MX2008005138A - Therapeutic agent for heart disease - Google Patents

Abstract

An anti-IL-6 receptor antibody is examined with respect to its amelioration effect on the state of an obstruction area in cardiac infarction and its inhibitory effect on left ventricular remodeling after cardiac infarction occurs. As a result, it is found that, when the anti-IL-6 receptor antibody is administered, the increase in MPO activity in an obstruction area in cardiac infarction can be significantly prevented and the expression of myocardial MCP-1 can also be prevented in both an obstruction area and a non-obstruction area. Based on the findings of an ultrasonographic examination and a histological examination of the heart, it is also found that hypercardia can be prevented.

Description

AGENTS FOR CARDIOPATITIS TREATMENT Field of the Invention The present invention relates to agents for the treatment of myocardial infarction comprising an inhibitor of IL-6 as an active ingredient and to uses thereof. Furthermore, the present invention relates to agents for suppressing left ventricular remodeling post-myocardial infarction comprising an inhibitor of IL-6 as an active ingredient and to uses thereof. Background of the Invention Myocardial infarction is one of the ischemic heart diseases. It is a disorder that causes myocardial necrosis where oppression of the cardiac coronary artery occurs due to arteriosclorosis and the like, and the blood circulation of the coronary artery is dramatically reduced or stopped. The expansion and / or deterioration of the infarcted area causes complications such as heart failure and / or severe arrhythmia of the induced ischemia and increases the risk of life. As the myocardial infarction progresses, the myocardial cells in the infarcted areas die and / or migrate and are replaced with fibrous tissues such as collagen fiber. These infarcted areas lack contractility and fail to withstand the intracardiac pressure that increases with cardiac contraction and then the fibrous wall extends little. As a result, to compensate for hypofunction, there is hypertrophy of the endocardial cavity and the left ventricle is enlarged in its entirety. This phenomenon is called left ventricular remodeling and is known to further decrease cardiac function and increase the incidence and mortality rate thereafter. Therefore, to improve the prognosis of myocardial infarction, it is considered important to suppress the progression of left ventricular remodeling as early as possible, and it is desirable to develop methods of treatment that are effective. IL-6 is a cytokine called B 2 cell stimulation factor (BSF2) or β2 interferon. IL-6 was discovered as a differentiation factor involved in the activation of B lymphocyte cells (Non-Patentable Document 1), and then it was revealed to be a multifunctional cytokine that influences the function of various cells (Non-Patentable Document 2) . It was reported that IL-6 induces the maturation of lymphocytic cells T (Non-Patentable Document 3). IL-6 transmits its biological activity through two types of proteins in the cell. One of the proteins is the IL-6 receptor, which is a ligand-binding protein with which IL-6 binds, and has a molecular weight of about 80 kDa (Taga, T. et al., J. Exp. Med. (198 /) 166, 967-981; and Yamasaki, K. et al., Science (1988) 241, 825-828). In addition to a membrane-bound form that crosses and is expressed in the cell membrane, the IL-6 receptor is present as a soluble IL-6 receptor, which mainly consists of the extracellular region of the form membrane bound. The other is the gp130 membrane protein, which has a molecular weight of approximately 130 kDa and participates in the transduction of non-ligand binding signals. The biological activity of IL-6 is transmitted to the cells by the formation of the IL-6 / IL-6 receptor complex by IL-6 and the IL-6 receptor, and the subsequent binding of the complex to gp130 (Document No Patentable). Inhibitors of IL-6 are substances that inhibit the transmission of the biological activity of IL-6. So far antibodies against IL-6 (anti-IL-6 antibodies), antibodies against IL-6 receptors (anti-IL-6 receptor antibodies), antibodies against gp130 (anti-gp130 antibodies), variants of IL-6, partial peptides of IL-6 or IL-6 receptors, and the like. There are several sources describing anti-IL-6 receptor antibodies (Non-Patentable Documents 7 and 8 and Patentable Documents 1-3). A humanized antibody PM-1, which was obtained through transplantation in a human of the complementarity termination region (CDR) of the mouse PM-1 antibody (Non-Patentable Document), which is one of the anti- body antibodies. IL-6 receptor. Until now, it has been suggested that IL-6 affects the function and structure of the heart in view of the fact that it negatively affects myocontractility (Non-Patentable Document 10), that the Cardiac hypertrophy develops in mice where gp130 is constantly activated due to overexpression of IL-6 and IL-6 receptors (Non-Patentable Document 11) and so on. After a myocardial infarction, IL-6 is expressed in the left ventricle, in particular, in the border area of reperfusion in myocardial infarction (Non-Patentable Document 12), and the level of expression is related to the size of the left ventricle (LV) after myocardial infarction (Non-Patentable Document 13). Furthermore, it was reported that myocardial cells generate IL-6 under tension with little oxygen (Non-Patentable Document 14), and that the expression of cytokines in non-muscle cells during post-infarct remodeling plays a regulatory role in the changes of the extracellular matrix (Non-Patentable Document 15). In addition, with respect to the relationship between myocardial infarction and IL-6, it is reported that the JAK / STAT system activated via IL-6 acts protectively in myocardial infarction (Non-Patentable Document 16). On the other hand, it is reported that according to an experiment where they used mice with IL-6 gene knockout, the IL-6 deficiency had no effect on the size of the infarcted area, on left ventricular remodeling or the like ( Non-Patentable Document 17). As described above, the role of IL_6 in myocardial infarction and in left ventricular remodeling after myocardial infarction was unknown.

References in the prior art to the present invention are shown below. [Non-Patentable Document 1] Hirano, T. et al., Nature (1986) 324, 73-76 [Non-Patentable Document 2] Akira, S. et al., Adv. in Immunology (1993) 54, 1-78 [Non-Patentable Document 3] Lotz, M. et al., J. Exp. Med. (1988) 167, 1253-1258 [Non-Patentable Document 4] Taga, T. et al. , J. Exp. Med. (1987) 166, 967-981 [Non-Patentable Document 5] Yamasaki, K. et al., Science (1988) 241, 825-828 [Non-Patentable Document 6] Taga, T. et al. ., Cell (1989) 58, 573-581 [Non-Patentable Document 7] Novick, D. et al., Hybridoma (1991) 10, 137-146 [Non-Patentable Document 8] Huang, Y. W. et al., Hybridoma (1993) 12,621-630 [Non-Patentable Document 9] Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906 [Non-Patentable Document 10] Finkel, M. S. et al., Science (1992) 257, 387-389 [Non-Patentable Document 11] Hirota, H. et al., Proc. Nati Acad. Sci. USA (1995) 92, 4862-4866 [Non-Patentable Document 12] Gwechenberger, M. et al., Circulation (1999) 99, 546-551 [Non-Patentable Document 13] Ono, K. et al., Circulation (1998) 98, 149-156 [Non-Patentable Document 14] Yamauchi-Takihara, K. et al., Circulation ( 1995) 91, 1520-1524 [Non-Patentable Document 15] Yue, P. et al., Am. J. Physiol. (1998) 275.H250-H258 [Non-Patentable Document 16] Negoro, S. et al., Cardiovasc. Res. (2000) 47, 797-805 [Non-Patentable Document 17] Fuchs M. et al., FASEB J. (2003) 17,2118-2120 [Patentable Document 1] WO 95/09873 [Patentable Document 2] Publication of French Patent Application No. FR 2694767 [Patentable Document 3] United States Patent No. 5216128 [Patentable Document 4] WO 92/19759 Description of the Invention Problems to be Solved by the Invention Until now, it had been suggested that IL-6 was involved in myocardial infarction and in posterior ventricular remodeling. However, its precise role was not clarified. In addition, it was not revealed what kind of effect can subsequently show the administration of the inhibitor IL-6 in myocardial infarction and in left ventricular remodeling later. The present invention was made under these circumstances, and an object of the present invention is to provide agents for the treatment of myocardial infarction, comprising an inhibitor of IL-6 as an active ingredient. Moreover, the present invention provides agents for the suppression of left ventricular remodeling post-myocardial infarction, which comprises an inhibitor IL-6 as an active ingredient. In addition, other objects of the present invention comprise providing methods for the treatment of myocardial infarction and methods for suppressing left ventricular remodeling post-myocardial infarction, both of which comprise the step of administering an IL-6 inhibitor to subjects who developed a myocardial infarction. Means for solving the problems To solve the problems raised above, the inventors investigated the effects of anti-IL-6 receptor antibodies to improve the condition of an infarcted area in myocardial infarction, and in the suppression of ventricular remodeling. Left post-myocardial infarction. First, the inventors of the present invention produced model myocardial infarct mice by ligating the left anterior descending branch of male Balb / c mice. Then, 500 μg of an anti-IL-6 receptor antibody (MR16-1) was administered into the abdominal cavity of the mouse infarct model. myocardium As a result, the increase in myeloperoxidase (MPO) activity in the area of myocardial infarction was significantly suppressed. Moreover, the expression of myocardial monocyte chemoattractant protein (MCP-1) was suppressed both in the infarcted area and in the non-infarcted area of the anti-IL-6 receptor antibody of the mice administered. In addition, both echocardiographic and histological examinations revealed that cardiac hypertrophy was suppressed with the anti-IL-6 receptor antibody of the mice administered. Then, the inventors of the present discovered for the first time that it is possible to improve the condition of an infarcted area in myocardial infarction and suppress left ventricular remodeling post-myocardial infarction by administering an anti-IL-6 receptor antibody and finally complete the present invention. Specifically, the present invention provides: [1] an agent for the treatment of myocardial infarction, comprising an inhibitor of IL-6 as an active ingredient, [2] the agent of [1], wherein the inhibitor of IL-6 is an antibody that recognizes IL-6; [3] the agent of [1], wherein the IL-6 inhibitor is an antibody that recognizes the IL-6 receptor; [4] the agent of [2] or [3], wherein the antibody is a monoclonal antibody; [5] the agent of [2] or [3], wherein the antibody is an antibody against human IL-6 or a human IL-6 receptor; [6] the agent of [2] or [3], wherein the antibody is a recombinant antibody; [7] the agent of [6], wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody; [8] an agent for the suppression of left ventricular remodeling post-myocardial infarction, comprising an inhibitor of IL-6 as an active ingredient; [9] the agent of [8], wherein the inhibitor IL-6 is an antibody that recognizes IL-6; [10] the agent of [8], wherein the inhibitor IL-6 is an antibody that recognizes the IL-6 receptor; [11] the agent of [9] or [10], wherein the antibody is a monoclonal antibody; [12] the agent of [9] or [10], wherein the antibody is an antibody against human IL-6 or a human IL-6 receptor; [13] the agent of [9] or [10], wherein the antibody is a recombinant antibody; [14] the agent of [13], wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody; [15] the agent of any of [8] to [14], which is used for the treatment of myocardial infarction; [16] a method for the treatment of myocardial infarction in a subject, comprising the step of administering an IL-6 inhibitor to a subject who developed myocardial infarction; [17] a method for the suppression of left ventricular remodeling post-myocardial infarction in a subject, comprising the step of administering an IL-6 inhibitor to a subject who developed myocardial infarction; [18] the method of [16] or [17], wherein the IL-6 inhibitor is an antibody that recognizes IL-6; [19] the method of [16] or [17], wherein the IL-6 inhibitor is an antibody that recognizes an IL-6 receptor; [20] the method of [18] or [19], wherein the antibody is a monoclonal antibody; [21] the method of [18] or [19], wherein the antibody is an antibody against human IL-6 or an antibody against the human IL-6 receptor; [22] the method of [18] or [19], wherein the antibody is a recombinant antibody; [23] the method of [22], wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody; [24] the use of an IL-6 inhibitor to obtain an agent for the treatment of myocardial infarction; [25] the use of the IL-6 inhibitor to produce an agent for the suppression of left ventricular remodeling after myocardial infarction;

[26] the use of [24] or [25], wherein the IL-6 inhibitor is an antibody that recognizes IL-6; [27] the use of [24] or [25], wherein the IL-6 inhibitor is an antibody that recognizes an IL-6 receptor; [28] the use of [26] or [27], wherein the antibody is a monoclonal antibody; [29] the use of [26] or [27], wherein the antibody is an antibody against human IL-6 or an antibody against the IL-6 receptor; [30] the use of [26] or [27], wherein the antibody is a recombinant antibody; and [31] the use of [30], wherein the antibody is a chimeric antibody or a humanized antibody or a human antibody. BEST MODE FOR CARRYING OUT THE INVENTION The present inventors discovered that the improvement of the condition of the infarcted area in myocardial infarction and the suppression of left ventricular remodeling post-myocardial infarction can be achieved by administering an anti-IL-6 receptor antibody. The present invention is based on these findings. The present invention relates to agents for the treatment of myocardial infarction and agents for left ventricular remodeling post-myocardial infarction, both of which comprise an inhibitor of IL-6 as an active ingredient.

In the present documentation, an "IL-6 inhibitor" is a substance that blocks IL-6 mediated signal transduction and inhibits the biological activity of IL-6. Preferably, the IL-6 inhibitor is a substance that has an inhibitory function against the binding of IL-6, the IL-6 receptor or gp130. The IL-6 inhibitors of the present invention include, in a non-limiting sense, for example, anti-IL-6 antibodies, anti-IL-6 receptor antibodies, anti-gp 130 antibodies, IL-6 variants, soluble variants of the IL-6 receptor and partial peptides of IL-6 or IL-6 receptor, and low molecular weight compounds that exhibit similar activities. Preferred IL-6 inhibitors of the present invention include antibodies that recognize IL-6 receptors. In the present invention, the source of the antibody is not particularly restricted; however, the antibody is preferably derived from mammals, and more preferably derives from humans. The anti-IL-6 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody through known means. In particular, monoclonal antibodies derived from mammals are preferred as the anti-IL-6 antibody used in the present invention. Monoclonal antibodies derived from mammals include those produced from hybridomas and those produced from hosts transformed with an expression vector comprising a gene of antibody, according to genetic engineering methods. Upon binding to IL-6, the antibody inhibits the binding of IL-6 to an IL-6 receptor, and blocks the transmission of the biological activity of IL-6 in the cell. These antibodies include MH166 (Matsuda, T. et al., Eur.

J. Immunol. (1988) 18, 951-956), the SK2 antibody (Sato, K. et al., Transaction of the 21st Meeting of the Japanese Society of Immunology (1991) 21, 166), and the like. Basically, it is possible to prepare hybridomas that produce the anti-IL-6 antibody using known techniques, as indicated below. Specifically, these hybridomas can be prepared using IL-6 as the sensitizing antigen to perform the immunization, according to a conventional immunization method, by fusing the obtained immune cells with known progenitor cells, according to a conventional cell fusion method, and searching for cells producers of monoclonal antibodies with a conventional analysis method. More specifically, it is possible to produce anti-IL-6 antibodies as indicated below. For example, it is possible to obtain human IL-6, used as a sensitizing antigen to obtain antibodies, using the gene and / or the amino acid sequence of IL-6 described in Eur. J. Biochem. (1987) 168, 543-550; J. Immunol. (1988) 140, 1534-1541; and / or Agr. Biol. Chem. (1990) 54, 2685-2698.

After transforming an appropriate host cell with a system of known expression vectors into which a genetic sequence of IL-6 has been inserted, the desired IL-6 protein is purified by a known method, from within a host cell or from the culture supernatant. This purified IL-6 protein can be used as a sensitizing antigen. Alternatively, one fusion protein of the IL-6 protein and another protein can be used as a sensitizing antigen. The anti-IL-6 receptor antibodies used in the present invention can be obtained as polyclonal or monoclonal antibodies according to known methods. In particular, the anti-IL-6 receptor antibodies used in the present invention are preferably monoclonal antibodies derived from mammals. Monoclonal antibodies derived from mammals include those produced from hybridomas and those produced from hosts transformed with an expression vector comprising the antibody gene, according to genetic engineering methods. By binding to an IL-6 receptor, the antibody inhibits the binding of IL-6 to the IL-6 receptor and blocks the transmission of the biological activity of IL-6 in the cell. These antibodies include the MR16-1 antibody (Tamura, T. et al., Proc, Nati, Acad. Sci. USA (1993) 90, 11924-11928), the PM-1 antibody (Hirata, Y. et al., J. Immunol. (1989) 143, 2900- 2906), the antibody AUK12-20, the antibody AUK64-7 and the antibody AUK146-15 (WO 92/19759), and the like. Among them, the PM-1 antibody can be mentioned as an example of a preferred monoclonal antibody against the human IL-6 receptor, and the MR16-1 antibody can be mentioned as a preferred monoclonal antibody against the mouse IL-6 receptor. Basically, it is possible to prepare hybridomas that produce an anti-IL-6 receptor monoclonal antibody using known techniques, as indicated below. Specifically, these hybridomas can be prepared using an IL-6 receptor as a sensitizing antigen to perform the immunization, according to a conventional immunization method, by fusing the immune cells obtained with known progenitor cells, according to a conventional cell fusion method, and looking for cells producing monoclonal antibodies with a conventional analysis method. More specifically, it is possible to produce IL-6 anti-receptor antibodies as indicated below. For example, it is possible to obtain a human IL-6 receptor or a mouse IL-6 receptor used as a sensitizing antigen to obtain antibodies, using the genes and / or amino acid sequences of IL-6 receptors described in Publication. of European Patent No. EP 325474 and Japanese Patent Application Publication Kokai No. (JP-A) Hei 3-155795, respectively.

There are two types of IL-6 receptor proteins, i.e., the proteins that are expressed on the cell membrane and the proteins separated from the cell membrane (soluble IL-6 receptor) (Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676). The soluble IL-6 receptor consists essentially of the extracellular region of the IL-6 receptor bound to the cell membrane, and differs from the IL-6 receptor bound to the membrane because it lacks the transmembrane region, or because it lacks the transmembrane and intracellular. It is possible to use any IL-6 receptor as the IL-6 receptor protein, provided that it can be used to run the sensitizing antigen to produce the anti-IL-6 receptor antibody used in the present invention. After transforming an appropriate host cell with a system of expression vectors with an inserted IL-6 receptor gene sequence, the desired IL-6 receptor protein is purified by a known method, from the interior of a host cell or from the culture supernatant. This purified IL-6 receptor protein can be used as a sensitizing antigen. Alternatively, a cell expressing the IL-6 receptor, or a fusion protein of the IL-6 receptor protein, and another protein as a sensitizing antigen can be used. The anti-gp130 antibodies used in the present invention can be obtained as polyclonal or monoclonal antibodies according to known methods. In particular, the anti-gp130 antibodies used in the present invention are preferably monoclonal antibodies derived from mammals. Monoclonal antibodies derived from mammals include those produced from hybridomas and those produced from hosts transformed with an expression vector comprising the antibody gene, according to genetic engineering methods. Upon binding to gp130, the antibody inhibits the binding of gp130 to the IL-6 / IL-6 receptor complex, and blocks the transmission of the biological activity of IL-6 in the cell. These antibodies include antibody AM64 (JP-A Hei 3-219894), antibody 4B11 and antibody 2H4 (US 5571513), antibody B-S12 and antibody B-P8 (JP-A Hei 8-291199), and similar. Basically, it is possible to prepare hybridomas that produce the anti-gp130 monoclonal antibody using known techniques, as indicated below. Specifically, these hybridomas can be prepared using gp130 as the sensitizing antigen to perform the immunization, according to a conventional immunization method, by fusing the obtained immune cells with known progenitor cells, according to a conventional cell fusion method, and looking for cells that produce monoclonal antibodies with a conventional analysis method. More specifically, the monoclonal antibody can occur as indicated below. For example, it is possible to obtain gp130 used as a sensitizing antigen to obtain antibodies, using the gene and / or the amino acid sequence of gp130 described in European Patent Application Publication No. EP 411946. After transforming an appropriate host cell with a A system of known expression vectors with an inserted gp130 genetic sequence, the desired gp130 protein is purified by a known method, either from within a host cell or from the culture supernatant. This purified gp130 protein can be used as a sensitizing antigen. Alternatively, a cell expressing gp130, or a fusion protein of the gp130 protein and another protein as a sensitizing antigen can be used. Mammals that can be immunized with a sensitizing antigen are not particularly limited, but are preferably selected taking into account compatibility with the progenitor cell used for cell fusion. In general rodents are used, such as mice, rats and hamsters. The immunization of the animals with a sensitizing antigen is carried out according to known methods. For example, as a general method, it is carried out by injecting the sensitizing antigen intraperitoneally or subcutaneously in mammals. Specifically, the sensitizing antigen preferably it is diluted or suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline or the like, mixed with an appropriate amount of a general adjuvant (eg, Freund's complete adjuvant), emulsified and then it is administered several times every 4 to 21 days to a mammal. In addition, it is possible to use a suitable vehicle for immunization with a sensitizing antigen. After this immunization, an increased level of the desired antibody in serum is confirmed, and then immune cells are obtained from the mammal to effect cell fusion. Preferred immune cells for cell fusion include, in particular, spleen cells. To use mammalian myeloma cells as a progenitor cell, ie, to be able to fuse a progenitor cell with the above immune cells, various strains of cells are appropriately used, for example, P3X63Ag8.653 (Kearney, JF 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, DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276 , 269-270), F0 (from St. Groth, SF et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, IS, J. Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), and the like.

Basically, it is possible to perform cell fusion of the aforementioned immune cell and myeloma cell using known methods, for example, the method of Milstein et al. (Kohler, G. and Milstein, C, Methods Enzymol. (1981) 73, 3-46), and the like. More specifically, the cell fusion mentioned above is carried out in a general culture medium with nutrients, in the presence of an agent that favors cell fusion. For example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used as agents that promote cell fusion. In addition, to improve the efficiency of the melt, auxiliary agents, such as dimethyl sulfoxide, can be added for use as needed. The ratio between the immune cells and myeloma cells used preferably is, for example, between 1 and 10 immune cells per each myeloma cell. The culture medium used for the cell fusion mentioned above is, for example, culture medium RPMI1640 or MEM, which are suitable for the proliferation of the aforementioned myeloma cells. A general culture medium can also be used to grow this type of cells. In addition, serum supplements may be used in combination, such as fetal calf serum (FCS). For cell fusion, the fusion cells (hybridomas) of interest are formed by mixing predetermined amounts of the immune cells and the aforementioned myeloma cells previously in the culture medium mentioned above, and then adding and mixing a concentration of between 30 and 60% (w / v) of PEG solution (for example, a PEG solution with an average molecular weight of between about 1000 and 6000) preheated to approximately 37 ° C. Subsequently, it is possible to eliminate cell fusion agents and the like, which are not suitable for the growth of the hybridoma, by repeating the steps of successive addition of an appropriate culture medium and removal of the supernatant by centrifugation. The above hybridomas are selected by culturing the cells in a general selection culture medium, for example, HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture medium is continued for a sufficient period of time, generally several days to several weeks, to kill the cells that are distinct from the hybridomas of interest (unfused cells). Then a conventional limited dilution method is applied to separate and clone the hybridomas that produce the antibody of interest. In addition to the method for immunizing a non-human animal with an antigen to obtain the aforementioned hybridomas, it is possible to obtain a desired human antibody having binding activity to a desired antigen or a cell expressing a desired antigen, sensitizing a human lymphocyte with a desired antigenic protein or a cell that expresses a desired in vitro antigen, and fusing the sensitized B lymphocyte with a human myeloma cell (e.g., U266) (see Japanese Patent Application Publication Kokoku No. (J PB) Hei 1 -59878 (patent application Japanese examined and approved, published for opposition)). Moreover, it is possible to obtain a desired human antibody by administering the antigen or cell expressing the antigen to a transgenic animal that has a repertoire of human antibody genes, and then implementing the above-mentioned method (see Application Publications). of International Patents No. WO 93/12227, WO 92/0391 8, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735). Hybridomas prepared in this way, which produce monoclonal antibodies, can be subcultured in a conventional culture medium and stored in liquid nitrogen for a prolonged period. To obtain monoclonal antibodies from the aforementioned hybidomas the following methods can be used: (1) a method in which the hybridomas are cultured according to conventional methods and the antibodies are obtained as a culture supernatant; (2) a method in which the hybridomas are proliferated by administering them to a compatible mammal, and the antibodies are obtained as ascites; and similar. The first method is used to obtain antibodies with a high purity, and the second method is more suitable for the production of antibodies on a large scale. For example, the preparation of hybridomas producing anti-IL-6 receptor antibodies can be carried out according to the method described in JP-A Hei 3-139293. The preparation can be carried out according to a method comprising injecting a hybridoma producing PM-1 antibody into the abdominal cavity of a BALB / c mouse, obtaining ascites, and then purifying the PM-1 antibody from the ascites, or the method comprising culturing the hybridoma in an appropriate medium (eg, RPMI1640 medium containing 10% fetal bovine serum and 5% BM-Condimed H1 (Boehringer Mannheim), medium for SFM hybridomas (GIBCO-BRL), medium PFHM -II (GIBCO-BRL), etc.), and then the PM-1 antibody can be obtained from the culture supernatant. A recombinant antibody can be used as a monoclonal antibody of the present invention, wherein the antibody is produced by genetic recombination techniques, by cloning an antibody gene from a hybridoma, inserting the gene into an appropriate vector, and subsequent introduction of the vector into a host (see, for example, Borrebaerck, CAK and Larrick, J. W, THERAPEUTIC MONOCLONAL ANDTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD., 1990). More specifically, mRNA encoding the variable region (V) of an antibody is isolated from a cell that produces the antibody of interest, such as a hybridoma. Isolation of the mRNA can be effected by preparing total RNA according to known methods, such as the guanidine ultracentrifugation method (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299) and the AGPC method (Chomczynski, P . and col., Anal.

Biochem. (1987) 162, 156-159), and the preparation of the mRNA can be carried out with a set of elements for purifying mRNA (Pharmacia), and the like. Alternatively, the mRNA can be prepared directly using the set of elements for purifying QuickPrep mRNA (Pharmacia). The cDNA of the V region of the antibody is synthesized from the mRNA obtained using reverse transcriptase. The synthesis of the cDNA can be carried out using the set of first strand reverse transcriptase cDNA synthesis elements, and the like. In addition, to synthesize and amplify the cDNA, the method of 5'-RACE (Frohman, MA et al., Proc. Nati. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al. ., Nucleic Acids Res. (1989) 17, 2919-2932), using the set of elements 5'-AmpliFINDER RACE (Clontech) and PCR. The DNA fragment of interest is purified from the obtained PCR products, and then it is ligated with a vector DNA. A recombinant vector is then prepared using the above DNA, and it is introduced into Escherichia coli or the like, and its colonies are selected to prepare the desired recombinant vector. The nucleotide sequence of the DNA of interest is confirmed, for example, with the dideoxi method. When a DNA encoding the V region of an antibody of interest is obtained, the DNA is ligated with a DNA encoding a desired antibody constant region (region C) and inserted into an expression vector. Alternatively, the DNA encoding the V region of the antibody can be inserted into an expression vector comprising the DNA of an antibody C region. To produce an antibody that can be used in the present invention, as will be described below, the antibody gene is inserted into an expression vector, so that it is expressed under the control of the expression regulatory region, eg, a enhancer and a promoter. Then, the antibody can be expressed by transforming a host cell with this expression vector. In the present invention, to reduce the heteroantigenicity against humans and the like, artificially genetically modified recombinant antibodies can be used, for example, chimeric antibodies, humanized antibodies or human antibodies. These modified antibodies can be prepared using known methods. It is possible to obtain a chimeric antibody by binding the DNA encoding the V region of the antibody, described above, with a DNA encoding a C region of a human antibody, by inserting the DNA into an expression vector and introducing it into a host to produce it (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92/19759). This known method can be used to obtain chimeric antibodies useful for the present invention. Humanized antibodies are also known as reformed human antibodies, and are antibodies in which regions of complementarity determination (CDR) of an antibody from a mammal other than human (e.g., a mouse antibody) are transferred to the CDR of a human antibody. General methods for this genetic recombination are also known (see European Patent Application Publication No. EP 125023 and International Patent Application Publication No. WO 92/19759). More specifically, a DNA sequence is synthesized by PCR, designed so that the CDRs of a mouse antibody are linked to the framework regions (FR) of a human antibody, from several oligonucleotides that were produced in a manner that contain superimposed portions at their ends. The DNA obtained is linked to a DNA encoding a C region of a human antibody, and then inserted into an expression vector. The expression vector is introduced into a host to produce the humanized antibody (see European Patent Application Publication No. EP 239400 and International Patent Application Publication No. WO) 92/19759). The FRs of the human antibodies that are desired to bind through the CDRs are selected so that the CDRs form an appropriate antigen binding site. The amino acid (s) within the FRs of the variable regions of the antibodies can be replaced as necessary, so that all the CDRs of the human reformed antibody form an appropriate antigen binding site (Sato, K., et al., Cancer Res. (1993) 53, 851-856). The C regions of the human antibody are used for the chimeric and humanized antibodies, and include Cy. For example, C? 1, C? 2, C? 3 or C? 4 can be used. In addition, to improve the stability of the antibody or its production, it is possible to modify the C regions of the human antibody. Chimeric antibodies consist of the variable region of an antibody derived from non-human mammals and a C region derived from a human antibody; and humanized antibodies consist of the CDRs of an antibody derived from non-human mammals and framework regions and C regions derived from a human antibody. Both have a reduced antigenicity in the human body, so they are useful as antibodies for use in the present invention. Preferred specific examples of humanized antibodies used in the present invention include a humanized antibody PM-1 (see Application Publication) of International Patent No. WO 92/19759). Furthermore, in addition to the method mentioned above for obtaining the human antibody, techniques for obtaining human antibodies comprising the use of a human antibody library are also known. For example, it is possible to express the variable regions of human antibodies on the phage surface as single chain antibodies (scFv) with the phage display method, and then select the phages that bind to antigens. By analyzing the genes of the selected phages, it is possible to determine the DNA sequences encoding the variable regions of the human antibodies that bind to the antigen. Once the DNA sequence of a scFV that binds to the antigen is revealed, an appropriate expression vector comprising the sequence can be constructed in order to obtain a human antibody. These methods are known, and it is possible to use the publications of WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388 as reference. The antibody gene constructed in advance can be expressed according to conventional methods. When a mammalian cell is used, the antibody gene can be expressed using a DNA in which the antibody gene to be expressed is functionally linked to a useful common promoter, and to a poly A 3 'signal of the gene of the antibody, or to a vector comprising the DNA. The examples of a promoter / enhancer include the early promoter / enhancer of human cytomegalovirus. In addition, other promoters / enhancers that can be used to express the antibody, which will be used in the present invention, include the viral promoters / enhancers of retroviruses, polyoma viruses, adenoviruses, simian viruses (SV40) and the like; and promoters / enhancers derived from mammalian cells, such as human elongation factor 1a (HEF1a). For example, when the SV40 promoter / enhancer is used, expression can be easily performed according to the method of Mulligan et al. (Mulligan, R C. et al., Nature (1979) 277, 108-114). Alternatively, in the case of an HEF1a promoter / enhancer, the method of Mizushima et al. (Mizushíma, S. and Nagata S., Nucleic Acids Res. (1990) 18, 5322). When E. coli is used, it is possible to express the antibody gene by functionally binding a conventional useful promoter, a signal sequence to secrete the antibody and the antibody gene that is desired to be expressed. Examples of promoters include the lacZ promoter, the araB promoter and the like. When the lacZ promoter is used, expression can be effected according to 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 according to the method from Better et al. (Better, M. et al., Science (1988) 240, 1041-1043). When the antibody is produced in the periplasm of E. coli, the signal sequence pei B (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379-4383) can be used as a signal sequence to secrete the antibody. The antibody produced in the periplasm is isolated and then used after properly folding the structure of the antibody (see, for example, WO 96/30394). As origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can be used. In addition, to improve the copy number of the gene in the host cell system, the expression vector may comprise the aminoglycoside phosphotransferase (APH) gene, the thymidine kinase (TK) gene, the xanthine-guanine phosphoribosyltramferase gene of E. coli (Ecogpt), the dihydrofolate reductase (dhfr) gene, or a similar gene, as a selection marker. Any production system can be used to prepare the antibodies that will be used in the present invention. Production systems for preparing the antibodies include in vitro and in vivo production systems. In vitro production systems include those in which eukaryotic cells or prokaryotic cells are used. Production systems in which eukaryotic cells are used include those in which cells are used animals, plant cells or fungal cells. These animal cells include (1) mammalian cells, eg, CHO, COS, myeloma cells, neonatal hamster kidney cells (BHK), HeLa, Vero and the like, (2) amphibian cells, eg, oocytes from Xenopus; and (3) insect cells, for example, sf9, sf21, Tn5 and the like. Known plant cells include cells derived from Nicotiana tabac? M, which can be grown as callus. Known fungal cells include yeasts, such as Sacchcromyces (e.g., S. cerevisiae), mold fungi, such as Aspergillus (e.g., A. niger), and the like. Production systems in which prokaryotic cells are used include those in which bacterial cells are used. Known bacterial cells include E. coli and Bacillus subtilis. Antibodies can be obtained by introducing a gene of an antibody of interest into these cells by transformation, and culturing the transformed cells in vitro. The cultivation is carried out according to known methods. For example, DMEM, MEM, RPMI1640, IMDM can be used as the culture medium, and it is possible to use serum supplements, such as FCS, in combination. In addition, a cell into which an antibody gene was introduced can be transferred to the abdominal cavity, or to a similar part of an animal, to produce an antibody in vivo.

On the other hand, in vivo production systems include those in which animals or plants are used. Production systems in which animals are used include those in which mammals or insects are used. Mammals that can be used include goats, pigs, sheep, mice, bovines and the like (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). In addition, the insects that can be used include silkworms. When plants are used, for example, tobacco can be used. In these animals or plants an antibody gene is introduced, an antibody is produced in the body of animals or plants, and then recovered. For example, the antibody gene is prepared as a fusion gene by inserting the gene into the medium of a gene encoding a protein, such as goat casein, which is produced only in milk. A DNA fragment comprising the inserted fusion of the antibody gene is injected into a goat embryo, and this embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic animal that is born from the goat that received the embryo, or is produced by the progeny of this animal. To increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones can be appropriately used in the transgenic goat (Ebers, K. M. et al., Bio / Technology (1994) 12, 699-702).

Furthermore, when a silkworm is used, it is infected with baculoviruses in which the desired antibody gene was inserted, and the desired antibody is obtained from the body fluid of this silkworm (Maeda, S. et al. , Nature (1985) 315, 592-594). Moreover, when tobacco is used, the desired antibody gene is inserted into a plant expression vector (e.g., pMON530), and this vector is introduced into bacteria such as Agrobacterium tumefaciens. This bacterium is used to infect tobacco (for example, Nicotiana tabacum), in order to obtain the desired antibody from the leaves of this tobacco (Julián, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138). When an antibody is produced in in vitro or in vivo production systems as previously described, the DNAs encoding the heavy chain (H chain) and the light chain (L chain) of the antibody can be inserted into separate expression vectors, and then a guest is co-transformed with the vectors. Alternatively, the DNAs can be inserted into a single expression vector to transform a host (see International Patent Application Publication No. WO). 94/11523). The antibodies used in the present invention can be antibody fragments or modified products based thereon, so long as they can be used appropriately in the present invention. For example, fragments of antibodies include Fab, F (ab ') 2, Fv and single chain Fv (scFv), where Fv of the H and L chains are joined through an appropriate connector. Specifically, antibody fragments are produced by treating an antibody with an enzyme, for example, papain or pepsin, or alternatively, genes encoding these fragments are constructed, introduced into expression vectors and expressed in appropriate host cells ( see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better M. &Horwitz, AH, 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, RE et al., TIBTECH (1991) 9, 132-137). It is possible to obtain a scFv by joining the V region of the H chain and the V region of the L chain of an antibody. In the scFv, the V region of the H chain and the V region of the L chain are linked by a linker, preferably via a peptide linker (Huston, JS et al., Proc. Nati. Acad. Sci. USA ( 1988) 85, 5879-5883). The V regions of the H and L chains in a scFv can be derived from any of the antibodies described previously. Peptide linkers for joining the V regions include, for example, a single arbitrary peptide chain consisting of between 12 and 19 amino acid residues. It is possible to obtain a DNA encoding a scFv using as a template the DNA encoding the H chain, or its V region, and the DNA encoding the L chain, or its V region, of the aforementioned antibodies, performing the PCR amplification of the DNA portion encoding the desired amino acid sequence in the template sequence, using primers that define the ends of the portion, and then further amplifying the amplified DNA portion with a DNA encoding a peptide linker portion, with a pair of primers that binds both ends of the linker with the H chain and the L chain. Also, once a DNA was obtained that encoding a scFv, an expression vector comprising the DNA and a host transformed with the vector can be obtained according to conventional methods. In addition, the scFv can be obtained according to conventional methods using the host. In a manner similar to that described above, it is possible to produce these antibody fragments from the host by obtaining and expressing their genes. In the present documentation, an "antibody" includes these antibody fragments. An antibody bound to various molecules, such as polyethylene glycol, can also be used as the modified antibody.

(PEG). In the present documentation, an "antibody" encompasses these modified antibodies. These modified antibodies can be obtained by modifying the antibodies obtained by chemical means. These methods are already established in the art.

The antibodies produced and expressed as described above can be isolated from the interior or exterior of the cell or the host, and can be purified until they are homogeneous. The isolation and / or purification of the antibodies used in the present invention can be carried out by affinity chromatography. Columns that can be used for affinity chromatography include, for example, the protein A column and the G protein column. Vehicles used for the protein A column include, for example, H iperD, PO ROS, Sepharose F. F. and similar. In addition to the above, other methods can be used to isolate and / or purify common proteins, and these are not limited in any way. For example, the antibodies used in the present invention can be isolated and / or purified by selecting and appropriately combining chromatographies, beyond affinity chromatography, filters, ultrafiltration, desalting, dialysis and the like. Chromatographies include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration and the like. These chromatographies can be applied to a high performance liquid chromatography (HPLC). Alternatively, reverse phase HPLC can be used. The concentration of the antibodies obtained as described above can be determined with absorbency measurements, ELI SA or the like. Specifically, the absorbance is determined by diluting the solution appropriately of antibody with PBS (-), measuring the absorbance at 280 nm and calculating the concentration (1.35 OD = 1 mg / ml). Alternatively, when using ELISA, the measurement can be performed as indicated below. Specifically, 100 μl of goat anti-human IgG (TAG), diluted to 1 μg / ml with 0.1 M bicarbonate buffer (pH 9.6), is added to a 96-well plate (Nunc), and incubated overnight at 4 ° C to immobilize the antibody. After blocking, add 100 μl of an appropriately diluted antibody of the present invention, or an appropriately diluted sample comprising the antibody, and human IgG (CAPPEL) as a reference, and incubate one hour at room temperature. After washing, 100 μl of alkaline phosphatase-labeled anti-human IgG (BIO SOURCE), diluted 5000x, is added and incubated at room temperature for one hour. After performing another wash, the substrate solution is added and incubated, and the absorbance is measured at 405 nm using the model 3550 microplate reader (Bio-Rad), to calculate the concentration of the antibody of interest. The IL-6 variants used in the present invention are substances that have the binding activity to an IL-6 receptor, and that do not transmit the biological activity of IL-6. That is, IL-6 variants compete with IL-6 for binding to IL-6 receptors, but they can not transmit the biological activity of IL-6, so they block IL-6-mediated signal transduction. .

IL-6 variants are produced by introducing one or more mutations, by substituting amino acid residues in the amino acid sequence of IL-6. The origin of IL-6 used as a base for IL-6 variants is not limited; however, human IL-6 is preferable, when considering its antigenicity, and similar factors. More specifically, amino acid substitution is performed by predicting the secondary structure of the amino acid sequence of IL-6 with known molecular modeling programs (eg, WHATIF; Vriend et al., J. Mol. Graphics (1990) 8, 52 -56), and further evaluating the influence of the one or more substituted amino acid residues in the entire molecule. Once the appropriate amino acid residue that you want to replace is determined, PCR methods are commonly practiced, using the nucleotide sequence encoding the human IL-6 gene as a template for introducing mutations, in order to substitute the amino acids, thereby obtaining a gene encoding a variant of IL-6. If necessary, this gene is inserted into an appropriate expression vector, and the IL-6 variant can be obtained by applying the previously mentioned methods to express, produce and purify the recombinant antibodies. Specific examples of IL-6 variants are described in 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. The partial peptides of IL-6 and the partial peptides of IL-6 receptors to be used in the present invention are substances that exhibit binding activity to IL-6 and IL-6 receptors, respectively, and that do not transmit the activity biological activity of IL-6. Namely, by binding and capturing an IL-6 or an IL-6 receptor, the partial peptide of IL-6 or the partial peptide of the IL-6 receptor specifically inhibits the binding of IL-6 to the IL-6 receptor. 6 As a result, the biological activity of IL-6 is not transmitted, so signal transduction mediated by IL-6 is blocked. The partial peptides of IL-6 or the IL-6 receptor are peptides that comprise part or all of the amino acid sequence of the region of the amino acid sequence of IL-6 or the participating IL-6 receptor. in the binding of IL-6 and the IL-6 receptor. These peptides commonly comprise between 10 and 80, preferably between 20 and 50, more preferably between 20 and 40 amino acid residues. The partial peptides of IL-6 or the partial peptides of the IL-6 receptor can be produced according to generally known methods, for example, genetic engineering techniques or peptide synthesis methods, with specification of the region of the amino acid sequence of IL-6 or the IL-6 receptor that participates in the binding of IL-6 and the IL-6 receptor, and using a portion or all of the amino acid sequence of the specified region. When a partial peptide of IL-6 or a partial peptide of an IL-6 receptor is prepared with a genetic engineering method, a DNA sequence encoding the desired peptide is inserted into an expression vector, and then the peptide by applying the aforementioned methods to express, produce and purify recombinant antibodies. To produce a partial peptide of IL-6 or a partial peptide of an IL-6 receptor through peptide synthesis methods, general-purpose peptide synthesis methods can be used, for example, solid-phase synthesis methods or synthesis methods in liquid phase. Specifically, the synthesis can be performed according to the method described in "Continuation of Development of Pharmaceuticals, Vol 14, Peptide Synthesis (in Japanese) (edited by Haruaki Yajima, 1991, Hirokawa Shoten)". As the solid phase synthesis method, for example, the following method can be used: the amino acid corresponding to the C-terminus of the peptide that is to be synthesized is attached to a support which is insoluble in organic solvents, then the peptide chain is elongated by repeating alternately (1) the condensation reaction of the amino acids whose α-amino groups and their functional branching chain groups are protected with appropriate protecting groups, one at a time, in a direction from the C-terminus to the N-terminus; and (2) the removal reaction of the groups protectors of the a-amino groups of the amino acid or the peptide bound to the resin. Peptide synthesis in solid phase is broadly classified into the Boc method and the Fmoc method, based on the type of protecting group used. Once the protein of interest has been synthesized as previously described, the deprotection reaction and the reaction to separate the peptide chain from the support are carried out. For the separation reaction of the peptide chain, in general, hydrogen fluoride or trifluoromethane sulphonic acid is used for the Boc method, and TFA for the Fmoc method. According to the Boc method, for example, the resin with the protected peptide mentioned above is treated in hydrogen fluoride in the presence of anisole. Then, the peptide is recovered by removing the protecting group and separating the peptide from the support. Upon freeze drying of the recovered peptide, a crude peptide can be obtained. On the other hand, in the Fmoc method, for example, it is possible to carry out the deprotection reaction and the reaction to separate the peptide chain from the support in TFA, according to a method similar to that previously described. The crude peptide obtained can be separated and / or purified by carrying out a HPLC. Elution can be carried out under optimal conditions, using a water-acetonitrile solvent system, which is generally used to purify proteins. The fractions corresponding to the peaks of the obtained chromatographic profile are collected and dried by freezing Accordingly, fractions of purified peptides are identified through molecular weight analysis, mass spectrum analysis, amino acid composition analysis, amino acid sequence analysis, or the like. Specific examples of partial peptides of IL-6 and partial peptides of IL-6 receptors are described in JP-A Hei 2-188600, JP-A Hei 7-324097, JP-A Hei 8-311098, and in the Publication of U.S. Patent No. US 5210075. The antibodies used in the present invention can also be conjugated antibodies, which are attached to various molecules, such as polylene glycol (PEG), radioactive substances and toxins. These conjugated antibodies can be obtained by chemically modifying the antibodies obtained. Methods for modifying antibodies are already established in the art. The "antibodies" of the present invention encompass these conjugated antibodies. Agents for the treatment of myocardial infarction and agents for the suppression of left ventricular remodeling post-myocardial infarction in the present invention can be used for myocardial infarction treatments. In the present documentation, "treatment of myocardial infarction" means the suppression or prevention of the symptoms of myocardial infarction, and heart failure and severe arrhythmia of induced ischemia, which occur as complications of myocardial infarction. The complication of symptoms of myocardial infarction includes arrhythmia (extrasystole, ventricular fibrillation and atrioventricular block), heart failure, rupture of the papillary muscle, rupture of the heart, ventricular aneurysm (which is formed at the apex of the heart as a result of infarction in the branch anterior descending artery of the left coronary artery), and post-myocardial infarction syndrome. The "agents for the treatment of myocardial infarction" of the present invention can suppress and prevent the symptoms described above. Meanwhile, in the present documentation, the term "suppression of left ventricular remodeling post-myocardial infarction" means suppressing or preventing myocardial hypertrophy (complete left ventricular hypertrophy) that occurs to compensate for the functional weakening of the infarcted area. Myocardial hypertrophy occurs when the cardiac muscle cells in the infarcted areas are replaced with fibrous tissue such as collagen fibers as a result of necrosis and / or exfoliation of the cells and the fibrous tissue is scarce. Then, the suppression and prevention of "displacement of the infarcted area with collagen fibers" and the "fibrous tissue extension", that is, the improvement of the condition of the infarcted area, is also included in the meaning of the "suppression of ventricular remodeling". left post-infarction of myocardium "described above If the symptoms of myocardial infarction and left ventricular remodeling post-myocardial infarction are suppressed or not, it can be determined using the myeloperoxidase (MPO) activity in infarcted and non-infarcted areas as an indicator of the cardiac muscle MPO is an enzyme present in the intercellular granules of neutrophils and its activity is known to be significantly elevated due to coronary artery diseases MPO activity increases when the infarcted areas spread and aggravate (necrosis and similar forms.) That is, when the MPO activity is suppressed with the administration of an agent of the present invention, the symptoms of myocardial infarction and left ventricular remodeling post-myocardial infarction can be considered to be suppressed. The activity of the MPO can be determined by known methods, including, for example, measurement methods described in FIG. points in the Examples. Alternatively, whether or not the symptoms of myocardial infarction or left ventricular remodeling post-myocardial infarction are suppressed, can also be determined using as an indicator the expression of MCP-1 (protein-1 chemoattractant monocyte) in the infarcted and non-infarcted areas of the heart muscle. MCP-1 is a chemokine that can cause heart failure by recruiting macrophages to the heart muscle and enhancing the expression of inflammatory cytokines. HE He knows that MCP-1 activates inflammation and induces fibrosis of the cardiac muscle and perivascular tissues. The extension and / or aggravation (necrosis and similar forms) of the infarcted area increases the expression of M PC-1. Specifically, when the expression of MCP-1 is suppressed, the symptoms of myocardial infarction and left ventricular remodeling post-myocardial infarction can be considered to be suppressed. The expression of MCP-1 can be measured by known methods for the measurement of expression of proteins which include, for example, Western blot analysis and ELISA. The phrases suppressing the "activity of the M PO" and "suppressing the expression of the MC P-1" also mean "improving the condition of the infarcted area" mentioned above. Moreover, the suppression of the symptoms of myocardial infarction and left ventricular remodeling after myocardial infarction can also be determined by measuring the end-diastolic dimension of the left ventricle and the ejection fraction by echocardiography, or Quantitative evaluation of the degree of fibrosis of the myocardium and hypertrophy of myocardial cells with a histological examination of the cardiac tissues. These measurements can be achieved using known methods. These measurements include, for example, methods that are described in the Examples. In the present invention, the activity of the inhibitors of IL-6 in the inhibition of IL-6 signal transduction can be evaluated with conventional methods. Specifically, IL-6 is added to cultures of IL-6 dependent human myeloma cell lines (S6B45 and KPMM2), the human Lennert lymphoid T cell line KT3 or the IL-6 dependent MH60 cell line. BSF2; and assimilation of 3H-thymidine by the IL-6 dependent cells in the presence of an inhibitor of IL-6 is measured. Alternatively, U266 cells expressing the IL-6 receptor are cultured, 25 L-labeled IL-6 and an IL-6 inhibitor are added to the culture at the same time; and then the 125 I-labeled IL-6 bound to the cells expressing the IL-6 receptor is quantified. In addition to the IL-6 inhibitor group, a negative control group is included that does not contain the IL-6 inhibitor in the test system previously described. The activity of the inhibitor of IL-6 in the inhibition of IL-6 can be evaluated by comparing the results of both groups. As will be indicated below in the Examples, it was found that administration of an anti-IL-6 receptor antibody suppressed the symptoms of myocardial infarction and left ventricular remodeling post-myocardial infarction. This finding suggests that IL-6 inhibitors, such as anti-IL-6 receptor antibodies, are useful as agents for the treatment of myocardial infarction and as agents for suppressing left ventricular remodeling post-myocardial infarction.

The subjects who will receive the administration of the agents of the present invention for the treatment of myocardial infarction and agents of the present invention for the suppression of left ventricular remodeling after myocardial infarction are mammals. Preferably, the mammals are human beings. The agents of the present invention for the treatment of myocardial infarction and the agents of the present invention for the suppression of left ventricular remodeling post-myocardial infarction can be administered as pharmaceutical substances, and can be administered systemically or locally, by an administration oral or parenteral For example, an intravenous injection may be selected, such as a drop infusion, an intramuscular injection, an intraperitoneal injection, a subcutaneous injection, a suppository, an enema, oral enteric tablets or the like. It is possible to select an appropriate method of administration depending on the age and symptoms of the patient. The effective dose per administration is selected in the range between 0.01 and 100 mg / kg body weight. Alternatively, the dose may be selected within the range of 1 to 1000 mg / patient, preferably within the range of 5 to 50 mg / patient. A preferred dose and method of administration are as follows: , example, when an anti-L-6 receptor antibody is used, the effective dose is a quantity such that the free antibody is present. present in the blood. Specifically, a dose of 0.5 to 40 mg / kg body weight / month (four weeks), preferably 1 to 20 mg / kg body weight / month is administered by intravenous injection, such as a drip infusion, a subcutaneous injection or similar, between one and several times per month, for example, twice a week, once a week, once every two weeks or once every four weeks. The administration schedule can be adjusted, for example, by extending the administration interval from twice a week or once a week to once every two weeks, once every three weeks or once every four weeks, while monitoring the condition after of the transplant and the changes in the values of the blood test. In the present invention, agents for the treatment of myocardial infarction and agents for suppressing left ventricular remodeling post-myocardial infarction may contain pharmaceutically acceptable carriers, such as preservatives and stabilizers. "Pharmaceutically acceptable vehicles" refer to materials that can be co-administered with a previously described agent, and they themselves may or may not produce the previously described effect of suppressing the symptoms of myocardial infarction and post-infarction left ventricular remodeling of myocardium. As an alternative, vehicles can be materials that do not have the effect of suppressing the symptoms of myocardial infarction and left ventricular remodeling post-myocardial infarction, but produce an additive or synergistic stabilizing effect when used in combination with an inhibitor of IL-6. These pharmaceutically acceptable materials include, for example, sterile water, physiological saline, stabilizers, excipients, buffers, preservatives, detergents, chelating agents (EDTA and the like) and binders. In the present invention, detergents include non-ionic detergents, and typical examples thereof include sorbitan esters of fatty acids, such as sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate; glycerin esters of fatty acids, such as glycerin monocaprylate, glycerin monomiristate and glycerin monostearate; polyglycerol esters of acids, such as decaglyceryl monostearate, decaglyceryl distearate and decaglyceryl monolinoleate; polyoxyethylene sorbitan fatty acid esters, such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate and polyoxyethylene sorbitan distearate; polyoxyethylene sorbit esters of fatty acids, such as polyoxyethylene sorbit tetrastearate and polyoxyethylene sorbit tetraoleate; esters polyoxyethylene glycerin acid fatty acids, such as polyoxyethylene glyceryl monostearate; polyethylene glycol esters of fatty acids, such as polyethylene glycol distearate; polyoxyethylene alkyl esters, such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene alkyl esters, such as polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether and polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylene alkyl phenyl esters, such as polyoxyethylene nonylphenyl ether; castor oils hardened with polyoxyethylene, such as polyoxyethylene castor oil and castor oil hardened with polyoxyethylene (polyoxyethylene hydrogenated castor oil); polyoxyethylene beeswax derivatives, such as polyoxyethylene sorbit beeswax; polyoxyethylene lanolin derivatives, such as polyoxyethylene lanolin; and polyoxyethylene amides of fatty acids and the like, with a HLB value of 6 to 18, such as polyoxyethylene amide stearic acid. Detergents also include anionic detergents, and typical examples thereof include, for example, alkyl sulfates having an alkyl group with 10 to 18 carbon atoms, such as sodium cetyl sulfate, sodium lauryl sulfate, and sodium oleyl sulfate.; the polyoxyethylene alkyl ether sulfates, wherein the alkyl group has 10 to 18 carbon atoms, and wherein the average molar amount of ethylene oxide added is from 2 to 4, such as sodium polyoxyethylene lauryl sulfate; salts of alkyl sufosuccinate esters that have a nyl group with 8 to 1 8 carbon atoms, such as the sodium lauryl sulfosuccinate ester; natural detergents, for example, lecithin; the glycerophospholipids; sphingospholipids, such as sphingomyelin; and the esters of sucrose and fatty acids, in which the fatty acids have 12 to 18 carbon atoms. One, two or more of the detergents described above may be combined, and may be added to the agents of the present invention. Detergents that are preferably used in the preparations of the present invention include polyoxyethylene sorbitan fatty acid esters, such as polysorbates 20, 40, 60 and 80. Polysorbates 20 and 80 are particularly preferred. Also preferred are polyoxyethylene polyoxypropylene glycols, such as poloxamer (Pluronic F-68® and the like). The amount of detergent added varies depending on the type of detergent used. When polysorbate 20 or 80 is used, the amount in general is in the range of 0.001 to 1000 mg / ml, preferably in the range between 0.003 and 50 mg / ml, more preferably, in the range between 0.005 and 2 mg / ml . In the present invention, the buffers include phosphate, citrate buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, capric acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid and other organic acids, and carbonic acid buffer, Tris buffer, histidine buffer and imidazole buffer. Liquid preparations can be formulated by dissolving the agents in aqueous buffers known in the field of liquid preparations. The concentration of the buffer is generally in the range of 1 to 500 mM, preferably in the range of 5 to 100 mM, more preferably in the range of 10 to 20 mm. The agents of the present invention may also comprise other low molecular weight polypeptides; proteins such as serum albumin, gelatin and immunoglobulin; amino acids; sugars and carbohydrates, such as polysaccharides and monosaccharides, sugar alcohols and the like. In the present documentation, the amino acids include basic amino acids, for example, arginine, lysine, histidine and ornithine, and inorganic salts of these amino acids (preferably hydrochloride salts and phosphate salts, namely, phosphate amino acids). When free amino acids are used, the pH is adjusted to a preferred value by adding buffer substances suitable for physiological use, for example, inorganic acids, in particular, hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid and formic acid, and salts of these. In this case, the use of phosphate is particularly beneficial, because it provides fairly stable products Dry them by freezing. The phosphate is particularly advantageous when preparations are made which substantially do not contain organic acids, such as malic acid, tartaric acid, citric acid, succinic acid and fumaric acid, or which do not contain the corresponding anions (malate ion, tartrate ion, citrate ion, ion succinate, fumarate ion and the like). The preferred amino acids are arginine, lysine, histidine and ornithine. In addition, it is possible to use acidic amino acids, for example, glutamic acid and aspartic acid, and salts thereof (preferably salts of amino acids); neutral amino acids, for example, isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine and alanine; and aromatic amino acids, for example, phenylalanine, tyrosine, tryptophan and its derivative, N-acetyl tryptophan. In the present documentation, 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 documentation, the sugar alcohols include, for example, mannitol, sorbitol and inositol. When the agents of the present invention are prepared as aqueous solutions for injection, the agents can be mixed, for example, with physiological saline and / or with an isotonic solution containing glucose or other auxiliary agents (such as D-sorbitol, D- mannose, D-mannitol and chloride of sodium). The aqueous solutions can be used in combination with suitable solubilizing agents, such as alcohols (ethanol and the like), polyalcohols (propylene glycol, PEG and the like), or non-ionic detergents (polysorbate 80 and HCO-50). In addition, if necessary, the agents may comprise diluents, solubilizers, substances for adjusting the pH, softening agents, reducing agents containing sulfur, antioxidants and the like. In the present documentation, sulfur-containing reducing agents include, for example, compounds comprising sulfhydryl groups, such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodolyol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione and thioalkanoic acids having between 1 and 7 carbon atoms. Moreover, antioxidants in the present invention include, for example, erythorbic acid, dibutylhydroxy toluene, butylhydroxy anisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbic acid palmitate, acid stearate L-ascorbic, sodium bisulfite, sodium sulfite, thiamyl gallate, propyl gallate, and chelating agents, such as disodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate. If necessary, the agents can be encapsulated in microcapsules (microcapsules of hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid] or the like), or they can be prepared as colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules and the like) (see "Remington's Pharmaceutical Science", 16th edition, edited by Oslo, 1980 , and similar). In addition, methods for preparing agents as sustained release agents are also known, and can be applied to the present invention (Langer et al., J. Biomed, Mater. Res. 1981, 15: 167-277; Langer, Chem Tech. 1992, 12: 98-105; US Patent No. 3773919; Patent Application European No. (EP) 58481; Sidman et al., Biopolymers 1983, 22: 547-556; and EP 133988). The pharmaceutically acceptable vehicles used are appropriately selected from those previously described, or are combined depending on the type of dosage form, in a non-limiting sense those. The present invention relates to methods for the treatment of myocardial infarction in subjects and methods for suppressing left ventricular remodeling post-myocardial infarction, both of which comprise the step of administering an IL-6 inhibitor to subjects who developed infarction of myocardium. In the present documentation, the "subject" refers to organisms and body parts of the organisms to which it is administered with an agent of the present invention for the treatment of myocardial infarction or an agent of the present invention to suppress left ventricular remodeling post-myocardial infarction. Organisms include animals (eg, humans, species of domestic animals and wild animals), but are not particularly limited to them. The "body parts of the organisms" are not particularly limited, but preferably include heart, heart muscle and infarcted and non-infarcted areas in myocardial infarcts. In the present documentation, "administration" includes oral and parenteral administrations. Oral administration includes, for example, the administration of oral agents. These oral agents include, for example, granule, powder, tablet, capsule, solution, emulsion and suspension. Parenteral administration includes, for example, the administration of injections. These injections include, for example, subcutaneous injections, intramuscular injections and intraperitoneal injections. Meanwhile, the effects of the methods of the present invention can be achieved by introducing genes comprising the oligonucleotides that it is desired to administer in living bodies, using gene therapy techniques. Alternatively, the agents of the present invention can be administered locally in the desired treatment areas. For example, the agents of the present invention can be administered through a local injection during the surgery, with the use of catheters, or resorting to direct administration of genes with DNA encoding a peptide of the present invention. The agents of the present invention can be administered in conjunction with treatment for the occurrence of myocardial infarctions, for example, catheter surgery (percutaneous transluminal coronary angioplasty (PTCA) and percutaneous coronary intervention (PCI), percutaneous transluminal coronary recanalization (PTCA)). Coronary artery bypass graft (IBAC) and the like When the methods of the present invention are put into practice, the agents of the present invention can be administered as part of pharmaceutical compositions, in combination with at least one known chemotherapeutic agent. Alternatively, the agents of the present invention can be administered in combination with at least one known immunosuppressant In one embodiment, the agents of the present invention and known chemotherapeutics can be practically administered at the same time.

All references of the prior art cited herein are incorporated by reference. EXAMPLES Next, the present invention will be specifically described with reference to the Examples, but should not be construed as being limited thereto.

Example 1 Preparation of a mouse model of myocardial infarction Male Balb / c mice (25 to 30g) were intubated through the trachea. The mice were on an artificial respirator and anesthetized by inhalation of 0.5 to 1.0% isoflurane. The right breast was opened. After ligating the left anterior descending coronary artery, the chest was closed. The mice were grouped in a group administered MR16-1 (group MR16-1) and a group without treatment (control group). The group MR16-1 was administered MR16-1 intraperitoneally in a dose of 500 μg / body. Example 2 Measurement of MPO activity The hearts of the mice were removed two days after the creation of myocardial infarctions (or coronary ligature). The hearts were divided into infarcted area and non-infarcted area, and were crushed. Then, the crushed heart muscle was combined with 10 volumes of 50 mM KPO4 buffered (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide. The crushed muscle was homogenized (POLYTRON, KINEMATICAAG, Lucerne, Switzerland) and then subjected to ultrasound treatment. The resulting extract was centrifuged at 13000 rpm for 10 minutes at 4 ° C. After mixing 50 μl of the resulting supernatant with 1.45 ml of substrate solution (50 mM KPO4 (pH 6.0), o-dianisidine dihydrochloride 0.167 mg / ml, and H2O2 0.005%) the changes in the color of the substrate solution by absorbance at 460 nm (extinction coefficient = 2,655). As a result, the MPO activity of the heart muscle showed no difference between the non-infarcted cardiac muscle and the cardiac muscle of the sham group but increased significantly, approximately four times, in the infarcted area (safety control 0.037 ± 0.006, risk control 0.122 ± 0.035; p <0.01). Meanwhile, the increase in MPO activity in the infarcted area was significantly suppressed in the group to which MR16-1 was administered (MR16-1-risk 0.034 ± 0.008, p <0.05 vs. risk control). EXAMPLE 3 Assay of MCP-1 Expression The hearts of the mice were removed two days after the creation of myocardial infarction. Hearts were divided into infarcted area and non-infarcted area and crushed. The crushed heart muscle was combined with the lysis buffer (2x PBS, NP-40 1%, sodium deoxycholate 0.5%, sodium dodecyl sodium hydroxide 0.1%, 1 mM PMSF, cocktail of protease inhibitor (Nacalai Tesque) 1%), and then homogenized. The resulting extract was centrifuged at 13000 rpm for 10 minutes at 4 ° C. The resulting supernatant was used as a total cell count to quantify the protein concentration by the Lowry method. Equal volumes of the protein solution were separated on a 12% polyacrylamide gel and the proteins were transferred to a Immun-BlotTM PVDF membrane. The membrane was then incubated with an anti-MCP-1 antibody (1:30, IBL Co.) as the primary antibody at 4 ° C overnight, and subsequently incubated with goat anti-rabbit IgG (1: 400; Signaling) as a secondary antibody at room temperature for 2 h. Expression of MCP-1 was detected by chemiluminescence using ECL (Amarsham Bioscience, Buckinghamshire, R.U.). The image analysis of the photograph was made using a computer program (Scion Image Frame Grabber Status). The results showed that MCP-1 cardiac muscle expression increased in both the infarcted area and in the non-infarcted area of the control group, but was much higher in the infarcted area. On the other hand, the increase in the expression of MCP-1 was suppressed in both areas in the group to which MR16-1 was administered. Example 4 Echocardiography Four weeks after the myocardial infarction was created, the hearts were examined with echocardiography under anesthesia to determine the end-diastolic diameter of the left ventricle and the fractional shortening (AF). The result of echocardiography four weeks after the creation of myocardial infarction showed that the end-diastolic diameter of the left ventricle in the control group increased significantly compared to the group simulated. This increase (in the left ventricular diameter) was significantly suppressed by the administration of MR16-1. Moreover, while the AF was reduced after myocardial infarction (generated), with the administration of MR 16-1 it improved significantly (control group 18.5 ± 2.9% vs. group MR16-1 28.5 ± 1.8%, p <0.05 ). Example 5 Histological evaluation The hearts of the mice were removed four weeks after the creation of myocardial infarction, fixed with 4% paraformaldehyde in phosphate buffer and then embedded in paraffin. The hearts were sectioned and then stained with Masson's trichrome to quantitatively evaluate the degree of cardiac muscle fibrosis and hypertrophy of the cardiac myocytes in the short axis section of the heart muscle in the non-infarcted area. Cardiac myocyte hypertrophy and stromal fibrosis were found as a result in the non-infarcted area in the control group. These symptoms, in contrast, were suppressed in the group to which MR16-1 was administered. Industrial Applicability The extension and / or aggravation of myocardial infarction can induce a complication of heart failure and / or severe arrhythmia induced by ischemia which increases the threat to life. The agents of the present invention for treating infarction at Myocardium, the agents to suppress left ventricular remodeling after myocardial infarction, and methods to treat or prevent myocardial infarction, can suppress the complication of myocardial infarct symptoms and achieve effective treatment. The index of involvement of left ventricular remodeling after myocardial infarction is also suggested to be related to the size of the area that suffered the infarction, and it is considered important to improve the condi- tion and prevent the area that suffered the Infarction increases to an early stage of the onset of myocardial infarction. In addition to complicating the symptoms of myocardial infarction, ventricular remodeling can be suppressed by administering an agent of the present invention comprising an inhibitor I L-6 as an active ing network to the patient with an early stage of myocardial infarction.

Claims (10)

1. An agent for the treatment of myocardial infarction, comprising an inhibitor IL-6 as an active ingredient.

2. The agent of claim 1, wherein the inhibitor IL-6 is an antibody that recognizes IL-6.

3. The agent of claim 1, wherein the IL-6 inhibitor is an antibody that recognizes the IL-6 receptor.

4. The agent of claim 2 or 3, wherein the antibody is a monoclonal antibody. The agent of claim 2 or 3, wherein the antibody is a human anti-IL-6 antibody or a human IL-6 receptor. 6. The agent of claim 2 or 3, wherein the antibody is a recombinant antibody. 7. The agent of claim 6, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. 8. An agent for the suppression of left ventricular remodeling post-myocardial infarction, comprising an inhibitor IL-6 as an active ingredient. 9. The agent of claim 8, wherein the inhibitor of IL-6 is an antibody that recognizes IL-6. 10. The agent of claim 8, wherein the IL-6 inhibitor is an antibody that recognizes the IL-6 receptor. eleven . The agent of claim 9 or 10, wherein the antibody is a monoclonal antibody. The agent of claim 9 or 10, wherein the antibody is a human anti-IL-6 antibody or a human IL-6 receptor. The agent of claim 9 or 10, wherein the antibody is a recombinant antibody. The agent of claim 13, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. 1

5. The agent of any of claims 8 to 14, which is used for the treatment of myocardial infarction. 1

6. A method for the treatment of myocardial infarction in a subject, comprising the step of administering an IL-6 inhibitor to a subject who developed myocardial infarction. 1

7. A method for the suppression of left ventricular remodeling post-myocardial infarction in a subject, comprising the step of administering an IL-6 inhibitor to a subject who developed myocardial infarction. 1

8. The method of claim 16 or 17, wherein the inhibitor of IL-6 is an antibody that recognizes IL-6. The method of claim 16 or 17, wherein the IL-6 inhibitor is an antibody that recognizes an IL-6 receptor. The method of claim 18 or 19, wherein the antibody is a monoclonal antibody. twenty-one . The method of claim 18 or 19, wherein the antibody is an anti-IL-6 antibody or an anti-human IL-6 receptor antibody. 22. The method of claim 18 or 19, wherein the antibody is a recombinant antibody. 23. The method of claim 22, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. 24. Use of an IL-6 inhibitor to produce an agent for the treatment of myocardial infarction. 25. Use of the IL-6 inhibitor to produce an agent for the suppression of left ventricular remodeling post-myocardial infarction. 26. The use of claim 24 or 25, wherein the inhibitor of IL-6 is an antibody that recognizes IL-6. 27. The use of claim 24 or 25, wherein the inhibitor of IL-6 is an antibody that recognizes an IL-6 receptor. 28. The use of claim 26 or 27, wherein the antibody is a monoclonal antibody. 2

9. The use of claim 26 or 27, wherein the antibody is an antibody against human IL-6 or an antibody against the human IL-6 receptor. 30. The use of claim 26 or 27, wherein the antibody is a recombinant antibody. 31 The use of claim 30, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.