Method for determining occurrence or risk of myocarditis

Abstract

Provided is a means that makes it possible to assess the onset of myocarditis or the risk thereof by a simple, minimally invasive method. A method for assessing the onset of myocarditis or the risk thereof, involving assessing myocarditis to be developing or assessing there to be a risk of the same when the concentration or expression level of ADAM28 in a body fluid sample collected from a subject is below a standard value.

Description

[Technical field]

[0001] The present invention relates to a method for determining the onset of or the risk of myocarditis. [Background technology]

[0002] Myocarditis is an inflammatory disease that mainly affects the myocardium, and when the inflammation extends to the pericardium, it is called myocarditis (Guidelines for the diagnosis and treatment of acute and chronic myocarditis (revised in 2009)). Because it is difficult to make a definitive diagnosis in mild cases, the detailed incidence and mortality rates in Japan are unknown, but there are reports that the incidence is 115 per 100,000 (Okada R, Kawai S, Kasuya H. Jpn Circ J 1989;53(1):40-48.), and that 0.6% of autopsy cases of non-cardiac death are asymptomatic myocarditis (Feeley KM, Harris J, Suvarna SK. J Clin Pathol 2000;53:147-149.). It is also said that 20% of sudden deaths in people under the age of 40 are due to myocarditis, so it is by no means a rare disease.

[0003] There are various types of myocarditis, and they can be divided into acute and chronic myocarditis, for example, based on the onset and progression. Among acute myocarditis, those that lead to cardiopulmonary crisis in the early stages of the disease are called fulminant myocarditis (Aoyama N, Izumi T, Hiramori K, et al. Circ J 2002;66:133-144.). On the other hand, chronic myocarditis can be either persistent or subclinical. Most cases are caused by infection, and the pathogens are mostly viruses, but others are known to be bacteria, rickettsia, spirochetes, mycoplasma, fungi, protozoa, and parasites (Guidelines for the diagnosis and treatment of acute and chronic myocarditis (revised 2009 edition)). Other causes include physical stimuli such as drugs, radiation, and heat, immune abnormalities such as metabolic disorders and autoimmune diseases, and pregnancy, but the cause is often unknown. When the cause cannot be identified, it is diagnosed as idiopathic myocarditis.

[0004] In many patients with acute myocarditis, cold-like symptoms (chills, fever, headache, muscle pain, general malaise) and digestive symptoms such as anorexia, nausea, vomiting, and diarrhea precede the onset of symptoms, followed by cardiac symptoms several hours to several days later (Guidelines for the diagnosis and treatment of acute and chronic myocarditis (revised in 2009)). Cardiac symptoms may be accompanied by (1) signs of heart failure (occurrence rate of approximately 70%), (2) chest pain due to pericardial irritation (approximately 44%), or (3) heart block or arrhythmia (approximately 25%). However, myocarditis rarely has obvious clinical symptoms or signs, and the initial symptoms and signs are nonspecific. After simple cold symptoms or digestive symptoms, patients may develop extreme shock, fainting, or difficulty breathing due to decreased cardiac function in a very short period of time, and may progress to fulminant myocarditis, which can be fatal. The mortality rate of fulminant myocarditis is said to be approximately 50%. In addition, widespread inflammation of the myocardium may lead to a state similar to dilated cardiomyopathy, with reduced cardiac function, leading to repeated heart failure. In the chronic stage, inflammation leads to widespread fibrosis of the pericardium, which reduces the pumping power of the heart, leading to a unique form of right heart failure called constrictive pericarditis.

[0005] As described above, myocarditis often does not have any prior symptoms, and the condition may rapidly deteriorate, leading to shock such as decreased blood pressure and impaired consciousness, or sudden death. Therefore, a simple diagnostic method that can differentiate it at an early stage is required. Physical examination should include fever, abnormal pulse (tachycardia, bradycardia, arrhythmia), and hypotension, and it is important to check for signs of pulmonary congestion such as a galloping rhythm (appearance of the third heart sound), moist rales, and signs of right heart failure such as jugular venous distension and leg edema. In cases with pericarditis, cardiac tamponade may occur. In addition to these symptoms and examination findings, myocarditis is diagnosed by chest X-rays, electrocardiograms, echocardiograms, cardiac MRI, blood tests, etc. Blood biochemistry tests include increases in C-reactive protein (CRP) and increases in the blood concentrations of myocardial proteins such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), myocardial specific creatine kinase (CK-MB), and cardiac troponin. However, CRP, AST, and LDH are also increased in other general inflammations besides myocarditis. CK-MB and cardiac troponin are specific and highly sensitive blood markers for myocardial injury because they are released into the blood due to myocardial necrosis and their blood concentrations rise, but they show high values ​​in widespread myocardial necrosis, so they are also high in myocardial infarction and acute coronary syndrome, and cannot be differentially diagnosed with myocarditis. CK-MB is mainly present in the myocardium, but there is also a small amount in skeletal muscle, so it also rises in skeletal muscle diseases such as rhabdomyolysis, polymyositis, and muscular dystrophy. For this reason, in order to confirm that the patient does not have coronary artery disease such as angina pectoris or myocardial infarction, endomyocardial biopsy (myocardial biopsy) may be performed during cardiac catheterization and even during cardiac catheterization. Although myocardial biopsy is an invasive and stressful test for the patient, it can confirm the diagnosis and diagnose the type of myocarditis, and is therefore useful for predicting the treatment plan and course of the disease. For this reason, if the condition persists, repeated myocardial biopsies may be performed to monitor the progress, despite the invasive nature of the procedure (Guidelines for the diagnosis and treatment of Class I, Level C, acute and chronic myocarditis (2009 revised edition)).In cases of decreased cardiac pumping function (systolic function) or cardiac expansion (diastolic function) failure, blood B-type natriuretic peptide (BNP) and N-terminal (NT)-proBNP are measured as indicators of cardiac load. If heart failure is suspected based on subjective symptoms and physical findings, it is recommended that blood tests, including BNP or NT-proBNP levels, urine tests, electrocardiograms, chest X-rays, etc. be performed to confirm the validity of the diagnosis (Cardiomyopathy Management Guidelines (2018 revised edition)). In particular, BNP, which has been established as an auxiliary diagnostic method for heart failure, is also useful in hypertrophic cardiomyopathy (Yoshibayashi M, Kamiya T, Saito Y, et al. N Engl J Med 1993;329:433-434.), and can predict not only left ventricular hypertrophy and left ventricular pressure difference (Mizuno Y, Yoshimura M, Harada E, et al. Am J Cardiol 2000;86:1036-1040.) (Hasegawa K, Fujiwara H, Doyama K, et al. Circulation 1993;88:372-380.), but also the severity of heart failure (Maron BJ, Tholakanahalli VN, Zenovich AG, et al. Circulation 2004;109:984-989.) and exercise tolerance (Thaman R, Esteban MT, Barnes S, et al. Am J Cardiol 2006;98:515-519.), and it has even been reported to be related to clinical prognosis (Cardiomyopathy Clinical Practice Guidelines (2018 revised edition)). The Acute and Chronic Heart Failure Clinical Practice Guidelines also recommend that in diagnosing heart failure, subjective symptoms, medical history, family history, physical findings, electrocardiogram, and chest X-ray should be considered first, and if chronic heart failure is suspected, the next test to be performed is measurement of blood BNP / NT-proBNP levels (Acute and Chronic Heart Failure Clinical Practice Guidelines (2017 revised edition)). However, blood BNP / NT-proBNP levels are indicators of heart failure, and their effectiveness in early disease states before cardiac function declines, such as myocarditis, is unclear.

[0006] On the other hand, if there are symptoms of heart failure (chest pain, shortness of breath, difficulty breathing) or symptoms of arrhythmia (palpitations, dizziness), an electrocardiogram is performed. An electrocardiogram is a simple diagnostic method with high sensitivity, and even if the initial electrocardiogram changes are slight, abnormal findings may become clear over time, so it is important to follow the progress of patients suspected of having myocarditis and repeat electrocardiograms. ST-T abnormalities are the most common, but they can present as localized ST elevations that closely resemble acute myocardial infarction, making it difficult to distinguish them from myocardial infarction. Recently, it has been reported that cardiac MRI is useful as an auxiliary diagnostic tool to examine the properties of the myocardium. However, tests using such modalities are time-consuming and expensive, so they are not suitable as primary tests. Summary of the Invention

[0007] Therefore, the present invention aims to provide a means that makes it possible to determine the onset or risk of myocarditis, particularly when the cardiomyopathy is mild and before it causes a decline in cardiac function, using a simple and minimally invasive technique.

[0008] The present inventors have conducted extensive research to solve the above problems. As a result, they have surprisingly clarified for the first time the relationship between the onset of myocarditis and ADAM28. Based on this finding, they have completed the present invention.

[0009] That is, according to one embodiment of the present invention, a method for determining the onset or risk of myocarditis is provided, which determines that a subject has myocarditis or is at risk of myocarditis if the concentration or expression level of ADAM28 in a body fluid sample collected from the subject is lower than a standard value.

[0010] In addition, according to another aspect of the present invention, there is provided a method for providing data for determining the onset or risk of myocarditis, which includes determining that myocarditis has occurred or there is a risk of myocarditis if the concentration or expression level of ADAM28 in a body fluid sample collected from a subject is lower than a standard value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] One aspect of the present invention relates to a method for determining the onset or risk of myocarditis, in which a subject is determined to have myocarditis or to be at risk of myocarditis if the concentration or expression level of ADAM28 in a body fluid sample collected from the subject is lower than a reference value.

[0012] Another aspect of the present invention relates to a method for providing data for determining the onset or risk of myocarditis, which comprises determining that a subject has myocarditis or is at risk of myocarditis if the concentration or expression level of ADAM28 in a body fluid sample collected from the subject is lower than a reference value.

[0013] According to the present invention, it is possible to determine the onset or risk of myocarditis, particularly when the cardiomyopathy is mild and before cardiac function declines, using a simple and minimally invasive technique.

[0014] Specifically, according to the method of one embodiment of the present invention, the onset of myocarditis or its risk in a subject can be easily determined. Moreover, according to the method of one embodiment of the present invention, even mild myocardial damage in which cardiac function is not impaired and BNP and NT-proBNP are not elevated can be detected. In addition, such a test can be easily performed since it is only necessary to measure the concentration or expression level of ADAM28 in a body fluid sample. When a blood sample is used as the body fluid sample, diagnosis is possible with a part of the blood collected in a normal blood test, and there is almost no burden on the subject. Therefore, according to the method of one embodiment of the present invention, the onset of myocarditis or its risk can be easily and minimally invasively determined without using expensive modalities, and data for the determination can be provided, and a significant improvement in the prognosis of myocardial damage can be expected.

[0015] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to the preferred embodiments, but the present invention is not limited to the following embodiments.

[0016] In this specification, the range "X to Y" means "X or more and Y or less." Furthermore, unless otherwise specified, the operations and measurements of physical properties are performed under conditions of room temperature (20 to 25°C) and relative humidity of 40 to 50% RH.

[0017] As used herein, the term "subject" refers to a subject that may develop myocarditis or myocardial injury, and is preferably a human subject. In another preferred embodiment, the subject is a patient who has been diagnosed with a disease involving myocarditis, or who has been treated for the disease after being diagnosed.

[0018] In the present specification, the term "body fluid sample" refers to any body fluid derived from a living organism (organism) in which the concentration or expression level of ADAM28 can be measured. The body fluid sample is preferably a blood sample. The blood sample may be whole blood, plasma, or serum. The blood sample may be blood (whole blood) collected from a subject as is, or may be a sample to which additives such as potassium EDTA, heparin, or sodium citrate have been added for the purpose of preventing coagulation. There is no particular restriction on the timing of collecting the body fluid sample from the subject. When the body fluid sample is a blood sample, the blood sample may be arterial blood or venous blood.

[0019] The body fluid sample used in the method of the present invention is preferably taken from the subject immediately after measurement, but may be stored. For example, there is no particular limitation on the method of storing blood samples, so long as the concentration or expression level of ADAM28 in the sample does not change. For example, freezing at -80°C is preferable for long-term storage.

[0020] In the method according to the present invention, the concentration or expression level of ADAM28 can be measured by a conventionally known method described below.

[0021] ADAM proteins (ADAMs: disintegrins and metalloproteinases) are multifunctional proteins involved in ectodomain shedding of transmembrane proteins, cell adhesion and invasion (Edwards DR, Handsley MM, Pennington CJ. Mol Aspects Med 2008;29(5): 258-289.) (Murphy G. Semin Cell Dev Biol 2009;20 (2):138-145.). The human genome contains 25 ADAMs, including 4 pseudogenes, of which 21 are composed of 13 proteolytic ADAMs and 8 non-proteolytic ADAMs that exhibit proteolytic activity (Edwards DR, Handsley MM, Pennington CJ. Mol Aspects Med 2008;29(5):258-289.) (Shiomi T, Lemaitre V, D'Armiento J, Okada Y. Pathol Int 2010;60(7):477-496.). Proteolytic ADAMs share the metalloproteinase domain of matrix metalloproteinases (MMPs), and typical proteolytic ADAMs contain propeptide, metalloproteinase, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane and intracellular domains. Many proteolytic ADAMs, including ADAM8, ADAM9, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM28, are known to be overexpressed in human cancers and are associated with cancer growth and progression. However, it has not been suggested that ADAM28 could be a useful indicator of myocardial inflammation and injury, especially in the early stages of myocarditis.

[0022] ADAM28 is a known protein, and its amino acid sequence and cDNA sequence are also known. There are two types of ADAM28: membrane type (ADAM28m) and secretory type (ADAM28s). Representative amino acid sequences and cDNA sequences of human membrane type ADAM28 (and its mature form), as well as representative amino acid sequences and cDNA sequences of human secretory type ADAM28 (and its mature form) are disclosed in WO 2016 / 143702. Note that "human membrane type ADAM28" means that the amino acid sequence or nucleotide sequence of membrane type ADAM28 has an amino acid sequence or nucleotide sequence identical or substantially identical to the amino acid sequence or nucleotide sequence of membrane type ADAM28 naturally expressed in humans. "Substantially identical" means that the amino acid sequence or nucleotide sequence of interest has 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more) identity with the amino acid sequence or nucleotide sequence of membrane-type ADAM28 naturally expressed in humans, and has the function of human membrane-type ADAM28. The same interpretation applies to organisms other than humans, and proteins, genes, and fragments thereof other than membrane-type ADAM28.

[0023] In the present specification, the "expression level" of ADAM28 includes both the expression of RNA (transcription product) complementary to the gene encoding ADAM28, and the expression of ADAM28 (translation product) itself. Therefore, in the present specification, the expression level of ADAM28 refers to the expression level or expression intensity of the transcription product or translation product. The expression level can usually be analyzed based on the production level of the transcription product, or the production level, activity, etc. of the translation product. In addition, the "concentration" of ADAM28 refers to the amount (expression level) of ADAM28 present per unit volume of a body fluid sample (e.g., a blood sample).

[0024] The expression level of ADAM28 may be measured by measuring the transcription product of the gene encoding ADAM28, i.e., mRNA, or by measuring the translation product of the gene, i.e., ADAM28 itself. Preferably, it is measured by measuring the translation product of the gene. The transcription product of the gene also includes cDNA obtained by reverse transcription from mRNA.

[0025] The expression level of the translation product can be measured by quantifying ADAM28 or measuring the activity of ADAM28. Quantification of ADAM28 can be performed, for example, by electrophoresis, Western blotting, or chromatography using affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, immunochromatography, or mass spectrometry, but can be easily and accurately measured using an antibody specific to ADAM28.

[0026] The antibody can be prepared by a known method. Alternatively, the antibody can be provided by, for example, MyBioSource, Invitrogen, Santa Cruz Biotechnology, Sigma-Aldrich, etc., and can be appropriately obtained and used. The antibody for detection may be a polyclonal antibody or a monoclonal antibody.

[0027] Detection of ADAM28 using an antibody can be performed by, but is not limited to, immunochromatography, Western blotting, EIA, ELISA, RIA, flow cytometry, and the like. The antibody can be labeled with a fluorescent label, a radioactive label, an enzyme, biotin, or the like, and a secondary antibody for detection that is labeled in this way can also be used. Incidentally, in clinical practice, the presence of myocardial injury is currently evaluated by detecting cardiac troponin T from a blood sample by immunochromatography (Trop T Sensitive, Roche Diagnostics). Therefore, in the method according to the present invention, similarly, by measuring ADAM28 by immunochromatography, it is expected to establish an evaluation method for myocarditis that can be measured in a minimally invasive and simple manner in clinical practice, can be universalized at low cost, has high accuracy and reproducibility, and allows for standardization of the measurement method. As a result, it is expected that appropriate treatment can be administered, and heart failure and sudden death can be prevented. Incidentally, when the above measurement is performed by immunochromatography, it is preferable to use an immunochromatographic test paper. The immunochromatographic test paper preferably has a sample pad area for receiving a blood sample, a conjugate pad area containing a labeled antibody capable of binding to ADAM28, a membrane body for developing a sample, and a test line on the membrane body on which an antibody capable of binding to ADAM28 is immobilized and capable of binding to ADAM28 bound to the labeled antibody in the conjugate pad area.More preferably, the immunochromatographic test paper has a control line that allows the amount of the label bound to the test line to be determined.More preferably, the label exhibits a concentration-dependent color development.

[0028] The expression level of the transcription product may be measured by, for example, measuring the level of gene expression in a sample using a nucleotide containing all or a part of the base sequence of mRNA as a probe or primer, and can be measured by, for example, a method using a microarray (microchip), a Northern blot method, a quantitative PCR method, etc. Quantitative PCR methods include agarose gel electrophoresis, polyacrylamide gel electrophoresis, a fluorescent probe method, a RT-PCR method, a real-time PCR method, an ATAC-PCR method, a Taqman PCR method, a SYVER (registered trademark) green method, a Body Map method, a Serial analysis of gene expression (SAGE) method, a Micro-analysis of Gene Expression (MAGE) method, etc., and these can be used appropriately. Evaluation may also be performed using a next-generation sequencer. Using these methods, the amount of mRNA can be measured by using a nucleotide probe or primer that hybridizes to the mRNA. The base length of the probe or primer used for measurement is preferably 10 to 50 mer, more preferably 15 to 25 mer.

[0029] When the expression of multiple genes, particularly the expression of multiple types of genes, is to be measured simultaneously, it is preferable to use a DNA microarray. A DNA microarray can be prepared by immobilizing nucleotides consisting of the base sequence of a gene or nucleotides containing a partial sequence thereof on a suitable substrate. Examples of the immobilization substrate include glass plates, quartz plates, and silicon wafers. Examples of the size of the substrate include 3.5 mm x 5.5 mm, 18 mm x 18 mm, and 22 mm x 75 mm, which can be set in various ways depending on the number of probe spots on the substrate and the size of the spots. As a method for immobilizing a polynucleotide or a fragment thereof, the charge of the nucleotide can be utilized to electrostatically bind the polynucleotide to a solid-phase carrier surface-treated with polycations such as polylysine, polyethyleneimine, and polyalkylamine, or a nucleotide having a functional group such as an amino group, an aldehyde group, a thiol group, or biotin can be covalently bound to a solid-phase surface having a functional group such as an amino group, an aldehyde group, or an epoxy group introduced therein. The immobilization can be performed using an array machine. At least one gene or a fragment thereof is immobilized on a substrate to prepare a DNA microarray, which is then contacted with mRNA or cDNA derived from a subject that has been labeled with a fluorescent substance and hybridized, and the type and amount of mRNA can be determined by measuring the fluorescence intensity on the DNA microarray.

[0030] In the method according to the present invention, the onset or risk of myocarditis can be determined by comparing the concentration or expression level of ADAM28 measured by the above-mentioned method with a reference value. That is, the present specification discloses a method for determining the onset or risk of myocarditis. The present specification also discloses a method for providing data for determining the onset or risk of myocarditis. In this case, as described above, when the concentration or expression level of ADAM28 is lower than the reference value, it is determined that myocarditis has occurred or there is a risk of myocarditis.

[0031] In this specification, the "reference value" is a value that can be a reference for determining an increase or decrease in the concentration or expression level of ADAM28 in the method of the present invention. Currently, cancer is known as a disease in which blood ADAM28 levels increase. Therefore, the measured value of the concentration or expression level of ADAM28 in a person who has not been diagnosed as having cancer (e.g., a healthy subject) can be used as the reference value. For example, Kuroda et al. have reported that when the cutoff value is set to 1.72 ng / mL, non-small cell lung cancer can be differentially diagnosed with a sensitivity of 72.5% and a specificity of 76.5% (Kuroda H, Mochizuki S, Shimoda M, et al. Int J Cancer 2010;127(8):1844-1856.). Since the measurement results differ depending on the measurement method and the standard used for the measurement, it is important to set a cutoff value for each measurement system. In addition, the concentration or expression level of ADAM28 measured in the same subject before the onset of cancer may be used as the reference value. The reference value is preferably the concentration or expression level of ADAM28 in a body fluid sample (eg, a blood sample) taken from a healthy subject.

[0032] According to yet another aspect of the present invention, a detection reagent for use in the method according to one aspect of the present invention described above is provided. Also, a kit for use in the method according to one aspect of the present invention described above, which includes the detection reagent, is provided. The detection reagent enables detection of the concentration or expression level of ADAM28 in the body fluid sample described above. Examples of such detection reagents include an antibody for specific binding to ADAM28 in a body fluid sample, or DNA or RNA for hybridization with the mRNA of the ADAM28 gene in a body fluid sample. Such DNA or RNA may be a probe capable of detecting hybridization by a fluorescent label or the like. Alternatively, the DNA or RNA may be a primer that can be used for amplifying mRNA. In addition to the above-mentioned detection reagent, the kit may contain other reagents necessary for detection, such as a buffer, various nucleotides, and other reagents necessary for hybridization or antibody binding. EXAMPLES

[0033] The effects of the present invention will be described using the following examples and comparative examples, although the technical scope of the present invention is not limited to the following examples.

[0034] [Preparation of myosin solution] Myosin (myosin, calcium-activated porcine heart, Sigma-Aldrich) was added with 0.01M phosphate buffer to prepare a 10mg / mL myosin solution. 20mg of Mycobacterium Tuberculosis H37 Ra, desiccated (Becton Dickinson Japan) was ground with a mortar and pestle and mixed with 2mL of Mycobacterium Tuberculosis H37 Ra, Complete Adjuvant in Oil (Becton Dickinson Japan) (hereafter referred to as CFA). 1mL each of myosin solution and CFA was placed in a 2mL Luer-lock type all-plastic disposable syringe and connected with a stopcock adapter for syringes. These two solutions were mixed in the syringe until it became cloudy to prepare the myosin solution to be administered.

[0035] [Creation of animal models] Myocarditis is often caused by infection with bacteria or viruses, but it is also known to be caused by autoimmune factors (Guidelines for the diagnosis and treatment of acute and chronic myocarditis (revised 2009)). On the other hand, it has been reported that viral myocarditis can progress to autoimmune myocarditis and aggravate myocardial damage (Richer MJ, et al. J Innate Immun 2009;1(5):421-434.) (Eriksson U, et al. Nat Med 2003;9(12):1484-1490.).

[0036] Myocarditis is thought to progress as follows (Martinez NE, et al. Future Virol 2012;7(6):593-608.). First, the virus infects and proliferates in the heart, triggering an innate immune response and damaging cardiomyocytes (first stage: viral myocarditis). If viral clearance is incomplete, myocardial damage occurs due to viral proliferation and antiviral immunity, leading to autoantibody and autoimmune T cell responses due to epitope spreading and molecular mimicry (second stage: immune myocarditis). Low-grade persistence of the virus and myocardial tissue damage in the first and second stages leads to cardiac remodeling and ultimately dilated cardiomyopathy (third stage: dilated cardiomyopathy).

[0037] Since myocarditis caused by infection can progress to autoimmune myocarditis and aggravate myocardial damage, autoimmune myocarditis has been studied using animal models. Myosin, actin, ADP / ATP carrier protein, SRA protein, laminin, etc. are known to cause myocarditis, and a model is often created by inducing immunity by administering cardiac myosin, the most abundant protein constituting myocardium, to Lewis rats (Izumi Tohru et al. Heart 1993;25(7):821-832). In this model, pericarditis and cardiac swelling begin about one week after myosin administration, and histologically, infiltration of inflammatory cells into the myocardium and myocardial necrosis begin to appear, and by the third week, myocarditis develops in all sensitized cases, and in some cases, the myocarditis spreads transmurally. Myocarditis is most severe on the 28th day, after which cellular infiltration subsides, and after 42 days it is almost entirely replaced by fibrosis (Izumi Toru et al. Heart 1993;25(7):821-832). In addition, in myosin-induced myocarditis models, it has been reported that cardiac function (left ventricular fractional shortening, left ventricular ejection fraction) declines 4-5 weeks after myosin administration (Nagaya N, et al. Circulation 2005;112(8):1128-1135.) (Mina Y, et al. J Cardiovasc Pharmacol Ther 2013;18(2):152-161.).

[0038] In the experiment, rats (Lewis, male, 7 weeks old, purchased from Charles River Japan Co., Ltd.) were quarantined and acclimatized for 6 days, and it was confirmed that all rats were in good health and had not lost weight before being used in the study. The rats were kept in a breeding environment with 12-hour lighting, a temperature of 20-26°C, and a humidity of 30-70%, and were allowed to freely access food and water.

[0039] In the myosin administration group, rats were anesthetized with 2% isoflurane inhalation and 0.1 mL of myosin solution (hereinafter referred to as "antigen") was administered to one hind footpad. One week after the first administration, the second antigen administration was performed. Two and five days after the first antigen administration, 0.1 mL of inactivated Bordetella pertussis suspension (200 billion / mL, Nacalai Tesque, Inc.) was administered intraperitoneally in awake rats. The abdomen was shaved beforehand. The control group was an untreated group, and the average value was used as the reference value.

[0040] The experiments were carried out in accordance with the guidelines for animal experiments at Terumo Corporation.

[0041] [Cardiac function test] Three weeks after the first antigen administration, under 2% isoflurane inhalation anesthesia, left ventricular short-axis images (papillary muscle level) were obtained using an ultrasound diagnostic device in M-mode, and LVDd (left ventricular end-diastolic diameter) and LVDs (left ventricular end-systolic diameter) were measured. Left ventricular ejection fraction (EF) was calculated using the Teichholtz method, and left ventricular fractional shortening (FS) was calculated using the formula (LVDd-LVDs) / LVDd. As a result, no significant differences were observed in either FS or EF, confirming that cardiac function had not yet deteriorated (Tables 1 and 2). Statistical analysis was performed using Student's t-test, with significance at p<0.05.

[0042] [Table 1]

[0043] [Table 2]

[0044] [Necropsy of rats] The day after the cardiac function test, the rats were weighed, and then the abdomen was opened under 2% isoflurane inhalation anesthesia, blood was collected from the abdominal aorta and abdominal vena cava into blood collection tubes containing EDTA-2K, and the rats were euthanized. The body weight at this time was significantly lower in the myosin-administered group (Table 3).

[0045] [Table 3]

[0046] The blood was centrifuged at 3000 rpm for 10 min at 4°C to collect plasma. The plasma was stored at -30°C and used to measure NT-proBNP and ADAM28. The hearts were washed with saline, weighed, and immersed in 10% neutral buffered formalin to fix them. No significant difference in heart weight was observed between the two groups. After fixation, the hearts were embedded in paraffin, and myocardial degeneration / necrosis and mononuclear cell infiltration were evaluated by HE staining, and fibrosis was evaluated by Masson's trichrome staining. Evaluation was scored into four grades, semi-quantitative, and statistically analyzed. Statistical analysis was performed using Student's t-test, with significance at p<0.05.

[0047] The grades and scores were set as shown in Table 4 below.

[0048] [Table 4]

[0049] As a result, myocardial degeneration / necrosis, mononuclear cell infiltration, and fibrosis were significantly observed in the myosin-administered group, confirming the onset of myocarditis. The results of the pathological findings are shown in Table 5.

[0050] [Table 5]

[0051] [NT-proBNP concentration measurement] The NT-proBNP concentration in plasma collected at autopsy was measured using Rat N-Terminal Pro-Brain Natriuretic Peptide ELISA (MyBioSource) according to the manual. The results are shown in Table 6. Statistical analysis was performed using Student's t-test, and p<0.05 was considered significant.

[0052] As a result, no significant difference was observed in plasma NT-proBNP concentrations between the myosin-administered group and the control group.

[0053] [Table 6]

[0054] [Determination of myocarditis] In the myosin-administered group, pathological findings revealed inflammation, but cardiac function was not impaired and plasma NT-proBNP concentrations were not significantly different from those in the control group, indicating that this was an early stage before heart failure.

[0055] [Measurement of ADAM28 concentration] The concentration of ADAM28 in plasma collected at autopsy was measured using a Rat A Disintegrin and Metalloprotease 28 (ADAM28) ELISA Kit (MyBioSource) according to the manual. The results are shown in Table 7. Statistical analysis was performed using Student's t-test, and p<0.05 was considered significant.

[0056] As shown in Table 7, the plasma ADAM28 concentration was significantly lower in the myosin-administered group compared to the control group. Therefore, when the concentration (or expression level) of ADAM28 is lower than that in a healthy subject, it can be determined that myocarditis has developed or that there is a risk of myocarditis.

[0057] In addition, when the Pearson correlation coefficient test was performed on the ADAM28 concentrations in arterial and venous blood collected from the same individual, the correlation coefficient was R = 0.87, p < 0.001. Based on these results, it was determined that there is no problem whether the sample was arterial or venous blood.

[0058] [Table 7]

[0059] This application is based on Japanese Patent Application No. 2020-044279 filed on March 13, 2020, the disclosure of which is incorporated by reference in its entirety.

Claims

1. A method for evaluating the onset or risk of myocarditis in a subject by comparing the concentration or expression level of ADAM28 in a body fluid sample collected from the subject with a criterion for indicating that the subject has myocarditis or is at risk of having myocarditis when the concentration or expression level is lower than a standard value.

2. A method for providing data for evaluating the onset or risk of myocarditis in a subject, comprising evaluating the onset or risk of myocarditis in a subject by comparing the concentration or expression level of ADAM28 in a body fluid sample collected from the subject with a standard value indicating that the subject has or is at risk of having myocarditis when the concentration or expression level is lower than a standard value.

3. The method according to claim 1 or 2, wherein the reference value is the concentration or expression level of ADAM28 in a body fluid sample collected from a healthy subject.

4. The method according to any one of claims 1 to 3, wherein the body fluid sample is a blood sample.

5. A detection reagent for use in the method according to any one of claims 1 to 4, which enables detection of the concentration or expression level of ADAM28 in the body fluid sample.

6. The detection reagent according to claim 5 , which is an antibody against ADAM28, or a DNA or RNA for hybridization with the mRNA of the ADAM28 gene.

7. A kit for use in the method according to any one of claims 1 to 4, comprising the detection reagent according to claim 6.

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