Peripartum cardiomyopathy treatment

Abstract

The present invention provides a pharmaceutical composition for use in the treatment or prevention of peripartum cardiomyopathy, the pharmaceutical composition comprising an IL-6 inhibitor as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating or preventing peripartum cardiomyopathy, and a method for treating and preventing peripartum cardiomyopathy.

Background Art

[0002] Dilated cardiomyopathy is a group of diseases characterized by (1) myocardial systolic dysfunction and (2) dilation of the left ventricular cavity among "idiopathic" cardiomyopathies. It is a disease with poor prognosis and progression characterized by chronic heart failure symptoms and repeated acute exacerbations, and sudden death due to lethal arrhythmia or arterial thromboembolism may also occur. Similar diseases that cause "left ventricular dilation" and "left ventricular systolic dysfunction" exist as in dilated cardiomyopathy, and specific cardiomyopathies with clear causes are diagnosed as secondary cardiomyopathies, distinguished from idiopathic (primary) dilated cardiomyopathy (Non-Patent Document 1).

[0003] Peripartum cardiomyopathy (PPCM, puerperal cardiomyopathy), known as secondary cardiomyopathy, is characterized by women without a history of heart disease and no other apparent cause of heart failure developing heart failure during pregnancy or the puerperium and presenting a pathological condition similar to dilated cardiomyopathy. More than half of peripartum cardiomyopathy patients normalize, but about 40% have residual myocardial function decline, and severe cases are fatal. Pregnancy and childbirth are considered to be involved in the onset and progression of this disease (Non-Patent Document 1). Reports on the treatment methods and pathological studies of peripartum cardiomyopathy have been published (Non-Patent Documents 2 and 3).

[0004] Interleukin-6 (IL-6) is a cytokine also known as B cell stimulatory factor 2 (BSF2) or interferon-β2. IL-6 was discovered as a differentiation factor involved in the activation of B lymphocyte lineage cells (Non-Patent Document 4), and later it became clear that it is a multifunctional cytokine that affects the functions of various cells (Non-Patent Document 5). It has been reported that IL-6 induces the maturation of T lymphocyte lineage cells (Non-Patent Document 6).

[0005] IL-6 transmits its biological activity through two proteins on the cell surface. One is the IL-6 receptor, a ligand-binding protein with a molecular weight of approximately 80 kD to which IL-6 binds (Non-Patent Documents 7 and 8). The IL-6 receptor exists not only as a membrane-bound type that penetrates the cell membrane and is expressed on the cell membrane, but also as a soluble IL-6 receptor consisting mainly of its extracellular region.

[0006] The other is the membrane protein gp130 with a molecular weight of approximately 130 kD that is involved in non-ligand-binding signal transduction. IL-6 and the IL-6 receptor form an IL-6 / IL-6 receptor complex, which then binds to gp130, thereby transmitting the biological activity of IL-6 into the cell (Non-Patent Document 9).

[0007] Research has been conducted on the relationship between IL-6 and various diseases. For example, reports have been made on the relationship between IL-6 and cardiac hypertrophy and the application of IL-6 inhibitors to the treatment of heart diseases (Non-Patent Documents 10 to 12, and Patent Documents 1 to 3)

[0008] Atrial Natriuretic Peptide (ANP) is a type of peptide consisting of 28 amino acids with biological activity. It is mainly biosynthesized and stored in the atrium and secreted into the blood as needed. ANP has vasodilatory and diuretic effects and controls circulatory homeostasis through a common receptor with Brain Natriuretic Peptide (BNP), NPR1 (Natriuretic Peptide Receptor 1). ANP and BNP have already been widely clinically applied as diagnostic and therapeutic agents for heart failure.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

[0010] [Non-Patent Document 1] Issued by the Japanese Circulation Society / Japanese Heart Failure Society, Guidelines for the Diagnosis and Treatment of Myocardial Disease (Revised Edition 2018); [Non-Patent Document 2] Bhattacharyya, A., Tex Heart Inst J. 2012; 39(1): 8-16. [Non-Patent Document 3] Kurdi, M., Front Immunol. 2018; 9: 3029 [Non-Patent Document 4] Hirano, T. et al., Nature (1986) 324, 73-76 [Non-Patent Document 5] Akira, S. et al., Adv. in Immunology (1993) 54, 1-78 [Non-Patent Document 6] Lotz, M. et al., J. Exp. Med. (1988) 167, 1253-1258 [Non-Patent Document 7] Taga, T. et al., J. Exp. Med. (1987) 166, 967-981 [Non-Patent Document 8] Yamasaki, K. et al., Science (1988) 241, 825-828 [Non-Patent Document 9] Taga, T. et al., Cell (1989) 58, 573-581 [Non-Patent Document 10] Shimizu I., J Mol Cell Cardiol. 2016; 97: 245-262 [Non-Patent Document 11] Chou, C.H. et al., Cardiovascular Research (2018) 114, 690-702 [Non-Patent Document 12] Kang, Y.M. et al., Circ Res. 2006, 99758-766. [Disclosure of the Invention] [Problems to be Solved by the Invention]

[0011] Both the maternal circulatory system and hormonal balance dynamically change during pregnancy, childbirth, and the postpartum period. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) produced by the heart control circulatory homeostasis via their common receptor, NPR1. To clarify the physiological and pathophysiological roles of the endogenous ANP / BNP-NPR1 system during the peripartum period, the phenotypes of Npr1 knockout mice during the peripartum period were examined, with a particular focus on maternal heart weight, blood pressure, and cardiac function. As a result, it was found that Npr1 knockout mice exhibited severe cardiac hypertrophy accompanied by peripartum cardiomyopathy-like fibrosis, and that Npr1 knockout mice serve as a model of postpartum cardiomyopathy (PPCM) without pregnancy-induced hypertension. Hereinafter, Npr1 knockout mice may be referred to as Npr1 - / - mice, and wild-type mice may be referred to as Npr1 + / + mice. Furthermore, upon further examination, the inventors confirmed an increase in interleukin-6 (IL-6) mRNA expression (hereinafter, the mRNA corresponding to IL-6 may be referred to as Il6) in the hearts of Npr1 - / - mice during or after lactation, and found that administration of an anti-IL-6 receptor antibody decreased cardiac hypertrophy in lactating Npr1 - / - mice, thus completing the present invention. The present invention provides the following compositions, drugs, methods, and uses.

[0012] [1-1] A pharmaceutical composition for use in the treatment or prevention of peripartum cardiomyopathy, containing an IL-6 inhibitor as an active ingredient. [1-2] The pharmaceutical composition according to [1-1], for use in a subject after childbirth or during lactation. The pharmaceutical composition according to [1-1] or [1-2], wherein the IL-6 inhibitor is an antibody that recognizes IL-6. The pharmaceutical composition according to [1-1] or [1-2], wherein the IL-6 inhibitor is an antibody that recognizes the IL-6 receptor. The pharmaceutical composition according to [1-3] or [1-4], wherein the antibody is a monoclonal antibody. The pharmaceutical composition according to any one of [1-3] to [1-5], wherein the antibody is an antibody against human IL-6 or an antibody against human IL-6 receptor. The pharmaceutical composition according to any one of [1-3] to [1-6], wherein the antibody is a recombinant antibody. The pharmaceutical composition according to any one of [1-3] to [1-7], wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. The pharmaceutical composition according to any one of [1-3] to [1-8], wherein the antibody is tocilizumab, satralizumab or sarilumab.

[0013] A pharmaceutical composition for use in suppressing or improving cardiac remodeling associated with peripartum cardiomyopathy, containing an IL-6 inhibitor as an active ingredient. The pharmaceutical composition according to [2-1], for use in cardiac remodeling in a subject after childbirth or during lactation. The pharmaceutical composition according to [2-1] or [2-2], wherein the IL-6 inhibitor is an antibody that recognizes IL-6. The pharmaceutical composition according to [2-1] or [2-2], wherein the IL-6 inhibitor is an antibody that recognizes the IL-6 receptor. The pharmaceutical composition according to [2-3] or [2-4], wherein the antibody is a monoclonal antibody. The pharmaceutical composition according to any one of [2-3] to [2-5], wherein the antibody is an antibody against human IL-6 or an antibody against human IL-6 receptor. The pharmaceutical composition according to any one of [2-3] to [2-6], wherein the antibody is a recombinant antibody. The pharmaceutical composition according to any one of [2-3] to [2-7], wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. The pharmaceutical composition according to any one of [2-3] to [2-8], wherein the antibody is tocilizumab, satralizumab or sarilumab. The pharmaceutical composition according to any one of [2-1] to [2-9], for use in the treatment or prevention of peripartum cardiomyopathy.

[0014] [3-1] An inhibitor of the IL-6 signaling pathway in the heart of a peripartum mother via the mineralocorticoid receptor, containing an IL-6 inhibitor as an active ingredient. The inhibitor according to [3-1], for use in a subject after childbirth or during lactation. The inhibitor according to [3-1] or [3-2], wherein the IL-6 inhibitor is an antibody that recognizes IL-6. The inhibitor according to [3-1] or [3-2], wherein the IL-6 inhibitor is an antibody that recognizes the IL-6 receptor. The inhibitor according to [3-3] or [3-4], wherein the antibody is a monoclonal antibody. The inhibitor according to any one of [3-3] to [3-5], wherein the antibody is an antibody against human IL-6 or an antibody against the human IL-6 receptor. The inhibitor according to any one of [3-3] to [3-6], wherein the antibody is a recombinant antibody. The inhibitor according to any one of [3-3] to [3-7], wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. The pharmaceutical composition according to any one of [3-3] to [3-8], wherein the antibody is tocilizumab, satralizumab or sarilumab. The inhibitor according to any one of [3-3] to [3-9], for use in the treatment of peripartum cardiomyopathy after childbirth or during lactation.

[0015] A method for treating or preventing peripartum cardiomyopathy, the method comprising administering an IL-6 inhibitor to a subject in need of said treatment or prevention. [4-2] The method according to [4-1], for treating or preventing peripartum cardiomyopathy that develops after childbirth or during lactation. [4-3] A method for suppressing or improving cardiac remodeling associated with peripartum cardiomyopathy, the method comprising administering an IL-6 inhibitor to a subject in need of said suppression or improvement. [4-4] The method according to [4-3], for suppressing or improving cardiac remodeling that occurs after childbirth or during lactation. [4-5] A method for inhibiting the IL-6 signaling pathway in the heart of a peripartum mother via the mineralocorticoid receptor, the method comprising administering an IL-6 inhibitor to a subject in need of said inhibition. [4-6] The method according to [4-5], for inhibiting the IL-6 signaling pathway in a subject after childbirth or during lactation. [4-7] The method according to any one of [4-1] to [4-6], wherein the IL-6 inhibitor is an antibody that recognizes IL-6. [4-8] The method according to any one of [4-1] to [4-6], wherein the IL-6 inhibitor is an antibody that recognizes the IL-6 receptor. [4-9] The method according to either [4-7] or [4-8], wherein the antibody is a monoclonal antibody. [4-10] The method according to any one of [4-7] to [4-9], wherein the antibody is an antibody against human IL-6 or an antibody against the human IL-6 receptor. [4-11] The method according to any one of [4-7] to [4-10], wherein the antibody is a recombinant antibody. [4-12] The method according to any one of [4-7] to [4-11], wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. [4-13] The method according to any one of [4-7] to [4-12], wherein the antibody is tocilizumab, satralizumab or sarilumab.

[0016] [5-1] Use of an IL-6 inhibitor in the manufacture of a medicament for use in the treatment or prevention of peripartum cardiomyopathy. Use of an IL-6 inhibitor in the manufacture of a medicament for suppressing or improving cardiac remodeling associated with peripartum cardiomyopathy. Use of an IL-6 inhibitor in the manufacture of a medicament for suppressing the IL-6 signaling pathway in the heart of a peripartum mother via the mineralocorticoid receptor. Use according to any one of [5-1] to [5-3], wherein the medicament is used for a peripartum subject. Use according to any one of [5-1] to [5-4], wherein the IL-6 inhibitor is an antibody that recognizes IL-6. Use according to any one of [5-1] to [5-4], wherein the IL-6 inhibitor is an antibody that recognizes the IL-6 receptor. Use according to any one of [5-1] to [5-6], wherein the antibody is a monoclonal antibody. Use according to any one of [5-1] to [5-7], wherein the antibody is an antibody against human IL-6 or an antibody against the human IL-6 receptor. Use according to any one of [5-1] to [5-8], wherein the antibody is a recombinant antibody. Use according to any one of [5-1] to [5-9], wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. Use according to any one of [5-1] to [5-10], wherein the antibody is tocilizumab, satralizumab or sarilumab.

Advantages of the Invention

[0017] The present invention provides a therapeutic or prophylactic agent for peripartum cardiomyopathy, particularly a therapeutic and prophylactic agent for peripartum cardiomyopathy during the postpartum or lactation period.

Brief Description of the Drawings

[0018]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 2G

Figure 2H

Figure 2I

Figure 2J

Figure 2K

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 3F

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4E

Figure 4F

Figure 4G

Mode for Carrying Out the Invention

[0019] The present invention relates to a therapeutic or prophylactic agent for peripartum cardiomyopathy containing an IL-6 inhibitor as an active ingredient.

[0020] In the present invention, the "IL-6 inhibitor" is a substance that blocks signal transduction by IL-6 and inhibits the biological activity of IL-6. The IL-6 inhibitor is preferably a substance having an inhibitory effect on the binding of any of IL-6, the IL-6 receptor, and gp130.

[0021] Examples of the IL-6 inhibitor of the present invention include, but are not particularly limited to, anti-IL-6 antibody, anti-IL-6 receptor antibody, anti-gp130 antibody, IL-6 variant, soluble IL-6 receptor variant, or partial peptide of IL-6 or IL-6 receptor, and low molecular weight substances showing the same activity as these. As the IL-6 inhibitor of the present invention, preferably, an antibody that recognizes the IL-6 receptor can be mentioned. The origin of the antibody in the present invention is not particularly limited, but preferably, it is derived from a mammal, and more preferably, an antibody derived from a human can be mentioned.

[0022] The anti-IL-6 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-IL-6 antibody used in the present invention, particularly, a monoclonal antibody derived from a mammal is preferable. Examples of the monoclonal antibody derived from a mammal include those produced by hybridomas and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. By binding to IL-6, this antibody inhibits the binding of IL-6 to the IL-6 receptor and blocks the intracellular transmission of the biological activity of IL-6. Examples of such antibodies include MH166 (Matsuda, T. et al., Eur. J. Immunol. (1988) 18, 951-956) and SK2 antibody (Sato, K. et al., Proceedings of the 21st General Meeting of the Japanese Society for Immunology, Academic Record (1991) 21, 166), etc.

[0023] The anti-IL-6 antibody-producing hybridoma can be prepared basically using known techniques as follows. That is, using IL-6 as a sensitizing antigen, immunize it according to a normal immunization method, fuse the obtained immune cells with known parent cells by a normal cell fusion method, and screen for monoclonal antibody-producing cells by a normal screening method to prepare it.

[0024] Specifically, the anti-IL-6 antibody can be prepared as follows. For example, human IL-6 used as the sensitizing antigen for antibody acquisition can be obtained by using the IL-6 gene / amino acid sequence disclosed in Eur. J. Biochem (1987) 168, 543-550, J. Immunol. (1988) 140, 1534-1541, or Agr. Biol. Chem. (1990) 54, 2685-2688.

[0025] After inserting the gene sequence of IL-6 into a known expression vector system and transforming an appropriate host cell, the target IL-6 protein can be purified from the host cell or the culture supernatant by a known method, and this purified IL-6 protein can be used as the sensitizing antigen. Alternatively, a fusion protein of IL-6 protein and another protein can be used as the sensitizing antigen.

[0026] The anti-IL-6 receptor antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-IL-6 receptor antibody used in the present invention, monoclonal antibodies derived from mammals are particularly preferred. Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. This antibody binds to the IL-6 receptor, inhibits the binding of IL-6 to the IL-6 receptor, and blocks the intracellular transmission of the biological activity of IL-6.

[0027] Examples of such antibodies include the MR16-1 antibody (Tamura, T. et al. Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928), the PM-1 antibody (Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906), the AUK12-20 antibody, the AUK64-7 antibody, or the AUK146-15 antibody (International Patent Application Publication No. WO 92-19759). Among these, the PM-1 antibody is exemplified as a preferred monoclonal antibody against the human IL-6 receptor, and the MR16-1 antibody is mentioned as a preferred monoclonal antibody against the mouse IL-6 receptor).

[0028] Anti-IL-6 receptor monoclonal antibody-producing hybridomas can basically be prepared by using known techniques as follows. That is, the IL-6 receptor is used as a sensitizing antigen, immunized according to a normal immunization method, the resulting immune cells are fused with known parent cells by a normal cell fusion method, and monoclonal antibody-producing cells are screened by a normal screening method).

[0029] Specifically, to produce an anti-IL-6 receptor antibody, the following steps can be taken. For example, the human IL-6 receptor used as the sensitizing antigen for antibody acquisition can be obtained by using the IL-6 receptor gene / amino acid sequence disclosed in European Patent Application Publication No. EP 325474, and the mouse IL-6 receptor can be obtained by using the IL-6 receptor gene / amino acid sequence disclosed in Japanese Patent Application Publication No. JP-A-3-155795).

[0030] The IL-6 receptor protein exists in two types: one that is expressed on the cell membrane and the other that is detached from the cell membrane (soluble IL-6 receptor) (Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676). The soluble IL-6 receptor is substantially composed of the extracellular region of the IL-6 receptor bound to the cell membrane, and is different from the membrane-bound IL-6 receptor in that it lacks the transmembrane region or both the transmembrane region and the intracellular region. Any IL-6 receptor may be used as long as it can be used as the immunizing antigen for the production of the anti-IL-6 receptor antibody used in the present invention.

[0031] After inserting the gene sequence of the IL-6 receptor into a known expression vector system to transform an appropriate host cell, the target IL-6 receptor protein can be purified from the host cell or the culture supernatant by a known method, and this purified IL-6 receptor protein can be used as the immunizing antigen. Also, cells expressing the IL-6 receptor or a fusion protein of the IL-6 receptor protein and another protein may be used as the immunizing antigen.

[0032] The anti-gp130 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-gp130 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferred. Examples of monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by hosts transformed with an expression vector containing an antibody gene by genetic engineering techniques. By binding to gp130, this antibody inhibits the binding of the IL-6 / IL-6 receptor complex to gp130 and blocks the transmission of the biological activity of IL-6 into the cell. Examples of such antibodies include the AM64 antibody (Japanese Patent Laid-Open No. 3-219894), the 4B11 antibody and the 2H4 antibody (US5571513), the B-S12 antibody and the B-P8 antibody (Japanese Patent Laid-Open No. 8-291199), and the like.

[0033] Hybridomas producing anti-gp130 monoclonal antibodies can basically be prepared by using known techniques as follows. That is, gp130 is used as the sensitizing antigen, immunized according to the usual immunization method, the obtained immune cells are fused with known parent cells by the usual cell fusion method, and monoclonal antibody-producing cells are screened by the usual screening method.

[0034] Specifically, to produce monoclonal antibodies, the following steps can be taken. For example, gp130 used as the sensitizing antigen for antibody acquisition can be obtained by using the gp130 gene / amino acid sequence disclosed in European Patent Application Publication No. EP 411946.

[0035] After inserting the gene sequence of gp130 into a known expression vector system to transform an appropriate host cell, the target gp130 protein can be purified from the host cell or the culture supernatant by a known method, and this purified gp130 protein can be used as the sensitizing antigen. Also, cells expressing gp130 or a fusion protein of gp130 protein and other proteins can be used as the sensitizing antigen.

[0036] The mammal immunized with the sensitizing antigen is not particularly limited, but it is preferably selected considering the compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, etc. are used.

[0037] To immunize an animal with the sensitizing antigen, it is carried out according to a known method. For example, as a general method, it is carried out by injecting the sensitizing antigen into the abdominal cavity or subcutaneous tissue of a mammal. Specifically, the sensitizing antigen diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, etc. is preferably mixed with an appropriate amount of a normal adjuvant such as Freund's complete adjuvant as desired, emulsified, and administered to the mammal several times every 4 - 21 days. Also, an appropriate carrier can be used during sensitizing antigen immunization.

[0038] After immunizing in this way and confirming that the desired antibody level has increased in the serum, immune cells are taken out from the mammal and subjected to cell fusion. Preferred immune cells to be subjected to cell fusion include, in particular, spleen cells.

[0039] Mammalian myeloma cells as the other parental cells to be fused with the immune cells are already various known cell lines, 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), FO (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., which can be appropriately used.

[0040] The cell fusion of the immune cells and myeloma cells can basically be carried out according to known methods, for example, the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3 - 46), etc.

[0041] More specifically, the cell fusion is carried out, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ), etc. are used, and furthermore, an auxiliary agent such as dimethyl sulfoxide can be added and used as desired to enhance the fusion efficiency.

[0042] The usage ratio of immune cells to myeloma cells is preferably, for example, 1 to 10 times the immune cells relative to the myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, which are suitable for the growth of the myeloma cell line, and other normal culture media used for this type of cell culture can be used. Furthermore, a serum supplement such as fetal calf serum (FCS) can also be used in combination.

[0043] For cell fusion, a predetermined amount of the immune cells and myeloma cells are well mixed in the culture medium, and a PEG solution pre-warmed to about 37°C, for example, a PEG solution with an average molecular weight of about 1000 to 6000, is usually added at a concentration of 30 to 60% (w / v) and mixed, whereby the target fusion cells (hybridomas) are formed. Subsequently, an appropriate culture medium is sequentially added, and the operation of centrifuging and removing the supernatant is repeated to remove cell fusion agents and the like that are not favorable for the growth of hybridomas.

[0044] The hybridomas are selected by culturing them in a normal selection culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). The culture in the HAT culture medium continues for a time sufficient for cells other than the target hybridomas (non-fused cells) to die, usually for several days to several weeks. Then, the normal limiting dilution method is carried out, and screening and cloning of the hybridomas that produce the target antibody are performed.

[0045] In addition to immunizing animals other than humans with an antigen to obtain the above-mentioned hybridoma, human lymphocytes can be sensitized in vitro with a desired antigen protein or antigen-expressing cells, and the sensitized B lymphocytes can be fused with human myeloma cells, such as U266, to obtain a desired human antibody having a binding activity to the desired antigen or antigen-expressing cells (see Japanese Patent Publication No. Sho 61-59878). Further, an antigen or antigen-expressing cells may be administered to a transgenic animal having a repertoire of human antibody genes, and a desired human antibody may be obtained according to the above-described method (see International Patent Application Publication Nos. WO 93 / 12227, WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO96 / 34096, WO96 / 33735). The hybridoma producing the monoclonal antibody thus prepared can be subcultured in a normal culture medium and can be stored in liquid nitrogen for a long period of time.

[0046] To obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a normal method and obtained as a culture supernatant thereof, or the hybridoma is administered to a compatible mammal for growth and obtained as ascites thereof. The former method is suitable for obtaining a high-purity antibody, while the latter method is suitable for mass production of the antibody.

[0047] For example, the production of a hybridoma producing an anti-IL-6 receptor antibody can be carried out by the method disclosed in Japanese Patent Laid-Open No. Hei 3-139293. A method of injecting a PM-1 antibody-producing hybridoma into the abdominal cavity of a BALB / c mouse to obtain ascites and purifying the PM-1 antibody from the ascites, or culturing the hybridoma in a suitable medium, such as RPMI1640 medium containing 10% fetal bovine serum and 5% BM-Condimed H1 (manufactured by Boehringer Mannheim), hybridoma SFM medium (manufactured by GIBCO-BRL), PFHM-II medium (manufactured by GIBCO-BRL), etc., and purifying the PM-1 antibody from the culture supernatant can be carried out.

[0048] In the present invention, as the monoclonal antibody, a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing this into a host, and using genetic recombination technology can be used (see, for example, Borrebaeck C. A. K. and Larrick J. W. THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0049] Specifically, mRNA encoding the variable (V) region of the antibody is isolated from cells that produce the target antibody, for example, hybridomas. Isolation of mRNA is carried out by known methods, for example, preparing total RNA by the guanidine ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294 - 5299), the AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156 - 159), etc., and preparing mRNA using an mRNA Purification Kit (manufactured by Pharmacia), etc. Also, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

[0050] Using reverse transcriptase, cDNA of the antibody V region is synthesized from the obtained mRNA. The synthesis of cDNA can be carried out using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit or the like. Also, for the synthesis and amplification of cDNA, the 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and the 5'-RACE method using PCR (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) can be used. The target DNA fragment is purified from the obtained PCR product and ligated to vector DNA. Furthermore, a recombinant vector is created therefrom, introduced into Escherichia coli or the like, and colonies are selected to prepare the desired recombinant vector. The nucleotide sequence of the target DNA is confirmed by a known method, for example, the dideoxy method.

[0051] Once DNA encoding the V region of the target antibody is obtained, it is ligated to DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector. Alternatively, DNA encoding the V region of the antibody may be incorporated into an expression vector containing DNA of the antibody C region.

[0052] To produce the antibody used in the present invention, as described below, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, host cells are transformed with this expression vector, and the antibody can be expressed.

[0053] In the present invention, genetically modified antibodies, for example, chimeric antibodies, humanized antibodies, human antibodies, which are artificially modified for the purpose of reducing heterologous antigenicity against humans or the like, can be used. These modified antibodies can be produced using known methods.

[0054] Chimeric antibodies can be obtained by ligating the DNA encoding the antibody V region obtained as described above to the DNA encoding the human antibody C region, incorporating this into an expression vector, introducing it into a host, and producing it (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92-19759). Using this known method, chimeric antibodies useful in the present invention can be obtained.

[0055] Humanized antibodies, also referred to as reshaped human antibodies or humanized antibodies, are those in which the complementarity-determining regions (CDRs) of non-human mammals, such as mouse antibodies, are transplanted into the complementarity-determining regions of human antibodies, and their general genetic recombination techniques are also known (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92-19759).

[0056] Specifically, a DNA sequence designed to ligate the CDRs of a mouse antibody to the framework region (FR) of a human antibody is synthesized by the PCR method from several oligonucleotides prepared to have overlapping portions at the ends. The resulting DNA is ligated to the DNA encoding the human antibody C region, then incorporated into an expression vector, introduced into a host, and produced (see European Patent Application Publication No. EP 239400, International Patent Application Publication No. WO 92-19759).

[0057] The FR of the human antibody ligated via the CDR is selected such that the complementarity-determining region forms a good antigen-binding site. If necessary, amino acids in the framework region of the variable region of the antibody may be substituted so that the complementarity-determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0058] For chimeric antibodies and humanized antibodies, a human antibody C region is used. Examples of the human antibody C region include Cγ, and for example, Cγ1, Cγ2, Cγ3, or Cγ4 can be used. Also, the human antibody C region may be modified to improve the stability of the antibody or its production.

[0059] A chimeric antibody consists of the variable region of an antibody derived from a non-human mammal and the C region derived from a human antibody, and a humanized antibody consists of the complementarity-determining region of an antibody derived from a non-human mammal and the framework region and C region derived from a human antibody. Since their antigenicity in the human body is reduced, they are useful as the antibodies used in the present invention.

[0060] A preferred specific example of the humanized antibody used in the present invention is the humanized PM-1 antibody (see International Patent Application Publication No. WO 92-19759). In addition to the methods described above for obtaining human antibodies, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the variable region of a human antibody can be expressed on the surface of a phage by the phage display method as a single-chain antibody (scFv), and phages that bind to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is clarified, an appropriate expression vector containing the sequence can be prepared to obtain a human antibody. These methods are already well-known, and reference can be made to WO92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, WO 95 / 15388.

[0061] The antibody gene constructed as described above can be expressed by a known method. When mammalian cells are used, it can be expressed by a commonly used useful promoter, the antibody gene to be expressed, DNA in which a polyA signal is functionally linked downstream of the 3'-side thereof, or a vector containing the same. For example, as the promoter / enhancer, the human cytomegalovirus immediate early promoter / enhancer can be mentioned.

[0062] In addition, as other promoter / enhancers that can be used for antibody expression in the present invention, virus promoters / enhancers such as retrovirus, polyomavirus, adenovirus, simian virus 40 (SV40), etc., or promoters / enhancers derived from mammalian cells such as human elongation factor 1α (HEF1α) can be used.

[0063] For example, when using the SV40 promoter / enhancer, it can be easily carried out according to the method of Mulligan et al. (Mulligan, R. C. et al., Nature (1979) 277, 108-114), and when using the HEF1α promoter / enhancer, it can be easily carried out according to the method of Mizushima et al. (Mizushima, S. and Nagata, S. Nucleic Acids Res. (1990) 18, 5322).

[0064] In the case of Escherichia coli, a useful promoter commonly used, a signal sequence for antibody secretion, and an antibody gene to be expressed can be functionally linked and expressed. For example, as the promoter, the lacZ promoter and the araB promoter can be mentioned. When using the lacZ promoter, 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) can be followed. When using the araB promoter, the method of Better et al. (Better, M. et al. Science (1988) 240, 1041-1043) can be followed.

[0065] As the signal sequence for antibody secretion, when producing in the periplasm of Escherichia coli, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379-4383) can be used. After separating the antibody produced in the periplasm, the structure of the antibody is appropriately refolded and used (for example, see WO96 / 30394).

[0066] As the origin of replication, those derived from SV40, polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can be used. Furthermore, for gene copy number amplification in the host cell line, the expression vector can contain, as a selectable marker, the aminoglycoside phosphotransferase (APH) gene, the thymidine kinase (TK) gene, the Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene, etc.

[0067] For the production of the antibody used in the present invention, any production system can be used. Production systems for antibody production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

[0068] When using eukaryotic cells, there are production systems that use animal cells, plant cells, or fungal cells. Examples of animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, etc., (2) amphibian cells such as Xenopus oocytes, or (3) insect cells such as sf9, sf21, Tn5, etc. As plant cells, cells derived from Nicotiana tabacum are known, and these can be cultured as callus. As fungal cells, yeast such as those belonging to the genus Saccharomyces, for example Saccharomyces cerevisiae, filamentous fungi such as those belonging to the genus Aspergillus, for example Aspergillus niger, etc. are known. When using prokaryotic cells, there are production systems that use bacterial cells. Examples of bacterial cells include Escherichia coli and Bacillus subtilis.

[0069] The target antibody gene is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain the antibody. The culture is carried out according to known methods. For example, as the culture medium, DMEM, MEM, RPMI1640, IMDM can be used, and serum supplements such as fetal calf serum (FCS) can also be used in combination. Alternatively, the cells into which the antibody gene has been introduced can be transferred into the abdominal cavity of an animal, etc., to produce the antibody in vivo.

[0070] On the other hand, in vivo production systems include production systems that use animals and production systems that use plants. When using animals, there are production systems that use mammals, insects, etc. As mammals, goats, pigs, sheep, mice, cows, etc. can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Also, as insects, silkworms can be used. When using plants, for example, tobacco can be used.

[0071] An antibody gene is introduced into these animals or plants to produce and recover the antibody in the body of the animal or plant. For example, the antibody gene is inserted into the middle of a gene encoding a protein that is specifically produced in milk, such as goat β-casein, to prepare a fusion gene. A DNA fragment containing the fusion gene into which the antibody gene has been inserted 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 goat or its offspring born from the goat that received the embryo. Hormones may be appropriately used for the transgenic goat in order to increase the amount of milk containing the desired antibody produced by the transgenic goat (Ebert, K.M. et al., Bio / Technology (1994) 12, 699-702).

[0072] Also, when using silkworms, a baculovirus into which the target antibody gene has been inserted is used to infect the silkworm, and the desired antibody is obtained from the body fluid of this silkworm (Maeda, S. et al., Nature (1985) 315, 592-594). Furthermore, when using tobacco, the target antibody gene is inserted into a plant expression vector, such as pMON530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. This bacterium is used to infect tobacco, such as Nicotiana tabacum, and the desired antibody is obtained from the leaves of this tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

[0073] When producing an antibody in an in vitro or in vivo production system as described above, the DNA encoding the antibody heavy chain (H chain) or light chain (L chain) may be separately incorporated into an expression vector to co-transform the host, or alternatively, the DNA encoding the H chain and L chain may be incorporated into a single expression vector to transform the host (see International Patent Application Publication No. WO 94-11523).

[0074] The antibodies used in the present invention may be full-length antibodies, antibody fragments, or their modified products, as long as they can be suitably used in the present invention. "Full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains". "Full-length antibody heavy chain" is a polypeptide consisting of, in the direction from the N-terminus to the C-terminus, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3) (abbreviated as VH-CH1-HR-CH2-CH3). "Full-length antibody light chain" is a polypeptide consisting of, in the direction from the N-terminus to the C-terminus, an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) (abbreviated as VL-CL). The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). The two full-length antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain, and between the hinge regions of the full-length antibody heavy chains. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. Examples of antibody fragments include, for example, Fab, F(ab') 2 , Fv, or single-chain Fv (scFv) in which the Fv of the H chain and L chain are linked by an appropriate linker.

[0075] Specifically, an antibody is treated with an enzyme, such as papain or pepsin, to generate antibody fragments, or a gene encoding these antibody fragments is constructed, introduced into an expression vector, and then 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, 663-66; Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

[0076] An scFv is obtained by linking the H-chain V region and the L-chain V region of an antibody. In this scFv, the H-chain V region and the L-chain V region are linked via a linker, preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H-chain V region and the L-chain V region in the scFv may be derived from any of those described as the above antibodies. As the peptide linker for linking the V regions, for example, any single-stranded peptide consisting of 12-19 amino acid residues is used.

[0077] The DNA encoding the scFv is obtained by using as a template the DNA encoding the H chain of the antibody or the DNA encoding the V region of the H chain, and the DNA encoding the L chain or the DNA encoding the V region of the L chain, amplifying the DNA portion encoding the desired amino acid sequence among those sequences by the PCR method using a primer pair that defines both ends thereof, and then further amplifying in combination with the DNA encoding the peptide linker portion and a primer pair that defines both ends thereof to be ligated to the H chain and the L chain, respectively.

[0078] Once the DNA encoding the scFv is prepared, an expression vector containing the same and a host transformed with the expression vector can be obtained according to conventional methods, and the scFv can be obtained according to conventional methods using the host. The genes of these antibody fragments can be obtained and expressed in the same manner as described above, and can be produced by a host. The "antibody" referred to in the present invention also includes these antibody fragments.

[0079] As a modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used. The "antibody" referred to in the present invention also includes these antibody modifications. To obtain such an antibody modification, it can be obtained by chemically modifying the obtained antibody. These methods have already been established in this field.

[0080] The antibody produced and expressed as described above can be separated from inside and outside the cells and purified to homogeneity from the host. The separation and purification of the antibody used in the present invention can be carried out by affinity chromatography. Examples of the column used for affinity chromatography include a protein A column and a protein G column. Examples of the carrier used for the protein A column include HyperD, POROS, Sepharose F.F., etc. In addition, separation and purification methods usually used for ordinary proteins may be used, and there is no limitation whatsoever.

[0081] For example, by appropriately selecting and combining chromatography other than the above affinity chromatography, filters, ultrafiltration, salting out, dialysis, etc., the antibody used in the present invention can be separated and purified. Examples of chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, etc. These chromatographies can be applied to HPLC (High performance liquid chromatography). Also, reverse phase HPLC may be used.

[0082] The concentration of the antibody obtained above can be measured by measuring absorbance or by ELISA or the like. That is, in the case of measuring absorbance, after appropriately diluting with PBS(-), the absorbance at 280 nm is measured, and 1 mg / ml is calculated as 1.35 OD. Also, in the case of ELISA, it can be measured as follows. That is, 100 μl of goat anti-human IgG (manufactured by TAG) diluted to 1 μg / ml with 0.1 M bicarbonate buffer (pH 9.6) is added to a 96-well plate (manufactured by Nunc) and incubated overnight at 4°C to immobilize the antibody. After blocking, 100 μl of the antibody used in the present invention or a sample containing the antibody appropriately diluted, or human IgG (manufactured by CAPPEL) as a standard is added and incubated at room temperature for 1 hour.

[0083] After washing, 100 μl of alkaline phosphatase-labeled anti-human IgG (manufactured by BIO SOURCE) diluted 5000-fold is added and incubated at room temperature for 1 hour. After washing, the substrate solution is added and incubated, and then the absorbance at 405 nm is measured using a MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate the concentration of the target antibody.

[0084] The IL-6 variant used in the present invention is a substance that has binding activity with the IL-6 receptor and does not transmit the biological activity of IL-6. That is, the IL-6 variant binds competitively to the IL-6 receptor with IL-6, but does not transmit the biological activity of IL-6, thus blocking signal transduction by IL-6.

[0085] The IL-6 variant is prepared by introducing mutations by substituting amino acid residues in the amino acid sequence of IL-6. The IL-6 serving as the basis for the IL-6 variant may be of any origin, but preferably human IL-6 in consideration of antigenicity and the like.

[0086] Specifically, it is carried out by predicting the secondary structure of the amino acid sequence of IL-6 using a known molecular modeling program, for example, WHATIF (Vriend et al., J. Mol. Graphics (1990) 8, 52-56), and further evaluating the influence on the entire amino acid residue to be substituted. After determining appropriate substituted amino acid residues, a gene encoding an IL-6 variant is obtained by introducing mutations so that the amino acids are substituted by the usual PCR method using a vector containing the nucleotide sequence encoding the human IL-6 gene as a template. This can be incorporated into an appropriate expression vector as needed, and an IL-6 variant can be obtained according to the methods for expression, production, and purification of the recombinant antibody.

[0087] Specific examples of the IL-6 variant are disclosed in Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, and Savino et al., EMBO J. (1994) 13, 1357-1367, WO96-18648, WO96-17869. The IL-6 partial peptide or IL-6 receptor partial peptide used in the present invention is a substance that has the binding activity to the IL-6 receptor or IL-6, respectively, and does not transmit the biological activity of IL-6. That is, the IL-6 partial peptide or IL-6 receptor partial peptide binds to the IL-6 receptor or IL-6 and specifically inhibits the binding of IL-6 to the IL-6 receptor by capturing them. As a result, since it does not transmit the biological activity of IL-6, it blocks the signal transduction by IL-6.

[0088] The IL-6 partial peptide or the IL-6 receptor partial peptide is a peptide consisting of a part or all of the amino acid sequence of the region involved in the binding of IL-6 and the IL-6 receptor in the amino acid sequence of IL-6 or the IL-6 receptor. Such a peptide usually consists of 10 to 80, preferably 20 to 50, more preferably 20 to 40 amino acid residues.

[0089] The IL-6 partial peptide or the IL-6 receptor partial peptide can be prepared by a commonly known method, such as a genetic engineering technique or a peptide synthesis method, based on a part or all of the amino acid sequence of the region identified as being involved in the binding of IL-6 and the IL-6 receptor in the amino acid sequence of IL-6 or the IL-6 receptor.

[0090] To prepare the IL-6 partial peptide or the IL-6 receptor partial peptide by genetic engineering techniques, a DNA sequence encoding the desired peptide can be incorporated into an expression vector and obtained according to the methods for the expression, production, and purification of the recombinant antibody.

[0091] To prepare the IL-6 partial peptide or the IL-6 receptor partial peptide by peptide synthesis methods, methods commonly used in peptide synthesis, such as solid-phase synthesis methods or liquid-phase synthesis methods, can be used.

[0092] Specifically, it may be carried out according to the method described in Volume 14 of the Development of Follow-up Pharmaceuticals, Peptide Synthesis, edited by Haruaki Yajima, Hirokawa Shoten, 1991. As the solid-phase synthesis method, for example, an amino acid corresponding to the C-terminus of the peptide to be synthesized is bound to a support insoluble in an organic solvent, and amino acids with their α-amino groups and side-chain functional groups protected by appropriate protecting groups are condensed one by one in the order from the C-terminus to the N-terminus, and the reaction of detaching the protecting group of the α-amino group of the amino acid or peptide bound on the resin is alternately repeated to extend the peptide chain. The solid-phase peptide synthesis method is roughly classified into the Boc method and the Fmoc method according to the type of protecting group used.

[0093] After synthesizing the target peptide in this way, a deprotection reaction and a cleavage reaction of the peptide chain from the support are carried out. For the cleavage reaction with the peptide chain, hydrogen fluoride or trifluoromethanesulfonic acid can usually be used in the Boc method, and TFA can usually be used in the Fmoc method. In the Boc method, for example, the protected peptide resin is treated in hydrogen fluoride in the presence of anisole. Then, the peptide is recovered by elimination of the protecting group and cleavage from the support. By freeze-drying this, a crude peptide is obtained. On the other hand, in the Fmoc method, for example, a deprotection reaction and a cleavage reaction of the peptide chain from the support can be carried out by the same operation as above in TFA.

[0094] The obtained crude peptide can be separated and purified by applying it to HPLC. For its elution, it may be carried out under optimal conditions using a water-acetonitrile solvent system commonly used for protein purification. Fractions corresponding to the peaks of the obtained chromatographic profile are collected and freeze-dried. The thus-purified peptide fraction is identified by molecular weight analysis by mass spectrometry, amino acid composition analysis, or amino acid sequence analysis, etc. Specific examples of the IL-6 partial peptide and the IL-6 receptor partial peptide are disclosed in JP-A-2-188600, JP-A-7-324097, JP-A-8-311098, and US Patent Publication US5210075.

[0095] The antibody used in the present invention may be a conjugate antibody bound to various molecules such as polyethylene glycol (PEG), radioactive substances, toxins, etc. Such a conjugate antibody can be obtained by subjecting the obtained antibody to a chemical modification. Note that the method for modifying the antibody has already been established in this field. The "antibody" in the present invention also includes these conjugate antibodies.

[0096] In the present invention, the IL-6 inhibitor is preferably an anti-IL-6 receptor antibody, and specific examples include tocilizumab, sarilumab, satralizumab, etc.

[0097] The therapeutic or prophylactic agent for peripartum cardiomyopathy and the inhibitor of cardiac remodeling associated with peripartum cardiomyopathy of the present invention can be used in the treatment of cardiomyopathy. "Peripartum cardiomyopathy" means heart failure that develops in women without a history of heart disease during pregnancy and the puerperium, showing a pathological condition similar to dilated cardiomyopathy, but is differentiated as a different disease from dilated cardiomyopathy. In the WHO definition and classification of cardiomyopathy, peripartum cardiomyopathy is classified as secondary cardiomyopathy. According to the definition of the American Heart Association (AHA), peripartum cardiomyopathy (puerperal cardiomyopathy) is regarded as an acquired type among primary cardiomyopathies (those with main lesions in the myocardium). In heart failure, as the disease progresses, left ventricular dilation, decreased contractility, and myocardial fibrosis occur, and this change is called "cardiac remodeling". "Heart failure" refers to a state in which the heart's function is insufficient, gradually causing palpitations, shortness of breath, fatigue, ankle swelling, etc., and if left untreated, eventually becoming dyspneic and unable to lie down. The degree and speed of progression vary from person to person, but generally worsen over time and become refractory. Sudden onset or rapid changes are not uncommon, and eventually, due to oxygen deficiency, arrhythmia and other diseases may occur, leading to a dangerous state of death.

[0098] In the present invention, "treatment or prevention of peripartum cardiomyopathy" means treating or preventing the decline in cardiac function caused by peripartum cardiomyopathy, as well as treating or preventing the acute heart failure symptoms (such as dyspnea, cough, edema, general fatigue, palpitations, shock, disturbance of consciousness, etc.) and chronic heart failure symptoms (such as shortness of breath during exertion, edema, palpitations, etc.) of peripartum cardiomyopathy.

[0099] In the present invention, "inhibition or improvement of cardiac remodeling" means stopping the progression of left ventricular dilation, decreased contractility, and myocardial fibrosis associated with the progression of the disease stage of heart failure, or improving these states compared with before drug administration.

[0100] The mineralocorticoid receptor (MR) is a receptor that has an equivalent affinity for mineralocorticoids (aldosterone) and glucocorticoids (cortisol), and is expressed in many tissues such as the kidney, colon, heart, central nervous system (hippocampus), brown adipose tissue, and sweat glands. In the present invention, the "IL-6 signaling pathway in the heart of the peripartum maternal body via the neuronal mineralocorticoid receptor" means a pathway in which the concentration of cardiac plasma aldosterone increases in the heart of the peripartum maternal body, the concentration of IL-6 in the heart tissue increases due to the activation of the central nervous system MR, and as a result, myocardial damage is caused.

[0101] In this specification, peripartum cardiomyopathy is diagnosed, for example, as new-onset heart failure symptoms within 5 months after delivery from the late pregnancy in women without a history of heart disease and without any other identifiable cause of heart failure. Examples of such symptoms include a decrease in the left ventricular ejection fraction (EF), a dilated cardiomyopathy-like condition such as less than 45%, and an increase in the blood concentration of brain natriuretic peptide (BNP). Peripartum cardiomyopathy can be diagnosed, for example, based on the measurement of BNP, chest Xp / CT, echocardiogram, and electrocardiogram examination. Furthermore, it can be diagnosed based on the results of cardiac CT, cardiac MRI, coronary angiography, myocardial biopsy, etc. to exclude other cardiomyopathies.

[0102] Delivery may be natural or by cesarean section. Natural delivery is roughly divided into three stages. In the first stage, contractions occur, the cervix gradually opens, and the fetus moves into the vagina. In the second stage, the fetus is delivered, and in the third stage, the placenta is delivered. "After delivery" refers to the time when the third stage of delivery is completed, and "after childbirth" refers to the time when the fetus is delivered by "after delivery" or cesarean section. In the present invention, the subjects to be treated include, for example, women from the late pregnancy (after 22 weeks of pregnancy) to within 5 months after childbirth. In one aspect of the present invention, women in the lactation period after childbirth are targeted. "Peripartum" refers to the period from 22 weeks of pregnancy to less than 7 days after birth, and "puerperium" refers to the period after childbirth until the mother recovers, usually 6 to 8 weeks after childbirth. "Lactation period" refers to the period from after childbirth until weaning, during which the infant is breastfed, which is about one year after birth. In one aspect of the present invention, women in the lactation period are targeted, and here, women in the lactation period include, regardless of whether they are actually breastfeeding or not, for example, women who have stopped breastfeeding during treatment. Also, in one aspect of the present invention, women within 5 months after delivery are included as subjects to be treated in the present invention.

[0103] As described below, the inventors have found that after delivery, particularly in the lactation period, hypertrophic changes occur in the mother's heart. In one aspect of the present invention, by targeting women in the above lactation period, it is possible to suppress the onset or exacerbation of peripartum cardiomyopathy during the lactation period. In another aspect of the present invention, treatment can also be continued after 5 months after delivery for chronic symptoms of peripartum cardiomyopathy. Another aspect of the present invention can be preferably used particularly when breastfeeding is continued even after 5 months after delivery.

[0104] As mentioned in the examples of this specification, NPR1, which is a receptor for ANP / BNP, is considered to be involved in the dynamic changes of the heart during pregnancy, delivery, and the period after delivery, Npr1 - / - Mice can be used as model animals for peripartum cardiomyopathy. Npr1 - / -The predicted mechanism of action for cardiac hypertrophy in mice is shown in Figure 1. When the ANP / BNP-NPR1 system is normal, the maternal heart develops reversible hypertrophy, accompanied by enhanced phosphorylation of ERK1 / 2 (extracellular signal-regulated kinase) protein. The mRNA expression of Nppa, Nppb, and Acta1 significantly increases during lactation. These hypertrophic changes in the maternal heart are thought to be due to increased plasma aldosterone levels and increased IL-6 production in the heart. However, when the ANP / BNP-NPR1 system is deficient, it causes excessive cardiac hypertrophy similar to peripartum cardiomyopathy, accompanied by fibrosis, significant cardiac dysfunction, and activation of the calcineurin-transcription factor (Nuclear factor of activated T-cells: NFAT) pathway in the heart during late pregnancy. These changes in the maternal heart are likely caused by a significant increase in plasma aldosterone and significant activation of the IL-6-dependent pathway in the heart. These results imply that the ANP / BNP-NPR1 system protects the maternal heart from lactation-induced cardiac remodeling. In Figure 1, MR indicates the mineralocorticoid receptor.

[0105] In the present invention, treatment by administration of an IL-6 inhibitor can be carried out in combination with other treatments. Examples of other treatments include, for example, drug therapy (administration of ACE inhibitors, angiotensin receptor blockers, β-blockers, diuretics, bromocriptine, etc.), and particularly in acute cases, artificial respiration management, intra-aortic balloon pumping (IABP), cardiopulmonary assist devices (PCPS, V-A bypass, ECMO), and the like.

[0106] In the present invention, administration of an IL-6 inhibitor can be used for the treatment of a subject diagnosed with peripartum cardiomyopathy. Further, in the present invention, administration of an IL-6 inhibitor can be used for preventing the exacerbation of the heart failure state of a subject diagnosed with peripartum cardiomyopathy. In one aspect of the present invention, administration of an IL-6 inhibitor can be performed as a prophylactic measure during the next pregnancy of a woman who was diagnosed with peripartum cardiomyopathy during the previous pregnancy. Here, the subjects of the prophylactic measure include, but are not limited to, women whose cardiac function has normalized by the time of the next pregnancy.

[0107] The IL-6 signal transduction inhibitory activity of the IL-6 inhibitor used in the present invention can be evaluated by a commonly used method. Specifically, IL-6-dependent human myeloma strains (S6B45, KPMM2), human Renner T lymphoma cell line KT3, or IL-6-dependent cells MH60.BSF2 are cultured, IL-6 is added thereto, and at the same time, an IL-6 inhibitor is coexisted to 3 measure the H-thymidine incorporation of the IL-6-dependent cells. Further, U266, which is an IL-6 receptor-expressing cell, is cultured, 125 I-labeled IL-6 is added, and at the same time, an IL-6 inhibitor is added, and the 125 I-labeled IL-6 bound to the IL-6 receptor-expressing cells is measured. In the above assay system, in addition to the group in which the IL-6 inhibitor is present, a negative control group not containing the IL-6 inhibitor is set, and by comparing the results obtained from both, the IL-6 inhibitory activity of the IL-6 inhibitor can be evaluated.

[0108] The drugs such as the pharmaceutical composition, therapeutic agent, and prophylactic agent of the present invention can be administered in the form of a pharmaceutical preparation and can be administered orally or parenterally, systemically or locally. For example, intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, enteric-coated oral preparation, etc. can be selected, and the administration method can be appropriately selected according to the age and symptoms of the patient. The effective dose ranges from 0.01 mg to 100 mg per kg of body weight per administration, preferably in the range of 1 mg to 2 mg per kg of body weight, preferably selected from 8 mg per kg of body weight and 12 mg to 2 mg per kg of body weight. Alternatively, a dose of 1 to 1000 mg per patient, preferably 100 to 200 mg, preferably 120 mg, 150 mg, 200 mg can be selected. Preferred doses and administration methods are, for example, in the case of an anti-IL-6 receptor antibody, the amount at which free antibody is present in the blood is the effective dose. As a specific example, 0.5 mg to 40 mg, preferably 1 mg to 20 mg per kg of body weight per month (4 weeks) is divided into one to several times, for example, 2 times / week, 1 time / week, 1 time / 2 weeks, 1 time / 4 weeks, etc. The administration schedule is adjusted by observing the post-transplantation status and the trend of blood test values and extending the administration interval from 2 times / week or 1 time / week to 1 time / 2 weeks, 1 time / 3 weeks, 1 time / 4 weeks, etc.

[0109] The drugs such as the pharmaceutical composition, therapeutic agent, and prophylactic agent of the present invention may be added with pharmaceutically acceptable carriers such as preservatives and stabilizers. A pharmaceutically acceptable carrier may be a material that itself has a therapeutic or prophylactic effect on the symptoms in peripartum cardiomyopathy and an inhibitory effect on cardiac remodeling associated with peripartum cardiomyopathy, or may be a material that does not have such an inhibitory effect, and means a material that can be administered together with the above drugs. Further, it may be a material that does not have a pharmacological effect and has a synergistic or additive stabilizing effect when used in combination with an IL-6 inhibitor. Examples of pharmaceutically acceptable materials include, for example, sterile water, physiological saline, stabilizers, excipients, buffers, preservatives, surfactants, chelating agents (such as EDTA), binders, and the like.

[0110] In the present invention, examples of the surfactant include nonionic surfactants, such as sorbitan fatty acid esters like sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monooleate; glycerin fatty acid esters like glycerin monocaprylate, glycerin monomyristate, glycerin monostearate; polyglycerin fatty acid esters like decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoerate; polyoxyethylene sorbitan fatty acid esters like polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate; polyoxyethylene sorbit fatty acid esters like polyoxyethylene sorbit tetra stearate, polyoxyethylene sorbit tetraoleate; polyoxyethylene glycerin fatty acid esters like polyoxyethylene glyceryl monostearate; polyethylene glycol fatty acid esters like polyethylene glycol distearate; polyoxyethylene alkyl ethers like polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene alkyl ethers like polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylene alkyl phenyl ethers like polyoxyethylene nonyl phenyl ether; polyoxyethylene hydrogenated castor oils like polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated ricinoleic oil); polyoxyethylene beeswax derivatives like polyoxyethylene sorbit beeswax; polyoxyethylene lanolin derivatives like polyoxyethylene lanolin; polyoxyethylene fatty acid amides like polyoxyethylene stearic acid amide, etc., having an HLB of 6 to 18, etc., can be cited as typical examples.

[0111] In addition, anionic surfactants can also be mentioned as surfactants. 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; polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl sulfate, having an average addition mole number of ethylene oxide of 2 to 4 and an alkyl group with 10 to 18 carbon atoms; alkyl sulfosuccinate salts such as sodium lauryl sulfosuccinate, having an alkyl group with 8 to 18 carbon atoms; natural surfactants such as lecithin and glycerophospholipids; sphingolipids such as sphingomyelin; sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms, etc. can be cited as typical examples.

[0112] One or more of these surfactants can be added in combination to the agent of the present invention. Preferred surfactants used in the preparation of the present invention are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60, or 80, and polysorbate 20 and 80 are particularly preferred. Also, polyoxyethylene polyoxypropylene glycols represented by poloxamers (such as Pluronic F-68 (registered trademark)) are also preferred.

[0113] The addition amount of the surfactant varies depending on the type of surfactant used. In the case of polysorbate 20 or polysorbate 80, generally it is 0.001 to 100 mg / mL, preferably 0.003 to 50 mg / mL, and more preferably 0.005 to 2 mg / mL.

[0114] In the present invention, examples of the buffer include phosphoric acid, citric acid, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, caprylic acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid, and other organic acids, or phosphate buffer (sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate hydrate), citrate buffer, carbonate buffer, Tris buffer, histidine buffer (L-histidine, L-histidine hydrochloride hydrate), imidazole buffer, etc.

[0115] Alternatively, a solution preparation may be prepared by dissolving it in an aqueous buffer known in the field of solution preparations. The concentration of the buffer is generally 1 to 500 mM, preferably 5 to 100 mM, and more preferably 10 to 20 mM.

[0116] In addition, the agent of the present invention may contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulins, amino acids, saccharides and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols.

[0117] In the present invention, examples of amino acids include basic amino acids such as arginine, lysine, histidine, ornithine, etc., or inorganic salts of these amino acids (preferably in the form of hydrochloride, phosphate, i.e., amino acid phosphate). When free amino acids are used, the preferred pH value is adjusted by the addition of a suitable physiologically acceptable buffer substance such as an inorganic acid, especially hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, formic acid or salts thereof. In this case, the use of phosphate is particularly advantageous in that a particularly stable lyophilized product can be obtained. It is particularly advantageous when the preparation does not substantially contain organic acids such as malic acid, tartaric acid, citric acid, succinic acid, fumaric acid, etc. or when the corresponding anions (malate ion, tartrate ion, citrate ion, succinate ion, fumarate ion, etc.) are absent. Preferred amino acids are arginine, lysine, histidine, or ornithine. Furthermore, acidic amino acids such as glutamic acid and aspartic acid, and their salt forms (preferably sodium salts), or neutral amino acids such as isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine, or alanine, or aromatic amino acids such as phenylalanine, tyrosine, tryptophan, or the derivative N-acetyltryptophan can also be used.

[0118] In the present invention, examples of saccharides and carbohydrates such as polysaccharides and monosaccharides include dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, raffinose and the like. In the present invention, examples of sugar alcohols include mannitol, sorbitol, inositol and the like.

[0119] When the agent of the present invention is made into an aqueous solution for injection, it can be mixed with, for example, physiological saline, glucose and other auxiliary drugs (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride) isotonic solutions. Further, the aqueous solution may be used in combination with a suitable solubilizing agent (for example, alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50, etc.)). If desired, it may further contain a diluent, solubilizing agent, pH adjuster, soothing agent, sulfur-containing reducing agent, antioxidant and the like.

[0120] In the present invention, examples of sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and those having a sulfhydryl group such as thioalkanoic acids having 1 to 7 carbon atoms.

[0121] In the present invention, examples of antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate or ethylenediaminetetraacetic acid disodium (EDTA), sodium pyrophosphate, sodium metaphosphate and other chelating agents.

[0122] Alternatively, if necessary, it can be encapsulated in microcapsules (such as microcapsules of hydroxymethylcellulose, gelatin, poly[methyl methacrylic acid], etc.), or made into a colloidal drug delivery system (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.) (see "Remington's Pharmaceutical Science 16th edition", Oslo Ed., 1980, etc.). Furthermore, methods for making the drug a sustained-release drug 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. (1982) 12, 98-105; U.S. Patent No. 3,773,919; European Patent Application Publication (EP) No. 58,481; Sidman et al., Biopolymers 1983, 22: 547-556; EP No. 133,988). The pharmaceutically acceptable carriers used are appropriately selected from the above or in combination according to the dosage form, but are not limited thereto.

[0123] The subjects to be treated in the present invention are not particularly limited, but include animals (for example, humans, domestic animal species, wild animals).

[0124] In the present invention, "administering" includes administering orally or parenterally. Oral administration can include administration in the form of an oral preparation, and as the oral preparation, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions, or suspensions can be selected.

[0125] Examples of parenteral administration include administration in the form of an injection, and examples of injections include subcutaneous injections, intramuscular injections, or intraperitoneal injections. In addition, the effects of the method of the present invention can be achieved by introducing a gene containing an oligonucleotide to be administered into a living body using a gene therapy technique. Further, the agent of the present invention can also be locally administered to the region to be treated. For example, it is also possible to administer by local injection during surgery, use of a catheter, or targeted gene delivery of DNA encoding the peptide of the present invention. The agent of the present invention may be administered simultaneously and in parallel with a prescription at the onset of cardiomyopathy, for example, catheter surgery methods (PTCA, PCI), thrombolytic therapy (PTCR), coronary artery bypass augmentation method (CABG), etc.

[0126] All prior art documents cited in this specification are incorporated herein by reference.

Examples

[0127] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0128] 1. Preparation of Mice All animal experiments were approved by the Animal Experiment Ethics Committee of the National Cerebral and Cardiovascular Center and were conducted in accordance with the guidelines of the Japanese Physiological Society. Npr1 - / - Mice were produced at the Howard Hughes Medical Institute (University of Texas Southwestern Medical Center, Dallas) (Nature. (1995) 378, 65-68. doi:10.1038 / 378065a0). All mice used in this study were of C57BL / 6 background. The mice were group-housed at 25°C under a 12:12 hour light:dark cycle, and access to food and water was unrestricted. Female mice (8 weeks old) were used for the experiment. Mating was performed with multiple females per male, and pregnant female mice were individually housed in separate cages. After parturition, they were housed in the same cage as the pups until the end of the lactation period, and the number of pups was not adjusted.

[0129] 2. Statistical analysis All data are presented as mean ± standard error of the mean. Animals were randomly assigned to experimental groups. Survival between mice was compared using Kaplan–Meier analysis followed by log-rank test. Pairwise comparisons were performed using two-sided unpaired Student's t-test. Differences among three or more groups were analyzed using one-way or two-way analysis of variance followed by Tukey–Kramer post hoc test. For comparisons between tissue-specific knockout mice, one-way analysis of variance was followed by Dunnett post hoc test. In microarray analysis, differential gene expression was determined using t-test. Adjustment for multiple testing was performed according to the Benjamini–Hochberg method, and the false discovery rate was set at 0.05. For all comparisons, statistical significance was indicated by a P-value of less than 0.05.

[0130] 3. Confirmation of PPCM-like cardiac remodeling in postpartum Npr1 knockout mice According to the experimental protocol shown in Fig. 2A, Npr1 - / - mice and wild-type mice (Npr1 + / + mice) were bred. The survival rate after five pregnancy-lactation cycles is shown in Fig. 2B. The survival rate of Npr1 - / - mice was significantly lower than that of Npr1 + / + mice over consecutive pregnancy-lactation cycles (Fig. 2B; P = 0.0008, vs. Npr1 + / + mice). Surprisingly, 75% of the Npr1 - / - female animals that died during consecutive pregnancy-lactation cycles died during lactation. After the fifth consecutive pregnancy-lactation cycle, the hearts of Npr1 - / - mice were significantly larger than those of Npr1 + / + mice, accompanied by an increase in lung weight, interstitial fibrosis, and increased mRNA expression of genes related to cardiac hypertrophy (Figs. 2C and 2D). The ratio of heart weight to tibial length (HW / TL) in the virgin state was higher in Npr1 + / + mice than in Npr1 - / -It was slightly but significantly higher in mice (Figures 2E and 2F). However, HW / TL after the first pregnancy-lactation cycle was Npr1 after parturition - / - significantly increased in mice (Figures 2E and 2F), and HW / TL (2PP) after the second consecutive pregnancy-lactation cycle was Npr1 after parturition - / - not only in mice, but also in Npr1 + / + mice was significantly increased (Figures 2E and 2F). Note that the increase in HW / TL means that cardiac hypertrophy has occurred.

[0131] Npr1 after parturition + / + In mice, cardiac hypertrophy in 1PP and 2PP completely recovered to the virgin level by 8 weeks later (Figure 2F). In contrast, cardiac hypertrophy in 2PP Npr1 - / - mice did not completely disappear by 8 weeks later (Figure 2F). The ratio of lung weight to tibia length (LuW / TL), which indicates pulmonary congestion due to heart failure, was almost the same between virgin Npr1 + / + mice and Npr1 - / - mice, and it hardly changed throughout the pregnancy-lactation cycle in Npr1 + / + mice, but it significantly increased in 1PP and 2PP Npr1 - / - mice (Figure 2G). Furthermore, the recovery of LuW / TL by 8 weeks after weaning tended to be hindered in 2PP Npr1 - / - mice (P = 0.064, vs. virgin Npr1 - / - mice; Figure 2G). In Npr1 after parturition + / + mice, the fibrotic area in the heart did not increase (Figures 2H and 2I), but cardiomyocytes were significantly hypertrophied (Figures 2J and 2K). Both the fibrotic area (Figures 2H and 2I) and cardiomyocyte size (Figures 2J and 2K) were significantly increased in both 1PP and 2PP in Npr1 - / - mice.

[0132] 4. Confirmation of induction of cardiac hypertrophy in mice by lactation Npr1 + / + mice and Npr1 - / -In mice, to clarify which process (i.e., pregnancy, parturition, or lactation) is the cause of cardiac hypertrophy, the maternal phenotypes during all three processes were examined. The experimental protocol is shown in Fig. 3A. Twenty-two Npr1 - / - mice showed higher blood pressure in the nulliparous state than Npr1 + / + mice (Fig. 3B). Although it has been reported that mice lacking proANP-converting enzyme develop pregnancy-induced hypertension (Chan, J.C., et al., Proc Natl Acad Sci U S A. (2005) 102, 785-790. doi: 10.1073 / pnas.0407234102), Npr1 - / - mice did not show that phenotype (Fig. 3B). In both Npr1 + / + mice and Npr1 - / - mice, maternal body weight was highest in the late pregnancy period (Fig. 3C). Plasma ANP peaked biphasically immediately after parturition and 2 weeks after lactation in Npr1 + / + mice, but peaked 2 weeks after parturition in Npr1 - / - mice (Fig. 3C). In contrast, in neither Npr1 + / + mice nor Npr1 - / - mice did HW / TL increase in the late pregnancy period or within 3 days after the first parturition (Fig. 3D). Furthermore, in neither Npr1 + / + mice nor Npr1 - / - mice did the expression of RCAN1 and the phosphorylation of ERK1 / 2 increase in the late pregnancy period (E18.5) (data not shown). These findings mean that cardiac hypertrophy in periparturient mice is not induced by pregnancy-related fluid volume overload.

[0133] In contrast, HW / TL increased significantly within 2 weeks of lactation in Npr1 + / + mice and Npr1 - / - mice after the first parturition (Fig. 3E). The mRNA expression levels of genes related to cardiac hypertrophy (Nppa, Nppb, and Acta1) were higher in Npr1 + / + mice and Npr1 - / -Significantly increased during lactation in both mouse strains (Figure 3F). In comparison, mRNA expression of fibrosis-related genes (Col3a1, Fn1, and Tgfb1) was significantly increased only in lactating Npr1 - / - mice (Figure 3F). However, systolic blood pressure two weeks after lactation was not different from that immediately after parturition in either Npr1 + / + mice or Npr1 - / - mice (data not shown).

[0134] After the first pregnancy-lactation cycle, cardiac hypertrophy that developed in Npr1 + / + mice or Npr1 - / - mice was not observed when pups were removed immediately after birth and lactation was not performed (Figure 3E; two weeks without lactation). Removal of pups and prevention of lactation decreased both hypertrophied cardiomyocytes and increased mRNA expression related to cardiac hypertrophy in primiparous Npr1 - / - mice (data not shown). However, preventing lactation did not affect cardiac function in either primiparous Npr1 + / + mice or Npr1 - / - mice (data not shown). These results indicate that lactation, rather than pregnancy, induces cardiac hypertrophy in mice. That is, these results indicate the importance of the ANP / BNP-NPR1 system in potentially suppressing hypertrophic cardiac remodeling during lactation.

[0135] 5. Effect of an IL-6 inhibitor on lactation-induced cardiac hypertrophy in mice Activation of brain MR by aldosterone is known to regulate cardiovascular inflammation, oxidative stress, and sympathetic nerve activity. Next, microarray analysis was performed using cardiac tissue-derived RNA and cDNA from virgin and two-week lactating Npr1 + / + mice and Npr1 - / - mice. Npr1 + / + mice and Npr1 - / -In the mouse heart, changes in the expression levels of more than twofold occurred with probes 3246 and 2336, respectively. The analysis results are shown in the following table. For the analysis, a t-test was used, and the P-value calculation was performed by asymptotic; multiple comparison correction and the Benjamini-Hochberg method. The adjusted P-value cutoff was set at 0.05, and the fold change cutoff was set at 2.0.

[0136]

Table 1

[0137]

Table 2

[0138]

Table 3

[0139]

Table 4

[0140] Furthermore, compared with lactating Npr1 + / + mice, in the hearts of lactating Npr1 - / - mice, significant (>1.5-fold) changes in gene expression occurred with 27 probes (data not shown). Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) to evaluate upstream regulatory factors. The evaluation results regarding cytokines are shown in the table.

[0141]

Table 5

[0142] Npr1 - / -In mice, it was revealed that cardiac inflammatory cytokines may contribute to lactation-induced cardiac hypertrophy. In addition, there are reports that IL-6 and IL-1β play important roles in the development of cardiac hypertrophy (Non-Patent Document 10), and lactating Npr1 in nulliparous and primiparous + / + and Npr1 - / - The expression levels of IL-6 and IL-1β mRNAs in the hearts of mice were examined. The experimental protocol is shown in Fig. 4A. Compared with nulliparous mice, the Il6 mRNA expression level in the heart tended to increase in lactating Npr1 + / + mice, but significantly increased in lactating Npr1 - / - mice (Fig. 4B). The mRNA level of Il1β tended to increase during lactation in primiparous Npr1 + / + mice and Npr1 - / - mice (Fig. 4B). In addition, the protein expression and phosphorylation level of signal transducer and activator of transcription 3 (STAT3), which is a downstream target of IL-6, were examined by Western blotting. The primary antibodies and secondary antibodies used are shown in the table.

[0143]

Table 6

[0144] Lactation significantly increased phosphorylated STAT3 (p-STAT3α) in the hearts of Npr1 - / - mice, but not in Npr1 + / + mice (Fig. 4C). Lactation did not affect the plasma concentration of IL-6 in either Npr1 + / + mice or Npr1 - / - mice, but the number of CD68-positive cells in the heart was higher in lactating Npr1 - / - mice than in lactating Npr1 + / + mice (data not shown). It was confirmed that administration of eplerenone decreased the Il6 expression in the hearts of primiparous Npr1 - / - mice (Fig. 4D).

[0145] Furthermore, the Npr1 of anti-IL-6 receptor antibody (MR16-1)+ / + Mouse and Npr1 - / - To confirm the effect in mice, the test was conducted according to the following procedure. Npr1 + / + Mouse and Npr1 - / - Mice were divided into a control IgG administration group (Npr1 + / + Mouse: n = 7; Npr1 - / - Mouse: n = 11) and an MR16-1 administration group (Npr1 + / + Mouse: n = 8; Npr1 - / - Mouse: n = 8). In all mice, control IgG or MR16-1 was intraperitoneally administered at 0.5 mg / mouse at the day of parturition (immediately after parturition) and 1 week after the start of lactation. The dosage was unified to 0.1 mL. Two weeks after the start of lactation, the heart weight and tibia length were measured, and the heart weight to tibia ratio (HW / TL) was calculated. Intraperitoneal injection of the anti-IL-6 receptor antibody (MR16-1) once a week was performed in Npr1 - / - mice, and a tendency to suppress lactation-induced cardiac hypertrophy was confirmed, while it was not suppressed in Npr1 + / + mice (Figure 4E).

[0146] Using the same method, administration tests of metoprolol (β1-adrenergic receptor antagonist), nicotine (α7-nicotinic acetylcholine receptor agonist), and tempol (radical scavenger) during lactation were also conducted. Pharmacological modification of sympathetic or parasympathetic nerve activity through any of these administrations did not suppress cardiac hypertrophy in Npr1 - / - mice (Figure 4F). Furthermore, administration of tempol (radical scavenger) to Npr1 - / - mice during lactation for 2 weeks did not suppress lactation-dependent cardiac hypertrophy (Figure 4F).

Industrial Applicability

[0147] The present invention provides a therapeutic or prophylactic agent for peripartum cardiomyopathy, particularly a therapeutic and prophylactic agent for peripartum cardiomyopathy after childbirth or during lactation.

Claims

1. A pharmaceutical composition for treating or preventing peripartum cardiomyopathy, comprising an IL-6 inhibitor as an active ingredient, wherein the IL-6 inhibitor is an anti-IL-6 receptor antibody.

2. 10. The pharmaceutical composition of claim 1 for use in a lactating subject.

3. 3. The pharmaceutical composition according to claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The pharmaceutical composition described in any one of claims 1 to 3, wherein the antibody is an antibody against human IL-6 receptor.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the antibody is a recombinant antibody.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody is tocilizumab, satralizumab or sarilumab.

Images