WO2007000924A1

WO2007000924A1 - Pharmaceutical composition comprising substance capable of inhibiting or promoting progranulin activity and screening method for substance capable of inhibiting or promoting progranulin activity

Info

Publication number: WO2007000924A1
Application number: PCT/JP2006/312428
Authority: WO
Inventors: Akifumi Matsuyama; Shizuya Yamashita; Yoshiki Sawa; Yuzuru Kanakura
Original assignee: Osaka University
Priority date: 2005-06-28
Filing date: 2006-06-21
Publication date: 2007-01-04

Abstract

A pharmaceutical composition for the treatment or prevention of a disease induced by the increased fat tissues/cells and/or a disease induced by the increased macrophage activity, the pharmaceutical composition comprising a substance capable of inhibiting a progranulin activity; a pharmaceutical composition for the treatment or prevention of a disease induced by the decreased fat tissues/cells and/or a disease induced by the decreased macrophage activity, the pharmaceutical composition comprising progranulin and/or a substance capable of promoting a progranulin activity; a method for screening a substance capable of inhibiting or promoting a progranulin activity; a substance provided by the method; and others.

Description

Specification

Pharmaceutical composition containing a substance that suppresses or promotes progranulin activity, and screening method for a substance that suppresses or promotes progranulin activity

Technical field

[oooi] The present invention relates to prodara-yulin activity for treating or preventing a disease caused by an increase or decrease in fat thread and tissue cells and a disease caused by an increase or decrease in Z or macrophage activity. A pharmaceutical composition comprising a substance that suppresses or promotes and a substance for treating or preventing a disease caused by an increase or decrease in adipose tissue 'cells and a disease caused by an increase or decrease in Z or macrophage activity. -A disease or its predisposition to which an increase or decrease in adipose tissue 'cells is characterized, characterized in that the method and the substance obtainable thereby, and the amount and Z or activity of prodara-urine are assessed Further, the present invention relates to a disease whose pathogenesis is an increase or decrease in Z or macrophage activity, or a method for examining the predisposition thereof.

Background art

[0002] In recent years, the prevalence of arteriosclerotic diseases such as coronary artery disease and cerebrovascular disorder secondary to metabolic syndrome has increased. This is associated with almost all arteriosclerotic conditions such as thrombosis and inflammation, where metabolic syndrome is obesity, impaired glucose tolerance associated with insulin resistance, abnormal lipid metabolism, hypertension, and elevated PAI-1 and CRP, respectively. Because. Visceral adipose tissue has been implicated in the onset of metabolic syndrome, and HDL (high-density lipoprotein) levels decline after the onset. Since visceral fat mass and HDL levels are inversely correlated in patients with metabolic syndrome, it was previously thought that a decrease in HDL levels was a sequelae of metabolic syndrome caused by visceral fat accumulation. (See Patent Documents 1 and 2). However, there was a contradiction between characterizing the decline in HDL as a sequelae of metabolic syndrome and the actual pathology.

[0003] Macrophages are cells that produce various substances and also infiltrate adipose tissue. Macrophage strength Among the substances produced, it is known that inflammatory site force-in is deeply involved in inflammatory diseases. Suppress the action of specific site force in to treat such diseases Drug development has been carried out. However, since such diseases are caused by many substances involved in various forms, it is considered that drugs that suppress the excessive action of the entire cyto-force-in producing cell are effective.

Non-patent document 1: Hopkins PN et al., Curr Opin Lipidol. 1996 Aug; 7 (4): 241-53 Non-patent document 2: Moller DE et al., Annu Rev Med. 2005; 56: 45-62

Disclosure of the invention

Problems to be solved by the invention

[0004] The problem to be solved by the present invention is to clarify the relationship between the decrease in HDL level and the accumulation of visceral fat and metabolic syndrome, and a substance useful for the treatment and prevention of metabolic syndrome and various diseases that accompany this, As well as the mechanism of macrophage activation, to find substances useful for the treatment and prevention of various diseases such as inflammatory diseases and arteriosclerosis, and to find screening methods for them. .

Means for solving the problem

[0005] As a result of intensive studies in view of the above circumstances, the present inventors have found that HDL suppresses visceral fat accumulation, thereby suppressing metabolic syndrome and suppressing macrophage activity. Specifically, macrophages produce prodranulin, and produralurin acts on macrophages to make them active and induce cytokines such as pro-inflammatory cytokines and physiologically active substances. It was found to secrete and induce differentiation of adipose precursor cells to adipocytes. Based on these findings, the present invention has been completed.

That is, the present invention provides:

(1) Adipose thread and tissue-Pharmaceutical composition containing a substance that suppresses produra-urine activity to treat or prevent diseases caused by increased cells and diseases caused by increased Z or macrophage activity object,

(2) The pharmaceutical composition according to (1), wherein the substance that suppresses prodara-urine activity is HDL or apolipoprotein AI.

(3) The pharmaceutical composition according to (1), wherein the substance that suppresses prodaranulin activity is an anti-progranulin antibody, (4) The disease is selected from the group consisting of obesity, diabetes, hypertension, hyperlipidemia, metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, cirrhosis. A pharmaceutical composition according to any one of (1), (3),

(5) Promote prodara-yulin and Z or prodara-yulin activity to treat or prevent diseases caused by a decrease in fat thread and texture 'cells and disease caused by decreased Z or macrophage activity A pharmaceutical composition comprising the substance

(6) The pharmaceutical composition according to (5), wherein the disease is selected from the group consisting of malnutrition, lupus, lipodystrophy, immune disease, and malignant tumor,

(7) Treatment or prevention of a disease caused by an increase in adipose tissue 'cells and a disease caused by an increase in Z or macrophage activity, characterized by administering a substance that suppresses prodara-urine activity Method,

(8) a substance that suppresses prodara-urine activity is HDL or apolipoprotein AI, or an anti-prodara-urin antibody, (7)

(9) The disease is selected from the group consisting of obesity, diabetes, hypertension, hyperlipidemia, metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, cirrhosis. The method according to any one of (7) and (8),

(10) A disease caused by a decrease in adipose tissue 'cells, characterized by administration of prodara-yulin and a substance that promotes Z or prodara-yulin activity, and caused by a decrease in Z or macrophage activity. A method of treating or preventing a disease,

(11) The method according to (10), wherein the disease is selected from the group consisting of malnutrition, lupus, lipodystrophy, immune disease, and malignant tumor,

(12) Fatty thread and tissue ・ Produralurin activity for the manufacture of a pharmaceutical thread and composition for treating or preventing diseases caused by increased cells and diseases caused by increased Z or macrophage activity. Use of inhibiting substances,

(13) A substance that suppresses prodara-urine activity HDL or apolipoprotein AI, or an anti-prodara-urine antibody, Use according to (12),

(14) The disease is selected from the group consisting of obesity, diabetes, hypertension, hyperlipidemia, metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, cirrhosis The use of either (12) or (13),

(15) Adipose and Woven · Prodara-Yulin and Z for the manufacture of a pharmaceutical composition for treating or preventing diseases caused by cell loss and diseases caused by decreased Z or macrophage activity. Or use of substances that promote prodara-urine activity,

(16) The use according to (15), wherein the disease is selected from the group consisting of malnutrition, leukemia, lipodystrophy, immune disease, and malignant tumor,

(17) A method for screening a substance that suppresses prodara-urine activity, comprising the following steps:

(a) culturing preadipocytes or macrophages in a medium containing prodara-yulin and a candidate substance;

(b) examining the differentiation of the preadipocytes into adipocytes or the activity of adipocytes; or

(c) Examining macrophage activity

Features

When the differentiation or the activity of the adipocytes, or the macrophage activity is suppressed as compared with the differentiation or activity when the adipose precursor cells or macrophages are similarly cultured in a medium not containing a candidate substance. , A method that indicates that the candidate substance is a substance that suppresses prodara-urine activity,

(18) A method for screening a substance that promotes prodara-urine activity, comprising the following steps:

(c) Examining macrophage activity

Features

The differentiation or the activity of the adipocytes, or the macrophage activity is the same as that obtained when the adipose precursor cells or the macrophages are similarly cultured in a candidate substance-free medium or A method that indicates that the candidate substance is a substance that promotes prodara-urine activity when promoted compared to activity,

(19) The method according to (17) or (18), wherein in step (a), Prodara-Yulin is supplied by macrophages,

(20) A substance that suppresses prodara-urine activity, which can be obtained by the method according to (17) or (19),

(21) A pharmaceutical composition for treating or preventing a disease caused by an increase in adipose tissue / cell and a disease caused by an increase in Z or macrophage activity, comprising the substance according to (20),

(22) a substance that promotes prodara-urine activity, obtainable by the method according to (18) or (19),

(23) A pharmaceutical composition for treating or preventing adipose tissue, a disease caused by a decrease in cells and a disease caused by a decrease in Z or macrophage activity, comprising the substance according to (22),

(24) A kit for screening for a substance that suppresses or promotes prodara-urine activity, which is used in the method according to any one of (17) to (19),

(25) A subject's adipose thread and tissueA disease or predisposition that is caused by an increase or decrease in cells, and a disease or predisposition that is caused by an increase or decrease in Z or macrophage activity. Obtaining a sample and assessing the amount and Z or activity of prodara-urine in the sample,

(26) If the disease is obesity, diabetes, hypertension, hyperlipidemia, metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, cirrhosis, malnutrition, illness, The method according to (25), which is selected from the group consisting of lipodystrophy, immune disease, and malignant tumor power,

Is to provide.

The invention's effect

According to the present invention, a prodrug-lily for treating or preventing a disease caused by an increase in fat thread and woven cells and a disease caused by an increase in Z or macrophage activity. Prodara-Yulin and Z for the treatment or prevention of pharmaceutical compositions containing substances that suppress the activity of adenine, adipose tissue, diseases caused by cell loss, and diseases caused by reduced Z or macrophage activity Or a pharmaceutical composition containing substances that promote prodara-urine activity, a method for screening these substances and substances obtainable thereby, and assessing prodara-urine amount and Z or activity, A disease or a predisposition thereof caused by an increase or decrease in adipose tissue / cell, and a method for examining a disease or predisposition thereof caused by an increase or decrease in Z or macrophage activity are provided.

Brief Description of Drawings

FIG. 1 shows the results of SDS-PAGE of purified macrophage-derived HDLZ apolipoprotein AI binding protein.

[FIG. 2] FIG. 2 is a graph showing the expression of prodara-yulin in macrophages by quantitative PCR.

FIG. 3 is an immunoplot showing the expression and secretion of prodaranulin in macrophages.

[FIG. 4] FIG. 4 is a graph showing the promotion of the differentiation of preadipocytes into adipocytes by co-culture with macrophages, and the suppression thereof by HDL and apolipoprotein AI.

FIG. 5 is a graph showing suppression of differentiation of preadipocytes into adipocytes by anti-progranulin antibody.

[FIG. 6] FIG. 6 is a graph showing the promotion of differentiation of preadipocytes into adipocytes by prodaranulin, and the suppression thereof by HDL and apolipoprotein AI.

FIG. 7 is a graph showing an increase in TNF-α expression in macrophages by prodaranulin using quantitative PCR and its suppression by HDL and apolipoprotein Α-I.

FIG. 8 is a graph showing the increase in MMP-9 expression in macrophages by prodaranulin using quantitative PCR and its suppression by HDL and apolipoprotein AI. [FIG. 9] FIG. 9 is a graph showing suppression of MMP-9 expression in macrophages by an anti-Prodara-Yulin antibody using quantitative PCR.

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] In one embodiment, the present invention relates to a proline activity for treating or preventing a disease caused by an increase in adipose tissue / cell and a disease caused by an increase in Z or macrophage activity. The present invention relates to a pharmaceutical composition containing an inhibitory substance. Adipose tissue

, Cells refer to adipose precursor cells, adipocytes, undifferentiated cells, stem cells, and the like, and Z or tissues containing these cells. The increase in adipose tissue and cells includes an increase in the number of adipose tissue 'cells, an increase in the volume of single adipocytes, and an increase in the activity of adipose tissue' cells. Adipose tissue Diseases caused by an increase in cysts can be caused directly or indirectly by an increase in adipose tissue cells, such as obesity, diabetes, hypertension, hyperlipidemia, Metabolic syndrome, arteriosclerotic disease, ischemic heart disease and the like can be mentioned.

[0010] Macrophage activity refers to phagocytosis of macrophages, function as antigen-presenting cells, and the action of producing or secreting Z or physiologically active substances such as MMP-9, TNF-a, IL 6, or cytokines This refers to any action that macrophages have, and its rise is, for example, an increase in the action of producing or secreting a physiologically active substance or cyto force-in. Diseases caused by increased macrophage activity may be those caused directly or indirectly by increased macrophage activity, such as inflammatory diseases, arteriosclerosis, ischemic heart disease, cirrhosis, etc. Can do.

[0011] As used herein, the term "prodara-urine activity" refers to the action of inducing or promoting the differentiation of produralurine into adipocytes of adipose precursor cells, and the action of increasing or maintaining the activity of adipocytes. And an action that increases or maintains macrophage activity. Thus, for example, the increase in “Prodara-Yulin activity” promotes the differentiation of preadipocytes into adipocytes to increase the number of adipocytes, or increase the activity of adipocytes, or MMP-9 from macrophages, Production and secretion of physiologically active substances such as TNF-a and IL-6 or cyto force-in may increase.

[0012] To "suppress" prodara-urine activity means that the above "prodara-urine activity" is incomplete or May be partly suppressed or completely extinguished. Specifically, reducing the promotion of differentiation rate from preadipocytes to adipocytes, blocking the induction of differentiation, and completely or incompletely stopping induced or promoted differentiation, and adipocytes This includes completely or incompletely blocking the action on macrophages, and further completely or incompletely blocking the action on macrophages. Therefore, suppression of prodara-urine activity reduces the number of adipocytes produced or reduces the activity of adipocytes, or bioactive substances such as MMP-9, TNF-a, IL6 Production and secretion of force-in may be reduced.

[0013] Substances that inhibit the activity of prodara-urine of the present invention include those capable of acting directly or indirectly on prodara-urine. The substance that suppresses produra-urine activity may be capable of suppressing the action of produralurin described above, for example, by binding directly or indirectly to produralurin. . For example, HDL and its main component apolipoprotein AI, anti-prodara-urine antibody, or a substance known as an antagonist / inhibitor of prodralurin, etc., preferably HDL Or apolipoprotein AI, or anti-prodara-urine antibody. Here, the anti-prodara-urin antibody may be a monoclonal antibody or a polyclonal antibody.

[0014] The pharmaceutical composition of the present invention generally contains a substance that suppresses prodara-urine activity, an excipient, an additive, and Z or a carrier. If desired, the pharmaceutical composition of the present invention may further contain one or more substances that inhibit prodara-urine activity. The dosage form of the pharmaceutical composition of the present invention is appropriately selected depending on the condition of the target, target site, etc. For example, it may be an oral preparation such as a powder, a tablet, a capsule or a syrup, or a liquid such as a liquid for injection or infusion. The amount of the substance that suppresses produra-urine activity in the pharmaceutical composition of the present invention depends on various conditions such as the age, sex, or history of the subject, and the type and severity of the disease to be treated or prevented. It is selected as appropriate.

[0015] In another aspect, the present invention relates to a disease whose pathogenesis is caused by a decrease in adipose tissue and cells. And a pharmaceutical composition comprising prodara-urine and a substance that promotes z- or prodara-urine activity for treating or preventing a disease caused by reduced z or macrophage activity. The reduction of adipose tissue / cell includes a decrease in adipose tissue / cell number, a decrease in the volume of a single adipocyte, and a decrease in adipose tissue 'cell activity. The disease caused by a decrease in adipose tissue or cells may be a disease caused directly or indirectly by a decrease in adipose tissue 'cells, for example, the ability to include malnutrition, anemia, lipodystrophy, etc. It is not restricted to these.

[0016] Decrease in macrophage activity refers to suppression of the action and function of macrophages, such as the production and secretion of bioactive substances such as macrophages such as MMP-9, TNF-a, and IL6, or cytodynamic force in. Or to disappear. The disease caused by a decrease in macrophage activity may be a disease caused directly or indirectly by a decrease in macrophage activity, such as an immune disease or a malignant tumor.

[0017] “Promoting” produralurin activity specifically means that the rate of differentiation from preadipocytes to adipocytes is further increased, the induction of differentiation is further promoted, and the effect of prodarurin on adipocytes And further enhancing the effect of prodarurin on macrophages. Therefore, by promoting produral urine activity, the number of resulting adipocytes increases or the activity of adipocytes increases, or macrophages such as MMP-9, TNF-a, IL-6, etc. The production and secretion of active substances or cytosines increases.

[0018] Substances that promote the activity of prodara-urine of the present invention include substances that can act directly or indirectly on prodara-urine. The substance that promotes produral-urine activity may be one that can promote the action of produralurin as described above, for example, by binding directly or indirectly to produralurin. . For example, prodara-yulinagost, proteolytic enzyme and the like.

[0019] The pharmaceutical composition of this aspect of the present invention comprises a product that promotes prodara-urine and Z or prodara-urine activity, and an excipient, additive, Z or carrier, and the like. The pharmaceutical composition of the present invention may further contain one or more substances that promote produralurine activity, if desired. Any dosage form of the pharmaceutical composition of the present invention can be used. It is appropriately selected depending on conditions such as a target state and a target site. For example, it may be a liquid such as an oral preparation such as a powder, a tablet, a capsule or a syrup, an injection or a drip solution. In the pharmaceutical composition of the present invention, the amount of the product that promotes prodara-urine and produralurin activity is determined by the age, sex, or history of the subject, and the type and severity of the disease to be treated or prevented. It is appropriately selected according to various conditions.

[0020] The present invention, in a further aspect, comprises administering a substance that suppresses prodara-urine activity, a disease caused by an increase in adipose tissue 'cells, and an increase in Z or macrophage activity The present invention provides a method for treating or preventing a disease. Substances that suppress prodaranulin activity, diseases caused by an increase in fat thread and tissue / cells, and diseases caused by an increase in macrophage activity are as described above. The dose, administration method, administration route, number of administrations, etc. of the substance that suppresses the product activity are appropriately selected depending on various factors such as the condition of the subject and the type of disease.

[0021] The present invention, in a still further aspect, comprises administering a product that promotes prodara-urin and Z or a product that promotes producturin activity, a disease caused by a decrease in adipose tissue 'cells, and Z Alternatively, the present invention provides a method for treating or preventing a disease caused by a decrease in macrophage activity. Substances that promote prodara-urine activity, diseases caused by a decrease in adipose tissue 'cells, and diseases caused by decreased macrophage activity are as described above. The dosage, administration method, administration route, number of administrations, etc. of the substance that promotes prodara-yulin and Z or prodara-yulin activity are appropriately selected according to various factors such as the condition of the subject, the type of disease, etc. .

[0022] In another embodiment, the present invention provides a disease in which an increase in adipose tissue · cells is caused, and

The present invention relates to the use of a substance that suppresses prodanranulin activity for the manufacture of a pharmaceutical composition for treating or preventing a disease caused by increased Z or macrophage activity. Substances that inhibit the activity of prodara-yulin may be added in any process for producing a pharmaceutical composition, or may be mixed with other components of the pharmaceutical composition at the time of use. Also good. The amount of the substance that suppresses prodara-urine activity to be added can be appropriately selected depending on, for example, the type and severity of the disease, the condition of the subject, and the like. [0023] In yet another aspect, the present invention provides a pharmaceutical thread for the treatment or prevention of a disease caused by a decrease in adipose tissue / cell and a disease caused by a decrease in Z or macrophage activity. It relates to the use of prodara-yulin and substances that promote prodara-yulin activity for the production of products. Prodara-Yulin and the substance that promotes Prodara-Yulin activity may be added in any step for producing the pharmaceutical composition, or may be mixed with other components of the pharmaceutical composition at the time of use. . The amount of prodara-urine and the substance that promotes prodara-urine activity to be added can be appropriately selected according to, for example, the type and severity of the disease, the condition of the subject, and the like.

[0024] In still another aspect, the present invention provides a method for screening a substance that suppresses prodara-urine activity, comprising the following steps:

(c) Examining macrophage activity

Features

When the differentiation or the activity of the adipocytes, or the macrophage activity is suppressed as compared with the differentiation or activity when the adipose precursor cells or macrophages are similarly cultured in a medium not containing a candidate substance. The present invention relates to a method for indicating that the candidate substance is a substance that suppresses prodara-urine activity.

[0025] There are various candidate substances, and examples include, but are not limited to, apolipoprotein A-I analogs, derivatives or mutants, or anti-progranulin antibody modifications or mutants. .

[0026] Adipose precursor cells include, but are not limited to, those isolated from visceral adipose tissue or subcutaneous adipose tissue, mesenchymal stem cells, and those derived from stem cells such as ES cells. . Methods for obtaining preadipocytes are well known to those skilled in the art. Macrophages can be obtained by appropriately selecting methods well known to those skilled in the art.

[0027] A method of culturing preadipocytes or macrophages in a medium containing prodara-urine The stage may be supplemented directly or indirectly with prodaranulin in the adipose precursor cell or macrophage culture system. For example, the product may be dissolved in a suitable solvent such as PBS and added to the medium, or the culture of host cells transduced with an expression vector containing the cDNA encoding product product-urine may be used. Kiyo may be added to the medium or supplied by macrophages. The addition of programming to the culture system of preadipocytes may be performed by co-culturing preadipocytes with macrophages or by adding a culture supernatant of macrophages. Macrophages infiltrated with adipose precursor cells and adipocytes are observed in adipose tissue in vivo. Therefore, in order to perform screening under conditions close to the environment in vivo, Prodara-Yulin was co-cultured with macrophages. The power of adding is more preferable. The addition of Prodara-Yulin to the macrophage culture system may be carried out by being produced by itself, or by adding a culture supernatant of macrophages cultivated separately. As the medium, RPMI 1640 medium, DMEMZF-12 medium and the like are known, and can be appropriately selected and used.

[0028] As means for examining differentiation from preadipocytes into adipocytes, for example, the number of fat cells is calculated by microscopic observation, and the size, number, fat content, etc. of fat droplets are observed by oil red O staining. Markers that are known to decrease or increase in expression from preadipocytes to adipocytes, such as aP2, CD36, adiponectin, resistin, GLUT4, TNF-a, PAI- Measurement of 1 etc. by quantitative PCR or immunoblot.

[0029] As a means of examining the activity of adipocytes, for example, immunization with substances known to be produced and secreted by adipocytes such as aP2, CD36, adiponectin, resistin, GLUT4, TNF-a, PAI-1 Examples include measuring by plotting, measuring the expression of genes encoding powerful substances by quantitative PCR, and quantifying the amount of fatty acid secreted by catecholamine supplements.

[0030] As a means for examining the macrophage activity, for example, it is performed by examining the production amount of a marker substance that changes the production amount of macrophage power when activated. Specifically, it is a powerful marker that production increases with macrophage activity. Investigate the amount of production of car substances such as MMP-9, TNF-a, IL-6, etc., or marker substances such as adiponectin, whose production is likely to decrease with macrophage activation. Is done. Examples of the means for examining the production amount of the marker substance include, but are not limited to, quantitative PCR, immunoblot, ELISA, bioassay and the like.

[0031] Substances obtainable by a powerful screening method are suitable, for example, for the treatment or prevention of diseases caused by increased adipose tissue 'cells and diseases caused by increased Z or macrophage activity. it is conceivable that.

[0032] Yet another embodiment of the present invention is a method for screening a substance that promotes prodara-urine activity. In other words, in the screening method described above, in the presence of a candidate substance, if preadipocyte force promotes differentiation into adipocytes or increases adipocyte activity, or increases macrophage activity, The substance is shown to be a substance that promotes Prodara-Yulin activity. There are various candidate substances, and examples thereof include, but are not limited to, proteolytic enzyme analogs, derivatives or mutants. Substances that can be obtained by powerful screening methods are considered to be suitable for the treatment or prevention of diseases that are caused by, for example, a decrease in fat tissue 'cells, and diseases that are caused by a decrease in Z or macrophage activity. .

[0033] Therefore, in a further aspect, the present invention relates to a substance that suppresses or promotes prodara-urine activity, which can be obtained by the above screening method.

[0034] In a still further aspect, the present invention provides, in a still further aspect, a disease whose pathogenesis is caused by an increase or decrease in adipose tissue 'cells, which comprises a substance that suppresses or promotes prodara-urine activity, which can be obtained by the screening method described above. The present invention relates to a pharmaceutical composition for treating or preventing a disease caused by an increase or decrease in Z or macrophage activity. In general, the pharmaceutical composition comprises a substance that suppresses or promotes the produra-urine activity that can be obtained by the screening method, and an excipient, an additive, and Z or a carrier. The pharmaceutical composition contains, in addition to the substance that suppresses or promotes the producturin activity obtainable by the above screening method, further one or more substances that inhibit or promote the producta-urine activity. . Agent of the pharmaceutical composition The shape has already been explained.

[0035] In another aspect, the present invention relates to a kit for screening for a substance that suppresses or promotes produralulin activity, which is used in the screening method. The kit of the present invention may include a medium, a culture vessel, a means for examining differentiation of preadipocytes into adipocytes, activity of fat cells, or macrophage activity, and the like. Usually, the instruction manual is attached to the kit. The above screening can be performed quickly and easily using a powerful kit.

[0036] The present invention, in another aspect, relates to a disease or predisposition to which an increase or decrease in adipose tissue cells of a subject is pathogenic and a disease or predisposition to which an increase or decrease in Z or macrophage activity is pathogenic The present invention relates to a test method comprising obtaining a sample from a subject and assessing the amount of produra-urine and Z or activity in the sample.

[0037] The disease may be caused directly or indirectly by an increase or decrease in the amount of prodara-urine and Z or activity. For example, obesity, diabetes, hypertension, hyperlipidemia, Examples include metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, cirrhosis, malnutrition, ulcer, lipodystrophy, immune disease, and malignant tumor. Preferred are obesity, metabolic syndrome, and arteriosclerotic disease.

[0038] The sample may be any sample as long as the target force is obtained. For example, serum, a puncture specimen of preadipose tissue, and the like.

[0039] The means for assessing the amount and Z or activity of prodara-urine may be directly assessing or indirectly assessing the amount and product or Z or activity of prodara-urine. Also good. For example, the expression of prodaranulin may be measured by ELISA, immunoplot, or quantitative PCR, or the preadipocytes are cultured in a medium supplemented with the sample, and differentiation from the preadipocytes into adipocytes is performed. It may be examined, or a substance in which the macrophage is cultured in a medium supplemented with the sample and production from macrophages is promoted or suppressed by prodara-ulin, such as MMP-9, TNF-a, IL The expression level or production / secretion level of 6 etc. may be measured.

[0040] The present invention will be described in more detail and specifically with reference to the following examples. It is not intended to limit the invention.

Example 1

[0041] Isolation and Identification of Macrophage-derived HDLZ Apolipoprotein A-I Binding Prodara-Yulin

Human monocyte-derived macrophages were cultured in RPMI 1640 (Gibco) containing 10% human serum for 7 days at 37 ° C, and then cultured in serum-free RPMI 1640 for 24 hours. Next, the culture supernatant was concentrated by 80% ammonium sulfate precipitation, and desalted using a PD-10 column (Amersham Pharmacia). The eluate was purified through an apolipoprotein A—I affinity column (Pierce). The purified protein was identified as a single protein of about 80 kDa by SDS-PAGE (see Figure 1). The internal amino acid sequence of this protein was determined by in-gel digestion with V8 peptidase and found to be prodara-urine (hereinafter also referred to as PEPI), a kind of pro-inflammatory site force in. I was strong.

[0042] Changes in Prodara-Yulin expression 'secretion during macrophage differentiation

A. Measurement of Prodara-Yulin expression in macrophages by RT-PCR

Human monocyte-derived macrophages were cultured in RPMI 1640 medium containing 10% human serum, and RNA was extracted at 0, 1, 3, 5, 7, and 10 days. Quantitative PCR (internal standard: GAP DH) was performed using TaqMan [registered trademark] probe (Applied Bio Systems) using powerful RNA as a recording model. The results are shown as relative values when the expression level on day 0 is taken as 1 (see FIG. 2). It was found that the expression of prodarulin increases with the macrophage distribution. As controls, CD14, whose expression decreases with macrophage differentiation, and SR-A and CD36, whose expression increases, were measured.

[0043] B. Production of Prodaranulin in Macrophages by Immunoplot Measurement of 'fraction ^' Human monocyte-derived macrophages were cultured in RPMI 1640 medium containing 10% human serum, 0, 1, 3, 5, 7, And the 10th mouse [serum-free RPMI 1640 medium] This culture medium was replaced, and the culture supernatant 24 hours later was subjected to immunoplot using anti-Prodara-Yulin antibody. The presence of Prodara-Yulin protein in the culture supernatant confirmed that Prodara-Yulin was secreted by macrophages and that the amount increased with the amount of macrophages (see Fig. 3). [0044] Induction and suppression of preadipocyte differentiation into adipocytes by prodaranulin

A. Promotion of preadipocyte differentiation into adipocytes by co-culture with macrophages and its suppression by HDLZ apolipoprotein AI

Human adipose precursor cells were co-cultured with macrophages for 7 days in DMEM / F-12 medium containing 66nM insulin, 0.033mM piotin, 0.017mM nontenthenic acid, 10% human serum, and then stained with oil red O. . As a control group, preadipocytes cultured in the absence of macrophages were used. Furthermore, in order to investigate the effects of HDL and apolipoprotein A—I on prodara-urine, a medium containing 20 gZm 1 HDL or 10 / zg / ml apolipoprotein A—I in the above-mentioned adipose precursor cells was used. And cultured. Differentiation of human preadipocytes into adipocytes was promoted by co-culture with macrophages and was shown to be blocked by HDL, apolipoprotein AI (see Fig. 4). It was speculated that produralurin promotes the differentiation of preadipocytes into adipocytes.

[0045] B. Inhibition of adipocytes of preadipocytes into adipocytes by anti-progranulin antibody

Human adipose precursor cells were co-cultured with macrophages in 10% human serum-containing DMEM / F-12 medium supplemented with 50 μg / ml anti-prodara-urine antibody (anti-PEPI antibody) for 7 days, followed by oil red O Staining was performed. Anti-Prodara-Yulin antibody suppresses the differentiation of adipocytes (see Fig. 5), so that Prodara-Yulin secreted by macrophages also promotes the differentiation of preadipocytes into adipocytes. I understood.

[0046] C. prodaranulin promotes differentiation of preadipocytes into adipocytes, and its suppression by HDL and apolipoprotein AI

Prodara-Yulin cDNA was ligated into the expression vector pcDNA3.1 (Invitorogen) and transfected into 293 cells. The preadipocytes were cultured for 7 days in DMEMZF-12 medium containing 10% ushi fetal serum supplemented with the obtained culture supernatant, and then oil red O staining was performed. As a control group, cultured preadipocytes in the absence of prodara-yulin were used. It was further confirmed that prodanranulin promotes the differentiation of preadipocytes into adipocytes (see Fig. 6). In addition, adipose precursor cells are cultured in a medium supplemented with HDL and apolipoprotein AI, and It was confirmed that the promotion of differentiation into adipocytes was suppressed by HDL and apolipoprotein AI (see Fig. 6).

Example 2

[0047] Increase in macrophage activity by prodara-yulin

Human monocyte-derived macrophages were cultured in DMEMZF-12 medium containing 10% human serum for 7 days. Next, after culturing in RPMI 1640 medium containing the culture supernatant of 293 cells transfected with an expression vector containing prodara-urine cDNA, macrophages were recovered and RNA was extracted. Quantitative PCR (internal standard: GAPDH) was performed using TaqMan (registered trademark) probe, and the expression of TNF-α and MMP-9 was measured. Expression of TNF-α and ΜΜΡ-9, which is expressed in activated macrophages and is known to induce differentiation of preadipocytes, is increased by prodara-yulin, and this increase is due to HDL, apolipoprotein It was found that it was suppressed by A—I (see Figures 7 and 8).

[0048] Reduction of macrophage activity by anti-Prodara-Yulin antibody

Human monocyte-derived macrophages are cultured in DMEMZF-12 medium containing 10% human serum containing anti-prodara-urine antibodies (M12, N19, and S15) for 1 day, RNA is extracted, and TaqMan (registered trademark) probe is used V and quantitative PCR (internal standard: GAPDH) were performed to measure the expression of MMP-9. It was found that anti-Prodara-Yulin antibody suppressed MMP-9 expression (see Fig. 9).

Industrial applicability

[0049] According to the present invention, prodara-urine activity for treating or preventing a disease caused by an increase or decrease in adipose tissue / cells and a disease caused by an increase or decrease in Z or macrophage activity is improved. To treat or prevent a pharmaceutical composition comprising a substance that suppresses or promotes, and a disease caused by an increase or decrease in fat thread and tissue cells and a disease caused by an increase or decrease in Z or macrophage activity A disease or predisposition to the pathogenesis of an increase or decrease in adipose tissue cells characterized by the method of screening the substance and the substance obtainable thereby, and prodara-urine amount and Z or activity , And increased Z or macrophage activity Or a method for testing a disease whose cause is a decrease or a predisposition thereof, etc., such as development of preventive, therapeutic or diagnostic agents for diseases such as obesity and metabolic syndrome It can be used in the field of manufacturing, the field of health foods, the research field of fat precursor cells or macrophages and related cells.

Claims

The scope of the claims

[I] Adipose thread and tissue · Pharmaceutical composition containing a substance that suppresses prodara-urine activity to treat or prevent diseases caused by increased cells and diseases caused by increased Z or macrophage activity object.

[2] HDL or apolipoprotein A—I is a substance that suppresses prodara-urine activity

The pharmaceutical composition according to claim 1.

[3] The pharmaceutical composition according to claim 1, wherein the substance that suppresses prodara-urin activity is an anti-prodara-urin antibody.

[4] The disease is selected from the group consisting of obesity, diabetes, hypertension, hyperlipidemia, metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, and cirrhosis. The pharmaceutical composition according to any one of claims 1 to 3.

[5] Includes prodara-yulin and a substance that promotes Z or prodaranulin activity to treat or prevent diseases caused by reduced adipose tissue 'cells and diseases caused by reduced Z or macrophage activity Pharmaceutical composition.

[6] The pharmaceutical composition according to claim 5, wherein the disease is selected from the group consisting of malnutrition, lupus, lipodystrophy, immune disease, and malignant tumor.

[7] A method for treating or preventing a disease caused by an increase in adipose tissue 'cells and a disease caused by an increase in Z or macrophage activity, which comprises administering a substance that suppresses prodara-urine activity.

[8] The method according to claim 7, wherein the substance is HDL or apolipoprotein AI, or an anti-prodara-urin antibody, which suppresses prodara-urine activity.

[9] The disease is selected from the group consisting of obesity, diabetes, hypertension, hyperlipidemia, metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, and cirrhosis. 9. A method according to any one of claims 7 or 8.

[10] Diseases caused by a decrease in adipose tissue 'cells, characterized by administration of prodara-yulin and Z or substances that promote prodara-yulin activity, and reduced Z or macrophage activity A method of treating or preventing a disease.

[II] Group of diseases consisting of malnutrition, lupus, lipodystrophy, immune disease, malignant tumor 11. The method of claim 10, wherein the method is more selected.

[12] Fatty thread and tissue · Progranulin activity for the manufacture of a pharmaceutical composition for treating or preventing diseases caused by increased cells and diseases caused by increased Z or macrophage activity. Use of inhibiting substances.

[13] The use according to claim 12, which is HDL or apolipoprotein AI, or an anti-prodara-urin antibody, which suppresses prodara-urine activity.

[14] The disease is selected from the group consisting of obesity, diabetes, hypertension, hyperlipidemia, metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, and cirrhosis. Use according to any of claims 12 or 13.

[15] Adipose tissue 'Programlin and Z for the manufacture of a pharmaceutical composition for the treatment or prevention of diseases caused by cell loss and diseases caused by reduced Z or macrophage activity. Or use of a substance that promotes Prodara-Yulin activity.

[16] The use according to claim 15, wherein the disease is selected from the group consisting of malnutrition, lupus, lipodystrophy, immune disease, and malignant tumor.

[17] A method for screening a substance that suppresses prodara-urine activity, comprising the following steps:

(c) Examining macrophage activity

Features

When the differentiation or the activity of the adipocytes, or the macrophage activity is suppressed as compared with the differentiation or activity when the adipose precursor cells or macrophages are similarly cultured in a medium not containing a candidate substance. A method wherein the candidate substance is a substance that suppresses prodara-urine activity.

[18] A method for screening a substance that promotes prodara-urine activity, comprising the following steps: (a) culturing preadipocytes or macrophages in a medium containing prodara-yulin and a candidate substance;

(c) Examining macrophage activity

Features

When the differentiation or the activity of the adipocytes, or the macrophage activity is promoted compared to the differentiation or activity when the adipose precursor cells or macrophages are similarly cultured in a medium not containing a candidate substance. A method wherein the candidate substance is a substance that promotes prodara-urine activity.

[19] The method according to [17] or [18], wherein in step (a), Prodara-Yulin is supplied by macrophages.

[20] A substance that suppresses prodara-urine activity, which can be obtained by the method according to claim 17 or 19.

[21] A pharmaceutical composition for treating or preventing a disease caused by an increase in adipose tissue / cell and a disease caused by an increase in Z or macrophage activity, comprising the substance according to claim 20.

[22] A substance that promotes prodara-urine activity, obtainable by the method according to claim 18 or 19.

[23] A pharmaceutical composition for treating or preventing an adipose tissue, a disease caused by a decrease in cells and a disease caused by a decrease in Z or macrophage activity, comprising the substance according to claim 22.

[24] A kit for screening for a substance that suppresses or promotes prodara-urine activity, which is used in the method according to any one of claims 17 to 19.

[25] A method for examining a disease or predisposition to which an increase or decrease in cells is a pathogenesis or a predisposition thereof, and a disease or a predisposition to an increase or decrease in Z or macrophage activity. A method comprising obtaining a sample from a subject and assessing the amount and Z or activity of prodara-urine in the sample. The disease is obesity, diabetes, hypertension, hyperlipidemia, metabolic syndrome, arteriosclerotic disease, ischemic heart disease, inflammatory disease, arteriosclerosis, liver cirrhosis, malnutrition, leukemia, lipodystrophy, immunity 26. The method according to claim 25, wherein the method is selected from the group consisting of disease and malignant tumor power.