP 125023; publication of international patent application No. WO 92/19759). This known method can be used in the n to obtain chimeric antibodies, used in the present invention.

Humanized antibodies are also referred to as a reconstructed human antibodies, and they are antibodies, where hypervariable sites (CDR) of an antibody of a mammal other than human (e.g., antibodies mouse), transferred to a CDR of a human antibodies. Common ways this recombination genes are also known (see European patent application No. EP 125023, publication of international patent application No. WO 92/19759).

More specifically, a DNA sequence designed such that CDR antibody mouse stitched to the frame regions (FR) of a human antibodies synthesized by PCR from several oligonucleotides that must be synthesized so as to contain overlapping parts at their ends. The resulting DNA bound to DNA that encodes a C-region of human antibodies, and then bring in expressing vector. Expressing the vector inserted in the host cell for producing gumanitarnogo antibody (see European patent application No. EP 125023, publication of international patent application No. WO 92/19759).

FR human antibodies are subjected to stapling through the CDR, is chosen so that the CDR form suitable antigennegative plot. The amino acid(s) in the FR of the variable regions of the antibodies may be for enema if necessary, so that what CDR reconstructed human antibodies form the corresponding antigennegative site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

With the field of human antibodies is used to produce chimeric and humanized antibodies, and they include γ. For example, can be used γ1, γ2, γ3 or γ4. In addition, to improve the stability of the antibody or its production, the field of human antibodies can be modified.

Chimeric antibodies consist of the variable regions of the antibodies produced by mammals other than man, and produced from With-human antibodies, and humanized antibodies consist of a CDR of the antibody produced by mammals other than man, and frame regions and P-regions derived from human antibodies. Both have reduced antigenicity in the human body, and therefore they are suitable as antibodies for use in the present invention.

Preferred specific examples of humanized antibodies used in the present invention include humanitariannet antibody PM-1 (see international patent application WO 92/19759).

Furthermore, in addition to the described method of obtaining human antibodies, methods for obtaining human antibodies “panning”-method using Bible the reading libraries of human antibodies are also known. For example, it is possible to Express the variable regions of human antibodies on the surface of the phage in the form of single-chain antibodies (scFv) using phage display, and then choose antigennegative phages. By analyzing the genes of the selected phages can be defined DNA sequences encoding the variable regions of human antibodies that bind to the antigen. Once the sequence of scFv DNA to bind to the antigen, is found, the corresponding containing the sequence of expressing the vector can be designed to obtain human antibodies. These methods are already known, and the publication WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388 can be used as a reference.

The above-constructed gene antibodies may be subjected expression by conventional methods. When using mammal cells, the antibody gene can be expressed with the use of DNA in which the gene of the antibody, which must be expressed functionally stitched with commonly used promoter and signal region polyadenylation in the forward direction antibody gene, or a vector containing DNA. Examples of system promoter/enhancer include pretani the promoter/enhancer of the human cytomegalovirus.

In addition, other promoters/enhancers, is the quiet can be used for the expression of antibodies which is supposed to be used in the present invention include viral promoters/enhancers from retrovirus, virus polyoma, adenovirus, simian virus SV40 and similar viruses; and produced by the cell of a mammal promoters/enhancers such as elongation factor 1α person (HEF1α).

For example, when using the promoter/enhancer of SV40 virus, expression can be easily carried out in a manner Mulligan et al. (Mulligan, R.C. et al., Nature (1979) 277, p.108-114). Alternatively, in the case of the promoter/enhancer HEF1α may be used in the method of Mizushima et al. (Mizushima, S. and Nagata, S., Nucleic Acids Res. (1990) 18, 5322).

In the case ofE. coligene antibodies can be expressed using functional linkage commonly used promoter, a signal sequence for secretion of antibodies and antibody gene, which must be expressed. Examples of the promoter include the lacZ promoter, the promoter araB, etc. In the case of promoter lacZ expression can be carried out by the method of Ward et al. (Ward, E.S. et al., Nature (1989) 341, 544-546; Ward, E.S. et al., FASEB J. (1992) 6, 2422-2427); and the araB promoter can be used by the method of Better et al. (Better, M. et al., Science (1988) 240, 1041-1043).

When antibody is produced in periplasmE. Colisignal sequence pel B (Lei, S.P. et al., J. Bacteriol. (1987) 169, 4379-4383) can be used as a signal by which sledovatelnot for secretion of antibodies. The antibody produced in periplasm, isolated and then used after the corresponding “refolding” structure of the antibody (see WO 96/30394).

As a site of replication initiation is used such that derived from SV40, a virus polyoma, adenovirus, papilloma virus in cattle (BVR), etc. in Addition, to increase the number of gene copies in the system of a host cell expressing the vector may contain a gene aminoglycosidetherapy (ARN), gene timedancing (TS), gene xanthine-guaninephosphoribosyltransferaseE. Coli(Ecogpt)gene digidrofolatreduktazy (dhfr) or similar gene as a selective marker.

Any producing system can be used to generate antibodies to be used in the present invention. Producing system to generate antibodies includein vitroandin vivoproducing system.In vitroproducing systems include systems utilizing eukaryotic cells or prokaryotic cells.

Producing systems that use eukaryotic cells include systems using animal cells, plant cells or cells of fungi. Such animal cells include (1) mammalian cells such as Cho, COS, myeloma, liver of hamsters (KSS), HeLa, Vero, etc.; (2) cells of amphibians, such as oocyteXenopus;and (3) insect cells such as the R sf9, sf21, Tn5 and other Known plant cells include cells derived fromNicotiana tabacum,which can be cultivated in the form of callus. Known cells of fungi include yeast, such asSaccharomyces (for example,S. cerevisiae), fungi, such asAspergillus (for example,A. Nigerand other

Producing systems that use cells prokaryotes include systems using bacterial cells. Known bacterial cells includeE. coliandBacillus subtilis.

Antibodies can be obtained by introducing the gene of interest antibodies in these cells by transformation and culturing the transformed cellsin vitro.The cultivation is carried out with known methods. For example, DMEM, MEM, RPMI1640, IMDM can be used as a culture medium and supplements whey, such as FCS, can be used in combination. In addition, the cage made genome antibodies can be transferred to the abdominal or other cavity of the animal to produce antibodiesin vivo.

On the other hand, producing systemin vivoinclude systems that use animals or plants. Producing system using animals include systems that use mammals or insects.

Mlekovita is s, which can be used include goats, pigs, sheep, mice, cattle, etc. (Vicki Glaser, SPECTRUM Biotechnology applications, 1993). Insects that can be used include also the silkworms. In the case of plants used, for example, tobacco.

Gene antibodies contribute to the specified animal or plant, and the antibody is produced in animals or in plants, and then removed. For example, the gene of the antibody receive as a fusion gene by introduction of a gene into the middle of the gene encoding the protein, such as β-casein goats, which is produced only in milk. The DNA fragment containing the fused gene, which is embedded in the gene of the antibody is injected into a goat embryo, and the embryo is transferred into the body of the female goats. The desired antibody is obtained from milk produced by transgenic animals, born from the goats that received an embryo, or produced by the offspring of the animal. To increase the amount of milk containing the desired antibody produced by the transgenic animals, transgenic goats can appropriately be used hormones (Ebert, K.M. et al., Bio/Technology (1994) 12, 699-702).

In addition, when using silkworm its infecting baculovirus introduced with the gene of the desired antibodies, and the desired antibody is obtained from the body fluid of the silkworm (Maeda, S. et al.,Nature (1985) 315, 592-594). In addition, when using tobacco, the gene of the desired antibody is administered in expressing vector plants (for example, pMON530), and the vector is introduced into the bacterium, such asAgrobacterium tumefaciens.Specified bacteria used for infection of tobacco (for example,Nicotiana tabacum)to obtain the desired antibody from the leaves of this tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

When antibody is produced in producing systemsin vitroorin vivoas described above, DNA encoding the heavy chain (H chain) and light chain (L chain) of antibody may be embedded in the individual expressing vectors, followed by the simultaneous transformation of the host vectors. Alternatively, DNA can be integrated in a single expressing vector for transformation of a host (see publication of international patent application No. WO 94/11523).

Antibodies used in the invention may be antibody fragments or modified products in cases where they can be appropriately used in the present invention. For example, fragments of antibodies include Fab, F(ab')2, Fv and single-chain Fv fragment (scFv)in which Fv H - and L-chains are linked via an appropriate linker.

More specifically, fragments of antibodies are produced during the processing of the antibody with an enzyme such as papain or pepsin, or, ka is este alternatives genes encoding these fragments, design, contribute to expressing the vectors that are expressed in an appropriate host cell (see, for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. &Horwitz, A.H., Methods in Enzymology (1989) 178, 476-496; Plueckthun, A. &Skerra, A., Methods in Enzymology (1989) 178, 497-515; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-666; Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

Fragment scFv can be obtained by linking the H chain V region and L-chain V-region of the antibody. In the fragment scFv, the H chain V region and L-chain V-region are linked via a linker, preferably via a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). V-region H - and L-chains in the scFv may be derived from any of the antibodies described above. Peptide linkers to link V-regions include, for example, an arbitrary single-chain peptide consisting of 12 to 19 amino acid residues.

DNA encoding scFv can be obtained using DNA encoding the H chain or the V region and the DNA encoding the L chain or the V region of the above antibody, as a matrix, amplification by PCR of the DNA that encodes the desired amino acid sequence in the matrix sequence in the presence of primers, indicating the part ends, and the subsequent amplification of the amplified DNA with a DNA that encodes a part of the peptide linker, and pairs of the primers, which connect both ends of the linker with the H-chain and L-chain.

In addition, after obtaining the DNA encoding scFv expressing a vector containing the DNA, and the owner, transformirovannykh vector, can be obtained by conventional methods. In addition, scFv can be obtained by conventional methods with the use of the owner.

Similarly to the above methods, these antibody fragments can be produced by the host by obtaining and expression of their genes. As used throughout this description, the term “antibody” encompasses these antibody fragments.

As modified antibodies can be used antibody associated with various molecules such as polyethylene glycol (PEG) (PEG). As used throughout this description, the term “antibody” encompasses these modified antibodies. Data modified antibodies can be obtained by chemical modification of the obtained antibodies. Such methods have already been created in this field.

Described above produced and expressed antibodies can be isolated from internal or external part of the cell or from the host organism and purified to a homogeneous state. Isolation and/or purification of the antibodies used in the present invention, can be carried out by affinity chromatography. The column, which can be used for affinity chromatography, including the indicate in themselves, for example, a column of protein a and a column of protein g Media used for the column with the protein And include, for example, HyperD, POROS, SepharoseF.F, etc. in Addition to the above methods, other methods used for isolation and/or purification of simple proteins, can be used without any restrictions.

As an example, antibodies used in the present invention, can be isolated and/or purified by appropriately selecting and combining methods chromatography, in addition to affinity chromatography, filtration, ultrafiltration, vysalivaniya, dialysis, etc. Chromatography include, for example, ion exchange chromatography, hydrophobic chromatography, gel-filtration and other Specified types of chromatography can be used in high-performance liquid chromatography (HPLC, HPLC). Alternatively can be used HPLC with reversed phase.

The concentration of antibodies, which are described above, can be determined by measuring the absorbance, using ELISA method or the like. More precisely, the absorption is determined by diluting appropriate solution of antibodies PBS(-), measuring the absorbance at 280 nm and calculating the concentration of 1.35 OD = 1 mg/ml). Alternatively, when using the ELISA method, the measurement can be carried out as follows. Specifically, 100 μl of goat antibodies protivoallergicheskogo IgG (TAG), diluted to a concentration of 1 μg/ml in 0.1 M bicarbonate buffer (pH 9,6), placed in a 96-well plate (Nunc) and incubated overnight at 4°C. to immobilize the antibody. After blocking, 100 μl of appropriately diluted antibody according to the invention or an appropriately diluted sample containing the antibody, and human IgG (CAPPEL) add as standard and incubated for one hour at room temperature.

After washing, 100 μl of 5000× diluted anti-human IgG labeled with alkaline phosphatase (BIO SOURCE), is added and incubated for one hour at room temperature. After another wash add substrate solution and incubated, and measure the absorbance at 405 nm using the device for microplate reader model 3550 (Bio-Rad)to calculate the concentration of interest antibodies.

Variants of IL-6 used in the present invention are substances which exhibit the ability to bind to the receptor of IL-6 and which do not transmit the biological activity of IL-6. That is, variants of IL-6 competes with IL-6 for binding to the receptors of IL-6, but does not transmit the biological activity of IL-6, therefore, block the transmission of the signal mediated by IL-6.

Variants of IL-6 are produced by introducing mutation(s) through the replacement of amino acid residues in aminokislotnoi sequence of IL-6. Source IL-6, used as the basis of variants of IL-6, is not limited; however, it is preferable IL-6 person, considering its antigenicity and other

Specifically, the replacement of amino acids is carried out by predicting the secondary structure of the amino acid sequence of IL-6 using known simulation program molecules (for example, WHATIF; Vriend et al., J. Mol. Graphics (1990) 8, 52-56), and then evaluating the impact of the replaced amino acid residue(s) to the entire molecule. After identifying the corresponding amino acid residue that is replaced, perform the conventional methods of PCR using the nucleotide sequence encoding the gene of IL-6 as the matrix for the introduction of mutations in such a way that amino acids are replaced and, thus, receive the gene encoding the variant IL-6. If necessary, this gene contribute to the appropriate expressing vector and IL-6 can be obtained using the methods described above expression, production and purification of recombinant antibodies.

Some examples of variants of IL-6 are described in publications Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, Savino et al., EMBO J. (1994) 13, 1357-1367, WO 96/18648 and WO 96/17869.

Partial peptides of IL-6 and a partial peptide of the receptor of IL-6 to be used in the present invention are substances which shows Aut ability to bind with the receptors of IL-6 and IL-6, respectively, and which do not transfer the biological activity of IL-6. Namely, when the binding and capture of the receptor of IL-6 or IL-6, partial peptides of IL-6 or a partial peptide of the receptor of IL-6 in a specific way prevents the binding of IL-6 receptor IL-6. In the biological activity of IL-6 is not transmitted, and therefore mediated IL-6 signaling is blocked.

Partial peptides of IL-6 or receptor of IL-6 are peptides containing partially or fully amino acid sequence region of IL-6 or the amino acid sequence of the receptor for IL-6, which is involved in the binding of IL-6 and receptor of IL-6. Typically, these peptides contain from 10 to 80, preferably from 20 to 50, more preferably from 20 to 40 amino acid residues.

Partial peptides of IL-6 or a partial peptide of the receptor of IL-6 can be obtained by known methods, for example, genetic engineering techniques or by peptide synthesis, by defining a region of IL-6 or the amino acid sequence of the receptor for IL-6, which is involved in the binding of IL-6 and receptor IL-6, and use partial or complete amino acid sequence of a specific region.

Upon receipt of a partial peptide of IL-6 or a partial peptide of the receptor of IL-6 gene engineering method, the DNA sequence encoding the desired peptide, straihgt in expressing vector, and then the peptide can be obtained in the ways described for the expression, production and purification of recombinant antibodies.

For a partial peptide of IL-6 or a partial peptide of the receptor of IL-6 by peptide synthesis, can be used conventional methods of synthesis, for example, the methods of solid-phase synthesis or methods of synthesis in the liquid phase.

More specifically, the synthesis can be carried out following the method described in the publication “Continuation of Development of Pharmaceuticals, Vol. 14, Peptide Synthesis (in Japanese) (ed. Haruaki Yajima, 1991, Hirokawa Shoten)”. As a method of solid-phase synthesis can be used, for example, the following way: the amino acid corresponding to the C-end of the synthesized peptide, associated with a carrier, which is not soluble in organic solvents, and then extend the peptide chain by alternately repeating (1) the condensation reaction of amino acids in which α-amino groups and functional groups of the branched chain protects the respective protective groups in turn, in the direction from the C-end N-end; and (2) the reaction of removing the protective group from the α-amino groups linked to the resin amino acid or peptide. Solid-phase peptide synthesis is classified into the BOC-and Fmoc method is a method based on the type of the protective group.

After synthesis of the protein of interest, as is written above, conduct the reaction unprotect and cleavage of the peptide chain from the media. For the implementation of the cleavage reaction of the peptide chain is mainly used hydrogen fluoride or triftormetilfullerenov for Vos-method and TFU - Fmoc method. According to Vos-method, for example, the above-described resin-protected peptide is treated with hydrogen fluoride in the presence of anisole. Then the peptide is extracted by removal of the protective groups and cleavage of the peptide from the carrier. The crude peptide can be obtained by lyophilization of the extracted peptide. On the other hand, in the Fmoc method, for example, the reaction of removing the protection and the reaction of cleavage of the peptide chain from the carrier can be carried out in TFU such as described above.

The crude peptide can be isolated and/or purified by HPLC. The elution can be performed in optimal conditions, using a solvent system of water-acetonitrile, which is usually used for protein purification. Fractions corresponding to the peaks obtained chromatographic profile, collect and lyophilizers. Thus, the purified peptide fractions identify molecular mass by mass spectral analysis, analysis of amino acid composition, analysis of amino acid sequence or other

Some examples of the partial peptides of IL-6 or a partial peptide cocktail recipes. who and IL-6 are described in patents JP-A Hei 2-188600, JP-A Hei 7-324097, JP-A Hei 8-311098 and the publication of the patent application U.S. No. US 5210075.

Antibodies used in the present invention may also be conjugated with antibodies that are associated with various molecules such as polyethylene glycol (PEG), radioactive substances and toxins. Such conjugated antibodies can be obtained by chemical modification of the obtained antibodies. Methods of modification of the antibody is already installed in this area. “Antibody” in accordance with the present invention cover these conjugated antibodies.

Means for treatment of myocardial infarction and means to suppress remodeling of the left ventricle after myocardial infarction in the present invention can be used for therapy of myocardial infarction.

Used in the description of the term “treatment of myocardial infarction” means the inhibition or prevention of the symptoms of myocardial infarction and heart failure and ischemia-induced severe arrhythmia, which are complications of myocardial infarction.

Symptoms, complicating myocardial infarction include arrhythmia (beats, ventricular fibrillation and atrioventricular block), heart failure, rupture of papillary muscle, cardiac rupture, ventricular aneurysm (which is formed in the apex of the heart as a result of a heart attack in front of the her descending branch of the left coronary artery) and post-infarction syndrome. “Tools for the treatment of myocardial infarction” according to the present invention can suppress or prevent the symptoms described above.

At the same time used in the description of the term “suppression of remodeling of the left ventricle after myocardial infarction” means the inhibition or prevention of myocardial hypertrophy (enlargement of the whole left ventricle)that occur to compensate for the deterioration of function of the affected infarct zone.

Myocardial hypertrophy occurs when the cells of the heart muscle in areas affected by heart attack, replaced by fibrous tissue, such as collagen fibers, resulting in necrosis and/or exfoliation of cells, and fibrous tissue gradually expands. Thus, suppression and prevention substitution zone of myocardial collagen fibers and proliferation of fibrous tissue, i.e. the improvement of the affected area of the heart, is also included in the meaning of “suppression of remodeling of the left ventricle after myocardial infarction”, described above.

Suppressed if the symptoms of myocardial infarction and remodeling of the left ventricle after myocardial infarction or are not suppressed, it is possible to determine the activity of myeloperoxidase (MPO) in areas of the heart muscle affected and not affected by the heart attack, as an indicator. MPO is an enzyme present in nutrilett is cnyh granules of neutrophils, and its activity, as it is known, is significantly increased in diseases of the coronary arteries. The MPO activity increases as the zone of infarction spread and worsen their condition (necrosis etc). That is, when the activity of MPO is suppressed by introduction means according to the present invention, it is possible to assume that the symptoms of myocardial infarction and remodeling of the left ventricle after myocardial infarction are suppressed. The activity of MPO can be defined by the known methods which include, for example, measurement methods described in the examples.

Alternatively, are suppressed if the symptoms of myocardial infarction and remodeling of the left ventricle after myocardial infarction or are not suppressed, can be identified by the expression of MCP-1 (chemoattractant protein-1 monocytes) as indicator in the affected and not affected by the infarct areas of the heart muscle. MCP-1 is a chemokine, which can cause heart failure, contributing to the influx of macrophages to the heart muscle and increasing the expression of inflammatory cytokines. It is known that MCP-1 activates inflammation and causes fibrosis of the heart muscle and okolososudistoe tissues. Distribution and/or deterioration (necrosis and other States affected by the infarct zone increases the expression of MCP-1. More precisely, we can assume that when the expression of MCP-1 suppressed the symptoms in which arcta infarction and remodeling of the left ventricle after myocardial infarction are suppressed. The expression of MCP-1 can be measured by the known methods used to measure protein expression, including, for example, Western blotting and ELISA method.

The phrase “inhibition of MPO activity and the suppression of the expression of MCP-1” also means “improvement of the condition of the heart”, as described above.

In addition, the suppression of symptoms of myocardial infarction and remodeling of the left ventricle after myocardial infarction can be determined by measuring end-diastolic size and ejection fraction of the left ventricle by echocardiography or by quantitative assessment of the degree of fibrosis of the myocardium and hypertrophy of cardiomyocytes histological verification of the heart tissue. Such measurement can be carried out with known methods. Such methods include, for example, described in the examples methods.

In the present invention, the ability of inhibitors of IL-6 inhibit the signaling of IL-6 can be estimated in the usual way. Specifically, IL-6 added to cultures of IL-6-dependent cell lines of human myeloma (S6B45 KPMM2), cell line CT-3 T-cell lymphoma, Lennert person or IL-6-dependent cell lines 60.BSF2; and consumption of3H-thymidine IL-6-dependent cells measured in the presence of an inhibitor of IL-6. Alternatively, U266 cells expressing the receptor of IL-6, cultivate, and culture add125I-labeled IL-6 and hibitor IL-6 at the same time; and then125I-labeled IL-6 associated with expressing the receptor of IL-6 cells, determine quantitatively. In addition to the group of inhibitors of IL-6, negative control group that does not contain the inhibitor of IL-6, include the system for analysis, described above. The ability of an inhibitor of IL-6 to inhibit IL-6 can be estimated by comparing results of both groups.

As shown in the examples below, the introduction of antibodies against the receptor for IL-6 was found to suppress the symptoms of myocardial infarction and remodeling of the left ventricle after myocardial infarction. The obtained data indicate that inhibitors of IL-6, such as antibodies against the receptor for IL-6, suitable as facilities used for the treatment of myocardial infarction, and the means used to inhibit remodeling of the left ventricle after myocardial infarction.

Entities which are supposed to accept the funds according to the invention for the treatment of myocardial infarction and means according to the invention to inhibit remodeling of the left ventricle after myocardial infarction, are mammals. Mammals are preferably human.

The means according to the invention for the treatment of myocardial infarction and means according to the invention to inhibit remodeling of the left ventricle after myocardial infarction can be introduced in the form of a pharmaceutical is the courthouse square and can be introduced systemically or locally by oral or parenteral administration. For example, can be selected intravenous injection, such as intravenous drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral tablets or other Appropriate route of administration may be selected depending on the patient's age and symptoms. Effective dose of one the introduction of choice in the field of from 0.01 to 100 mg/kg of body weight. Alternatively, the dose can be selected in the range from 1 to 1000 mg/patient, preferably in the range of from 5 to 50 mg/patient. The preferred dose and route of administration are as follows: for example, using antibodies against the receptor for IL-6, the effective dose is the amount at which the free antibody present in the blood. More precisely, the dose from 0.5 to 40 mg/kg body weight/month (four weeks), preferably from 1 to 20 mg/kg body weight/month is administered through intravenous injection, such as intravenous drip infusion, subcutaneous injection or other, from once to several times a month, for example, twice a week, once a week, once every two weeks or once every four weeks. The injection mode can be adjusted, for example, by increasing the interval of administration, comprising the range from two times a week or once a week to once every two weeks, one of razgadai three weeks or once every four weeks, while monitoring status after transplantation and change the test values in the blood.

In the present invention means for treatment of myocardial infarction and means to suppress remodeling of the left ventricle after myocardial infarction may contain pharmaceutically acceptable carriers, such as preservatives and stabilizers. “Pharmaceutically acceptable carriers” refers to substances that can be introduced together with the above-described means and may or may not themselves provide the above-described effect of suppressing the symptoms of myocardial infarction and remodeling of the left ventricle after myocardial infarction. Alternatively, the media can be a substance that does not have the effect of suppression of symptoms of myocardial infarction and remodeling of the left ventricle after myocardial infarction, but produce additive or synergistic stabilizing effect when used in combination with an inhibitor of IL-6.

Such pharmaceutically acceptable substances include, for example, sterile water, saline, stabilizers, fillers, buffers, preservatives, detergents, chelating means (EDTA etc.) and binder.

In the present invention, the detergents include non-ionic detergents, and typical examples of such detergents include esters of the IRNA acids and sorbitan, such as servicemanagement, sorbitanoleat and servicemanagement; esters of fatty acids and of glycerol, such as glycerylmonostearate, glycerylmonostearate and glycerylmonostearate; esters of fatty acids and polyglycerol, such as decapitalization, demilitarization and decapitationalbany; esters of polyoxyethylenesorbitan and fatty acids, such as polyoxyethylenesorbitan, polyoxyethylene sorbitan monooleate, polyoxyethylenesorbitan, polyoxyethylenesorbitan, polyoxyethylenesorbitan and polyoxyethylenesorbitan; esters of polyoxyethylenesorbitan and fatty acids, such as polyoxyethylenesorbitan and polyoxyethylenesorbitan; esters of polyoxyethylenesorbitan and fatty acids, such as poliauxietilenglikola; polietilenglikolya esters of fatty acids, such as polietilenglikolmonostearat; polyoxyethyleneglycol ethers, such as polyoxyethylenesorbitan ether; polyoxyethylene-polyoxypropylene ethers, such as polyoxyethylene-polyoxypropyleneglycol, polyoxyethylene-polyoxypropylene ether and polyoxyethylene-Polyoxypropylenediamine ether; polyoxyethylene ethers of alkyl phenols, such as polyacetate enemy ether of Nonylphenol; polyoxyethylene derivatives hardened castor oils such as polyoxyethylene derivative of castor oil and polyoxyethylene derived cured castor oil (polyoxyethylene hydrogenated castor oil); polyoxyethylene derived beeswax, such as polyoxyethylenesorbitan beeswax; polyoxyethylene lanolin derivatives, such as polyoxyethylenated; and polyoxyethylene derivatives of fatty acid amides, etc., with HLB of 6 to 18, such as the amide polyoxyethyleneglycol acid.

Detergents include anionic detergents, and typical examples of such detergents include, for example, alkyl sulphates containing alkyl group with 10 to 18 carbon atoms, such as cityswift sodium, sodium lauryl sulfate and reinsulate sodium sulfate polyoxyethyleneglycol ethers in which the alkyl group contains from 10 to 18 carbon atoms, and the average number of moles of added ethylene oxide is 2 to 4, such as polyoxyethyleneglycol sodium; salt alkylsulfonates of ester containing an alkyl group with 8-18 carbon atoms, such as sodium salt laurylsulphate ether; natural detergents, such as lecithin; the glycerophospholipids; sphingophospholipids, such as sphingomyelin; and with whom you esters of sucrose and fatty acids, in which the fatty acids contain from 12 to 18 carbon atoms.

One, two or more detergents described above can be combined and add to the means according to the invention. Detergents, which are preferably used in the preparations according to the invention, include polyoxyethylene esters sorbitan and fatty acids, such as Polysorbate 20, 40, 60 and 80. Polysorbate 20 and 80 are particularly preferred. Polyoxyethyleneglycol, such as poloxamer (Pluronic F-68®etc), are also preferred.

The number of added detergent varies depending on the type of detergent. In the case of Polysorbate 20 or 80 quantity were made mainly in the range from 0.001 to 100 mg/ml, preferably in the range from 0.003 to 50 mg/ml, more preferably in the range of 0.005 to 2 mg/ml

In the present invention buffers include phosphate, citrate buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, capric acid, desoxycholic acid, salicylic acid, triethanolamine, fumaric acid and other organic acids; and a carbon dioxide buffer, Tris buffer, his-tag buffer and imidazole buffer.

Liquid preparations can be prepared by Rast is orenia funds in aqueous buffer solutions, known in the field of cooking liquid preparations. The concentration of the buffer is generally from 1 to 500 mm, preferably from 5 to 100 mm, more preferably from 10 to 20 mm.

The means according to the invention can also contain other low molecular weight polypeptides; proteins, such as serum albumin, gelatin, and immunoglobulins; amino acids, sugars and carbohydrates such as polysaccharides and monosaccharides, sugar alcohols, etc.

In this specification, amino acids include basic amino acids such as arginine, lysine, histidine and ornithine, and inorganic salts of these amino acids, preferably hydrochloric salts and phosphate salts, namely, phosphate, amino acids). When using free amino acids the pH is adjusted to a preferred value by adding the corresponding physiologically acceptable for tebufelone substances, for example, inorganic acids, in particular hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid and formic acid and their salts. In this case, the use of phosphate is particularly favorable, because it is quite stable lyophilized products. Phosphate is particularly preferred when the products do not contain largely organic acids such as malic acid, tartaric acid, lim is fair acid, succinic acid and fumaric acid, or does not contain relevant anions (ion malate, tartrate ion, citrate ion, succinate ion, ion fumarata etc). Preferred amino acids are arginine, lysine, histidine and ornithine. You can also use acidic amino acids, for example, glutamic acid and aspartic acid and their salts (preferably sodium salt); neutral amino acids, such as isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine, and alanine; and aromatic amino acids, such as phenylalanine, tyrosine and tryptophan and derivatives thereof, N-acetyltryptophan.

In this invention sugars and carbohydrates such as polysaccharides and monosaccharides include, for example, dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose and raffinose.

In the present invention the sugar alcohols include, for example, mannitol, sorbitol and Inositol.

In the case of the preparation of the agents according to the invention in the form of aqueous solutions for injection, they can be mixed, for example, saline solution and/or isotonic solution containing glucose or other auxiliary means (such as D-sorbitol, D-mannose, D-mannitol and sodium chloride). Aqueous solutions can be used in combination with appropriate stabilizers such as alcohols (ethanol the other), polyhydric alcohols (propylene glycol, PEG, etc) or non-ionic detergents (Polysorbate 80, HCO-50).

Such agents may also contain, if necessary, diluents, solubilizing means of regulating the pH of the substances, sedatives, sulfur-containing reducing agents, antioxidants, etc.

The present invention sulfur-containing reducing agents include, for example, compounds containing sulfhydryl groups, such as N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, toorbit, thioglycolic acid and their salts, sodium thiosulfate, glutathione, and Tolkunova acid having from 1 to 7 carbon atoms.

In addition, the antioxidants in the present invention include, for example, erythorbate acid, dibutylaminoethanol, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, palmitate, L-ascorbic acid, stearate, L-ascorbic acid, sodium bisulfite, sodium sulfite, tremellales, propylgallate and chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate and metaphosphate sodium.

If necessary, these agents may be enclosed in microcapsules (microcapsules of hydroxymethylcellulose, gelatin, poly[methylmethacrylic acid] and so on) or prepared in the form of kolloidnyi systems for drug delivery (liposome, microspheres of albumin, microemulsion, nanoparticles, nanocapsules and the like) (see “Remington''s Pharmaceutical Science 16thedition”, Oslo Ed., 1980, etc). In addition, methods of preparation of such agents in the form of long-acting drugs are also known and applicable to the present invention (Langer et al., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982, 12: 98-105; U.S. patent No. 3773919; European patent application No. (EP) 58481; Sidman et al., Biopolymers 1983, 22: 547-556; and European patent application EP 133988).

Used pharmaceutically acceptable carriers appropriately selected from the above carriers or combined, depending on the type of dosage form, but they are not limited as outlined in the media.

The present invention relates to a method of treatment of myocardial infarction in subjects and methods of suppressing remodeling of the left ventricle after myocardial infarction, comprising the stage of introduction of the inhibitor of IL-6 subjects who have suffered a myocardial infarction.

In this description, the term “subject” refers to organisms and body parts of organisms, which is expected to enter the tool according to the invention for the treatment of myocardial infarction or means according to the invention to inhibit remodeling of the left ventricle after myocardial infarction. The organisms include animals (e.g., human, homemade the animals and wild animals), but not limited to.

“Body parts of organisms” is not limited, but preferably include the heart, the heart muscle and the affected and not affected by the infarct zone during myocardial infarction.

In this description, “introduction” includes oral and parenteral administration. Oral administration includes, for example, the introduction of oral means. Such oral tools include, for example, granule, powder, tablet, capsule, solution, emulsion and suspension.

Parenteral administration includes, for example, injection. Such injections include, for example, subcutaneous injection, intramuscular injection, and intraperitoneal injection. At the same time, the effects of the methods according to the invention can be achieved by using the introduced genes containing oligonucleotides, which must be entered in living organisms using methods of gene therapy. On the other hand, [drug] means according to the invention can be introduced locally in the intended treatment zone. For example, such means may be imposed by local injection during surgery, using catheters or by directional transgenes DNA that encodes a peptide according to the invention. Such means according to the invention can be introduced along with the treatment of myocardial infarction, for example by a catheter which organizations (percutaneous transluminal coronary angioplasty (RTSA) and percutaneous intervention for coronary vessels (PCI)), percutaneous transluminal coronary recanalization (PTCR), coronary artery bypass (CABG) and other

When implementing the methods of the present invention [therapeutic] the means according to the invention can be introduced as part of a pharmaceutical composition in combination with at least one known chemotherapeutic agent. As a variant, the means according to the invention can be introduced in combination with at least one known immunosuppressant. In one embodiment, the invention [therapeutic] the means according to the invention and known chemotherapeutic agents can be introduced almost simultaneously.

All references of the prior art cited in the description are incorporated by reference.

[Example]

As described below, the present invention will be more precisely described with reference to examples, but they should not be construed as limiting the invention.

[Example 1] a model of myocardial infarction mouse

Mice of Balb/c (25 to 30 g) was introduced into the trachea tube. Mice were kept on the ventilator and perform anesthesia by inhalation of 0.5 to 1.0% izoflurana. The chest on the left was open. After ligating the left anterior descending coronary artery chest was closed. The mice were divided into group introduced the first antibody MR16-1 (MR16-1 group) and untreated group (control group). The group processed MR16-1, were subjected to intraperitoneal introduction of MR16-1 in a dose of 500 mg/body.

[Example 2] the Measurement of MPO activity

The hearts were removed from the mice two days after the creation of models of myocardial infarction (or coronary ligation). The hearts were divided on the affected infarct zone and in the region of the heart, not stricken by a heart attack, and crushed. Then crushed the heart muscle was combined with 10 volumes of 50 mm CRO4buffer (pH 6.0)containing 0.5% hexadecyltrimethylammonium. Shredded muscle homogenized (transmitter station, KINEMATICAAG, Luzern, Switzerland) and then subjected to the action of ultrasound. The extract obtained was centrifuged at 13000 rpm for 10 minutes at 4°C. thereafter, 50 μl of the resulting supernatant was mixed with a 1.45 ml of substrate solution (50 mm CRO4(pH 6.0), 0,167 mg/ml o-danishdairyboard and 0.005% N2About2), changes in the coloring substrate solution was monitored by measuring the absorbance at 460 nm (absorption coefficient = 2,655).

In the result, it was shown that MPO activity of the heart muscle did not differ in the area of the heart muscle is not affected by the heart attack, and cardiac muscle falsely operated group, but in the infarction zone activity increased significantly, by about four times (the risk that is not associated with the unreliability of control procedures, 0,037 ± 0,006; p is, IC, associated with the unreliability of control procedures, 0,122 ± 0,035; p<0,01). At the same time, this increase of MPO activity in the affected infarct zone was largely suppressed in the group with the antibody MR16-1 (risk associated with the use of MR16-1, 0,034 ± 0,008; p<0,05 against the risks associated with unreliable control procedures).

[Example 3] Analysis of the expression of MCP-1

The hearts were removed from the mice two days after creating a model of myocardial infarction. The hearts were divided on the affected infarct zone and in an area stricken by a heart attack, and crushed. Then crushed the heart muscle was mixed with buffer for lysis (2× PBS, 1% NP-40, 0.5% deoxycholate sodium, 0.1% sodium dodecyl sulfate, 1 mm PMSF, 1% cocktail of protease inhibitors (Nacalai Tesque) and homogenized. The extract was centrifuged at 13000 rpm for 10 minutes at 4°C. the Obtained supernatant was used as a full cell lysate for estimation of protein concentration by the method of Lowry. Equal volumes of protein solution were separated on 12% polyacrylamide gels and proteins transferred to the membrane Immun-BlotTMPVDF. Then the membrane was incubated with anti-MCP-1 antibody (1:30; IBL Co.) as the first antibody at 4°C overnight and then incubated with anti-rabbit goat IgG (1:400; Cell Signaling) as the second antibody at room temperature for 2 hours. Express the MCP-1 was determined by chemiluminescence, using ECL (Amersham Bioscience, Buckinghamshire, U.K.). Analysis of the images on the pictures was performed using a computer program (Scion Image Frame Grabber Status).

The result showed that the expression of MCP-1 cardiac muscle was increased in the infarction area, and is not affected by the infarction area in the control group, but was significantly higher in the affected area. On the other hand, the increased expression of MCP-1 was suppressed in both zones in the group with the antibody MR16-1.

[Example 4] Echocardiography

Four weeks after creation of the model of myocardial infarction of the heart was examined echocardiographic method under anesthesia to determine end-diastolic diameter and fraction shortening (FS) of the left ventricle.

The result echocardiography four weeks after you create a model of myocardial infarction showed that end-diastolic diameter of the left ventricle in the control group was significantly increased compared with falsely operated by the group. This increase (diameter of the left ventricle) were considerably reduced with the introduction of MR16-1. In addition, although FS was reduced after myocardial infarction (created), it was greatly improved with the introduction of MR16-1 (the control group of 18.5 ± 2.9% vs. MR16-1 group of 28.5 ± 1,8%; p<0,05).

[Example 5] Histological evaluation

The hearts were removed from the mice two days after the creation of m is Delhi myocardial infarction, was fixed with 4% paraformaldehyde-phosphate buffer and then embedded in paraffin. Hearts were cut and then stained with trichrome Masson for the quantitative assessment of the degree of fibrosis of the heart and hypertrophy of cardiomyocytes on the short axis section of the heart muscle in the area not affected by the heart attack.

In the hypertrophy of cardiomyocytes and stromal fibrosis were found in areas not affected by the heart attack, in the control group. In contrast, these symptoms were suppressed in the group with the antibody MR16-1.

Industrial applicability

The development of myocardial infarction and/or the deterioration can cause complications, such as heart failure and/or induced ischemia severe arrhythmia, which increase the danger to life. The means according to the invention, intended for the treatment of myocardial infarction, means to suppress remodeling of the left ventricle after myocardial infarction and methods of treatment or prevention of myocardial infarction can suppress symptoms, complicating myocardial infarction, and can contribute to effective treatment.

The extent of remodeling of the left ventricle after myocardial infarction, as I believe, is also related to the size of the affected area and an important factor for improving health and preventing took the value of the affected myocardial area at an early stage of occurrence of myocardial infarction. In addition to complicating myocardial infarction symptoms, remodeling of the left ventricle can be suppressed by introducing means according to the invention containing the inhibitor of IL-6 as an active ingredient, a patient at an early stage of myocardial infarction.

1. For the treatment of myocardial infarction, containing the antibody that recognizes the receptor for IL-6, as an active ingredient.

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

3. The tool according to claim 1, where the antibody is an antibody against the receptor of IL-6 persons.

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

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

6. Means for suppressing remodeling of the left ventricle after myocardial infarction containing the antibody that recognizes the receptor for IL-6, as an active ingredient.

7. The tool according to claim 6, where the antibody is a monoclonal antibody.

8. The tool according to claim 6, where the antibody is an antibody against the receptor of IL-6 persons.

9. The tool according to claim 6, where the antibody is a recombinant antibody.

10. The tool according to claim 9, where the antibody is a chimeric antibody, humanitariannet antitail human antibody.

11. The tool according to claim 6, which is used for the treatment of myocardial infarction.

12. A method of treating myocardial infarction in a subject, comprising the stage of introduction of antibodies that recognize the receptor for IL-6, the subject, who had myocardial infarction.

13. The method of suppressing remodeling of the left ventricle after myocardial infarction in a subject, comprising the stage of introduction of antibodies that recognize the receptor for IL-6, the subject, who had myocardial infarction.

14. The method according to item 12 or 13, where the antibody is a monoclonal antibody.

15. The method according to item 12 or 13, where the antibody is an antibody against the receptor of IL-6 persons.

16. The method according to item 12 or 13, where the antibody is a recombinant antibody.

17. The method according to clause 16, where the antibody is a chimeric antibody, humanitariannet antibody or human antibody.

18. The use of antibodies that recognize the receptor for IL-6, to obtain funds for the treatment of myocardial infarction.

19. The use of antibodies that recognize the receptor for IL-6, to obtain the means to suppress remodeling of the left ventricle after myocardial infarction.

20. Use p or 19, where the antibody is a monoclonal antibody.

21. Use p or 19, where the antibody is an antibody against the receptor of IL-6 persons.

22. Use p or 19, where the antibody is a recombinant antibody.

23. The application of article 22, where the antibody is a chimeric antibody, humanitariannet antibody or human antibody.



 

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41 cl, 11 dwg, 162 ex

FIELD: medicine.

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17 cl, 11 tbl, 8 ex

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3 cl, 9 ex, 7 tbl

FIELD: medicine.

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39 cl, 20 dwg, 4 tbl, 8 ex

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28 cl, 3 dwg, 4 tbl, 22 ex

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20 cl, 10 tbl, 8 ex

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5 cl, 6 tbl

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11 cl, 2 ex, 1 tbl

FIELD: chemistry.

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36 cl, 8 dwg, 3 tbl, 3 ex

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19 cl, 8 dwg, 3 tbl, 4 ex

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6 tbl, 3 ex

FIELD: medicine, pharmaceutics.

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41 cl, 22 dwg, 3 tbl, 4 ex

FIELD: medicine, pharmaceutics.

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4 cl, 1 tbl, 2 dwg, 13 ex

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118 cl, 19 dwg, 12 tbl, 33 ex

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10 cl, 1 tbl, 2 dwg, 1 ex

FIELD: genetic engineering, immunology, medicine.

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EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

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