JPWO2018101398A1 - Method for treating inflammatory bowel disease and pharmaceutical composition for use therein - Google Patents

Abstract

The present invention provides a pharmaceutical composition for use in the treatment or prevention of inflammatory bowel disease (IBD). More specifically, a pharmaceutical composition containing apelin or an apelin receptor agonist is provided. A pharmaceutical composition containing apelin or an apelin receptor agonist may also be useful in the treatment of other diseases caused by inflammatory macrophages, such as collagen disease.

Description

  This application is an application that enjoys the benefit of the priority of Japanese Patent Application No. 2006-234062 (filing date: December 1, 2016), which is incorporated herein by reference in its entirety.

  The present invention relates to a method for treating inflammatory bowel disease (IBD) and a pharmaceutical composition for use therein. More particularly, it relates to a method for treating inflammatory bowel disease (IBD) comprising administering a pharmaceutical composition containing apelin or an apelin receptor agonist. The present invention also relates to a method for treating a disease caused by inflammatory macrophages and a pharmaceutical composition for use therein.

  Inflammatory Bowel Disease (hereinafter referred to simply as “IBD”) is an inflammatory disease that causes intractable inflammation and ulceration in the mucous membrane of the digestive tract such as the large intestine and small intestine and has a chronic course. Details are not known (for example, see Non-Patent Document 1). This IBD is narrowly defined as ulcerative colitis with diffuse non-specific inflammation of unknown origin that mainly forms erosions and ulcers in the colonic mucosa, and all layers with nonspecific granulomas in the entire digestive tract It refers to two diseases of Crohn's disease, which induces sexual inflammation and causes ulceration, abscess, fibrosis, and thereby stenosis and fistula. In Japan, IBD, which was once considered a disease in Europe and the United States, has been steadily increasing in number in recent years, and currently there are 170,000 people including ulcerative colitis and Crohn's disease. It occurs especially in young people in their 10s and 20s, and relapses and remissions are repeated, greatly affecting growth, study, work, pregnancy, childbirth, and so on. In the absence of radical treatment, patients are forced to continue treatment throughout their lives and are intractable diseases that suffer from physical, mental, social and economic burdens.

  Although the full pathogenesis and pathogenesis of IBD has not yet been elucidated, various inflammatory mediators such as tumor necrosis factor (hereinafter referred to as “TNF”) and macrophage migration inhibitory factor are onset, relapse, prolongation, and severe It has become clear that it is involved in conversion (see, for example, Non-Patent Documents 2 to 5).

  The current main treatment options for IBD include 5-aminosalicylic acid (simply referred to as “5-ASA”) preparations, steroid preparations, immunomodulators and the like. However, there are many patients who are resistant to these treatments and have difficulty in withdrawal, and serious side effects are also a problem due to long-term treatment. Recently, anti-TNF monoclonal antibody preparations have been developed as novel therapies (see, for example, Non-Patent Documents 6 and 7). This treatment is expected to suppress TNF activity and rapidly improve inflammation in patients with Crohn's disease and ulcerative colitis. However, serious side effects such as cases in which this therapy is resistant or secondary ineffective, infections such as tuberculosis, and malignant tumors such as malignant lymphoma frequently occur (see, for example, Non-Patent Documents 8 and 9). ), Which makes it difficult to continue treatment with anti-TNF antibodies. Therefore, the development of new treatments for IBD is urgent, but unlike current therapies that suppress systemic immune responses in particular, strategies that select treatment targets specific to the pathological condition are considered essential in the future. .

Podolsky DK. Inflammatory bowel disease.N Engl J Med 2002; 347: 417-29. Sartor RB.Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases. Am J Gastroenterol 1997; 92 (12 Suppl.): 5S-11S. Papadakis KA, Targan SR.Role of cytokines in the pathogenesis of inflammatory bowel disease.Annu Rev Med 2000; 51: 289-98. de Jong YP, Abadia-Molina AC, Satoskar AR, et al. Development of chronic colitis is dependent on the cytokine MIF. 2001; 2: 1061-66. Ohkawara T, Nishihira J, Takeda H, et al.Amelioration of dextran sulfate sodium-induced colitis by anti-macrophage migration inhibitory factor antibody in mice.Gastroenterology 2002; 123: 256-70. van Dullemen HM, van Deventer SJ, Hommes DW, et al Treatment of Crohn's disease with anti-tumor necrosis factor chimeric monoclonal antibody (cA2) .Gastroenterology 1995; 109: 129-35. Targan SR, Hanauer SB, van Deventer SJ, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn ’s disease.Crohn ’s Disease cA2 Study Group.N Eng J Med 1997; 337: 1029-35. Bickston SJ, Lichtenstein GR, Arseneau KO, et al. The relationship between infliximab treatment and lymphoma in Crohn's disease.Gastroenterology 1999; 117: 1433-37. Keane J, Gershon S, Wise RP, et al. Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent.N Engl J Med 2001; 345: 1098-104. Michio Onizawa, Takashi Nagaishi et al. Signaling pathway via TNF-α / NF-κB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis. Am J Physiol Gastrointest Liver Physiol. 2009 Apr; 296 (4): G850-9 . Suzana Brozovic, Takashi Nagaishi1 et al. CD1d function is regulated by microsomal triglyceride transfer protein.Nature Medicine 10, 535-539 (2004) Hideki Iijima, Markus F. Neurath, Takashi Nagaishi et al. Specific Regulation of T Helper Cell 1-mediated Murine Colitis by CEACAM1. J Exp Med. 2004 Feb 16; 199 (4): 471-82. Osamu Yamaji, Takashi Nagaishi et al. The Development of Colitogenic CD4 + T Cells Is Regulated by IL-7 in Collaboration with NK Cell Function in a Murine Model of Colitis.J Immunol. 2012 Mar 15; 188 (6): 2524-36. Watanabe, M., Y. Ueno, T. Yajima, S. Okamoto, T. Hayashi, M. Yamazaki, Y. Iwao, H. Ishii, S. Habu, M. Uehira, et al 1998. Interleukin 7 transgenic mice develop chronic colitis with decreased interleukin 7 protein accumulation in the colonic mucosa.J. Exp. Med. 187: 389-402. Berg DJ, Davidson N, Kuhn R, Muller W, Menon S, Holland G, Thompson-Snipes L, Leach MW, Rennick D. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine Production and CD4 (+ ) TH1-like responses. J Clin Invest 1996; 98: 1010-20

  An object of the present invention is to provide a pharmaceutical composition for use in the treatment or prevention of inflammatory bowel disease (IBD) and a method for treating or preventing inflammatory bowel disease (IBD) using the same. . Another object of the present invention is to provide a pharmaceutical composition for use in treating or preventing a disease caused by inflammatory macrophages, and a method for treating or preventing a disease caused by inflammatory macrophages using the same. I will.

（式中、ＲはＬＲＢ（Lissamine Rhodamine B）またはＰＢ（Patent Blue）を表し、ｎは０または１である。）から成る群より選択される、［１］記載の医薬組成物。
［６］炎症性腸疾患（ＩＢＤ）が潰瘍性大腸炎である、［１］〜［５］のいずれか一項記載の医薬組成物。
［７］炎症性腸疾患（ＩＢＤ）がクローン病である、［１］〜［５］のいずれか一項記載の医薬組成物。The present inventors have found that inflammatory bowel disease (IBD) can be treated by stimulation of the apelin-APJ system. The present invention is based on such findings and includes the following aspects:
[1] A pharmaceutical composition for use in the treatment or prevention of inflammatory bowel disease (IBD), comprising apelin or an apelin receptor agonist.
[2] The medicament according to [ ¹ ], wherein the apelin is selected from the group consisting of apelin-36, apelin-17, apelin-13, [Pyr ¹ ] -apelin-13, apelin-12, and variants thereof. Composition.
[3] The pharmaceutical composition according to [2], wherein the apelin is [Pyr ¹ ] -Apelin-13.
[4] The pharmaceutical composition according to [1], wherein the apelin receptor agonist is a low molecular compound, a plant extract, or an agonist antibody.
[5] The apelin receptor agonist is TODDLER / ELABELA, E339-3D6, MM07, (S) -N- (1- (butylamino) -5-methyl-1-oxohexane-3-yl) -5- ( 2,6-dimethoxyphenyl) -1-isobutyl-1H-pyrazole-3-carboxamide, (S) -3-{[1- (1-ethyl-propyl) -2-thiophen-2-ylmethyl-1H-benzimidazole -5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -3-{[2-furan-2-ylmethyl-1- (2-methyl-cyclohexyl) -1H-benzimidazole-5-carbonyl ] -Amino} -5-methyl-hexanoic acid, (S) -5-methyl-3-{[1-((1R, 2R) -2-methyl-cyclohexyl) -2-thia 5-ylmethyl-1H-benzimidazol-5-carbonyl] -amino} -hexanoic acid, (S) -3-{[1- (1-ethyl-propyl) -2-thiazol-5-ylmethyl-1H -Benzimidazol-5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -5-cyclohexyl-3- (5- (2,6-dimethoxyphenyl) -1- (4-fluorophenyl)- 1H-pyrazole-3-carboxamide) pentanoic acid, pine extract, saffron extract, kina extract, comfrey extract, gardenia extract, crocetin, and a compound represented by the following formula:

(Wherein R represents LRB (Lissamine Rhodamine B) or PB (Patent Blue), and n is 0 or 1). The pharmaceutical composition according to [1].
[6] The pharmaceutical composition according to any one of [1] to [5], wherein the inflammatory bowel disease (IBD) is ulcerative colitis.
[7] The pharmaceutical composition according to any one of [1] to [5], wherein the inflammatory bowel disease (IBD) is Crohn's disease.

（式中、ＲはＬＲＢ（Lissamine Rhodamine B）またはＰＢ（Patent Blue）を表し、ｎは０または１である。）から成る群より選択される、［８］記載の治療または予防方法。
［１３］炎症性腸疾患（ＩＢＤ）が潰瘍性大腸炎である、［８］〜［１２］のいずれか一項記載の治療または予防方法。
［１４］炎症性腸疾患（ＩＢＤ）がクローン病である、［８］〜［１２］のいずれか一項記載の治療または予防方法。[8] A method for treating or preventing inflammatory bowel disease (IBD) in a subject, comprising a step of administering apelin or an apelin receptor agonist to the subject.
[9] The treatment according to [8], wherein the apelin is selected from the group consisting of apelin-36, apelin-17, apelin-13, [Pyr ¹ ] -apelin-13, apelin-12, and variants thereof. Or prevention methods.
[10] The method of treatment or prevention according to [9], wherein the apelin is [Pyr ¹ ] -Apelin-13.
[11] The method of treatment or prevention according to [8], wherein the apelin receptor agonist is a low molecular compound, a plant extract, or an agonist antibody.
[12] The apelin receptor agonist is TODDLER / ELABELA, E339-3D6, MM07, (S) -N- (1- (butylamino) -5-methyl-1-oxohexane-3-yl) -5- ( 2,6-dimethoxyphenyl) -1-isobutyl-1H-pyrazole-3-carboxamide, (S) -3-{[1- (1-ethyl-propyl) -2-thiophen-2-ylmethyl-1H-benzimidazole -5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -3-{[2-furan-2-ylmethyl-1- (2-methyl-cyclohexyl) -1H-benzimidazole-5-carbonyl ] -Amino} -5-methyl-hexanoic acid, (S) -5-methyl-3-{[1-((1R, 2R) -2-methyl-cyclohexyl) -2-thi Zol-5-ylmethyl-1H-benzimidazol-5-carbonyl] -amino} -hexanoic acid, (S) -3-{[1- (1-ethyl-propyl) -2-thiazol-5-ylmethyl-1H- Benzimidazol-5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -5-cyclohexyl-3- (5- (2,6-dimethoxyphenyl) -1- (4-fluorophenyl) -1H -Pyrazole-3-carboxamide) pentanoic acid, pine extract, saffron extract, kina extract, comfrey extract, gardenia extract, crocetin, and a compound represented by the following formula:

(Wherein R represents LRB (Lissamine Rhodamine B) or PB (Patent Blue), and n is 0 or 1). The treatment or prevention method according to [8].
[13] The treatment or prevention method according to any one of [8] to [12], wherein the inflammatory bowel disease (IBD) is ulcerative colitis.
[14] The treatment or prevention method according to any one of [8] to [12], wherein the inflammatory bowel disease (IBD) is Crohn's disease.

（式中、ＲはＬＲＢ（Lissamine Rhodamine B）またはＰＢ（Patent Blue）を表し、ｎは０または１である。）から成る群より選択される、［１５］記載の使用。
［２０］炎症性腸疾患（ＩＢＤ）が潰瘍性大腸炎である、［１５］〜［１９］のいずれか一項記載の使用。
［２１］炎症性腸疾患（ＩＢＤ）がクローン病である、［１５］〜［１９］のいずれか一項記載の使用。[15] Use of apelin or an apelin receptor agonist in the manufacture of a medicament for treating or preventing inflammatory bowel disease (IBD).
[16] The use according to [15], wherein the apelin is selected from the group consisting of apelin-36, apelin-17, apelin-13, [Pyr ¹ ] -apelin-13, apelin-12, and variants thereof. .
[17] Use according to [16], wherein the apelin is [Pyr ¹ ] -apelin-13.
[18] The use according to [15], wherein the apelin receptor agonist is a low molecular compound, a plant extract, or an agonist antibody.
[19] The apelin receptor agonist is TODDLER / ELABELA, E339-3D6, MM07, (S) -N- (1- (butylamino) -5-methyl-1-oxohexane-3-yl) -5- ( 2,6-dimethoxyphenyl) -1-isobutyl-1H-pyrazole-3-carboxamide, (S) -3-{[1- (1-ethyl-propyl) -2-thiophen-2-ylmethyl-1H-benzimidazole -5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -3-{[2-furan-2-ylmethyl-1- (2-methyl-cyclohexyl) -1H-benzimidazole-5-carbonyl ] -Amino} -5-methyl-hexanoic acid, (S) -5-methyl-3-{[1-((1R, 2R) -2-methyl-cyclohexyl) -2-thi Zol-5-ylmethyl-1H-benzimidazol-5-carbonyl] -amino} -hexanoic acid, (S) -3-{[1- (1-ethyl-propyl) -2-thiazol-5-ylmethyl-1H- Benzimidazol-5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -5-cyclohexyl-3- (5- (2,6-dimethoxyphenyl) -1- (4-fluorophenyl) -1H -Pyrazole-3-carboxamide) pentanoic acid, pine extract, saffron extract, kina extract, comfrey extract, gardenia extract, crocetin, and a compound represented by the following formula:

The use according to [15], wherein R is selected from the group consisting of RRB (Lissamine Rhodamine B) or PB (Patent Blue), and n is 0 or 1.
[20] The use according to any one of [15] to [19], wherein the inflammatory bowel disease (IBD) is ulcerative colitis.
[21] The use according to any one of [15] to [19], wherein the inflammatory bowel disease (IBD) is Crohn's disease.

[22] A pharmaceutical composition for treating or preventing a disease caused by inflammatory macrophages, comprising apelin or an apelin receptor agonist.
[23] Diseases caused by inflammatory macrophages include systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease (MCTD) ), Selected from the group consisting of Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, and sarcoidosis, [22] The pharmaceutical composition according to [22].
[24] A method for treating or preventing a disease caused by inflammatory macrophages in a subject, comprising a step of administering apelin or an apelin receptor agonist to the subject.
[25] Diseases caused by inflammatory macrophages are systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyperiarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease (MCTD) ), Selected from the group consisting of Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, and sarcoidosis, [24] The treatment or prevention method according to [24].
[26] Use of apelin or an apelin receptor agonist in the manufacture of a medicament for treating or preventing a disease caused by inflammatory macrophages.
[27] Diseases caused by inflammatory macrophages are systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease (MCTD) ), Selected from the group consisting of Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, and sarcoidosis, [26] Use as described.

[28] A food composition for treating, ameliorating or preventing inflammatory bowel disease (IBD), comprising apelin or an apelin receptor agonist.
[29] A supplement for use in the treatment, symptom improvement or prevention of inflammatory bowel disease (IBD), comprising apelin or an apelin receptor agonist.
[30] A food composition for adjusting the intestine, improving the intestinal environment, suppressing intestinal inflammation, or preventing intestinal inflammation, comprising apelin or an apelin receptor agonist.
[31] A supplement for adjusting the intestine, improving the intestinal environment, suppressing intestinal inflammation or preventing intestinal inflammation, comprising apelin or an apelin receptor agonist.
[32] A food composition for treatment, symptom improvement or prevention of a disease caused by inflammatory macrophages, comprising apelin or an apelin receptor agonist.
[33] Diseases caused by inflammatory macrophages are systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyperiartritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease (MCTD) ), Selected from the group consisting of Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, and sarcoidosis, [32] The food composition according to [32].
[34] A supplement containing apelin or an apelin receptor agonist for use in the treatment, symptom improvement or prevention of a disease caused by inflammatory macrophages.
[35] Diseases caused by inflammatory macrophages are systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyperiartritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease (MCTD) ), Selected from the group consisting of Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, and sarcoidosis, [34] The supplement according to [34].

[36] A method of controlling the function of inflammatory macrophages and / or T cells by apelin or an apelin receptor agonist.
[37] The method according to [36], comprising a step of contacting apelin or an apelin receptor agonist with inflammatory macrophages and / or T cells in vitro or in vivo.
[38] The method according to [36] or [37], wherein the control of the function of inflammatory macrophages and / or T cells is suppression of production of cytokines and / or chemokines that induce inflammation.
[39] The method according to [36] or [37], wherein the control of inflammatory macrophage and / or T cell function is suppression of production of TNF, CCL2, CCL5 and / or CXCL10.

[40] The pharmaceutical composition according to any one of [1] to [7], which suppresses inflammation.
[41] The pharmaceutical composition according to any one of [1] to [7] and [40], which acts on inflammatory macrophages and / or T cells.
[42] The pharmaceutical composition according to any one of [1] to [7] and [40] to [41], wherein the apelin receptor agonist is MM07.

(B) Toll-like receptor (TLR) 4 expression of (A) F4 / 80 ⁺ , CD11b ⁺ macrophage population in the lamina propria of the intestinal tract of Rag2 ^{− / −} mice, and (C) of TLR4 ^high and TLR4 ^low macrophages It is the figure shown about production of IL-10 and TNF of a culture supernatant. * P <0.05. It is the figure which showed about (A) expression of TLR4 of a macrophage in the large intestine mucosa lamina propria of a T cell transfer enteritis model, and (B) expression of apelin receptor (APJ) in a macrophage of TLR4 ^low and TLR4 ^high . In (A), TLR4 expression in macrophages of Rag2 ^{− / −} mice that did not develop enteritis was used as a control, and TLR4 ^low and TLR4 ^high populations were fractionated based on this expression. It is the figure which showed about IL-6, MIP-2, TNF, CCL2, CCL5, and CXCL10 production in the apelin addition culture supernatant in the inflammatory macrophage isolated from the large intestine mucosa lamina propria of T cell transfer enteritis model. It is the figure shown about the survival rate of the apelin administration group in a T cell transfer enteritis model, and a control group. It is the figure shown about the typical colon pathological findings and pathological score of an apelin administration group and a control group in a T cell transfer enteritis model. * P <0.05. It is the figure shown about the influence on the weight loss rate (A) and the clinical score (B) by apelin deficiency of a T cell transfer enteritis model. It is the figure shown about the influence on the survival rate by apelin deficiency of a T cell transfer enteritis model. It is the figure which showed about the influence on the typical colon pathological findings and pathological score by apelin deficiency of the T cell transfer enteritis model. * P <0.01. It is the figure shown about the influence on the weight curve by apelin deficiency of a DSS enteritis model. It is the figure which showed about the influence on macrophage intramucosal macrophage TLR4 expression by apelin deficiency of a DSS enteritis model. It is the figure which showed about the influence on the TLR4 (B) and CCL2 (C) expression of a macrophage mucosa macrophage number (A) by apelin deficiency of a T cell transfer enteritis model. * P <0.05. It is the figure shown about the influence on TNF, CCL2, CCL5, and CXCL10 production of the macrophage in a large intestine mucosa by apelin deficiency of a T cell transfer enteritis model. * P <0.05.

  As described above, the present inventors have found that stimulation of the apelin-APJ system can treat inflammatory bowel disease (IBD) and other diseases caused by inflammatory macrophages. The present invention is described in detail below.

Apelin (APL)
Apelin-36 is a bioactive peptide consisting of 36 amino acids identified in 1998 in the bovine stomach as an endogenous ligand for APJ, an orphan receptor (Biochem. Biophys. Res. Commun., 251, 471 (1998). The apelin gene has been identified in various species such as humans, dogs, cows, rats, mice, rhesus monkeys, and zebrafish, and encodes a 77-amino acid apelin preproprotein (also called apelin (1-77)). Yes. Apelin and APJ are found in brain, kidney, heart, lung, adipose tissue, gastrointestinal tract, mammary gland and the like. Apelin has a wide range of activities including effects on cardiac contractility, blood pressure, appetite, drinking behavior, hypothalamic-pituitary-adrenal system, gastric acid secretion, insulin and gallbladder constrictor (CCK) secretion. The base and amino acid sequences of apelin are known and can be obtained from databases such as GenBank:

APLN apelin [Homo sapiens (human)]
Gene ID: 8862, NM_017413.4, NP_059109.3

In the processing of apelin preproprotein, the signal peptide corresponding to residues 1 to 22 of the apelin preproprotein is cleaved, and as a result, a 55 amino acid (residues from 23 to 77) apelin proprotein (apellin). (Also called (23-77)). Apelin-36 (also called apelin-36 (42-77)) is a 36 amino acid long peptide obtained from the 55 amino acid long apelin (23-77) proprotein (Tatemoto et al., Biochem. Biophys. Res). Comm., 251: 471-476, 1998), corresponding to residues 42 to 77 of the preproprotein. Apelin-17 and apelin-13 are derived from the carboxyl (C) terminal side of apelin. Apelin-17 corresponds to residues 61-77 of the apelin proprotein and is also referred to as apelin-17 (61-77). Apelin-13 corresponds to residues 65-77 of the apelin proprotein and is also referred to as apelin-13 (65-77). Furthermore, [Pyr ¹ ] -apelin-13, in which glutamine (Gln) at the N-terminal of apelin-13 is replaced with pyroglutamic acid (Pyr), is equivalent to apelin-13 and has about 60 times stronger activity than apelin-36. Known (Tatemoto, K. et al., Biochem. Biophys. Res. Commun. 251: 471-476, 1998 and Zhang et al, Bioorganic & Medicinal Chemistry, Vol.22, 1 June 2014, pp.2992- 2997). Apelin-12 is also known to exist. In the present specification, apelin-like peptides having agonist activity of the APJ receptor are collectively referred to as apelin. Therefore, apelin includes apelin-36, apelin-17, apelin-13, [Pyr ¹ ] -apellin- 13, apelin-12, and variants thereof. Variants of apelin (hereinafter sometimes referred to as modifications) introduce, for example, 1 to several (for example, within 25%) amino acid substitutions, deletions or insertions into wild-type apelin peptides Can be obtained. A modified apelin can be obtained, for example, by amidating or acetylating the N-terminal of an apelin peptide, or by PEGylating an apelin peptide. The modification can be produced by any method known to those skilled in the art.

  Regarding apelin and its apelin variants (modifications), for example, WO99 / 33976, WO00 / 18793, WO01 / 70769, and Zhang et al, Bioorganic & Medicinal Chemistry, Vol.22, 1 June 2014, pp.2992- There is a disclosure in 2997. The contents described in these documents are expressly incorporated herein by reference. As the apelin used in the present invention, the modified apelin disclosed in these documents can be appropriately selected and used.

Apelin receptor (APJ)
The apelin receptor (APJ) was cloned in 1993 as an orphan G protein-coupled receptor (GPCR). The apelin receptor (APJ) is a member of the seven-transmembrane G protein receptor family and is structurally related to the angiotensin II type I receptor (ATIR). However, none of the known peptidergic ligands for the angiotensin receptor, including angiotensin, activates APJ. The APJ receptor gene is present on human chromosome 11, and its transmembrane region has 40-50% homology with the angiotensin (AT1) receptor (Gene, 136, 355 (1993)). The mRNA is expressed in several tissues such as the central nervous system, blood mononuclear cells, lung, heart, and placenta. The base and amino acid sequences of the apelin receptor (APJ) are known and can be obtained from databases such as GenBank:

APLNR apelin receptor [Homo sapiens (human)]
Gene ID: 187, NM_005161.4, NP_005152.1

  Apelin-36 (42-77), apelin-17 (61-77), and apelin-13 (65-77) all have a phenylalanine residue at the C-terminus and can bind to the APJ receptor. it can. In (Ala-13) apelin-13, a peptide antagonist version of apelin-13 (65-77), the C-terminal phenylalanine is replaced with alanine (Lee et al., Endocrinology, 146: 231-236, 2005). ). (Ala-13) -Apelin-13 binds to the APJ receptor but does not stimulate the activity of the APJ receptor.

  APJ has been shown to function as an endogenous antagonist system that inhibits angiotensin II's blood pressure-increasing action by forming a heterodimer with angiotensin II receptor AT1. In addition, the apelin-APJ system exhibits a strong proliferative action on vascular endothelial cells, and is involved in various physiological angiogenesis and angiogenesis during disease states.

Apelin Receptor Agonist Apelin Receptor (APJ) agonists are typically apelin peptides and variants thereof, as long as they can stimulate apelin receptor activity, other agonist antibodies, small molecules, etc. It may be a substance.

（式中、ＲはＬＲＢ（Lissamine Rhodamine B）またはＰＢ（Patent Blue）を表し、ｎは０または１である。）
アペリンを長期的に作用させるとデセンシタイゼーションが起こるが、バイアスドアゴニストはデセンシタイゼーションが起こらないため、医薬品として有用である。BrameらもアペリンのサイクリックペプチドＭＭ０７がバイアスドアゴニストであることも報告している（Brame et al. Hypertension 2015: 65, 834-840）。本明細書においては、アペリンバイアスドアゴニストもアペリンアゴニストに含まれる。ＷＯ２０１５１４７６４１は、アペリンのサイクリックペプチドとその使用について開示している。ＷＯ２０１２１２５４０８は、ＰＥＧ化したアペリンを開示している。ＥＰ１９０３０５２とＵＳ２０１４／００９４４５０もＡＰＪ受容体リガンドを開示している。アペリンアゴニストに関する総論としては、Narayanan et al, J Med Chem. 2015 Oct 22-58(20):791327があり、さまざまな化合物が記述されている。Narayanan et al, Bioorg. Med Chem. 2016 24; 3758-3770にもＡＰＪ受容体アゴニストが開示されている（例えば、（Ｓ）−５−シクロヘキシル−３−（５−（２，６−ジメトキシフェニル）−１−（４−フルオロフェニル）−１Ｈ−ピラゾール−３−カルボキサミド）ペンタン酸）また、アペリンとは異なる別の内因性リガンドとして、ＴＯＤＤＬＥＲ／ＥＬＡＢＥＬＡが同定されている（Wang et al, Sci. Rep. 2015, 5, 8170）。これらの公知の化合物を適宜選択し、必要に応じて改変を加えて、アペリン受容体アゴニストとして使用することができる。ＷＯ２０１４０９９９８４には、野生型アペリン−１３と比較して増加した安定性を有するＡＰＪアゴニストであって、アゴニストのＮ末端またはＣ末端で半減期を延長する基によって修飾され、かつ少なくとも１個のアミノ酸が、非標準アミノ酸によって置換されている、ＡＰＪアゴニストが開示されている。半減期を延長する基としては、ＰＥＧ、Ｆｃドメイン、ＩｇＧ、ＨＳＡ、ＰＥ、Ａｂ、ペプチド、脂質、ポリ（Ｏ−２−ヒドロキシエチル）デンプン（ＨＥＳ）、およびナノ構造体などが挙げられる。本発明においては、これらのアペリン受容体アゴニストも使用されうる。For example, WO2015188807A1 and WO20140444738A1 include an apelin receptor (APJ) agonist compound (eg, (S) -N- (1- (butylamino) -5-methyl-1-oxohexane-3) based on an imidazole skeleton. -Yl) -5- (2,6-dimethoxyphenyl) -1-isobutyl-1H-pyrazole-3-carboxamide, (S) -3-{[1- (1-ethyl-propyl) -2-thiophene-2 -Ylmethyl-1H-benzimidazol-5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -3-{[2-furan-2-ylmethyl-1- (2-methyl-cyclohexyl) -1H -Benzimidazol-5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -5-methyl-3-{[1- (1R, 2R) -2-methyl-cyclohexyl) -2-thiazol-5-ylmethyl-1H-benzimidazol-5-carbonyl] -amino} -hexanoic acid, (S) -3-{[1- (1- Ethyl-propyl) -2-thiazol-5-ylmethyl-1H-benzimidazol-5-carbonyl] -amino} -5-methyl-hexanoic acid). Have also disclosed a non-peptide APJ agonist (E339-3D6) containing a polycyclic fluorophore (Iturrioz et al, FASEB J. 2010; 24: 1506-1517). Margathe et al. Synthesized a derivative from E339-3D6 and found a biased agonist having a cAMP production inhibitory activity stronger than the receptor internalization activity (Margathe et al., J. Med. Chem. 2014: 2908-2919). Such compounds include, for example, the following:

(In the formula, R represents LRB (Lissamine Rhodamine B) or PB (Patent Blue), and n is 0 or 1).
Desensitization occurs when apelin is allowed to act for a long period of time, but biased agonists are useful as pharmaceuticals because desensitization does not occur. Brame et al. Also reported that the cyclic peptide MM07 of apelin is a biased agonist (Brame et al. Hypertension 2015: 65, 834-840). In the present specification, an apelin biased agonist is also included in the apelin agonist. WO2015147641 discloses apelin cyclic peptides and their use. WO2012125408 discloses PEGylated apelin. EP1903052 and US2014 / 0094450 also disclose APJ receptor ligands. For a general review on apelin agonists, see Narayanan et al, J Med Chem. 2015 Oct 22-58 (20): 791327, which describes various compounds. Narayanan et al, Bioorg. Med Chem. 2016 24; 3758-3770 also discloses APJ receptor agonists (eg (S) -5-cyclohexyl-3- (5- (2,6-dimethoxyphenyl) -1- (4-fluorophenyl) -1H-pyrazole-3-carboxamide) pentanoic acid) TODLER / ELABELA has also been identified as another endogenous ligand different from apelin (Wang et al, Sci. Rep. 2015, 5, 8170). These known compounds can be appropriately selected and modified as necessary to be used as an apelin receptor agonist. WO2014099944 is an APJ agonist having increased stability compared to wild-type apelin-13, modified with a group that increases half-life at the N-terminus or C-terminus of the agonist, and at least one amino acid is Disclosed are APJ agonists that are substituted by non-standard amino acids. Groups that extend half-life include PEG, Fc domain, IgG, HSA, PE, Ab, peptides, lipids, poly (O-2-hydroxyethyl) starch (HES), and nanostructures. In the present invention, these apelin receptor agonists can also be used.

  Furthermore, WO2013 / 146891 teaches pine extract, saffron extract, kina extract and comfrey extract, and crocetin, which is the main component of saffron extract, as a drug having the same function as apelin. These plant-derived extracts (extracts) can be obtained by any method known to those skilled in the art. It is known that crocetin is also contained in gardenia fruit.

Agonist antibodies can be obtained by any method known to those skilled in the art. Methods for producing monoclonal antibodies are known to those skilled in the art, and agonist antibodies can be obtained by selecting anti-APJ monoclonal antibodies having agonist activity. The animal from which the antibody is derived is not particularly limited, and human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies, and the like can be used. When used against humans, the antibody can be any of a human antibody, a humanized antibody, and a chimeric antibody, but is preferably a human antibody. Any antibody having agonistic activity against APJ need not be full length, and even antibody fragments can be used. Therefore, Fab, Fab ′, Fab′-SH, F (ab ′) ₂ , Fv, V _H , V _L , Fab-Fv, scFv, minibody, Nanobody, and the like can also be used as agonist antibodies. Further, the antibody or antibody fragment may contain a peptide sequence identified by screening by a phage display method or the like. Furthermore, aptamers composed of nucleic acids can be used in the same manner as ordinary antibodies. The antibody may form a complex with a small molecule.

Inflammatory bowel disease (IBD)
Inflammatory bowel disease (IBD) is a general term for chronic diseases that cause inflammation in the colon and small intestine. Crohn's disease (CD) and ulcerative colitis (UC) are the main types of inflammatory bowel disease, but in a broad sense also includes intestinal tuberculosis and Behcet's disease. Crohn's disease has symptoms not only in the small and large intestines, but also in the mouth, esophagus, stomach, and anus, while ulcerative colitis is primarily observed in the colon and rectum.

Crohn's disease (CD)
Crohn's disease is a type of inflammatory bowel disease (IBD) that can cause symptoms in any part of the gastrointestinal tract, from the mouth to the anus. The signs and symptoms include abdominal pain, diarrhea, fever, and weight loss. Other complications such as anemia, rash, arthritis, redness of eyes, fatigue, etc. may occur outside the gastrointestinal tract. It is not uncommon for bowel obstruction to occur and patients with this disease are at increased risk of intestinal cancer. As therapeutic agents for Crohn's disease, 5-aminosalicylic acid (mesalazine), corticosteroids, anti-TNF receptor antagonists and the like are used.

Ulcerative colitis (UC)
Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease (IBD) that causes inflammation and ulcers in the colon. Symptoms vary from mild to severe and usually manifest in young adults. The course is unpredictable, but is likely to be a lifetime illness. A prominent symptom of active disease is bloody diarrhea. UC has much in common with Crohn's disease, which is another IBD, but unlike Crohn's disease, UC shows symptoms only in the colon and rectum, not in the entire gastrointestinal tract. UC is an intermittent disease, in which the time when symptoms worsen and the time when there are relatively no symptoms alternately occur. The symptoms of UC sometimes disappear spontaneously, but usually require treatment before remission. As therapeutic agents for ulcerative colitis, 5-aminosalicylic acid (mesalazine), corticosteroids, immunosuppressants, anti-TNF receptor antagonists and the like are used.

Animal model of inflammatory bowel disease (IBD) A wide variety of models have been devised for the study of inflammatory bowel disease, including colitis models based on chemical and immunological mechanisms and spontaneous colitis models based on genetic modifications. Has been. Models derived from chemicals include sodium dextran sulfate (DSS), 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone) and 2,4,6-trinitrobenzenesulfonic acid (TNBS). ) An induced enteritis model is used (for example, see Non-Patent Documents 10 to 12). Further, as a chronic enteritis model induced by effector T cells, a method (T cell transfer enteritis model) in which naive T cells are transferred to immunodeficient mice such as Rag-deficient mice and SCID mice is known (Non-patent Document 13). ). Various models such as IL-7 transgenic (Tg) mice (Non-Patent Document 14) and IL-10-deficient (KO) mice (Non-Patent Document 15) are known as models for spontaneous development of chronic enteritis.

Disease macrophages caused by inflammatory macrophages can be broadly classified into tissue-resident macrophages and monocyte-derived macrophages. The inventors have shown that monocyte-derived inflammatory macrophages are increased in the lamina propria of the mouse inflammatory bowel disease (IBD) model, and that APJ expression is resident in inflammatory macrophages. It was found to be higher than macrophages. APJ-apelin stimulation suppresses the symptoms of animal models of inflammatory bowel disease (IBD). These data suggest that diseases caused by at least inflammatory macrophages can be treated by administration of apelin.

  Accordingly, one aspect of the present invention relates to a pharmaceutical composition for use in the treatment of diseases caused by inflammatory macrophages, comprising apelin or an apelin receptor agonist. Examples of the diseases caused by inflammatory macrophages include systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease (MCTD). ), Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, sarcoidosis Not.

Pharmaceutical Composition One aspect of the present invention relates to a pharmaceutical composition for use in the treatment or prevention of inflammatory bowel disease (IBD) comprising apelin or an apelin receptor agonist. The apelin can be apelin-36, apelin-17, apelin-13, apelin-12, or a variant thereof, but is preferably [Pyr ¹ ] -apelin-13. The apelin receptor agonist can be a small molecule compound, a plant extract, or an agonist antibody. Also, as an apelin receptor agonist, pine extract, saffron extract, kina extract, comfrey extract, gardenia extract, or crocetin can be used. The content ratio of apelin or apelin receptor agonist in 100% by weight of the pharmaceutical composition can be appropriately set within the range of 0.001 to 99.99% by weight.

  Inflammatory bowel disease (IBD) can be ulcerative colitis or Crohn's disease. Moreover, another aspect of the present invention relates to a pharmaceutical composition for use in the treatment of diseases caused by inflammatory macrophages, comprising apelin or an apelin receptor agonist. Diseases caused by inflammatory macrophages are collagen disease, more specifically systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed Sexual connective tissue disease (MCTD), Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, or sarcoidosis sell.

  The other components in the pharmaceutical composition according to the present invention are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include pharmaceutically acceptable carriers and additives. The carrier or additive is not particularly limited and can be appropriately selected depending on, for example, the dosage form. Any carrier, diluent, excipient, suspension, lubricant, adjuvant, vehicle, delivery system, Emulsifiers, tablet disintegrants, absorbents, preservatives, surfactants, colorants, flavorings, or sweeteners may be included. There is no restriction | limiting in particular also about content in the pharmaceutical composition based on this invention, According to the objective, it can select suitably.

  There is no restriction | limiting in particular as a dosage form of the pharmaceutical composition based on this invention, According to the desired administration method, it can select suitably, For example, an injection (Solution, suspension, solid agent for use, etc.) And solid preparations (tablets, capsules, suppositories, powders, etc.). As an injection, for example, a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the composition, and subcutaneous, intramuscular, intravenous, etc. are added by a conventional method. Injectables can be produced. Examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride. The solid agent may be provided with an enteric coating.

  There is no restriction | limiting in particular as an administration method of the pharmaceutical composition based on this invention, For example, according to the dosage form of a pharmaceutical composition, a patient's condition, etc., either local administration or systemic administration can be selected. Administration can be performed by, for example, intravenous administration, subcutaneous administration, intramuscular administration, oral administration, enteral administration, enema administration, tube feeding, and the like. Enteral administration is not limited to administration via the anus, but also includes administration via a tube inserted into the digestive tract from outside the individual, such as a gastric fistula. The insertion position is not limited to the intestine, but includes the esophagus, stomach, small intestine, large intestine and the like.

  The subject of administration of the pharmaceutical composition according to the present invention is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs, cats and the like. Is preferably a human patient, particularly a human patient who develops IBD. In addition, the pharmaceutical composition according to the present invention may be administered for the purpose of preventing the onset of IBD, and particularly for the purpose of preventing recurrence.

  The dosage of the pharmaceutical composition according to the present invention is not particularly limited, and can be appropriately selected depending on the dosage form, the age and weight of the administration subject, the degree of desired effect, and the like. The dose of apelin is, for example, 100 to 1,000,000 nmol per day, preferably 150 to 100,000 nmol, and the administration frequency can be, for example, 1 to 100 times per month.

  The administration time of the pharmaceutical composition according to the present invention is not particularly limited and may be appropriately selected depending on the purpose. For example, it may be administered prophylactically to a patient susceptible to the above-mentioned diseases, It may be administered therapeutically to patients presenting with symptoms. Moreover, there is no restriction | limiting in particular as frequency of administration, According to the age of an administration subject, a body weight, the grade of a desired effect, etc., it can select suitably.

Methods of treatment and use in the manufacture of a medicament One aspect of the present invention relates to a method of treating or preventing inflammatory bowel disease (IBD) in a patient. Such methods include the step of administering apelin or an apelin receptor agonist to the subject. The apelin can be apelin-36, apelin-17, apelin-13, apelin-12, or a variant thereof, but is preferably [Pyr ¹ ] -apelin-13. The apelin receptor agonist can be a small molecule compound, a plant extract, or an agonist antibody. Also, as an apelin receptor agonist, pine extract, saffron extract, kina extract, comfrey extract, gardenia extract, or crocetin can be used. Inflammatory bowel disease (IBD) can be ulcerative colitis or Crohn's disease. Another aspect of the present invention relates to a method for treating or preventing a disease caused by inflammatory macrophages in a patient. Such methods include the step of administering apelin or an apelin receptor agonist to the subject. Diseases caused by inflammatory macrophages are collagen disease, more specifically systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed Sexual connective tissue disease (MCTD), Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, or sarcoidosis sell.

One aspect of the present invention relates to the use of apelin or an apelin receptor agonist in the manufacture of a medicament for use in the treatment or prevention of inflammatory bowel disease (IBD). The apelin can be apelin-36, apelin-17, apelin-13, apelin-12, or a variant thereof, but is preferably [Pyr ¹ ] -apelin-13. The apelin receptor agonist can be a small molecule compound, a plant extract, or an agonist antibody. Also, as an apelin receptor agonist, pine extract, saffron extract, kina extract, comfrey extract, gardenia extract, or crocetin can be used. Inflammatory bowel disease (IBD) can be ulcerative colitis or Crohn's disease. Another aspect of the present invention relates to the use of apelin or an apelin receptor agonist in the manufacture of a medicament for use in the treatment or prevention of diseases caused by inflammatory macrophages. Diseases caused by inflammatory macrophages are collagen disease, more specifically systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed Sexual connective tissue disease (MCTD), Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, or sarcoidosis sell.

Food Compositions and Supplements One aspect of the present invention relates to food compositions or supplements containing apelin or an apelin receptor agonist, particularly for use in the treatment, symptom improvement or prevention of inflammatory bowel disease (IBD). The present invention relates to a food composition or supplement, or a food composition or supplement (also referred to as nutritional supplement or health supplement) for intestinal regulation, intestinal environment improvement, intestinal inflammation suppression or intestinal inflammation prevention. One embodiment of the present invention also relates to a food composition or supplement containing apelin or an apelin receptor agonist for use in the treatment, symptom improvement or prevention of a disease caused by inflammatory macrophages. Diseases caused by inflammatory macrophages are collagen disease, more specifically systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed Sexual connective tissue disease (MCTD), Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, or sarcoidosis sell. Apelin was initially isolated from the bovine stomach. Therefore, animal organ extracts (eg, kidney, heart, lung, adipose tissue, gastrointestinal tract) and the like can be used as a food composition or supplement containing apelin. As an apelin receptor agonist, for example, pine extract, saffron extract, kina extract, comfrey extract, gardenia extract, crocetin and the like can be used, but are not limited thereto.

  Examples of the food composition include general foods, foods for specific health including conditional foods for specified health, nutritional supplements, functional foods, foods for the sick, and the like. The specific form of the food composition is not particularly limited. For example, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, milk drinks and other drinks; ice cream, ice sherbet, shaved ice and other frozen desserts; Candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, etc .; noodles such as buckwheat, udon, harusame, Chinese noodles, instant noodles; -Livestock processed foods; Dairy products such as processed milk and fermented milk; processed oils and fats such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing; seasonings such as sauces and sauces; soups, stews, Examples include salads, side dishes, sprinkles, pickles, bread, and cereals. In the case of food for specified health use, dietary supplement, functional food, etc., forms such as powder, granule, capsule, troche, tablet, syrup and the like can be mentioned. The supplement according to the present invention can take any form such as a tablet, powder, liquid and the like. Moreover, a supplement can be prepared as what is orally ingested 1-3 times a day, before a meal, during a meal, and after a meal, for example.

Controlling the function of inflammatory macrophages One aspect of the present invention relates to a method of controlling the function of inflammatory macrophages with apelin or an apelin receptor agonist. Such methods can include contacting apelin or an apelin receptor agonist with inflammatory macrophages in vitro or in vivo. Here, the control of the function of inflammatory macrophages is, for example, suppression of production of cytokines and / or chemokines that induce inflammation, and preferably suppression of production of TNF, CCL2, CCL5 and / or CXCL10. Such control of the function of inflammatory macrophages can be useful in the treatment and / or prevention of diseases involving inflammatory macrophages.

  Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.

<Example 1: Experimental method>
(1) Mice C57BL / 6 (Claire Japan) as wild-type (WT) mice, donated by Rag2 deficient (Rag2 ^{− / −} ) mice (Taconic), apelin deficient (APL ^{− / −} ) mice (Vienna, IMBA) ) Was used for cell preparation and DSS enterocolitis and T cell transfer enterocolitis models. In addition, apelin / Rag2 double deficient (APL ^{− / −} Rag2 ^{− / −} ) mice were prepared by crossing Rag2 ^{− / −} and APL ^{− / −} mice. For the experiments in the examples, female mice aged 8 to 12 weeks were used.

(2) Antibody and cell analysis For staining of cells, anti-F4 / 80 antibody (BM8: eBioscience), anti-CD11b antibody (M1 / 70: BD Biosciences or eBioscience), anti-CD11c antibody (HL3: BD Biosciences), anti-TLR4 Antibody (MTS510: abcam or BioLegend), anti-CD4 antibody (RM4-5: BD Biosciences, eBioscience, or BioLegend), anti-CD62L antibody (MEL-14: BD Biosciences, eBioscience or BioLegend), anti-CD44 antibody (IM7: BD Biosciences) Or BioLegend), anti-apelin receptor (APJ) antibody (H-300: Santa Cruz), anti-CCR2 antibody (475301: R & D Systems), and FACSCalibur (BD Biosciences) or FACSCanto for flow cytometry analysis (FACS) II (BD Biosciences), FA for cell isolation by FACS method Saria II a (BD Biosciences), Cell If necessary, the analysis software Quest (BD Biosciences), FACS Diva (BD Biosciences), was used FlowJo (TreeStar).

(3) Preparation of T cell transfer enteritis model The preparation of T cell transfer enteritis model was performed by Nagaishi et al (Takashi Nagaishi et al. SHP1 Phosphatase-Dependent T Cell Inhibition by CEACAM1 Adhesion Molecule Isoforms. Immunity. 2006 Nov; 25 (5): 769-81.). Specifically, spleen cells were collected from WT mice, hemolyzed using ACK buffer, and CD4-positive T cells were isolated using Pan T CELL Isolation Kit II (Miltenyi Biotec) and CD8 microbead beads (Miltenyi Biotec). Staining was carried out with anti-CD4 antibody, anti-CD44 antibody and anti-CD62L antibody in PBS buffer containing 2% FCS. From these cell suspensions, a naive T cell fraction of CD4 ⁺ CD44 ⁻ CD62L ⁺ was isolated using FACSAria II. 2 × 10 ⁵ isolated naive T cells were suspended in PBS and transferred into the abdominal cavity of Rag2 ^{− / −} or APL ^{− / −} Rag2 ^{− / −} mice. These mice are known to develop enteritis 6 to 8 weeks after transfer.

(4) Isolation and analysis of macrophages from intestinal mucosal lamina propria The isolation from intestinal mucosa lamina was performed according to the method described in Non-Patent Document 11. That is, after recovering the large intestine from a euthanized Rag2-deficient mouse and removing feces, it was immersed in HBSS containing 1 mM dithiothreitol (DTT: Sigma), 5 mM EDTA (Nacalai Tesque) and 10% FCS at 37 ° C. Was processed. Tissues other than the supernatant were collected as pellets, washed with PBS, immersed in HBSS containing 10% FCS, 0.2 mg / ml collagenase type IV (Sigma), and 10 μg / ml DNase I (Sigma), and treated with The supernatant was recovered and filtered through a 40 μm cell strainer, and then centrifuged again to recover the pellet. The leukocyte fraction was recovered from this by specific gravity centrifugation using Percoll (GE). The collected cells were stained with anti-F4 / 80 antibody, anti-CD11b antibody, and anti-CD11c antibody, and the F4 / 80 ⁺ CD11b ⁺ CD11c ⁻ macrophage fraction was isolated and cultured using FACSAria II.

(5) Measurement of cytokines and chemokines Macrophages and T cells isolated from the lamina propria of mouse intestinal tract are RPMI1640 medium containing 1% non-essential amino acid, 10 mM HEPES, 500 U / ml penicillin, 100 μg / ml streptomycin, 10% FCS. And 0 to 1000 nM of synthetic apelin-13 (APL-13) was added as necessary and cultured at 37 ° C. for 48 hours. The culture supernatant was collected, and IL-6 (BD Biosciences), MIP-2 (BD), TNF (BD Biosciences), CCL2 (BD Biosciences), CCL5 (R & D Systems), CXCL10 (R & D Systems), IFN-γ The concentrations of various cytokines and chemokines such as IL-2 were measured by ELISA.

<Example 2: Expression system of inflammatory macrophages in the lamina propria>
When cells were collected from the lamina propria of the colonic mucosa of Rag2 ^{− / −} mice and the macrophage population of F4 / 80 ⁺ CD11b ⁺ CD11c ⁻ was analyzed (FIG. 1A), a group with high expression of TLR4, which is a marker of inflammatory macrophages ( There were TLR4 ^high ) and a low group (TLR4 ^low ) (FIG. 1B). TLR4 ^high macrophage group and TLR4 ^low macrophage group were isolated using FACSAria II and cultured for 48 hours. Production of IL-10 and TNF in the culture supernatant was measured by ELISA. As a result, in the culture supernatant of the TLR4 ^high macrophage fraction, IL-10 production was significantly lower and TNF production was higher than that of the TLR4 ^low macrophage culture supernatant (FIG. 1C). Thus, it was suggested that the TLR4 ^high fraction reflects inflammatory (M1) and the TLR4 ^low fraction reflects non-inflammatory (M2) macrophages.

<Example 3: Expression of apelin receptor in intestinal mucosa macrophage of IBD model>
Naive T cells derived from the spleen of WT mice were isolated by FACS method and transferred into the abdominal cavity of Rag2 ^{− / −} mice. This was used as a T cell transfer group, and a control group was prepared by administering only the solvent (PBS) intraperitoneally without transferring T cells. In mice of the control group and the T cell transfer group, cells in the lamina propria of the large intestine were collected 6 to 8 weeks after cell transfer, and TLR4 expression of the macrophage population was analyzed in the same manner as in Example 2 above. As a result, it was confirmed that the proportion of TLR4 ^high inflammatory (M1) macrophages was increased in macrophages in the T cell transfer group that developed enteritis compared to the control group that did not develop enteritis (FIG. 2A). ).

  Furthermore, the macrophage fraction of the T cell transfer group was identified using the TLR4 expression level of each M1 / M2 macrophage fraction in the control group as an index, and the expression of apelin receptor (APJ) in each fraction was analyzed by the FACS method. As a result, it was confirmed that the APJ expression was relatively increased in the M1 fraction in the T cell transfer group as compared to the M2 fraction (FIG. 2B). From these results, it was suggested that APJ is highly expressed in inflammatory macrophages in the lesion area of the IBD model.

<Example 4: Action of apelin receptor agonist on inflammatory macrophages>
The colonic mucosa lamina propria macrophage of the T cell transfer enteritis model induced by the method described in Example 1 (3) above was isolated using FACSAria II, cultured in the presence of 0 to 1000 nM APL-13 for 48 hours, and the culture Production of cytokines and chemokines such as IL-6, MIP-2, TNF, CCL2, CCL5, CXCL10 in the supernatant was measured by ELISA. As a result, the production of TNF, CCL2, CCL5, and CXCL10 was suppressed depending on the addition concentration of APL-13, although the production of IL-6, MIP-2, and the like was not affected (FIG. 3). From these results, it was confirmed that the production of chemokine cytokines such as TNF, CCL2, CCL5, and CXCL10 that induce inflammation can be suppressed by allowing an apelin receptor agonist such as APL-13 to act on macrophages in the inflamed area.

CCL2 (MCP-1) is known to be associated with diseases associated with the onset of monocyte infiltration such as psoriasis, rheumatoid arthritis, and atherosclerosis, and mainly monocytes, memory T cells, dendritic cells It has chemotactic activity against leukocytes such as basophils, and has the effect of attracting leukocytes to the local area of inflammation. References related to CCL2 functions include:
− Xia M, Sui Z (Mar 2009). “Recent developments in CCR2 antagonists”. Expert Opinion on Therapeutic Patents. 19 (3): 295-303.doi: 10.1517 / 13543770902755129
− Xu LL, Warren MK, Rose WL, Gong W, Wang JM (Sep 1996). "Human recombinant monocyte chemotactic protein and other CC chemokines bind and induce directional migration of dendritic cells in vitro". Journal of Leukocyte Biology. 60 (3 ): 365-71. PMID 8830793.
− Mehrabian M, Sparkes RS, Mohandas T, Fogelman AM, Lusis AJ (Jan 1991). "Localization of monocyte chemotactic protein-1 gene (SCYA2) to human chromosome 17q11.2-q21.1". Genomics. 9 (1) : 200-3.doi: 10.1016 / 0888-7543 (91) 90239-B.PMID 2004761.

  CCL5 (RANTES) is known to be associated with diseases such as rheumatoid arthritis and has chemotactic activity on leukocytes such as T cells, eosinophils, and basophils, and attracts leukocytes to the inflamed area. It is known to have an action to cause. CXCL10 (IP-10) is a chemokine discovered in psoriasis caused by infiltration of neutrophils, has an action of enhancing the adhesion of T cells to endothelial cells, monocytes, macrophages, NK cells, activated T cells This contributes to the migration of B cells to the local area of inflammation. CXCL10 is thought to be important in delayed type hypersensitivity reactions, and recently it has been reported that it binds to TLR4 and is also associated with cell damage in diabetes.

<Example 5: Administration effect of apelin receptor agonist in IBD model animal>
In the T cell transfer enteritis model induced by the method described in Example 1 (3) above, 7.5 μg of APL-13 per mouse over 9 weeks from 1 week before T cell transfer to 8 weeks after transfer, or A control solution (PBS) was administered intraperitoneally three times a week to serve as an apelin administration group and a control group, respectively. As a result, the survival rate of the control group mice at 8 weeks after the transfer was about 60%, compared with about 90% in the apelin-administered group (FIG. 4).

  Further, the large intestine of mice 8 weeks after transfer was collected, fixed and sliced, and then stained with hematoxylin and eosin. Histopathological analysis was performed on evaluation items such as epithelial injury, intestinal wall thickening, lymphocyte infiltration, and granulocyte infiltration. As a result, the apelin-administered group showed a tendency to improve enteritis pathologically compared to the control group (FIG. 5). From this result, it was shown that apelin receptor agonists such as APL-13 can be applied to the treatment of inflammatory diseases such as IBD.

  In the T cell transfer enteritis model induced by the method described in Example 1 (3) above, 7.5 μg of MM07 or control solution per mouse over 9 weeks from 1 week before T cell transfer to 8 weeks after transfer (PBS) was administered intraperitoneally three times a week to serve as a MM07 administration group and a control group, respectively. Further, after collecting the large intestine of mice 8 weeks after transfer and isolating T cells, IFN-γ, IL- which are inflammatory cytokines in the T cell culture supernatant by the method described in Example 1 (5) above. 2. When the amount of TNF was measured by ELISA, the production of IFN-γ, IL-2, and TNF was lower in the culture supernatant of T cells isolated from the MM07 administration group than in the control group. This result suggests that MM07 administration has an inhibitory effect on inflammatory cytokines in an enteritis model, and that apelin receptor agonists such as MM07 can be applied to the treatment of inflammatory diseases such as IBD.

<Example 6: IBD exacerbation effect in apelin-deficient mice>
In order to further investigate the effect of apelin in the IBD model, analysis was performed using apelin-deficient (APL ^{− / −} ) mice. A T cell transfer enteritis model was induced in APL ^{− / −} Rag2 ^{− / −} or Rag2 ^{− / −} mice by the method described in Example 1 (3) above. As a result, in APL ^{− / −} , as compared with Rag2 ^{− / −} , exacerbation of weight loss at 5 and 6 weeks after cell transfer (FIG. 6A), exacerbation of clinical symptoms such as diarrhea, bloody stool, glans back and dermatitis (Fig. 6B), a decrease in survival rate (FIG. 7), and deterioration of pathological findings (FIG. 8) were confirmed.

In addition to the above model, APL ^{− / −} mice were also evaluated by the DSS enteritis model. In practice, 2% DSS solution was administered to WT or APL ^{− / −} mice with free drinking for 5 days to induce inflammation of the large intestine, and then replaced with normal drinking water to evaluate enteritis. As a result, weight loss significantly worsened with APL ^{− / −} compared with WT mice (FIG. 9). Furthermore, macrophages infiltrating the lamina propria of the intestinal tract were isolated from WT or APL ^{− / −} mice 8 days after replacement with normal drinking water by the method described in Example 1 (4) above, and the expression of TLR4 was compared. As a result, TLR4 expression was increased in colon macrophages of APL ^{− / −} mice compared to WT (FIG. 10).

<Example 7: Analysis of inflammatory macrophage dynamics in the lamina propria of the intestinal mucosa of apelin-deficient mice>
After inducing T cell transfer enteritis in APL ^{− / −} Rag2 ^{− / −} and Rag2 ^{− / −} mice by the method described in Example 1 (3) above, the intestinal mucosa lamina propria by the method described in Example 1 (4) above macrophages infiltrating isolated, was compared the number of cells, Rag2 - / ^- as compared to APL ^- / - ^Rag2 - / - significant increase was observed in the number of cells macrophages infiltrating in mice (FIGS. 11A and 11B). Further, Rag2 ^{- / -} as compared to ^{APL - /} - Rag2 - / - are TLR4 and CCL2 expressed was increased on colonic mucosa murine macrophages (Fig. 11C, D).

Furthermore, in the supernatant obtained by isolating macrophages from the lamina propria of Rag2 ^{− / −} and APL ^{− / −} Rag2 ^{− / −} mice and culturing for 48 hours, the production of TNF, CCL2, CCL5, and CXCL10 was significantly enhanced ( FIG. 12). These results also revealed that apelin receptor agonists have a function of suppressing the inflammation-inducing action on inflammatory macrophages.

  While preferred embodiments of the invention are shown herein, it will be apparent to those skilled in the art that such embodiments are provided for purposes of illustration only, Various modifications, changes and substitutions may be made without departing from the invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the invention. In addition, the contents described in all publications, including patents and patent application documents referred to in this specification, are incorporated by reference in the same manner as the contents specified in this specification. Should be interpreted.

  The present inventors have revealed that apelin or an apelin receptor agonist can be used to treat or prevent inflammatory bowel disease (IBD). Apelin or an apelin receptor agonist can also be used to treat diseases such as collagen disease caused by inflammatory macrophages.

Claims (16)

  A pharmaceutical composition for use in the treatment or prevention of inflammatory bowel disease (IBD), comprising apelin or an apelin receptor agonist. Apelin, apelin -36, apelin -17, apelin-13 [Pyr ^1] - apelin -13 apelin -12, and is selected from the group consisting of variants pharmaceutical composition of claim 1. The pharmaceutical composition according to claim 2, wherein the apelin is [Pyr ¹ ] -apelin-13.   The pharmaceutical composition according to claim 1, wherein the apelin receptor agonist is a low molecular compound, a plant extract, or an agonist antibody. The apelin receptor agonist is TODDLER / ELABELA, E339-3D6, MM07, (S) -N- (1- (butylamino) -5-methyl-1-oxohexane-3-yl) -5- (2,6 -Dimethoxyphenyl) -1-isobutyl-1H-pyrazole-3-carboxamide, (S) -3-{[1- (1-ethyl-propyl) -2-thiophen-2-ylmethyl-1H-benzimidazole-5 Carbonyl] -amino} -5-methyl-hexanoic acid, (S) -3-{[2-furan-2-ylmethyl-1- (2-methyl-cyclohexyl) -1H-benzimidazol-5-carbonyl] -amino } -5-methyl-hexanoic acid, (S) -5-methyl-3-{[1-((1R, 2R) -2-methyl-cyclohexyl) -2-thiazole 5-ylmethyl-1H-benzimidazol-5-carbonyl] -amino} -hexanoic acid, (S) -3-{[1- (1-ethyl-propyl) -2-thiazol-5-ylmethyl-1H-benzimidazole -5-carbonyl] -amino} -5-methyl-hexanoic acid, (S) -5-cyclohexyl-3- (5- (2,6-dimethoxyphenyl) -1- (4-fluorophenyl) -1H-pyrazole -3-carboxamide) pentanoic acid, pine extract, saffron extract, kina extract, comfrey extract, gardenia extract, crocetin, and compounds represented by the following formula:

The pharmaceutical composition according to claim 1, wherein R is selected from the group consisting of RRB (Lissamine Rhodamine B) or PB (Patent Blue), and n is 0 or 1.   The pharmaceutical composition according to any one of claims 1 to 5, wherein the inflammatory bowel disease (IBD) is ulcerative colitis.   The pharmaceutical composition according to any one of claims 1 to 5, wherein the inflammatory bowel disease (IBD) is Crohn's disease.   The pharmaceutical composition according to any one of claims 1 to 7, which suppresses inflammation.   The pharmaceutical composition according to any one of claims 1 to 8, which acts on inflammatory macrophages and / or T cells.   The pharmaceutical composition according to any one of claims 1 to 9, wherein the apelin receptor agonist is MM07.   A method for treating or preventing inflammatory bowel disease (IBD) in a subject, comprising a step of administering apelin or an apelin receptor agonist to the subject.   Use of apelin or an apelin receptor agonist in the manufacture of a medicament for treating or preventing inflammatory bowel disease (IBD).   A food composition for suppressing intestinal inflammation, comprising apelin or an apelin receptor agonist.   A pharmaceutical composition for use in the treatment or prevention of a disease caused by inflammatory macrophages, comprising apelin or an apelin receptor agonist.   Diseases caused by inflammatory macrophages are systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis and polymyositis, nodular polyarteritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease (MCTD), Wegener 15. Selected from the group consisting of granulomatosis, Takayasu arteritis, temporal arteritis, eosinophilic fasciitis, adult Still's disease, ankylosing spondylitis, psoriatic arthritis, Behcet's disease, and sarcoidosis. The pharmaceutical composition as described.   A method of controlling the function of inflammatory macrophages and / or T cells by apelin or an apelin receptor agonist.

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