JPWO2017142055A1 - Predictive factors and testing methods for the development of arteriosclerotic diseases - Google Patents

Abstract

  The present invention provides a diagnostic marker for an arteriosclerotic disease and a method for evaluating the onset risk. More specifically, the step of adopting C5a protein in a blood sample as a diagnostic marker for arteriosclerotic disease, measuring the amount of C5a protein in a blood sample obtained from a subject in vitro, and C5a protein Provided is a method for evaluating the risk of developing an arteriosclerotic disease, comprising the step of evaluating the risk of developing arteriosclerosis or vascular inflammation in a subject using the amount as an index. This method is useful for evaluating the risk of developing arteriosclerotic disease at an extremely early stage.

Description

Cross-reference of related applications

  This application is an application that enjoys the benefit of the priority of Japanese Patent Application No. 2016-27867 (filing date: February 17, 2016), which is incorporated herein by reference in its entirety.

  The present invention relates to a method and kit for assessing the risk of developing arteriosclerotic diseases. More specifically, the present invention relates to a method and a kit for evaluating the risk of developing an arteriosclerotic disease by measuring the amount of C5a protein in blood. The present invention also relates to a composition for use in the treatment or prevention of arteriosclerotic diseases, which contains a substance that suppresses the activity of C5a protein.

  Arteriosclerosis is a serious disease that causes fatal myocardial infarction and cerebral infarction. Arteriosclerosis is a basic pathology of lifestyle-related diseases such as hypertension, dyslipidemia, and diabetes, which impairs the quality of life and is often fatal. Known methods for monitoring arteriosclerosis throughout the body include MRI (cerebral white matter lesion), vascular echo (carotid intima-media complex thickening), pulse wave velocity (PWV) measurement, etc. It is difficult to predict the onset of sexual disease at an early stage (Non-patent Document 1).

  Conventionally, it is known that lipoproteins such as LDL and HDL in blood are related to the development of atherosclerosis (Non-patent Document 2). In addition, it has been reported that CRP, IL-5 and other molecules as inflammatory markers are associated with obstructive arteriosclerosis and coronary arteriosclerosis (Patent Document 1, Non-Patent Document 3). Excessive lipid intake is said to be involved in the onset of arteriosclerosis, and it is known that high blood cholesterol levels increase the risk of arteriosclerosis and myocardial infarction (Non-patent Document 4). However, at present, there is no reliable early marker for arteriosclerosis, and there is a need for such an early marker for disease.

WO2012 / 067165

London GM et al., Arterial functions: how to interpret the complex physiology. Nephrol Dial Transplant. 25 (12): 3815-23. 2010. Babiak J. et al., Lipoproteins and atherosclerosis. Baillieres Clin Endocrinol Metab. 1 (3): 515-50. 1987. Ait-Oufella Het al., Recent advances on the role of cytokines in atherosclerosis. Arterioscler Thromb Vasc Biol. 31 (5): 969-79. 2011. Martin SS et al., Dyslipidemia, coronary artery calcium, and incident atherosclerotic cardiovascular disease: implications for statin therapy from the multi-ethnic study of atherosclerosis. Circulation. 129 (1): 77-86. 2014.

  An object of the present invention is to provide a method and a kit for evaluating the risk of developing an arteriosclerotic disease. Another object of the present invention includes providing a composition for use in the treatment or prevention of arteriosclerotic diseases.

  The inventors have found that complement component C5a is elevated in the blood before vascular inflammation occurs before the onset of arteriosclerosis. Increased C5a activates neutrophils, induces neutrophil adhesion to the intima and leukocyte accumulation, and further increases other inflammatory factors (such as MCP-1). Moreover, it was confirmed that suppression of C5a attenuates neutrophil adhesion, leukocyte accumulation, and expression of inflammatory factors. Since this increase in C5a occurs prior to the increase in other inflammatory factors and markers and is specific before onset, it is possible to use arteriosclerosis and blood vessels that can be used at an earlier stage compared to conventional examination methods. Those skilled in the art will appreciate that they are useful as predictors of the risk of developing inflammation. It is also understood by those skilled in the art that arteriosclerosis can be treated or prevented by suppressing the activity of C5a present in the blood before the onset of atherosclerosis or at an extremely early time point. The present invention is based on these findings, and more specifically, relates to the following matters.

[1] A method for evaluating the risk of developing an arteriosclerotic disease, the step of measuring the amount of C5a protein in a blood sample obtained from a subject in vitro, and the test using the amount of C5a protein as an index A method comprising evaluating the risk of developing an arteriosclerotic disease in a person.
[2] The method according to [1], wherein when the measured value of the amount of the C5a protein is statistically significantly larger than a reference value, the risk of developing an arteriosclerotic disease in the subject is high. .
[3] The subject has one or more factors in blood concentration that are not statistically significantly increased compared to a reference value, and the factors are TNF-α, IL-1α, IL The method according to [1] or [2], which is one or more factors selected from the group consisting of -1β, IL-7, and IL-11.
[4] The arteriosclerotic disease is atherosclerosis, obstructive arteriosclerosis, myocardial infarction, cerebral infarction, stroke, angina pectoris, cerebral thrombus, cerebral hemorrhage, or renal failure, [1] to [3] The method as described in any one of.
[5] The method according to any one of [1] to [4], wherein the C5a protein is a polypeptide having the sequence shown in SEQ ID NO: 1, or a variant or fragment thereof.
[6] The method according to any one of [1] to [5], wherein the measurement is performed using a substance capable of specifically binding to C5a protein.
[7] The method according to [6], wherein the substance capable of binding is an antibody.
[8] The method according to any one of [1] to [7], wherein the blood sample is serum, plasma, or whole blood.
[9] A kit for evaluating the risk of developing arteriosclerotic disease, comprising an anti-C5a antibody, a fragment thereof, and / or a chemically modified derivative thereof.
[10] The kit according to [9], which is for use in the method according to any one of [1] to [8].
[11] The kit according to [9] or [10], wherein the antibody, fragment or chemically modified derivative is bound to a solid phase carrier.
[12] The kit according to [11], wherein the solid phase carrier is a test strip, a plastic tube, a microplate, or a glass bead.
[13] A composition for use in the treatment or prevention of an arteriosclerotic disease, comprising a substance that inhibits binding between a C5a protein and a C5a receptor as an active ingredient.
[14] The composition according to [13], wherein the substance that inhibits the binding between the C5a protein and the C5a receptor is a C5a receptor antagonist.
[15] The method according to any one of [1] to [8], wherein the subject is 75 years old or older.
[16] The method according to any one of [1] to [8] or [15], wherein plaque is not formed or is not developed in the subject.

FIG. 6 is an image and graph showing that HFD induces leukocyte migration in the femoral artery. The upper part of the left image shows blood flow in the femoral artery of a mouse fed with a normal diet (NC) for 1 week (1w) to 8 weeks (8w). The lower part of the left image shows blood flow in the femoral artery of a mouse fed a high fat diet (HFD) for 1 week (1w) to 8 weeks (8w). Arrows indicate leukocytes adhered to the endothelium and rolling leukocytes. The graph on the right represents the number of adherent leukocytes and rolling leukocytes in the femoral artery. It is the graph which compared the number of circulating leukocytes (Circulating leukocytes), neutrophils (Neutrophil), monocytes (Monocytes), and lymphocytes (Lymphocytes) between NC and HFD. It has been shown that neutrophils are significantly increased. It is the figure shown about the time schedule and flow cytometry analysis of a neutrophil depletion experiment. Wild-type mice were treated with neutrophil-depleting antibody 1A8 or control IgG 22 and 24 days after the start of HFD. In these mice, leukocyte migration was observed 28 days after the start of HFD. 1A8 clearly reduced neutrophils in peripheral blood compared to control IgG. FIG. 6 is an image and graph showing leukocyte migration in the femoral artery of HFD-fed wild type mice treated with 1A8 (right image) or IgG (left image). The graph shows that leukocyte migration is significantly inhibited by neutrophil depletion (n = 4 each). It is the image and graph which showed about monocyte depletion in the blood of a LysM-eGFP mouse | mouth using clodronic acid liposome. The highest expression of GFP in peripheral leukocytes was observed in neutrophils, but monocytes were also intermediately positive. Therefore, monocytes were removed using clodronic acid liposomes. Clodronic acid liposome treatment depleted GFP ⁺ Ly-6C ^hi leukocytes as monocytes in peripheral blood, but does not remove GFP ⁺ Ly6G ⁺ leukocytes as neutrophils. Images show leukocyte recruitment in HFD fed LysM-eGFP mice treated with clodronate liposomes to remove monocytes. Clodronic acid liposomes were administered 24 hours prior to observation. The left image shows the femoral artery when treated with solvent alone, and the right image shows the femoral artery when treated with clodronate liposomes. Leukocyte migration did not change with monocyte depletion (graph) (n = 4 each). It is a graph which shows the result of having analyzed the serum of the mouse | mouth which gave HFD for 4 weeks by the cytokine array. It was shown that the expression of C5a is the largest increase among various cytokines. It is a graph which shows that the C5a level of serum rises when HFD is given for 4 weeks. The result of the ELISA is shown (left). Similarly, the mRNA level of C5 (precursor of C5a) in the liver is also increased (right). It is an image and a graph which show that leukocyte migration is observed when C5a protein is administered to the mouse | mouth which received the normal diet. veh: Control. C5a: C5a administration. It is a graph which shows that neutrophils are induced when mouse C5a is administered. veh: Control. C5a: C5a administration. It is an image and a graph which show that administration of a C5aR antagonist (C-terminal peptide of C5a) does not produce HFD-induced leukocyte migration. veh: Control. antagonist: administration of antagonist. FIG. 6 is a graph showing that HFD increases MCP-1 expression. When HFD was given for 4 weeks, MCP-1 levels in the serum of mice increased significantly. NC: normal diet. HFD: high fat diet. It is a graph which shows the analysis result of the MCP-1 mRNA level in various tissues. HFD significantly increased MCP-1 mRNA in circulating leukocytes. FIG. 5 is a graph showing that HFD increases MCP-1 mRNA levels in neutrophils, which are peripheral blood leukocytes. It is a graph which shows the result of having measured the amount of MCP-1 in the serum at the time of administering a C5aR antagonist. C5aR antagonist significantly reduced serum MCP-1 levels increased by HFD. FIG. 6 is a graph showing MCP-1 mRNA levels in neutrophil-like HL-60 treated with 3 nM recombinant C5a. C5a increased MCP-1 expression levels compared to controls (N = 5 each). FIG. 4 is a diagram and graph showing flow cytometric analysis of leukocytes in the femoral artery of wild type or CCR2 ^{− / −} mice fed NC or HFD. CD45 ⁺ cells were gated at the box (top). In the wild-type mice given HFD, intimal leukocytes were significantly increased compared to the NC group, whereas in CCR2 ^{− / −} mice, there was no change in CD45 positive cells between HFD and NC (each Group n = 7). These leukocytes were positive for CD11b, CD11c, and CCR2 (performed 8 independent experiments). As a negative control, an isotype control antibody was used (gray part). FIG. 4 is a graph showing flow cytometric analysis of a single cell suspension of a femoral artery of a wild type mouse given HFD for 4 weeks when peripheral blood neutrophils were depleted with a specific antibody (1A8). . Neutrophil depletion significantly reduced femoral artery intimal leukocytes compared to controls (IgG) (n = 6 each). FIG. 6 is a graph showing flow cytometric analysis of a single cell suspension of a femoral artery of a wild type mouse treated with C5a for 4 weeks. C5a significantly increased femoral artery leukocytes. veh: Control. It is the figure which showed the mechanism proposed about the vascular inflammation by HFD. HFD-feeding increases C5 expression levels and serum C5a levels in the liver (Liver). C5a induces an increase in neutrophils (Neutrophilia) and neutrophil recruitment in the femoral artery. Migration of neutrophils contribute to accumulation of CD11c ⁺ leukocytes in the femoral artery intima (CD11c ⁺ leukocyte). It is the figure which showed the analysis result of the blood C5a density | concentration in a coronary artery disease person (CAD (+)) and a non-patient (CAD (-)). From left, the graph shows non-patients under 75 years old (CAD (-)), coronary artery disease patients under 75 years old (CAD (+)), non-patients over 75 years old (CAD (-)), coronary arteries over 75 years old Diseased person (CAD (+)). The vertical axis is the concentration of C5a. In the group with stenosis in the coronary artery over 75 years of age, the blood C5a concentration is significantly increased. Data are mean ± SD. ^* P <0.05.

  The present invention is described in detail below. It should be noted that 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.

Method for evaluating risk of developing arteriosclerotic disease One aspect of the present invention relates to a method for evaluating the risk of developing arteriosclerotic disease. Such an evaluation method includes, for example, (i) a step of measuring the amount of C5a protein in a blood sample obtained from a subject in vitro, and (ii) an artery in the subject using the amount of C5a protein as an index. The method may include assessing the risk of developing sclerosis or vascular inflammation.

Arteriosclerotic diseases Atherosclerotic diseases include atherosclerosis, obstructive arteriosclerosis, myocardial infarction, cerebral infarction, stroke, angina, cerebral thrombus, cerebral hemorrhage, renal failure. Arteriosclerosis refers to thickening, hardening and loss of elasticity of the arterial wall. As arteriosclerosis progresses, blood flow to organs and tissues is gradually restricted. Atherosclerosis is a form of arteriosclerosis caused by the accumulation of fat plaque, cholesterol, and other substances in the arterial wall and can cause serious diseases such as cerebral infarction and myocardial infarction. According to the present invention, it is possible to evaluate the risk of developing an arteriosclerotic disease in a subject using the amount of C5a protein in a blood sample obtained from the subject as an index. In particular, the method of the present invention can be suitably used for evaluating the risk of developing atherosclerosis.

Vascular inflammation It is thought that there is a close relationship between arteriosclerosis and vascular inflammation. We have found that a high fat diet induces acute inflammation, which further induces chronic inflammation. According to the present invention, the risk of developing vascular inflammation as a pre-stage of arteriosclerosis in a subject can also be evaluated using the amount of C5a protein in a blood sample obtained from the subject as an index.

Blood Sample In the method of the present invention, a blood sample obtained from a subject is used for measurement. As used herein, the “subject” is a mammal, preferably a human. The step of collecting blood from a human subject can be excluded from the steps of the method of the present invention. As for blood derived from a subject, blood collected from the subject may be immediately subjected to the method of the present invention, or blood that has been refrigerated or frozen directly after collection or directly after appropriate treatment. You may use it, returning to room temperature, before using for the method of invention. Appropriate treatment before refrigeration or freezing includes, for example, adding heparin or the like to whole blood for anticoagulation, or separating it as plasma or serum. These processes may be performed based on techniques known in the art.

The C5a protein C5a is a protein fragment that is released when the complement component C5 is cleaved into C5a and C5b fragments by a C5 converting protease. C5b is important in later events in the complement cascade, while C5a acts as a highly inflammatory peptide. Although the origin of C5 is hepatocytes, its synthesis is also found in macrophages, which can cause a local increase in C5a. C5a has chemotaxis and anaphylatoxin and is essential for innate immunity, but is also related to adaptive immunity. Increased production of C5a has been associated with many inflammatory diseases.

The gene sequences of human C5 and C5a are known and can be obtained from databases such as GenBank:
C5 complement component 5 [Homo sapiens (human)]
Gene ID: 727
mRNA: NM_001317163.1
Protein: NP_001304092.1

  C5a corresponds to the 74 amino acid residues at amino acid positions 678-751 of C5. C5a is further rapidly metabolized by carboxypeptidase B to the 73 amino acid C5a des-Arg lacking the C-terminal arginine. Therefore, in the present application, the term “amount of C5a protein” can be understood to mean the total amount of C5a and C5a des-Arg. For example, when an antibody is used for measurement of the amount of C5a protein, those skilled in the art will recognize that both C5a and C5a des-Arg are recognized if the antibody recognizes the N-terminal side of C5a. Any antibody that recognizes the C-terminus of C5a can specifically recognize either C5a or C5a des-Arg, but in the present invention, it is not necessary to distinguish between them. Moreover, the amount of C5a protein can also be indirectly evaluated by measuring the amount of C5a des-Arg.

Anti-C5a antibody In the method of the present invention, the anti-C5a antibody used for measuring the amount of C5a protein may be a polyclonal antibody or a monoclonal antibody. The anti-C5a antibody can be produced using a method known to those skilled in the art. Further, as the anti-C5a antibody, a commercially available antibody may be purchased and used. The isotype of the antibody is not limited, and any of IgG, IgM, IgA, IgE, and IgD may be used. There is no limitation on the animal species from which the antibody is derived. In addition, fragments of these antibodies (for example, Fab, F (ab ′) ₂ , Fab, Fv, Facb, Fd) can also be used. These antibodies and antibody fragments may contain modifications such as labels useful for detection. The anti-C5a antibody used in the present invention may similarly recognize C5 protein, C5a des-Arg, a fragment of C5a protein, and / or a C5a protein variant having a mutation in part. .

Variant of C5a protein In the present specification, the term “mutant” of C5a protein refers to an amino acid sequence of C5a protein, preferably a human-derived wild-type C5a protein represented by SEQ ID NO: 1 or a partial sequence thereof. Above, preferably about 1 to several amino acid deletions, substitutions, additions or insertions, or the amino acid sequence or a partial sequence thereof, about 80% or more, for example about 85% or more, preferably about 90% More preferably, it means a variant exhibiting% identity of about 95% or more, such as about 97% or more, about 98% or more, or about 99% or more. As used herein, “several” refers to an integer of about 10 or less, such as 9, 8, 7, 6, 5, 4, 3 or 2. In addition, “% identity” means that the amino acid sequence of the protein of SEQ ID NO: 1 and the amino acid sequence of the mutant have the highest degree of coincidence, and at this time a gap is introduced or no gap is introduced. Preferably, when the two amino acid sequences are aligned by introducing a gap, the ratio of the number of matched amino acid residues to the total number of amino acid residues (including the number of gaps when a gap is introduced) (% And can be determined with or without introducing a gap using a BLAST or FASTA protein search system. Specific examples of C5a protein mutants include polymorphisms (including SNPs) based on the race or individual of the subject, splice mutations, and the like. That is, the C5a protein of the subject in the method of the present invention does not necessarily have the amino acid sequence shown in SEQ ID NO: 1, and may be a variant thereof.

C5a Protein Fragment As used herein, the term “fragment” of C5a protein refers to a wild-type C5a protein, preferably a human-derived wild-type C5a protein represented by SEQ ID NO: 1, or at least five amino acids constituting a variant thereof. Less than total number, at least 7 or more and less than total number, preferably at least 8 or more and less than total number, for example, at least 10 or more and less than total number, at least 15 or more and less than total number, more preferably at least 20 or more and less than total number, at least 25 or more A polypeptide fragment comprising one or more epitopes consisting of less than the total number, even more preferably at least 35 or more and less than the total number, at least 40 or more and less than the total number, at least 50 or more and less than the total number. . Such a fragment can bind immunospecifically to the antibody or fragment thereof used in the present invention. The reason for including such a peptide fragment in the C5a protein is that the object of the present invention can be achieved if the C5a protein in blood can be quantified even if it is fragmented, and the above-mentioned wild-type C5a protein ( Preferably, a human-derived wild-type C5a protein represented by SEQ ID NO: 1) or a full-length polypeptide thereof is fragmented by a hydrolase such as a protease or peptidase present in blood. Because there is. That is, the C5a protein to be measured in the method of the present invention does not necessarily have a full-length sequence, and may be a fragment thereof.

Amount of C5a protein As used herein, “amount of C5a protein” refers to the amount of C5a protein present in blood (whole blood, serum, or plasma) derived from a subject. In the present invention, the blood sample is preferably serum. The quantity may be either an absolute quantity or a relative quantity. In the case of an absolute amount, it corresponds to the mass or volume of the C5a protein contained in a predetermined blood volume. In the case of a relative amount, it refers to a relative value represented by a measured value of C5a protein derived from a subject with respect to a specific measured value, and examples thereof include concentration, fluorescence intensity, and absorbance. The amount of the C5a protein can be measured using a known in vitro measurement method, for example, a method of measuring using a substance that can specifically bind to the C5a protein such as an antibody.

In vitro measurement As a method for measuring the amount of C5a protein in vitro, any method known to those skilled in the art, for example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay, luminescence immunoassay, radioimmunoassay (RIA), immunoturbidimetric method, latex agglutination reaction, latex turbidimetry, erythrocyte agglutination reaction, particle agglutination reaction or Western blotting can be used (for example, see the measurement method described in JP-A-2015-155795). ). A series of measurement steps can also be performed using, for example, a fully automatic ELISA system.

  When carrying out the method of the present invention using an immunoassay using a label such as an enzyme immunoassay, a fluorescence immunoassay, a radioimmunoassay, or a luminescence immunoassay, an anti-C5a antibody is immobilized. Alternatively, it is preferable to immobilize components in the sample and perform their immunological reaction. As solid phase carriers, beads made of materials such as polyethylene, polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, cellulose, sepharose, glass, metal, ceramics or magnetic materials Insoluble carriers in the form of microplates, test tubes, sticks or test pieces can be used. The solid phase can be formed by binding a solid phase carrier and the anti-C5a antibody or sample component according to a known method such as a physical adsorption method, a chemical binding method, or a combination thereof.

  In the present invention, the reaction is directly detected by labeling the anti-C5a antibody, or indirectly by using a labeled secondary antibody. From the viewpoint of sensitivity, it is preferable to use the latter indirect detection (for example, sandwich method) in the method of the present invention.

As the labeling substance, in the case of enzyme immunoassay, alkaline phosphatase, peroxidase (POD), β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase or biotin-avidin complex, etc. may be used. it can. In the case of fluorescence immunoassay, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, Alexa, AlexaFluoro, or the like can be used. In the case of a radioimmunoassay, tritium, iodine 125, iodine 131, or the like can be used. Further, the light emitting immunoassay, NADH, FMNH _2, luciferase system, luminol - hydrogen peroxide -POD system can be used acridinium ester or dioxetane compound system or the like.

  For binding of the labeling substance and the antibody, a known method such as glutaraldehyde method, maleimide method, pyridyl disulfide method or periodate method is used in the case of enzyme immunoassay, and chloramine T is used in the case of radioimmunoassay. A known method such as the Bolton Hunter method can be used. The measurement operation method may follow a known method. For example, when the anti-C5a antibody is directly labeled, a component in blood is solid-phased and brought into contact with the labeled anti-C5a antibody to form a C5a protein-anti-C5a antibody complex. Unbound labeled antibody can be washed and separated, and the amount of C5a protein in the blood can be measured from the amount of bound labeled antibody or the amount of unbound labeled antibody.

  For example, when a labeled secondary antibody is used, the antibody of the present invention is reacted as a primary antibody with a sample (primary reaction), and the labeled secondary antibody is further reacted with the primary antibody (secondary reaction). The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. By the primary reaction and the secondary reaction, a solid-phased C5a-anti-C5a antibody-labeled secondary antibody complex or a solid-phased anti-C5a antibody-C5a-labeled secondary antibody complex is formed. The unbound labeled secondary antibody can be washed and separated, and the mass of C5a in the sample can be measured from the amount of bound labeled secondary antibody or the amount of unbound labeled secondary antibody.

  Specifically, in the case of enzyme immunoassay, a substrate is reacted with a labeled enzyme under the optimum conditions, and the amount of the reaction product may be measured by an optical method. In the case of fluorescent immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of radioimmunoassay, the amount of radioactivity by the radioactive substance label is measured. In the case of the luminescence immunoassay, the amount of luminescence by the luminescence reaction system may be measured.

  In addition, the formation of immune complex aggregates by immunoturbidimetry, latex agglutination, latex turbidimetry, erythrocyte agglutination or particle agglutination can be measured by measuring the transmitted light and scattered light using optical methods or visually. It can also be implemented by a measuring method. In this case, a phosphate buffer, glycine buffer, Tris buffer, Good buffer, or the like can be used as a solvent, and a reaction accelerator such as polyethylene glycol or a nonspecific reaction inhibitor is included in the reaction system. Also good.

An example of an embodiment of the measurement method of the present invention includes the following procedure. First, the antibody of the present invention is immobilized on an insoluble carrier as a primary antibody. Preferably, the solid surface on which no antigen is adsorbed is blocked with a protein unrelated to the antigen (calf serum, bovine serum albumin, gelatin, etc.). Subsequently, the immobilized primary antibody is brought into contact with the test sample. Next, a labeled secondary antibody that reacts with C5a at a site different from the primary antibody is contacted, and a signal from the label is detected. The “secondary antibody that reacts with C5a at a site different from that of the primary antibody” used herein is not particularly limited as long as it is an antibody that recognizes a site other than the binding site between the primary antibody and C5a protein. Any of polyclonal antibodies, antisera, and monoclonal antibodies may be used, and fragments (also referred to as fragments) of these antibodies (for example, Fab, F (ab ′) ₂ , Fab, Fv, Facb, Fd, etc.) should be used. You can also. Furthermore, multiple types of monoclonal antibodies may be used as secondary antibodies.

  On the contrary, the antibody of the present invention is labeled to give a secondary antibody, and an antibody that reacts with C5a at a site different from the antibody of the present invention is immobilized on an insoluble carrier as a primary antibody. The detected primary antibody may be brought into contact with a test sample, and then the antibody of the present invention labeled as a secondary antibody may be contacted to detect a signal from the label.

  In the method of the present invention, a test strip for immunochromatography known in the art (for example, see the description in JP-A-2015-155795. The description of this document is explicitly incorporated herein by reference. ) May be used. An immunochromatographic test strip (also referred to as a test strip) is, for example, a sample receiving part made of a material that easily absorbs a sample, a reagent part containing an antibody, a developing part in which a reaction product of the sample and the antibody moves, and development. It is composed of a labeling section for coloring the reaction product, a presentation section for developing the colored reaction product, and the like, and can be in the same form as a pregnancy diagnostic agent. First, when a sample is given to the sample receiving portion, the sample receiving portion absorbs the sample and causes the sample to reach the reagent portion. Subsequently, in the reagent part, a reaction between C5a and anti-C5a antibody in the sample occurs, and the reacted complex moves through the developing part and reaches the labeling part. In the labeling part, the reaction between the reaction complex and the labeled secondary antibody occurs, and when the reaction product with the labeled secondary antibody develops to the presentation part, coloration is recognized.

Substance that can specifically bind to C5a protein In the in vitro measurement described above, not only anti-C5a antibody but also other substances can be used as long as they can specifically bind to C5a protein. As used herein, “specifically bindable” means that a substance can substantially bind only to the C5a protein. In this case, nonspecific binding may be present to the extent that it does not affect the detection of the C5a protein. The “substance capable of specifically binding to C5a protein” used in the present invention also recognizes C5 protein, C5a des-Arg, C5a protein fragment, and / or C5a protein having a mutation in part. You may do.

  Examples of the “specifically bindable substance” include C5a binding protein. More specifically, in addition to the above-mentioned anti-C5a antibody, for example, C5a receptor protein or fragment thereof, phage display method and the like The C5a-binding polypeptide obtained by screening in the above, or a chemically modified derivative thereof may be used. Here, the “chemically modified derivative” is used for functional modification necessary to acquire or retain the specific binding activity of the substance with the C5a protein, or for labeling necessary for detecting the substance. Including any of the modifications.

  Functional modifications include, for example, glycosylation, deglycosylation, PEGylation. Modifications on the label include, for example, fluorescent dyes (FITC, rhodamine, Texas red, Cy3, Cy5), fluorescent proteins (eg, GFP, EGFP, RFP, YFP, CFP, PE, APC,), enzymes (eg Labeling with horseradish peroxidase, alkaline phosphatase, glucose oxidase) or biotin, avidin, streptavidin.

Combination of Other Markers One of the embodiments of the present invention further includes a step of measuring in vitro the amount of other markers (for example, other known inflammatory markers) other than C5a in a blood sample obtained from a subject. May be included.

As the reference value reference value, for example, a measurement value corresponding to a healthy person can be used. “Healthy person” refers to an individual, preferably a healthy individual, who is not suffering from at least obvious arteriosclerosis. Further, the healthy person needs to be the same species as the subject. For example, when the subject to be detected is a human subject, the healthy subject must also be a human. The physical condition of a healthy person is preferably the same as or close to that of the subject. For example, in the case of a human, the physical condition corresponds to race, sex, age, height, weight, and the like. Alternatively, measured values in the past of the subject can also be used. As the measurement values in the past, for example, those measured at regular or irregular medical examinations can be used. Evaluation of measured values over time can be useful in evaluating the risk of developing arteriosclerotic diseases.

Statistical significance “Statistically significant” includes, for example, a case where the risk value (significance level) of the obtained value is less than 5%, 1%, or 0.1%. Therefore, “statistically significant” in terms of measured value means that, for example, when the quantitative difference of C5a protein obtained from each of a subject and a healthy person is statistically processed, It means that there is a significant difference and the amount of the protein of the subject is relatively large compared to that of the healthy subject. For example, regarding the amount of C5a protein in the blood, this corresponds to the case where the subject is twice or more, preferably 3 times or more, more preferably 4 times or more, most preferably 5 times or more that of a healthy person. If the quantitative difference is 3 times or more, the reliability is high, and it can be said that there is a significant statistical amount. The statistical processing test method may be appropriately selected from known test methods capable of determining the presence or absence of significance, and is not particularly limited. For example, Student t test method, multiple comparison test method, Kruskal-Wallis test Dunn's multiple analysis can be used.

  The amount of C5a protein in the blood of a healthy subject is preferably measured by the same method as the method for measuring the amount of C5a protein in the blood of the subject described above. The amount of C5a protein in the blood of a healthy person can be measured every time the amount of C5a protein in the blood of a subject is measured, but the amount of C5a protein measured in advance can also be used. it can. In particular, if the amount of C5a protein in various physical conditions of a healthy person is measured in advance and the value is input to a computer and stored in a database, the physical condition of the subject can be input to the computer. It would be convenient because the amount of C5a protein of a healthy person who has the optimal physical condition for comparison with the subject can be used immediately.

Assessment of risk of development If the amount of C5a protein in the blood of a subject is statistically significantly greater than the amount of C5a protein in the blood of a healthy subject, the subject is at risk of developing arteriosclerotic disease. Evaluate as high. In particular, the present invention has an advantage in that it can be evaluated even for early arteriosclerotic diseases. An early stage arteriosclerotic disease refers, for example, to plaques that are not formed or have not yet developed. Further, from the viewpoint of marker protein, it means that CRP or IL-1 is not detected. In particular, according to the method of the present invention, even in a subject in which an increase in part or all of TNF-α, IL-1α, IL-1β, IL-7 and IL-11 is not detected, atherosclerotic disease The risk of onset can be assessed. The subject subject can also specify as an index that blood vessel parameters such as IMT (blood vessel echo) and PWV (pulse wave velocity) are in a normal range.

Kit One aspect of the present invention relates to a kit for evaluating the risk of developing an arteriosclerotic disease. The kit according to the present invention is a substance that can specifically bind to C5a protein in order to measure the amount of C5a protein in a blood sample obtained from a subject in vitro, such as an anti-C5a antibody, a fragment thereof, and / or Or chemically modified derivatives thereof. Such antibodies, fragments or chemically modified derivatives may be bound to a solid support. As the solid phase carrier, a test strip, a plastic tube, a microplate, a glass bead, or the like can be used. In addition to the above elements, this kit includes, for example, a labeled secondary antibody, as well as a substrate necessary for detection of the label, a carrier, a washing buffer, a sample diluent, an enzyme substrate, a reaction stop solution, and a purified standard substance. C5a protein, instructions for use, and the like.

Composition for treating or preventing arteriosclerotic disease One of the embodiments of the present invention is a therapeutic or therapeutic agent for arteriosclerotic disease comprising a substance that inhibits the binding between C5a protein and C5a receptor (C5aR) as an active ingredient. It relates to a composition for use in prevention. The substance that inhibits the binding between the C5a protein and the C5a receptor can be, for example, a C5a receptor antagonist, an anti-C5a antibody, an anti-C5aR antibody, or a soluble C5aR fragment. As the C5a receptor antagonist, for example, a C5a C-terminal peptide can be used. In addition, CCX168 (Avacopan), W-54011, PMX-53, etc. can be used. Since the complement component C5a is elevated in the blood before vascular inflammation before the onset of arteriosclerosis occurs, the pharmaceutical composition according to the present invention has potential arteriosclerotic properties at the stage where plaque is not formed or is not developed. It is thought to be particularly useful for disease prevention.

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

Example 1: High Fat Diet Increases Leukocyte Migration in the Femoral Artery of Wild Type Mice Using Biological Microscopy (IVM), Wild Type Mice Feeded with High Fat Diet (HFD) or Normal Diet (NC) Leukocyte migration in the femoral artery was observed. Wild-type mice (C5lBL / 6J, male, 7 weeks old) purchased from Charles river Labo were used. Seven-week-old wild-type mice were fed a high fat diet (HFD: 1.25% cholesterol, 20% lipid contained) or a normal diet (NC). Thereafter, leukocyte migration in the femoral artery was observed by IVM after 1, 2, 4 and 8 weeks for wild-type mice. Pentobarbital is administered intraperitoneally to mice and anesthetized. During observation, mechanical ventilation is performed to maintain normal blood pH, and a thermal pad and infrared lamp are irradiated to maintain body temperature at 36-37 ° C. went. The femoral artery and vein are exposed by incising the skin of the thigh, and rhodamine 6G (0.3 mg / kg in 300 ul PBS (-)) is administered from the right femoral vein to 20 times the left upper femoral artery bifurcation. Observation was performed with a fluorescence microscope (BX51WI) equipped with a water immersion objective lens (Rhodamine 6G emits fluorescence by taking blood cells other than red blood cells into the cells). Recording was performed at a frame rate of 5 frames / second, and the number of leukocytes to be adhered or rolled was calculated using analysis software (MetaMorph). Adhesion was performed with white blood cells left standing for 3 seconds or more, and rolling was performed in the next frame.

As shown in FIG. 1a, the time-dependent leukocyte migration in mice given HFD was observed from 1 to 8 weeks, and the number of vascular adhesive leukocytes was observed at 4 and 8 weeks. Image analysis showed a significant increase compared to NC (FIG. 1a). When HFD is given to mice, especially the number of peripheral neutrophils (CD11b ⁺ Ly-6G ⁺ ) is increased (HFD: 22.7 ± 1.0%, n = 11; NC: 7.7 ± 0). .70%, n = 10; P <0.001), the total number of leukocytes was unchanged (FIG. 1b). There was no significant change in the number of other types of cells, such as monocytes and lymphocytes.

Peripheral blood flow cytometry and white blood cell count were performed as follows. Blood was collected from the heart, 200 μl of blood was added to 1.8 ml of a Lysing buffer containing the following antibodies, and reacted at room temperature for 15 minutes.
Rat anti-mouse CD11b-FITC
Rat anti-mouse CD45 PerPC
Rat anti-mouse Ly-6G-APC
Rat anti-mouse Ly-6C-APC
Rat anti-mouse CD3-PE
Rat anti-mouse CD19-PE
The fluorescently stained leukocytes were fixed with 1% paraformaldehyde and analyzed. Flow cytometry was performed on 10,000 leukocytes using BD FACS Calibur, and the data was analyzed with FlowJo7.6. At the time of analysis, neutrophils were CD45 ⁺ Ly6G ⁺ CD11b ⁺ , monocytes were CD45 ⁺ Ly6C ^hi CD11b ⁺ , and lymphocytes were CD45 ⁺ CD3 ⁺ and CD45 ⁺ CD19 ⁺ . The white blood cell count was counted using XT-2000iV.

Example 2: Role of neutrophils in femoral artery HFD-induced leukocyte migration To confirm the involvement of neutrophils in HFD-induced leukocyte migration, neutrophils were injected by injection of Ly-6G specific antibody 1A8. IVM analysis was performed after depletion. The neutrophil removal (depletion) experiment was performed as follows. In order to remove neutrophils in peripheral blood, 250 μg of rat anti-Ly6G antibody was intraperitoneally administered to wild type mice on the 22nd and 24th days after loading with a high fat diet. A normal rat IgG2a administration group was used as a control group. On the 28th day after loading with a high fat diet, IVM was performed, and flow cytometry of peripheral blood and measurement of MCP-1 level in plasma were performed by ELISA.

  Flow cytometry analysis of peripheral blood confirmed neutrophil depletion (removal) (FIG. 2a). Depletion of neutrophils significantly reduced HFD-induced leukocyte migration (FIG. 2b). In lysozyme M (LysM) -eGFP mice, in which neutrophils abundantly express GFP, when the depletion of monocytes was induced by intraperitoneal injection of clodronate liposomes, the liposomes were clearly monocytes in peripheral blood However, there was no change in leukocyte migration in the femoral artery (FIGS. 2c and 2d). These results show that neutrophils, not monocytes, were mobilized to the femoral artery by week 4 in HFD.

  The monocyte removal (depletion) experiment was performed as follows. LysM-eGFP (LysM is a protein called lysozyme M) fed with a high fat diet to identify the leukocyte fraction that migrates with the high fat diet, and is expressed in neutrophils, monocytes, and macrophages. Incorporating the eGFP gene into the LysM protein was fluorescently labeled). LysM-eGFP mice used were those transferred from Dr. Y. Inomata. Since this mouse expresses the highest level of eGFP among leukocytes, it can be said that if adhesion or rolling is observed, it is a neutrophil. However, since monocytes are also expressed about 1/10 of that, 200 μl of clodronate was used to perform IVM of LysM-eGFP mice fed with a high fat diet to eliminate the possibility of monocytes under monocyte removal. Liposomes were administered from the tail vein 24 hours before IVM (clodronic acid liposomes killed by phagocytosis of monocytes and macrophages).

Example 3: HFD increases C5a levels in the liver and mobilizes neutrophils to understand how activating HFD induces neutrophils to produce MCP-1 Serum levels of various cytokines were measured in mice given NC for 4 weeks. Serum was collected from mice fed a high fat diet or a normal diet for 4 weeks, and 8 mice were mixed for each group and used for the cytokine array. In the cytokine array used, specific antibodies against about 20 types of cytokines are spotted on the membrane, and when the serum is reacted therewith, the spots chemiluminescent according to the concentration in the serum. The intensity of the spot was quantified with an LAS-1000 image analyzer, and the relative value of the light emission amount of the high fat diet group was calculated with the normal diet group as 1.00.

  As shown in FIG. 3a, the mice that received HFD had the highest relative expression level of C5a in serum (NC: 1.00, HFD: 2.81). This result was also confirmed by C5a ELISA as shown in FIG. 3b (HFD: 18.15 ± 1.53 ng / ml, n = 4; NC: 11.95 ± 0.52 ng / ml). , N = 4; P <0.01). C5a, an activated form of C5, is a potent neutrophil chemoattractant produced in the liver. Subsequent quantitative RT-PCR revealed that CFD levels in the liver were increased by HFD (HFD: 2.57 ± 0.52, n = 8; NC: 1.00 ± 0.13). n = 8; P <0.05; FIG. 3b).

  The analysis by ELISA was performed as follows. Blood was collected from the heart at 4 weeks after loading with a high fat diet, and the concentrations of MCP-1 and C5a in the serum were quantified by ELISA. The primary antibody is added to a 96-well plastic microplate and left overnight at room temperature. Thereafter, the plate is washed 3 times with PBS (-) containing 0.02% Tween 20, and PBS (-) containing 1% BSA is added. Let stand for 30 minutes, wash 3 times with PBS (-) containing 0.02% Tween 20, and add serum or reference solution. After standing at room temperature for 1.5 hours, washed with 0.02% Tween20-containing PBS (−) three times, a biotin-labeled secondary antibody is added, and the mixture is allowed to stand for 1.5 hours. Wash 3 times with PBS (-) containing 0.02% Tween 20, add HRP-streptavidin, and allow to stand for 30 minutes. Wash 3 times with PBS (-) containing 0.02% Tween 20, add TMB substrate, and after 15 minutes, stop the reaction with 0.6 N sulfuric acid. The absorbance at 450 nm is measured, and the concentration of MCP-1 or C5a in the sample is calculated from the calibration curve.

  Quantitative RT-PCR was performed as follows. PBS (-) was perfused from the left ventricle of a wild-type mouse fed with a high fat diet or a normal diet for 4 weeks for blood removal, and aorta, liver, spleen, bone marrow, and visceral fat were collected. Peripheral blood leukocytes were collected and lysed, and 4 mice were combined into one, and three neutrophils were combined into one to collect RNA from each tissue. CDNA was synthesized from the collected RNA and subjected to real-time PCR. The relative expression level was calculated by the standard curve method or the ΔΔCt method using the endogenous control as 18S ribosomal RNA.

Example 4: Important role of C5a in HFD-induced neutrophil migration Since C5a can activate neutrophils, IVM analysis was performed in mice treated with C5a without HFD. 2 μg / kg of mouse C5a was administered from the tail vein, and after 90 minutes, observation of leukocyte migration by IVM and flow cytometry for peripheral blood leukocytes were performed. Administration of C5a significantly induced leukocyte migration in the femoral artery of mice (FIG. 3c). Also, treatment with C5a significantly increased the number of peripheral neutrophils in wild-type mice (solvent only: 7.38 ± 0.17%, n = 4; C5a: 12.41 ± 1.55% , N = 4; P <0.05; FIG. 3d). These data indicate the importance of the role C5a plays in HFD-induced neutrophil migration in the femoral artery. Furthermore, infusion of C5a receptor antagonist (C5aR) significantly reduced HFD-induced leukocyte migration (FIG. 3e). Wild-type mice were fed a high fat diet and 0.3 mg / kg / day of C5aR antagonist or control was intraperitoneally administered daily. Four weeks later, leukocyte migration by IVM was observed.

Example 5: Increased MCP-1 expression in neutrophils by HFD Persistence of HFD increases serum MCP-1 levels. In mice given HFD, serum MCP-1 levels were significantly increased as early as 4 weeks compared to mice given NC (HFD: 268.1 ± 40.6 pg / ml, n = 8; NC: 115.1 ± 10.43 pg / ml, n = 8; P <0.01; FIG. 4a). When the expression level of MCP-1 in various tissues was measured, the MCP-1 level was very high in leukocytes after giving HFD for 4 weeks (FIG. 4b). Since neutrophils did not express CCR2, presumed to be a receptor for MCP-1, the inventors hypothesized that neutrophils produced MCP-1 in response to HFD. To confirm this hypothesis, the expression level of MCP-1 was measured separately in neutrophils (Ly6g ⁺ ) and monocytes / lymphocytes (LY-6G ⁻ ). As shown in FIG. 4c, HFD significantly upregulated MCP-1 expression in neutrophils but not in monocytes / lymphocytes (Ly-6G ⁻ , NC: 1. 00 ± 0.15, HFD: 1.21 ± 0.71; Ly-6G ⁺ , NC: 1.49 ± 0.73, HFD: 5.01 ± 1.10, n = 3; P <0.05 ). C5aR antagonist also significantly reduced MCP-1 levels in the serum of HFD fed mice (solvent only: 330.4 ± 49.92 pg / ml, n = 4; C5aR: 189.8 ± 11.72 pg). / Ml, n = 4; P <0.05; FIG. 4d). Wild-type mice were fed a high fat diet and 0.3 mg / kg / day of C5aR antagonist or control was intraperitoneally administered daily. After 4 weeks, blood MCP-1 concentration was measured by ELISA.

  To show the direct effect of C5a on MCP-1 expression in neutrophils, the inventors examined MCP-1 expression under C5a treatment in HL60 differentiated to neutrophils. In order to differentiate HL-60 cells into neutrophils, the cells were cultured for 5 days in RPMI 1640 containing 10% fetal bovine serum 1.3% DMSO. 3 nM C5a was added to the differentiated cells and recovered 1 hour later to isolate mRNA. Real-time PCR was performed to examine MCP-1 expression. The expression of MCP-1 under C5a treatment was significantly increased in HL60 differentiated into neutrophils compared to the control (control: 1.00 ± 0.24, n = 5; C5a: 2. 58 ± 1.15, n = 5; P <0.05; FIG. 4e). In contrast, the expression level of MCP-1 in undifferentiated HL-60 was not different between C5a treatment and control (control: 1.00 ± 0.24, n = 5; C5a : 0.62 ± 0.27, n = 4), indicating that C5a acts on differentiated neutrophils. These results indicate that C5a induces MCP-1 expression in neutrophils.

Example 6: Single cells of a femoral artery sample to monitor inflammation in the intima and medial areas of blood vessels where neutrophil-derived MCP-1 is required for HFD-induced increase in intimal leukocytes Flow cytometric analysis of the suspension was performed. Analysis was performed on leukocytes adhering to the leukocytes accumulated in the intima. Wild-type mice or CCR2 ^{− / −} mice were fed a high fat diet or a normal diet for 4 weeks, and blood was removed by refluxing PBS (−) from the left ventricle. CCR2 ^{− / −} mice were those assigned by Dr. K. Egashira. The femoral artery was collected and the two animals were combined into one sample. The collected blood vessel was made into a single cell suspension by collagenase treatment and centrifuged through a nylon mesh. The obtained cells were reacted with the following antibody and stained, and flow cytometry was performed.
Anti-mouse CD11b-FITC
Anti-mouse CD11c-PE
Anti-mouse CD45-PerPC
Anti-mouse / rat CCR2-APC

When HFD was given for 4 weeks, the number of CD45 positive cells in the femoral artery was significantly increased compared to when NC was given (HFD: 7.21 ± 0.67%, n = 7; NC: 3 47 ± 0.50%, n = 7; P <0.0001; FIG. 5a). When CCP2-deficient (CCR2 ^{− / −} ) mice were used to inhibit MCP-1 / CCR2 signaling, a decrease in CD45 positive cells associated with HFD was seen in the femoral artery (HFD: 2.40 ± 0.38%). , N = 7; NC: 2.62 ± 0.51%, n = 7; FIG. 5a). As shown in FIG. 5a, the majority of CD45 positive cells were positive for CD11b and CD11c. To confirm the importance of neutrophils in these intimal leukocytes, neutrophil depletion was performed in wild-type mice given HFD for 4 weeks. As shown in FIG. 5b, neutrophil depletion significantly reduced HFD-induced intimal CD45 positive cells in the femoral artery (control IgG: 11.38 ± 0.82%, n = 6; 1A8: 8.96 ± 0.69%, n = 6; P <0.05). Furthermore, when mouse C5a was administered to wild-type mice for 28 days, the number of intimal CD45-positive cells in the femoral artery was significantly increased compared to the control (solvent only: 5.38 ± 0.69%, n = 7; C5a: 8.41 ± 1.04%, n = 8; P <0.05; FIG. 5c). These results indicate that serum MCP-1 upregulation, peripheral neutrophils, and serum C5a play an important role in the increase of intimal CD45 positive cells by HFD.

Example 7: Analysis of a blood sample derived from a subject Blood is collected from a subject using EDTA, sodium citrate or heparin as an anticoagulant, and plasma or serum is separated. The 96-well plastic microplate to which the primary antibody has been adsorbed is washed three times with 0.02% Tween20-containing PBS (−), and 1% BSA-containing PBS (−) is added. Allow to stand for 30 minutes, wash 3 times with PBS (-) containing 0.02% Tween 20, and add sample or reference solution. After standing at room temperature for 1.5 hours, washed with 0.02% Tween20-containing PBS (−) three times, a biotin-labeled secondary antibody is added, and the mixture is allowed to stand for 1.5 hours. Wash 3 times with PBS (-) containing 0.02% Tween 20, add HRP-streptavidin, and allow to stand for 30 minutes. Wash 3 times with PBS (-) containing 0.02% Tween 20, add TMB substrate, and after 15 minutes, stop the reaction with 0.6 N sulfuric acid. The absorbance at 450 nm is measured, and the C5a concentration in the sample is calculated from the calibration curve.

Example 8: Analysis of blood C5a concentration in patients with coronary artery disease and non-patients In 99 patients with cardiac catheterization (88 men, 11 women; average age 67.2 ± 0.8 years) C5a concentration was measured. About plasma obtained from the subject, the C5a concentration in blood was measured using ELISA. Anti-C5a antibody was added to a 96-well plate and allowed to stand overnight. The antibody was adsorbed on the bottom of the plate, washed, and then PBS (-) solution containing 5% BSA was added for blocking. Subsequently, the prepared C5a reference solution and plasma were added to each well and allowed to react. Thereafter, the plate was washed and biotin-conjugated anti-C5a antibody was added for reaction, and after washing, avidin-HRP was added. After washing, 3,3 ′, 5,5′-tetramethylbenzidine (TMB) solution, which is a chromogenic substrate, was added for color development, and the reaction was stopped with H ₂ SO ₄ solution. Thereafter, the absorbance at 450 nm was measured using a microplate reader, a standard curve was created from the standard substance, and the C5a concentration of each sample was calculated.

  After the measurement of the blood C5a concentration, statistical processing was performed. The cardiac catheterization tester was divided into two groups, under 75 years old and older, and a comparison test of blood C5a concentration was performed in each group with and without coronary artery stenosis. A Kruskal-Wallis test was performed, and Dunn's multiple analysis was performed based on the subsequent results. A P value of less than 0.05 was considered significant. As a result, it was found that the blood C5a concentration was significantly increased in the group with stenosis in the coronary artery over 75 years old.

  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.

  As described above, the inventors have found that the complement component C5a is increased in the blood before vascular inflammation occurs before the onset of arteriosclerosis. Increased C5a activates neutrophils, induces neutrophil adhesion to the intima and leukocyte accumulation, and further increases other inflammatory factors (MCP-1). Moreover, it was confirmed that suppression of C5a attenuates neutrophil adhesion, leukocyte accumulation, and expression of inflammatory factors. Since this increase in C5a occurs prior to the increase in other inflammatory factors and markers and is specific before the onset, the onset of arteriosclerotic disease that can be used at an earlier stage compared to conventional testing methods Useful as a predictor of risk. In addition, those skilled in the art can also treat or prevent arteriosclerotic diseases by suppressing the activity of C5a present in the blood before the onset of atherosclerotic diseases or at an extremely early time point. Understood.

Claims (14)

A method for assessing the risk of developing arteriosclerotic disease,
Measuring the amount of C5a protein in a blood sample obtained from the subject in vitro, and evaluating the risk of developing an arteriosclerotic disease in the subject using the amount of C5a protein as an index,
The subject has one or more factors in the blood concentration that are not statistically significantly increased compared to a reference value, the factors being TNF-α, IL-1α, IL-1β, One or more factors selected from the group consisting of IL-7 and IL-11,
A method of determining that the risk of developing an arteriosclerotic disease in the subject is high when a measured value of the amount of the C5a protein is statistically significantly greater than a reference value.   The method of claim 1, wherein the subject is 75 years of age or older.   The method according to claim 1, wherein plaques are unformed or undeveloped in the subject.   The atherosclerotic disease is atherosclerosis, obstructive arteriosclerosis, myocardial infarction, cerebral infarction, stroke, angina pectoris, cerebral thrombosis, cerebral hemorrhage, or renal failure. The method described in 1.   The method according to any one of claims 1 to 4, wherein the C5a protein is a polypeptide having the sequence shown in SEQ ID NO: 1, or a variant or fragment thereof.   The method according to any one of claims 1 to 5, wherein the measurement is performed using a substance capable of specifically binding to C5a protein.   The method according to claim 6, wherein the bindable substance is an antibody.   The method according to any one of claims 1 to 7, wherein the blood sample is serum, plasma or whole blood.   A kit for evaluating the risk of developing arteriosclerotic disease, comprising an anti-C5a antibody, a fragment thereof, and / or a chemically modified derivative thereof.   The kit according to claim 9 for use in the method according to any one of claims 1-8.   The kit according to claim 9 or 10, wherein the antibody, fragment or chemically modified derivative is bound to a solid phase carrier.   The kit according to claim 11, wherein the solid phase carrier is a test strip, a plastic tube, a microplate, or a glass bead.   A composition for use in the treatment or prevention of arteriosclerotic diseases, comprising a substance that inhibits the binding between C5a protein and C5a receptor as an active ingredient.   14. The composition according to claim 13, wherein the substance that inhibits binding between the C5a protein and a C5a receptor is a C5a receptor antagonist.

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