Troponin marker combinations for early identification of type 2 vs. type 1 acute myocardial infarction

Abstract

The present invention relates to a method for assessing myocardial infarction comprising the steps of: determining the amount of a first biomarker in a sample of a subject, wherein said first biomarker is a cardiac troponin; determining the amount of a second biomarker in a sample of a subject, wherein said second biomarker is selected from the group consisting of BMP10 type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, cardiac myosin binding protein C (cMyBPC) and ANG2 (angiopoietin 2); comparing the amount of the biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker; and assessing the subject based on the comparison and / or calculation. The present invention also relates to the use of a first biomarker, which is a cardiac troponin, and a second biomarker selected from the group consisting of BMP10 type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, cardiac myosin binding protein C (cMyBPC) and ANG2 (angiopoietin 2), or at least one detection agent for said first biomarker and at least one detection agent for said second biomarker, for assessing myocardial infarction. Furthermore, the present invention further relates to a computer-implemented method for assessing myocardial infarction, as well as a device and a kit for assessing myocardial infarction.

Description

[Technical Field]

[0001] The present invention relates to a method for assessing myocardial infarction, comprising the steps of: determining the amount of a first biomarker in a sample of a subject, wherein the first biomarker is cardiac troponin; determining the amount of a second biomarker in a sample of a subject, wherein the second biomarker is selected from the group consisting of BMP10 peptide (bone morphogenesis protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP peptide, cardiac myosin-binding protein C (cMyBPC), and ANG2 (angiopoietin 2); comparing the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker; and assessing the subject based on the comparison and / or calculation. The present invention also relates to the use of a first biomarker, which is cardiac troponin, and a second biomarker selected from the group consisting of BMP10 peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP peptide, cMyBPC, and ANG2 (angiopoietin 2), or at least one detection agent for the first biomarker and at least one detection agent for the second biomarker for assessing myocardial infarction. Furthermore, the present invention further relates to a computer-implemented method for assessing myocardial infarction, as well as a device and a kit for assessing myocardial infarction. [Background technology]

[0002] The goal of modern medicine is to provide personalized or individualized treatment regimens. These are treatment regimens that take into account the individual needs or risks of the patient. When it is necessary to determine a possible treatment regimen within a short period of time, personalized or individualized treatment regimens should also be considered. In such situations, measuring circulating biomarkers by blood tests can help diagnose disease conditions (e.g., cardiomyocyte damage by troponin testing), understand disease risks mechanistically (e.g., elevated lipids and atherosclerosis), identify the biological pathways involved (e.g., protective effect of BMP10 in atherosclerotic plaques; Upton PD et al., J Cell Sci 2020;133:jcs239715;doi:10.1242 / jcs.239715), and improve treatment outcomes.

[0003] Diagnostic examinations for patients suspected of having AMI (acute myocardial infarction) require admission to the ED, registration of a 12-lead electrocardiogram (ECG), blood tests to diagnose or rule out myocardial injury, assessment of clinical signs and medical history, physical examination, and other diagnostic tests to diagnose AMI or differential diagnosis.

[0004] According to the Universal Definition of AMI (Thygesen K et al., Circulation 2018;138:e618-e651.doi:10.1161 / CIR.0000000000000617.), five different types of AMI are defined based on the different mechanistic pathways underlying AMI, with types 1 and 2 myocardial infarction being described in more detail below. According to the guidelines, types 3 through 5 of AMI are clinically well distinguishable by those skilled in the art for three specific clinical situations: type 3 ("death before it becomes possible to obtain blood for cardiac biomarker measurement, or the patient may die immediately after the onset of symptoms before an increase in biomarker levels occurs"), type 4 (PCI and stent-related AMI), and type 5 (CABG-related AMI). Therefore, this invention aims at the most common situations for diagnosing suspected AMI (types 1 and 2), the distinction (type 1 vs. type 2) being highly relevant to the choice of treatment:

[0005] Type 1 myocardial infarction (T1MI): Coronary atherothrombosis caused by plaque rupture and erosion in one or more coronary arteries and / or distal emboli leads to intraluminal thrombosis and subsequent reduction in myocardial perfusion and necrosis.

[0006] Type 2 myocardial infarction (T2MI) is secondary to a sudden imbalance between the supply and demand of myocardial oxygen. Reduced myocardial perfusion can result from stable coronary atherosclerosis (without plaque rupture), coronary artery spasm, microvascular dysfunction, coronary embolism or dissection, and systemic hemodynamic disorders including hypotension, hypertension, tachycardia, or hypoxemia (DeFilippis et al, Circulation 2019:140;1661-1678).

[0007] Type 2 MI is often associated with the presence of other underlying comorbidities (Thygesen et al, Eur Heart J 2018:40(3);246-247), tends to be more frequent in women, and is reported to have higher short- and long-term mortality rates than type 1 MI. The 5-year survival rate for type 2 MI patients is reported to be low, below 40% (McCarthy et al, JAMA 2018:320(5);433).

[0008] The management of type 1 and type 2 myocardial infarction differs substantially from that of type 2 MI, as the myocardial damage in type 2 MI is not due to atherosclerosis and unstable coronary artery disease (CAD). Therefore, management should address the root cause and underlying cause (e.g., oxygen therapy in the case of hypoxemia or volume replacement in the case of hypotension). This is in contrast to type 1 myocardial infarction, which requires immediate invasive treatment or fibrinolysis depending on the extent of the infarction and the availability of percutaneous coronary intervention (PCI). In type 2 MI, coronary artery angiography may be useful in determining whether underlying CAD is present (Thygesen et al, Eur Heart J 2018:40(3);246-247). The default treatment of type 2 MI with specific therapies for type 1 MI is currently not supported by clinical evidence and may even have unfavorable effects on outcomes (Collinson et al, ACC May 18, 2016, Diagnosing Type 2 Myocardial Infarction), thus making this distinction all the more important. While troponin peak levels tend to be higher in T2 MI than T1 MI, their discriminatory power in clinical practice remains insufficient (Smilowitz et al, Coronary Artery Disease 2018;29(1);46-52). Therefore, identifying and differentiating patients with type 2 MI versus type 1 MI (Nagele 2020 Circulation.2020;141:1431-1433.DOI:10.1161 / CIRCULATIONAHA.119.044996) is a critical unmet need.

[0009] Myocardial necrosis biomarkers, including troponin, were observed at different concentrations in patients with type 1 and type 2 AMI (see, e.g., Nestelberger et al., 2021 JAMA Cardiol.doi:10.1001 / jamacardio.2021.0669).

[0010] However, while elevated troponin levels are specific to myocardial injury, not all elevated troponin levels indicate myocardial infarction (MI). Therefore, additional information is still needed to enable reliable and early assessment of myocardial infarction (Collet JP et al, Eur Heart J 2021;42:1289-1367.doi:10.1093 / eurheartj / ehaa575), and especially to better differentiate type 1 MI from type 2 MI. [Overview of the project]

[0011] Therefore, the present invention provides means and methods that meet these needs.

[0012] Advantageously, in the research that forms the basis of this invention, it was found that a combination of a first biomarker and a second biomarker, preferably a third biomarker, enables reliable early assessment of myocardial infarction. The study surveyed patients presenting to the emergency department. For this purpose, patients with confirmed myocardial infarction were subdivided into those with type 1 myocardial infarction and those with type 2 myocardial infarction. The amounts of various biomarkers were determined, and the biomarkers were analyzed and mathematically combined via logistic regression analysis. The area under the receiver operating characteristic curve (AUC) was used to evaluate biomarker performance. The AUC value is the mathematical integral of the function f(x) within the interval [a][b]. AUC was also examined for biomarker pairs and triplets. Together, biomarker combinations that showed improved AUC compared to the best single biomarker AUC were identified. The results are described in the appended examples below.

[0013] In particular, if a patient suspected of having a myocardial infarction is, for example, in an emergency unit, early assessment of the patient is crucial in determining when to initiate therapeutic measures, including drug administration, physical or other therapeutic interventions. It is especially important to distinguish between type 1 and type 2 myocardial infarction, as the treatment for type 1 and type 2 myocardial infarction differs significantly (see above). Thanks to the present invention, the identification of AMI patients can be assessed by early biomarker determination, which is important for preventing myocardial damage (Collet JP et al, Eur Heart J 2021;42:1289-1367.doi:10.1093 / eurheartj / ehaa575), thus preventing life-threatening onsets. The biomarker pairs and triplets identified in the research underlying the present invention provide a reliable basis for medical decisions, and assessment can be performed in a time- and cost-effective manner.

[0014] The present invention is a method for assessing myocardial infarction in a subject, (a) Determining the amount of a first biomarker in the subject sample, wherein the first biomarker is a cardiac troponin such as cardiac troponin T or I, (b) Determining the amount of a second biomarker in the sample of the subject, wherein the second biomarker is BMP10 type peptide (bone morphogenesis protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, such as NT-proBNP or BNP, cardiac myosin-binding protein C (cMyBPC), or ANG2 (angiopoietin-2), (c) Comparing the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker, (d) A method comprising assessing myocardial infarction based on the comparison and / or calculation performed in step (c).

[0015] In one embodiment of the method of the present invention, the method further includes determining the amount of a third biomarker. In particular, in step (b) of the method of the present invention, (i) If the amount of BMP10 type peptide is determined as the second biomarker, the method may further include determining the amount of cMyBPC (cardiac myosin-binding protein C), at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6, as a third biomarker, or (ii) If the amount of FGF23 is determined to be the second biomarker, the method may further include determining the amount of at least one lipid biomarker, e.g., cMyBPC, CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2, or IGFBP7, as a third biomarker, or (iii) If the amount of BNP-type peptide is determined as the second biomarker, the method may further include determining the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, as a third biomarker, or (iv) If the amount of ANG2 is determined to be the second biomarker, the method may further include determining the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7, as a third biomarker.

[0016] Therefore, the present invention is a method for assessing myocardial infarction in a subject, (a) A step of determining the amount of a first biomarker in the sample of the subject, wherein the first biomarker is cardiac troponin, and determining the amount of the first biomarker in the sample of the subject; (b) Determining the amounts of the second and third biomarkers in the (said) sample of the subject; (c) Comparing the amount of the biomarker with a reference for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker; (d) Assessing myocardial infarction based on the comparison and / or calculation performed in step (c). A method is related to this.

Mode for Carrying Out the Invention

[0017] As used in this specification and the claims, it should be understood that "a" or "an" can mean one or more depending on the context in which it is used. Thus, for example, reference to "an" element can mean that at least one element can be utilized.

[0018] As used hereinafter, the terms "have", "comprise", or "include", or any grammatical variations thereof, are used non-exclusively. Thus, these terms may refer to both situations where no additional features exist in the entity being described in this context, in addition to the features introduced by these terms, and situations where one or more additional features exist. By way of example, the expressions "A has B", "A comprises B", and "A includes B" may all refer to situations where, in addition to B, no other elements exist in A (i.e., the situation where A consists solely and exclusively of B), and situations where, in addition to B, one or more additional elements such as element C, elements C and D, or even further elements exist in entity A. The term "comprising" includes embodiments where only the recited items exist, i.e., has a limiting meaning in the sense of "consisting of".

[0019] Furthermore, when used hereinafter, the terms "in particular", "more specifically", "typically", and "more typically", or similar terms, are used in conjunction with additional / alternative features without restricting the possibility of alternatives. Thus, the features introduced by these terms are additional / alternative features and are not intended to limit the scope of the claims in any way. The present invention may be implemented by using alternative features, as would be understood by a person skilled in the art. Similarly, features introduced by expressions such as "in one embodiment of the present invention" or similar expressions are intended to be additional / alternative features without any limitation regarding alternative aspects of the present invention, without any limitation regarding the scope of the present invention, and without any limitation regarding the possibility of combining such introduced features with other additional / alternative or non-additional / alternative features of the present invention.

[0020] Furthermore, as used herein, the term “at least one” will be understood to mean that one or more of the items referred to following this term may be used in accordance with the invention. For example, where this term indicates that at least one sampling unit should be used, this may be understood to mean one sampling unit or two or more sampling units, i.e., two, three, four, five, or any other number. Depending on the item referred to by this term, a person skilled in the art will understand which upper limit this term may refer to, if such an upper limit exists.

[0021] As used herein, the term “about” means that with respect to any number following the term, there exists an interval precision within which the technical effect can be achieved. Thus, “about” as referred herein preferably means the exact number or a range around that exact number of ±20%, preferably ±15%, more preferably ±10%, or even more preferably ±5%.

[0022] Furthermore, terms such as “first,” “second,” and “third” in the specification and claims are used to distinguish similar elements and are not necessarily used to indicate a chronological or sequential order. For example, the amount of the second biomarker may be determined before the amount of the first biomarker.

[0023] The method of the present invention may consist of the steps described above, or may include additional steps such as a step for further evaluation of the assessment obtained in step (d), or a step for recommending or initiating therapeutic measures such as treatment. Furthermore, the method of the present invention may include steps prior to step (a), such as a step relating to sample pretreatment. However, preferably, the method described above is assumed to be an ex vivo method that does not require any steps performed on the human or animal body. Therefore, the method is an in vitro method. Furthermore, the method can be assisted by automation. Typically, the determination of biomarkers may be assisted by robotic instruments, and comparison and assessment may be assisted by data processing equipment such as computers.

[0024] According to the present invention, myocardial infarction is assessed. The term "myocardial infarction" is well known in the art. As used herein, the term refers to acute myocardial infarction (abbreviated as "AMI"). The clinical diagnostic criteria for acute myocardial infarction are the presence of acute myocardial injury (i.e., cardiac troponin (cTn) values ​​above the upper limit of the 99th percentile (URL), including elevated and / or decreased cTn values) in the context of evidence of acute myocardial ischemia. Evidence of acute myocardial ischemia includes clinical signs and symptoms, ischemic ECG changes, the occurrence of pathological Q waves, and imaging evidence of new loss of viable myocardium or new focal wall motion abnormalities in a pattern consistent with ischemic etiology (Thygesen et al, Eur J Heart 2019:40(3);237-269, DOI:10.1093 / eurheartj / ehy462).

[0025] In one embodiment of the present invention, the term "assessing myocardial infarction" refers to the distinction between type 1 and type 2 myocardial infarction. Therefore, it is determined whether the subject has type 1 or type 2 myocardial infarction.

[0026] Therefore, the present invention is a method for distinguishing between type 1 myocardial infarction and type 2 myocardial infarction, (a) A step of determining the amount of a first biomarker in the target sample, wherein the first biomarker is cardiac troponin, (b) Determining the amount of the second biomarker and optionally a third biomarker in the subject (the) sample, (c) Comparing the amount of the biomarker with a standard for the biomarker and / or calculating a score for distinguishing between type 1 myocardial infarction and type 2 myocardial infarction based on the amount of the biomarker, (d) A method comprising distinguishing between type 1 myocardial infarction and type 2 myocardial infarction based on the comparison and / or calculation performed in step (c).

[0027] The term "type 1 myocardial infarction" is well known in the art. As used herein, this term refers to an ischemic myocardial infarction (AMI) with an underlying ischemic etiology of atherosclerotic plaque rupture / erosion accompanied by occlusive or nonocclusive thrombus formation in one or more coronary arteries. Accordingly, the diagnostic criteria for type 1 AMI include the aforementioned criteria for AMI, including, for example, the identification of coronary thrombi by angiography, including intra-coronary imaging or autopsy, as evidence of acute myocardial ischemia (Thygesen et al, Eur J Heart 2019:40(3);237-269, DOI:10.1093 / eurheartj / ehy462).

[0028] The term "type 2 myocardial infarction" is well known in the art. As used herein, this term refers to an ischemic myocardial infarction (AMI) with an underlying ischemic etiology resulting from either a) increased oxygen demand (e.g., persistent tachyarrhythmia or severe hypertension) or b) decreased oxygen supply (e.g., hypotension, bradyarrhythmia, respiratory failure, severe anemia, coronary vasospasm, coronary artery dissection, or microvascular dysfunction). Accordingly, the diagnostic criteria for type 2 AMI include the aforementioned diagnostic criteria for AMI, which include evidence of an imbalance between myocardial oxygen supply and demand unrelated to acute coronary atherothrombosis (Thygesen et al, Eur J Heart 2019:40(3);237-269, DOI:10.1093 / eurheartj / ehy462).

[0029] In another embodiment of the present invention, the term “assessing myocardial infarction” relates to the diagnosis of type 2 myocardial infarction.

[0030] Therefore, the present invention is a method for diagnosing type 2 myocardial infarction, (a) A step of determining the amount of a first biomarker in the target sample, wherein the first biomarker is cardiac troponin, (b) Determining the amount of the second biomarker and optionally a third biomarker in the subject (the) sample, (c) Comparing the amount of the biomarker with a standard for the biomarker and / or calculating a score for diagnosing type 2 myocardial infarction based on the amount of the biomarker, (d) A method comprising diagnosing type 2 myocardial infarction based on the comparison and / or calculation performed in step (c).

[0031] As used herein, the term “diagnose” means to determine whether or not a subject referred to according to the method of the present invention has type 2 myocardial infarction.

[0032] In another embodiment of the present invention, the term “assessing myocardial infarction” preferably relates to the assessment of treatment or management of myocardial infarction in an affected subject. Based on the present invention, a treatment decision can be made, and the subject can be treated accordingly. For example, it can be determined whether a patient will receive treatment aimed at treating type 2 myocardial infarction or type 1 myocardial infarction.

[0033] As used herein, the term “diagnose” means to determine whether or not a subject referred to according to the method of the present invention has type 2 myocardial infarction.

[0034] As will be understood by those skilled in the art, assessments such as distinctions or diagnoses made in accordance with the present invention are preferably correct for 100% of the subjects investigated, but are usually not 100% correct. This term typically requires that the statistically significant portion of the subjects be accurately assessed. Whether a portion is statistically significant can be further easily determined by those skilled in the art using various well-known statistical assessment tools, such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. Further details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically, assumed confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%. P-values ​​are typically 0.2, 0.1, and 0.05.

[0035] According to the present invention, the assessment of myocardial infarction is typically understood as an aid in the assessment of myocardial infarction, for example, as an aid in distinguishing between type 2 and type 1 myocardial infarction, or as an aid in diagnosing type 2 myocardial infarction. Therefore, the methods and uses of the present invention may be part of a complete assessment. A complete assessment may include assessment of further markers, clinical parameters and / or ECG (which may contribute to the score, for example). The final assessment (distinction, etc.) is, in principle, performed by a physician.

[0036] As used herein, the term “subject” refers to an animal, preferably a mammal, more typically a human. The subjects investigated by the method of the present invention are those suspected of having myocardial infarction or those who have had myocardial infarction. Preferably, the subjects have myocardial infarction such as non-ST-elevation myocardial infarction (NSTEMI). In one embodiment, the subject is male. In another embodiment, the subject is female.

[0037] However, subjects to be tested are preferably those who do not have type 3, type 4, or type 5 myocardial infarction. An overview of these types of AMI is incorporated herein by reference, Thygesen K et al. Circulation 2018;138:e618-e651.doi:10.1161 / CIR.0000000000000617. Type 3 (defined as "death before it becomes possible to obtain blood for cardiac biomarker measurement, or the patient may die shortly after the onset of symptoms before an increase in biomarker levels occurs"), type 4 (PCI and stent-related AMI), and type 5 (CABG-related AMI). Therefore, subjects shall have type 1 or type 2 myocardial infarction, or subjects shall be suspected of having type 1 or type 2 myocardial infarction.

[0038] In a preferred embodiment, the subjects are diabetic patients, i.e., those suffering from diabetes. For example, the subjects may have type 1 or type 2 diabetes. Typically, the diabetic subjects have type 2 diabetes. Table 5 of the Examples shows preferred markers and marker combinations for patients suffering from diabetes. Preferably, the second marker in diabetic patients is ANG2.

[0039] The term “sample,” as used herein, refers to any sample containing the first, second, and / or third biomarkers referred herein under physiological conditions. More typically, a sample is a body fluid sample, such as a blood sample or a sample derived therefrom (e.g., serum or plasma), a urine sample, a saliva sample, an interstitial fluid sample, a lymph sample, etc. Most typically, such a sample is a blood, serum, or plasma sample.

[0040] Furthermore, the blood sample is assumed to be a dried blood spot sample. A dried blood spot sample can be obtained by coating a droplet of blood onto an absorbent filter. The blood is completely saturated onto paper and air-dried for several hours. The blood may also be collected from the subject to be tested, for example, from a finger using a lancet.

[0041] In a preferred embodiment, the sample is a blood (i.e., whole blood), serum, or plasma sample. Serum is the liquid fraction of whole blood obtained after blood has been allowed to clot. To obtain serum, the blood clot is removed by centrifugation, and the supernatant is collected. Plasma is the cell-free fluid portion of blood. To obtain a plasma sample, whole blood is collected in an anticoagulant-treated tube (e.g., a citrate-treated or EDTA-treated tube). Cells are removed from the sample by centrifugation, and the supernatant (i.e., plasma sample) is obtained.

[0042] Blood samples include capillary blood samples. Such samples can be obtained, for example, from a finger puncture.

[0043] Preferably, the subject has a myocardial infarction at the time the sample is obtained, but may not have been diagnosed with myocardial infarction at that time. Diagnosis may be made later (for example, based on the determination of cardiac troponin in a second sample obtained from the subject 1 to 3 hours after the first sample). Diagnostic methods for myocardial infarction are well known in the art and are described, for example, in Collet JP et al. (ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. Eur Heart J. 2020 Aug 29:ehaa575.doi:10.1093 / eurheartj / ehaa575.Epub ahead of print.PMID:32860058). Protocols currently available for the diagnosis of non-ST-elevation ACS combine biomarker concentrations at presentation and at later time points such as 1 hour, 2 hours, or 3 hours later.

[0044] Advantageously, the method of the present invention enables early assessment of myocardial infarction. The assessment referred to herein can be reliably performed based on the amounts of first, second, and third biomarkers in a sample (preferably a single sample) obtained from the subject at the time of presentation. Thus, in one embodiment, the sample is a sample obtained from the subject during an emergency department examination.

[0045] According to the present invention, the amounts of at least three biomarkers, namely the first biomarker, the second biomarker, and optionally the third biomarker, are determined in a sample (preferably a single sample) obtained from the subject at the time of presentation.

[0046] The first biomarker is cardiac troponin, a well-known marker in the diagnostic field. The term "cardiac troponin" typically refers to human cardiac troponin T or cardiac troponin I.

[0047] In one embodiment, cardiac troponin is troponin T (measured in the Examples section).

[0048] In an alternative embodiment, cardiac troponin is cardiac troponin I.

[0049] The second marker is at least one biomarker selected from the group consisting of BMP10 type peptide (bone morphogenesis protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, e.g., NT-proBNP or BNP; cardiac myosin-binding protein C (cMyBPC); and ANG2 (angiopoietin-2). Preferred second biomarkers are, in preferred order, BMP10 type peptide, FGF23, and ANG2.

[0050] In one embodiment, the second biomarker is a BMP10-type peptide (bone morphogenesis protein type 10 peptide). The BMP10-type peptide is preferably NT-proBMP10, proBMP10, or BMP, more preferably NT-proBMP10 or proBMP10, and most preferably NT-proBMP10.

[0051] BMP10 type peptides are well known in the art. Preferred BMP10 type peptides are disclosed, for example, in Susan-Resiga et al. (J Biol Chem. 2011 Jul 1;286(26):22785-94), whose entirety is incorporated herein by reference (see, for example, Figure 3A of Susan-Resiga et al., or U.S. Patent Application Publication No. 2012 / 0213782).

[0052] In one embodiment, the BMP10-type peptide is unprocessed preproBMP10. In another embodiment, the BMP10-type peptide is the propeptide proBMP10. This marker includes the N-terminal prosegment and BMP10. In another embodiment, the BMP10-type peptide is the N-terminal prosegment of BMP10 (the N-terminus is proBMP10). In yet another embodiment, the BMP10-type peptide is BMP10.

[0053] In one embodiment, the BMP10 type peptide is part of a homodimer or heterodimer complex.

[0054] Human preproBMP10 (i.e., unprocessed preproBMP10) has a length of 424 amino acids. The amino acid sequence of human preproBMP10 is shown, for example, in Sequence ID No. 1 of International Publication No. 2020 / 035605A1 or in Figure 3 of U.S. Patent Application Publication No. 2012 / 0213782, the entire sequence of which is incorporated herein by reference. Sequence ID No. 1 of International Publication No. 2020 / 035605A1 is the same sequence as Sequence ID No. 2 in the sequence listing of this application.

[0055] Furthermore, the amino acid sequence of preproBMP10 can be determined by Uniprot (see sequence of accession number O95393-1). Human preproBMP10 contains a short signal peptide (amino acids 1-21) that is enzymatically cleaved to release proBMP10. Thus, human proBMP10 contains amino acids 22-424 of human preproBMP10 (i.e., a polypeptide having the sequence shown in SEQ ID NO: 1 of International Publication No. 2020 / 035605A1). Human proBMP10 is further cleaved into the N-terminal prosegment of BMP10 and the active (non-glycosylated) BMP10. The N-terminal prosegment of BMP10 contains amino acids 22-316 of a polypeptide having the sequence shown in SEQ ID NO: 1 of International Publication No. 2020 / 035605A1 (i.e., human preproBMP10). BMP10 contains amino acids 317-424 of a polypeptide having the sequence shown in Sequence ID No. 1 of International Publication No. 2020 / 035605A1.

[0056] The preferred BMP10-type peptides are BMP10 and N-terminal proBMP10. After cleavage of proBMP10, BMP10 and N-terminal proBMP10 remain structurally close together to form a homodimer or heterodimer of BMP10, or remain combined with other BMP family proteins (Yadin et al., CYTOGFR 2016, 27(2016)13-34). Dimerization occurs by the formation of Cys-Cys crosslinks or strong adhesion at the C-terminal peptides of both binding partners. Thus, a structure consisting of two subunits is formed.

[0057] Since proBMP10 is cleaved in equimolar proportions into BMP10 and the N-terminal prosegment, the amount of BMP10 reflects the amount of the N-terminal prosegment. Therefore, the amount of BMP10 can be determined by determining the amount of the N-terminal prosegment, and vice versa.

[0058] Preferably, the amount of BMP10 type peptide is determined using one or more antibodies (or their antigen-binding fragments) that specifically bind to the BMP10 type peptide.

[0059] For example, one or more antibodies that specifically bind to the N-terminal prosegment of BMP10 can be used. Since such antibodies (or fragments) also bind to proBMP10 and preproBMP10, the sum of the amounts of the N-terminal prosegment of BMP10, proBMP10, and preproBMP10 is determined in step a) of the method of the present invention. Therefore, the expression "determine the amount of the N-terminal prosegment of BMP10" also means "the step of determining the sum of the amounts of the N-terminal prosegment of BMP10, proBMP10, and preproBMP10."

[0060] For example, structural predictions based on findings from other BMP-type proteins such as BMP9 indicate that BMP10 remains in complex with proBMP10, and therefore, the detection of the N-terminal prosegment also reflects the amount of BMP10.

[0061] For example, one or more antibodies that specifically bind to BMP10 can be used. Since such antibodies (or fragments) also bind to proBMP10 and preproBMP10, the sum of the amounts of BMP10, proBMP10, and preproBMP10 is determined in step a) of the method of the present invention. Therefore, the expression “determine the amount of BMP10” also means “the step of determining the sum of the amounts of BMP10, proBMP10, and preproBMP10.”

[0062] For example, structural predictions based on findings from other BMP-type proteins such as BMP9 indicate that BMP10 remains in a complex with proBMP10, and therefore the detection of BMP10 also reflects the amount of the N-terminal prosegment.

[0063] Furthermore, it is assumed that the total amount of all four BMP10-type peptides mentioned above—namely BMP10, the N-terminal prosegment of BMP10, proBMP10, and preproBMP10—will be determined.

[0064] Therefore, according to the present invention, the following amounts of BMP10 type peptide can be determined: • Amount of BMP10 • Amount of the N-terminal prosegment of BMP10 • amount of proBMP10 • Amount of preproBMP10 • The sum of the amounts of BMP10, proBMP10, and preproBMP10. · The sum of the amounts of the N-terminal prosegment of BMP10, proBMP10, and preproBMP10, or • Total amount of BMP10, N-terminal prosegment of BMP10, proBMP10, and preproBMP10

[0065] In one embodiment, the amount of BMP10-type peptide is determined as described in International Publication No. 2020 / 035605A1, which is incorporated herein by reference in its entirety. In another embodiment, the amount of BMP10-type peptide is determined as described in International Publication No. 2021 / 165465A1, which is incorporated herein by reference in its entirety. In the Examples section of this application, the amount of BMP10-type peptide was determined by using an antibody that binds to NT-proBMP10.

[0066] In another embodiment, the second biomarker is FGF23. The biomarker fibroblast growth factor-23 (abbreviated as "FGF-23") is well known in the art. FGF-23 plays an important role in regulating calcium phosphate and vitamin D metabolism and plays a causal role in the pathogenesis of LV hypertrophy and renal disease, which are major determinants of cardiovascular events. Preferably, FGF-23 is human FGF-23. The sequence of human FGF-23 is well known in the art; for example, the amino sequence can be determined by GenBank accession number NM_020638.1 GI:10190673. Furthermore, the sequence is also disclosed in Shimada et al., 2001, PNAS, vol.98(11), pp. 6500-6505.

[0067] In another embodiment, the second biomarker is a BNP-type peptide. Preferably, the BNP-type peptide is NT-proBNP, proBNP, or BNP, more preferably NTproBNP or BNP, and most preferably NT-proBNP. The BNP-type peptide may be glycosylated (as described elsewhere in this specification) or not.

[0068] Brain natriuretic peptide-type peptides (also referred to herein as BNP-type peptides) are preferably selected from the group consisting of pre-proBNP, proBNP, NT-proBNP, and BNP. The prepropeptide (134 amino acids in the case of pre-proBNP) contains a short signal peptide, which is enzymatically cleaved to release a propeptide (108 amino acids in the case of proBNP). The propeptide is further cleaved into an N-terminal propeptide (NT-propeptide, 76 amino acids in the case of NT-proBNP) and an active hormone (32 amino acids in the case of BNP). Preferably, the brain natriuretic peptides according to the present invention are NT-proBNP, BNP, and their variants. BNP is an active hormone and has a shorter half-life than its inactive counterpart, NT-proBNP. Preferably, the brain natriuretic peptide-type peptide is BNP (brain natriuretic peptide), more preferably NT-proBNP (the N-terminus of the prohormone brain natriuretic peptide).

[0069] In one embodiment, the BNP-type peptide is BNP.

[0070] In another embodiment, the BNP-type peptide is NT-proBNP.

[0071] NT-proBNP may be "total NT-proBNP" (also referred to herein as tNT-proBNP) or "non-glycosylated NT-proBNP," for example, NT-proBNP that is non-glycosylated at the S44 position.

[0072] The sequence of human NT-proBNP is well known in the art and has already been described in detail in the prior art, for example, International Publication No. 02 / 089657, International Publication No. 02 / 083913, and Bonow 1996, New Insights into the cardiac natriuretic peptides. Circulation 93:1946-1950. Preferably, human NT-proBNP has the amino acid sequence shown in Sequence ID No. 1.

[0073] NT-proBNP and its precursor proBNP can be O-glycosylated. The summary concerns the O-glycosylation of NT-proBNP and proBNP, and is provided, for example, by Schellenberger et al. and Halfinger et al., both of which are incorporated by reference in relation to the entirety of their disclosure (Schellenberger et al., Arch Biochem Biophys 2006;451; Halfinger et al., Clinical Chemistry 63:1,359-368(2017)).

[0074] The term "O-linked glycosylation" is well known in the art (and is also referred to herein as "glycosylation"). O-linked glycosylation is the binding of sugar molecules, i.e., carbohydrates, to serine or threonine residues. This is a post-translational modification that occurs after protein synthesis. How NT-proBNP and proBNP are glycosylated is described, for example, in Schellenberger et al. and Halflinger et al. (cited above).

[0075] An overview of the O-glycosylation site of NT-proBNP is provided below in this specification. The following sequence is a human NT-proBNP sequence (SEQ ID NO: 1, which serves as the reference sequence). The O-glycosylation site is underlined. [ka]

[0076] Therefore, human NT-proBNP (as shown in SEQ ID NO: 1) contains at least the following glycosylation sites: T36, S37, S44, T48, S53, T58, and T71.

[0077] Preferably, as used herein, the term “non-glycosylated NT-proBNP” refers to NT-proBNP that is not glycosylated (i.e., not O-glycosylated) at one or more positions selected from the group consisting of T36, S37, S44, T48, S53, T58, and T71 of human NT-proBNP. Thus, at least one of the following amino acid residues of human NT-proBNP: T36, S37, S44, T48, S53, T58, and T71 is not glycosylated, i.e., does not contain O-glycosylation.

[0078] In some embodiments, the term “non-glycosylated NT-proBNP” refers to NT-proBNP in which at least two of the aforementioned amino acid residues (i.e., T36, S37, S44, T48, S53, T58, and T71) are not glycosylated. In some embodiments, the term “non-glycosylated NT-proBNP” refers to NT-proBNP in which at least three of the aforementioned amino acid residues are not glycosylated. In some embodiments, the term “non-glycosylated NT-proBNP” refers to NT-proBNP in which at least three, at least four, at least five, at least six, or all of the aforementioned amino acid residues are not glycosylated. In some embodiments, the term “non-glycosylated NT-proBNP” refers to NT-proBNP in which at least the serine residue at position 44 (i.e., S44) is not glycosylated. Thus, non-glycosylated NT-proBNP can be non-glycosylated at position S44 (i.e., Ser44).

[0079] In a preferred embodiment, determining the amount of nonglycosylated NT-proBNP in a sample from a subject involves contacting the sample with an antibody or antigen-binding fragment that specifically detects nonglycosylated NT-proBNP. Preferably, the antibody or antigen-binding fragment that specifically detects nonglycosylated NT-proBNP specifically binds to an epitope of NT-proBNP whose epitope contains a glycosylation site but is not glycosylated at the glycosylation site. The complex formed between the antibody (or fragment) and the biomarker is proportional to the amount of nonglycosylated NT-proBNP.

[0080] In some embodiments, an antibody or antigen-binding fragment that specifically detects non-glycosylated NT-proBNP specifically binds to an epitope of NT-proBNP containing a T36 amino acid residue, and the T36 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP containing a glycosylated T36 amino acid residue.

[0081] In some embodiments, the antibody or fragment specifically binds to an epitope of NT-proBNP containing an S37 amino acid residue, and the S37 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP containing a glycosylated S37 amino acid residue.

[0082] In some embodiments, the antibody or fragment specifically binds to an epitope of NT-proBNP containing the S44 amino acid residue, and the S44 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP containing the glycosylated S44 amino acid residue. In some embodiments, the epitope of the antibody or its antigen-binding fragment contains amino acid residues 42-46 of human NT-proBNP (as shown in SEQ ID NO: 1).

[0083] In preferred embodiments, the antibody for specifically detecting nonglycosylated NT-proBNP is the monoclonal antibody MAB 1.21.3 disclosed in International Publication No. 2004099253A1, or an antibody comprising the six CDRs of said antibody. Furthermore, the use of the antigen-binding fragment of said antibody is also envisioned.

[0084] In some embodiments, the antibody or fragment specifically binds to an epitope of NT-proBNP containing a T48 amino acid residue, and the T48 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP containing a glycosylated T48 amino acid residue.

[0085] In some embodiments, the antibody or fragment specifically binds to an epitope of NT-proBNP containing an S53 amino acid residue, and the S53 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP containing a glycosylated S53 amino acid residue.

[0086] In some embodiments, the antibody or fragment specifically binds to an epitope of NT-proBNP containing a T58 amino acid residue, and the T58 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP containing a glycosylated T58 amino acid residue.

[0087] In some embodiments, the antibody or fragment specifically binds to an epitope of NT-proBNP containing a T71 amino acid residue, and the T71 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP containing a glycosylated T71 amino acid residue.

[0088] Preferably, as used herein, the term “glycosylated NT-proBNP” refers to NT-proBNP that is glycosylated (i.e., O-glycosylated) at one or more positions (i.e., amino acid residues) selected from the group consisting of T36, S37, S44, T48, S53, T58, and T71 of human NT-proBNP. Thus, human NT-proBNP includes glycosylation, i.e., O-glycosylation, where at least one of the following amino acid residues of human NT-proBNP is glycosylated: T36, S37, S44, T48, S53, T58, and T71.

[0089] In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least two of the aforementioned amino acid residues (i.e., T36, S37, S44, T48, S53, T58, and T71) are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least three of the aforementioned amino acid residues are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least three, at least four, at least five, at least six, or all of the aforementioned amino acid residues are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least the serine residue at position 44 (i.e., S44) is glycosylated.

[0090] "Total amount of NT-proBNP" preferably refers to the amounts of glycosylated and non-glycosylated NT-proBNP. Therefore, this term refers to the sum of the amounts of glycosylated and non-glycosylated NT-proBNP.

[0091] Preferably, the determination of the amount of total NT-proBNP involves contacting the sample with an antibody or antigen-binding fragment that specifically detects total NT-proBNP. More preferably, the antibody or antigen-binding fragment that specifically detects total NT-proBNP binds to a region of human NT-proBNP that cannot be glycosylated. Therefore, the antibody (or fragment) specifically binds to a region of NT-proBNP that does not support a glycosylation site, i.e., an O-glycosylation site, particularly to a region of human NT-proBNP. The complex formed between the antibody (or fragment) and the biomarker is proportional to the amount of total NT-proBNP.

[0092] In particular, the antibody (or fragment thereof) specifically binds to regions of NT-proBNP that do not support the T36, S37, S44, T48, S53, T58, or T71 glycosylation sites (of human NT-proBNP). For example, the first 35 amino acid residues, i.e., N-terminal amino acid residues 1 to 35, are known not to have O-glycosylation sites. Preferably, the antibody or antigen-binding fragment that specifically detects total NT-proBNP binds to an epitope located within the first 35 amino acids of NT-proBNP, more preferably to an epitope located within the first 20 amino acids of NT-proBNP, and most preferably to an epitope located within amino acid residues 10 to 20 of human NT-proBNP. In a preferred embodiment, the epitope of the antibody or its antigen-binding fragment includes amino acid residues 13 to 16 of human NT-proBNP. The sequence of human NT-proBNP is shown above (see Sequence ID No. 1).

[0093] In preferred embodiments, the antibody for specifically detecting total NT-proBNP is the monoclonal antibody MAB 17.3.1 disclosed in International Publication No. 2004099253A1, or an antibody comprising the six CDRs of said antibody. Furthermore, the use of the antigen-binding fragment of said antibody is also envisioned.

[0094] In another embodiment, the second biomarker is ANG2. The biomarker angiopoietin-2 (abbreviated as "ANG2," often also called ANGPT2) is well known in the art. It is an antagonist to both naturally occurring Ang-1 and TIE2 (see, e.g., Maisonpierre et al., Science 277 (1997) 55-60). The protein can induce tyrosine phosphorylation of TEK / TIE2 in the absence of ANG-1. In the absence of angiogenesis-inducing substances such as VEGF, loosening of cell matrix contact via ANG2 can induce endothelial cell apoptosis with resulting vascular regression. In conjunction with VEGF, it can promote endothelial cell migration and proliferation, and thus function as a permissive angiogenesis signal. The sequence of human angiopoietin is well known in the art. Uniprot lists three isoforms of angiopoietin-2: isoform 1 (Uniprot identifier: O15123-1), isoform 2 (identifier: O15123-2), and isoform 3 (O15123-3). In a preferred embodiment, the total amount of angiopoietin-2 is determined. This total amount is preferably the sum of the amounts of complex and free angiopoietin-2.

[0095] In another embodiment, the second biomarker is cMyBPC (cardiac myosin-binding protein C). Myosin-binding protein C is a myosin-related protein found in the cross-linking region (C region) of the A band of striated muscle.

[0096] In accordance with this invention, the amount of cardiac isoform, namely cMyBPC (cardiac myosin-binding protein C), is determined (also known as MYBPC3, CMD1MM, CMH4, FHC, LVNC10, MYBP-C, myosin-binding protein C, heart, cMyBP-C, and myosin-binding protein C3). cMyBPC is produced in the cardiac muscle. It is encoded by the MYBPC3 gene. Further information on human cMyBPC can be found in the UniProtKB database under accession number Q14896 (MYPC3_HUMAN).

[0097] As described above, the amount of the third biomarker can be determined in accordance with the present invention.

[0098] For example, if the amount of BMP10 type peptide is determined as the second biomarker, the method may further include determining, in one embodiment, the amount of cMyBPC (cardiac myosin-binding protein C), at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6, as a third biomarker. Preferred third biomarkers are cMyBPC, at least one lipid biomarker, and hsCRP.

[0099] If the amount of FGF23 is determined as the second biomarker, the method may further include, in one embodiment, determining the amount of at least one lipid biomarker, such as cMyBPC, CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, such as sFlt1, ANG2, or IGFBP7, as a third biomarker. Preferred third biomarkers are cMyBPC and at least one lipid biomarker.

[0100] If the amount of BNP-type peptide is determined as the second biomarker, the method further includes determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, such as FGF23 or ANG2, as a third biomarker.

[0101] If the amount of ANG2 is determined as a second biomarker, the method may, in one embodiment, include determining the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7, or a third biomarker, particularly cMyBPC, LDL, or CHOL. Alternatively, the third biomarker may be IGFBP7, apolipoprotein A-1, or LDL. The use of IGFBP7, APOAT, or LDL, particularly LDL, as a third biomarker is advantageous in diabetic patients, for example, in combination with cardiac troponins such as cardiac troponin T and ANG2.

[0102] The term “Growth Differentiation Factor-15” or “GDF-15” refers to a polypeptide that is a member of the transforming growth factor (TGF)-cytokine superfamily. The terms polypeptide, peptide, and protein are used interchangeably throughout this specification. GDF-15 was initially cloned as macrophage inhibitory cytokine 1 and subsequently identified as placental transforming growth factor-15, placental osteomorphocyte protein, nonsteroidal anti-inflammatory drug activator gene 1, and prostate-derived factor (Bootcov loc cit; Hromas, 1997 Biochim Biophys Acta 1354:40-44; Lawton 1997, Gene 203:17-26; Yokoyama-Kobayashi 1997, J Biochem (Tokyo), 122:622-626; Paralkar 1998, J Biol Chem 273:13760-13767). The amino acid sequence of GDF-15 is disclosed in International Publication No. 99 / 06445, International Publication No. 00 / 70051, International Publication No. 2005 / 113585, etc., as well as in Bottner 1999, Gene 237:105-111, Bootcov loc.cit, Tan loc.cit., Baek 2001, Mol Pharmacol 59:901-908, Hromas loc cit, Parakar loc cit, and Morrish 1996, Placenta 17:431-441.

[0103] As used herein, the term "sFlt-1" refers to the polypeptide that is the soluble form of fms-like tyrosine kinase 1. The polypeptide is also referred to in the art as soluble VEGF receptor 1 (sVEGF R1) (see, e.g., Sunderji 2010, Am J Obstet Gynecol 202:40e1-7). Soluble Flt-1 was identified in conditioned culture medium of human umbilical vein endothelial cells. The endogenous sFlt1 receptor is chromatographically and immunologically similar to recombinant human sFlt1 and binds [125I]VEGF with comparable high affinity. Human sFlt1 has been shown in vitro to form a VEGF-stabilized complex with the extracellular domain of KDR / Flk-1. Preferably, sFlt1 refers to human sFlt1 as described in Kendall 1996, Biochem Biophs Res Commun 226(2):324-328, and for the amino acid sequence, for example, Genebank accession number P17948, GI:125361 for human and BAA24499.1, GI:2809071 for mouse sFlt-1 (Genbank is available from the NCBI in the United States under www.ncbi.nlm.nih.gov / entrez).

[0104] Interleukin-6 (abbreviated as IL-6), secreted by T cells and macrophages, is an interleukin that stimulates an immune response, for example, during infection and post-traumatic stress, particularly during burns or other tissue injuries leading to inflammation. It acts as both a pro-inflammatory and anti-inflammatory cytokine. In humans, it is encoded by the IL-6 gene. The sequence of human IL-6 can be assessed by GenBank (see NM_000600.3 for the polynucleotide sequence and NP_000591.1 for the amino acid sequence). IL-6 transmits signals via a cell surface type I cytokine receptor complex consisting of the ligand-bound IL-6Rα chain (CD126) and the signaling component gp130 (also called CD130). CD130 is a common signaling molecule for several cytokines, including leukemia suppressor (LIF), ciliary neurotropic factor, oncostatin M, IL-11, and cardiotrophin-1, and is expressed almost ubiquitously in most tissues. In contrast, CD126 expression is limited to specific tissues. When IL-6 interacts with its receptor, it triggers gp130 and IL-6R proteins to form a complex, thus activating the receptor. These complexes bring together the intracellular domain of gp130 to initiate a signaling cascade via specific transcription factors, Janus kinase (JAK), and signaling / activating transcription factors.

[0105] CRP (C-reactive protein) is an acute-phase protein that was discovered more than 75 years ago as a blood protein that binds to the C-polysaccharide of Streptococcus pneumoniae. Known as a reactive inflammatory marker, CRP is produced by distal organs (i.e., the liver) in response to or in reaction to chemokines or interleukins originating from the primary lesion site. CRP is known to consist of five single subunits linked non-covalently and assembled as a cyclic pentamer with a molecular weight of approximately 110–140 kDa. Preferably, the CRP used herein refers to human CRP. The sequence of human CRP is well known and has been disclosed, for example, by Woo et al. (J. Biol. Chem. 1985. 260(24), 13384-13388). CRP levels are usually low in normal individuals but can increase 100–200 times or more due to inflammation, infection, or injury (Yeh (2004) Circulation. 2004; 109: 11-11-11-14). CRP is known to be an independent predictor of cardiovascular risk. CRP can be measured by immunoassays that are well known in the art and commercially available, such as ELISA. In preferred embodiments, the CRP is hsCRP (high-sensitivity CRP).

[0106] IGFBP-7 (insulin-like growth factor-binding protein 7) is a 30 kDa modular glycoprotein known to be secreted from endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono, Y., et al, Biochem Biophys Res Comm 202 (1994) 1490-1496). Preferably, the term "IGFBP-7" refers to human IGFBP-7. The protein sequence is well known in the art and can be accessed, for example, via UniProt (Q16270, IBP7_HUMAN) or GenBank (NP_001240764.1). A detailed definition of the biomarker IGFBP-7 is provided, for example, in International Publication No. 2008 / 089994, which is incorporated herein by reference in its entirety. IGFBP-7 has two isoforms, isoform 1 and 2, which are produced by alternative splicing. In one embodiment of the present invention, the total amount of both isoforms is measured (see UniProt database entries Q16270-1 and Q16270-2 for the sequences).

[0107] The biomarker endothelial cell-specific molecule 1 (abbreviated as ESM-1) is well known in the art. Biomarkers are often also called endocans. ESM-1 is a secreted protein and is mainly expressed in endothelial cells of human lung and kidney tissue. Public domain data suggest that it is expressed not only in the thyroid, lung, and kidney, but also in cardiac tissue. See, for example, the entry for ESM-1 in the Protein Atlas database (Uhlen M. et al., Science 2015;347(6220):1260419). The expression of this gene is regulated by cytokines. ESM-1 is a proteoglycan composed of a 20 kDa mature polypeptide and a 30 kDa O-linked glycan chain (Bechard D et al., J Biol Chem 2001;276(51):48341-48349). In a preferred embodiment of the present invention, the amount of human ESM-1 polypeptide is determined in a sample from a subject. The sequence of the human ESM-1 polypeptide is well known in the art (see, for example, Lassale P. et al., J. Biol. Chem. 1996;271:20458-20464), and can be assessed via the Uniprot database, for example, see entry Q9NQ30(ESM1_HUMAN). Two isoforms of ESM-1 are produced by alternative splicing: isoform 1 (having Uniprot identifier Q9NQ30-1) and isoform 2 (having Uniprot identifier Q9NQ30-2). Isoform 1 is 184 amino acids long. Isoform 2 is missing amino acids 101 to 150 of isoform 1. Amino acids 1 to 19 form a signal peptide (which can be cleaved).

[0108] In a preferred embodiment, the amount of isoform 1 of the ESM-1 polypeptide is determined, i.e., isoform 1 has the sequence shown under UniProt acceptance number Q9NQ30-1.

[0109] In another preferred embodiment, the amount of isoform 2 of the ESM-1 polypeptide is determined, i.e., isoform 2 has the sequence shown under UniProt accession number Q9NQ30-2.

[0110] In another preferred embodiment, the amounts of isoform-1 and isoform-2 of the ESM-1 polypeptide, i.e., the total ESM-1, are determined.

[0111] According to the present invention, the amount of one or more lipid biomarkers can be determined.

[0112] The lipid biomarkers referred to herein are preferably selected from the group consisting of cholesterol (also known herein as "CHOL"), TAG (a triglyceride also known herein as "TRIGLY"), LDL (low-density lipoprotein), HDL (high-density lipoprotein), and apolipoprotein A-1 (also known herein as "APOAT"). Thus, one or more markers can be determined from the aforementioned group of markers.

[0113] In one embodiment, the lipid biomarker is cholesterol. Cholesterol is a steroid with a secondary hydroxyl group at the C3 position. It is synthesized in many types of tissues, but particularly in the liver and intestinal wall. About three-quarters of cholesterol is newly synthesized, and one-quarter comes from dietary intake. Cholesterol assays are used to screen for the risk of atherosclerosis, as well as to diagnose and treat disorders involving elevated cholesterol levels and impaired lipid and lipoprotein metabolism.

[0114] In another embodiment, the lipid biomarker is apolipoprotein A-1. Apolipoproteins are the protein components of lipoproteins. Lipoproteins are classified according to their ultracentrifugal densities. Apolipoprotein A-1 is the major protein component of high-density lipoprotein (HDL). Apolipoprotein A-1 activates the enzyme lecithin-cholesterol-acyltransferase (LCAT), which catalyzes the esterification of cholesterol, thereby enhancing the lipid-carrying capacity of lipoproteins. Further information on apolipoprotein A-1 is available under UniProt-Accession-Number UniProtKB-P02647(APOA1_HUMAN).

[0115] In yet another embodiment, the lipid biomarker is triglycerides. Triglycerides are esters of the trihydric alcohol glycerol with three long-chain fatty acids. They are partially synthesized in the liver and partially obtained in food. Triglyceride determination is used in the diagnosis and treatment of patients with diabetes mellitus, nephropathy, hepatic obstruction, lipid metabolism disorders, and numerous other endocrine disorders.

[0116] In yet another embodiment, the lipid biomarker is LDL. Therefore, the LDL content is determined. Low-density lipoprotein (LDL) plays a crucial role in causing and influencing the progression of atherosclerosis, particularly coronary artery disease. LDL is one of five major groups of lipoproteins that transport all lipid molecules around the body in extracellular water. Blood tests generally report the amount of cholesterol estimated to be contained in LDL particles on average by using the LDL-cholesterol:Friedewald formula. Furthermore, LDL content can be assessed through the cholesterol content in the LDL.

[0117] In yet another embodiment, the lipid biomarker is HDL. HDL is synthesized in the intestines and liver. High-density lipoprotein (HDL) is responsible for the reverse transport of cholesterol from peripheral cells to the liver. Monitoring HDL cholesterol in serum or plasma is clinically important because HDL cholesterol levels are important in assessing the risk of atherosclerosis.

[0118] Preferably, at least one vascular biomarker referred to herein is selected from the group consisting of FGF23 (fibroblast growth factor 23), sFlt-1 (soluble fms-like tyrosine kinase-1), GDF15 (growth and differentiation factor 15), ESM-1 (endothelial cell-specific molecule 1), ANG2 (angiopoietin 2), and IGFBP7 (insulin-like growth factor-binding protein 7).

[0119] In one embodiment, the vascular biomarker is FGF23.

[0120] In another embodiment, the vascular biomarker is sFlt1.

[0121] In yet another embodiment, the vascular biomarker is GDF15.

[0122] In yet another embodiment, the vascular biomarker is ESM1.

[0123] In yet another embodiment, the vascular biomarker is ANG2.

[0124] In yet another embodiment, the vascular biomarker is IGFBP7.

[0125] Preferably, at least one inflammatory biomarker referred to herein is CRP, for example, hsCRP (highly sensitive C-reactive protein) and / or IL6 (interleukin-6).

[0126] In one embodiment, the inflammatory biomarker is CRP. In an alternative embodiment, the inflammatory biomarker is IL-6.

[0127] In one embodiment, the first marker is cardiac troponin, and the second marker is a BMP10 type peptide.

[0128] In one embodiment, the first marker is cardiac troponin, and the second marker is FGF23.

[0129] In one embodiment, the first marker is cardiac troponin, and the second marker is cMyBPC.

[0130] In one embodiment, the first marker is cardiac troponin, and the second marker is ANG2.

[0131] In one embodiment, the first marker is cardiac troponin, and the second marker is a BNP-type peptide, such as NT-proBNP or total NT-proBNP. Preferred combinations of two or three biomarkers are shown in Table 2 of the Examples section. Further preferred combinations of markers are as follows:

[0132] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is cMyBPC.

[0133] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is CHOL.

[0134] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is FGF23.

[0135] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is LDL.

[0136] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is TRIGLY.

[0137] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is APOAT.

[0138] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is HDL.

[0139] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is sFLT1.

[0140] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is GDF15.

[0141] In one embodiment, the first marker is cardiac troponin, the second marker is a BMP10 type peptide, and the third marker is CRP, for example, hsCRP.

[0142] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is IL6.

[0143] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is ESM1.

[0144] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is IGFBP7.

[0145] In one embodiment, the first marker is cardiac troponin, the second marker is BMP10 type peptide, and the third marker is ANG2.

[0146] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is cMyBPC.

[0147] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is TRIGLY.

[0148] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is CHOL.

[0149] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is LDL.

[0150] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is HDL.

[0151] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is APOAT.

[0152] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is ANG2.

[0153] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is sFLT1.

[0154] In one embodiment, the first marker is cardiac troponin, the second marker is FGF23, and the third marker is IGFBP7.

[0155] In one embodiment, the first marker is cardiac troponin, the second marker is NTproBNP, and the third marker is cMyBPC.

[0156] In one embodiment, the first marker is cardiac troponin, the second marker is NTproBNP, and the third marker is FGF23.

[0157] In one embodiment, the first marker is cardiac troponin, the second marker is NTproBNP, and the third marker is ANG2.

[0158] In one embodiment, the first marker is cardiac troponin, the second marker is NTproBNP, and the third marker is CHOL.

[0159] In one embodiment, the first marker is cardiac troponin, the second marker is NTproBNP, and the third marker is LDL.

[0160] In one embodiment, the first marker is cardiac troponin, the second marker is NTproBNP, and the third marker is TRIGLY.

[0161] In one embodiment, the first marker is cardiac troponin, the second marker is NTproBNP, and the third marker is HDL.

[0162] In one embodiment, the first marker is cardiac troponin, the second marker is NTproBNP, and the third marker is APOAT.

[0163] In one embodiment, the first marker is cardiac troponin, the second marker is tNTproBNP, and the third marker is cMyBPC.

[0164] In one embodiment, the first marker is cardiac troponin, the second marker is tNTproBNP, and the third marker is CHOL.

[0165] In one embodiment, the first marker is cardiac troponin, the second marker is tNTproBNP, and the third marker is LDL.

[0166] In one embodiment, the first marker is cardiac troponin, the second marker is tNTproBNP, and the third marker is TRIGLY.

[0167] In one embodiment, the first marker is cardiac troponin, the second marker is tNTproBNP, and the third marker is HDL.

[0168] In one embodiment, the first marker is cardiac troponin, the second marker is tNTproBNP, and the third marker is APOAT.

[0169] In one embodiment, the first marker is cardiac troponin, the second marker is ANG2, and the third marker is cMyBPC.

[0170] In one embodiment, the first marker is cardiac troponin, the second marker is ANG2, and the third marker is LDL.

[0171] In one embodiment, the first marker is cardiac troponin, the second marker is ANG2, and the third marker is CHOL.

[0172] In one embodiment, the first marker is cardiac troponin, the second marker is ANG2, and the third marker is ESM1.

[0173] The following combinations of biomarkers can be advantageously applied to patients with diabetes.

[0174] In one embodiment, the first marker is cardiac troponin, and the second marker is ANG2.

[0175] In one embodiment, the first marker is cardiac troponin, the second marker is ANG2, and the third marker is IGFBP7.

[0176] In one embodiment, the first marker is cardiac troponin, the second marker is ANG2, and the third marker is LDL.

[0177] In one embodiment, the first marker is cardiac troponin, the second marker is ANG2, and the third marker is APOAT.

[0178] As used herein, the term “determine” refers to the qualitative and quantitative determination of a biomarker referred to in accordance with the present invention, that is, the term includes the determination of the presence or absence of such biomarker or the determination of its absolute or relative quantity.

[0179] As used herein, the term “quantity” refers to the absolute amount, relative amount, or concentration of a compound referred to herein, as well as any value or parameter that correlates with or can be derived from them. Such values ​​or parameters include intensity signal values ​​derived from all specific physical or chemical properties obtained from the compound by direct measurement, e.g., intensity values ​​in mass spectra or NMR spectra. Furthermore, it includes all values ​​or parameters obtained by indirect measurement as explicitly stated elsewhere herein, e.g., compounds obtained from specifically bound ligands or response levels determined by biological readout systems in response to intensity signals. It should be understood that values ​​correlated with the above-mentioned quantities or parameters can also be obtained by all standard mathematical operations.

[0180] The determination of the quantity in the method of the present invention may be carried out by any technique that enables the detection of the presence or absence or quantity of the second molecule upon its release from the first molecule. The appropriate technique depends on the properties of the molecule and the characteristics of the biomarker, which will be discussed in more detail elsewhere in this specification.

[0181] Typically, the amount of a biomarker referred to in accordance with the present invention can be determined by an immunoassay using sandwich, competitive, or other assay formats, particularly when the biomarker is a protein biomarker (e.g., BNP-type peptide, BMP10-type peptide, cardiac troponin, GDF-15, sFlt-1, ANG2, CRP, IL6, ESM1, or IGFBP7, APOAT, etc.). The assay generates a signal indicating the presence or absence or amount of the biomarker. More suitable methods include measuring physical or chemical properties specific to the biomarker, such as its exact molecular mass or NMR spectrum. Such methods preferably include analytical instruments such as biosensors, optical devices associated with immunoassays, biochips, mass spectrometers, NMR analyzers, surface plasmon resonance analyzers, or chromatography devices. Furthermore, the methods include microplate ELISA-based methods and fully automated or robotic immunoassays (e.g., available from Roche). Appropriate measurement methods according to the present invention may also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electrogenerated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), electrochemiluminescence sandwich immunoassay (ECLIA), dissociation-enhanced lantanide fluoroimmunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetry, turbidimetric analysis, latex-enhanced turbidimetry or turbidimetric analysis, or solid-phase immunoassay. Further methods known in the art, such as gel electrophoresis, 2D gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blotting, are also known. More typically, techniques specifically envisioned for determining the biomarkers mentioned herein are described in the following accompanying examples.

[0182] The biomarkers determined according to the present invention are well known in the art. Furthermore, methods for determining the amount of biomarkers are known. For example, the biomarkers can be measured as described in the Examples section (see Example 1).

[0183] Some of the biomarkers are lipid biomarkers such as HDL, LDL, cholesterol, and triglycerides. The amounts of these biomarkers can be determined, for example, enzymatically.

[0184] The enzymatic determination of the amount of (total) triglycerides is preferably performed as follows: a) A step of contacting the sample with lipase under conditions and for a time sufficient to enable conversion to glycerol and free fatty acids, b) A step of contacting a glycerol-containing sample with glycerokinase under conditions and for a time sufficient to enable conversion to glycerol 3-phosphate, c) A step of contacting a sample containing glycerol-3-phosphate with glycerophosphate oxidase under conditions and for a time sufficient to allow conversion to dihydroxyacetone phosphate and H2O2, d) The process includes determining the amount of H2O2 produced enzymatically or chemically, thereby determining the amount of triglycerides.

[0185] Therefore, the detection agents for triglycerides are enzymes: glycerokinase, glycerophosphate oxidase, and glycerophosphate oxidase. Preferably, these enzymes are used in combination.

[0186] The amount of cholesterol can be assessed by the amount of cholesterol esters in the sample. Enzymatic determination of the amount of cholesterol esters is preferably performed by, a) A step of contacting the sample with cholesterol esterase under conditions and for a sufficient time to enable conversion to cholesterol, b) A step of contacting a cholesterol-containing sample with cholesterol oxidase under conditions and for a time sufficient to enable the generation of H2O2, c) The process includes determining the amount of H2O2 produced enzymatically or chemically, thereby determining the amount of cholesterol.

[0187] Therefore, the detection agents for cholesterol esters are the enzymes cholesterol esterase and cholesterol oxidase. Preferably, these enzymes are used in combination.

[0188] As described above, the amount of LDL can be determined by determining LDL cholesterol and cholesterol esters. The enzymatic determination of the amounts of LDL cholesterol and cholesterol esters is preferably performed as follows: a) A step of contacting the sample under conditions and for a sufficient time to enable conversion to cholesterol, in the presence of a nonionic surfactant that selectively solubilizes LDL with cholesterol esterase, b) A step of contacting a cholesterol-containing sample with cholesterol oxidase under conditions and for a time sufficient to enable the generation of H2O2, c) The process includes determining the amount of H2O2 produced enzymatically or chemically, thereby determining the amount of LDL cholesterol and cholesterol esters, and therefore the amount of LDL.

[0189] Therefore, the detection agents for HDL are the enzymes cholesterol esterase and cholesterol oxidase. Preferably, these enzymes are used in combination.

[0190] As described above, the amount of HDL can be assessed by determining the amount of HDL cholesterol and cholesterol esters, i.e., the amount of cholesterol and cholesterol esters present in HDL. The enzymatic determination of the amount of LDL cholesterol and cholesterol esters is preferably performed as follows: a) A step of contacting the sample under conditions and for a sufficient time to enable conversion to cholesterol, in the presence of a nonionic surfactant that selectively solubilizes LDL with cholesterol esterase, b) A step of contacting a cholesterol-containing sample with cholesterol oxidase under conditions and for a time sufficient to enable the generation of H2O2, c) The process includes determining the amount of H2O2 produced enzymatically or chemically, thereby determining the amount of LDL cholesterol and cholesterol esters, and therefore the amount of LDL.

[0191] Therefore, the detection agents for LDL are the enzymes cholesterol esterase and cholesterol oxidase. Preferably, these enzymes are used in combination.

[0192] The amount of H2O2 can be enzymatically determined, for example, by converting it in a further step (step e or d) in which the sample is brought into contact with a peroxidase (such as horseradish peroxidase) and a chromogenic substrate. The substrate is typically oxidized by the peroxidase using H2O2 as the oxidizing agent. The catalytic reaction in the presence of H2O2, peroxidase, and substrate typically results in characteristic changes detectable by spectrophotometrics. For example, the peroxidase catalyzes the conversion of the chromogenic substrate to a colored product and, when acting on a chemiluminescent substrate, generates light. For example, DAOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline) and 4-aminoantipyrine in the presence of H2O2 and peroxidase results in an oxidative coupling of DAOS and 4-aminoantipyrine to form a blue pigment. This pigment can be detected, for example, by measuring the absorbance of light at approximately 590 nm. Alternatively, 10-acetyl-3,7-dihydroxyphenoxazine can be used as a substrate for peroxidase, enabling the detection of H2O2. This non-fluorescent reagent reacts with H2O2 to produce resolphin, a fluorescent compound. Another option is that 4-aminophenazone + 4-chlorophenol in the presence of H2O2 and peroxidase results in the formation of 4-(p-benzoquinone-monoimino)-phenazone (a red product), which can be determined by photometric means.

[0193] It should be understood that the present invention is not limited to the above-mentioned markers (first, second, and third markers). Rather, the present invention may encompass the determination of additional markers.

[0194] As used herein, the term “criterion” preferably means a quantity or score that enables the assignment of subjects to either a group of subjects suffering from a disease or condition, such as type 2 myocardial infarction or type 1 myocardial infarction (MI). Such a criterion may be a threshold (or cutoff) quantity or score that separates these groups from each other. Thus, the criterion shall be a quantity or score that enables the assignment of subjects to either a group of subjects suffering from type 1 or type 2 myocardial infarction. For example, the criterion shall be a quantity or score that enables the assignment of subjects to either a group of subjects suffering from type 2 myocardial infarction or a group of subjects not suffering from type 2 myocardial infarction.

[0195] In one embodiment, the criterion is a reference quantity. In another embodiment, the criterion is a reference score.

[0196] The term "at least one object" refers to one object or two or more objects, for example, at least 10, 50, 100, 200, or 1000 objects.

[0197] Reference values ​​can, in principle, be calculated for a cohort of subjects based on a given parameter, such as the mean or mean value of biomarker levels, by applying standard statistical methods. In particular, the accuracy of tests, such as those intended for diagnosing events, is best described by receiver operating characteristics (ROC) (see, in particular, Zweig 1993, Clin. Chem. 39:561-577). An ROC graph is a plot of all sensitivity / specificity pairs resulting from continuously varying the decision threshold across the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to accurately assign subjects to a particular prognosis or diagnosis. An ROC plot shows the overlap between two distributions by plotting sensitivity versus 1-specificity over the entire range of thresholds suitable for making distinctions. On the y-axis is sensitivity, or true positivity, defined as the ratio of the number of true positive test results to the product of the number of true positive test results and the number of false negative test results. This is also called positivity in the presence of disease or symptoms. The y-axis is calculated only from the affected subgroup. On the x-axis is the false positive rate, or 1-specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negatives and the number of false positive results. The x-axis is an index of specificity and is calculated only from the unaffected subgroup. Since the true positive rate and false positive rate are calculated entirely separately by using test results from two different subgroups, the ROC plot is independent of the prevalence of events in the cohort. Each point on the ROC plot represents a susceptibility / -specificity pair corresponding to a specific decision threshold. A fully distinguishable test (no overlap between the two distributions of results) will have an ROC plot passing through the upper left corner, with a true positive rate of 1.0, or 100% (fully susceptible) and a false positive rate of 0 (fully specific). The theoretical plot for an indistinguishable test (identical distributions of results for the two groups) will be a 45° diagonal from the lower left corner to the upper right corner. Most plots will fall between these two extremes. If the ROC plot falls completely below the 45° diagonal, this can be easily corrected by swapping the criteria for "positive rate" from "higher" to "lower," and vice versa.Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the study. Depending on the desired confidence interval, the threshold can be derived from the ROC curve, which enables the diagnosis of the disease with an appropriate balance of sensitivity and specificity. For example, the reference used in the aforementioned method of the present invention, i.e., the threshold that enables the distinction between type 1 and type 2 myocardial infarction, can usually be generated by establishing the ROC for the cohort as described above and deriving the threshold amount therefrom. Depending on the desired sensitivity and specificity for the diagnostic method, the ROC plot can derive an appropriate threshold.

[0198] It should be understood that the criteria are intended to enable the assessments referenced herein. For example, they are intended to enable the distinction between type 1 and type 2 myocardial infarction, or the determination of appropriate treatment.

[0199] Step c) of the method of the present invention includes comparing the amount of a biomarker (i.e., a first biomarker, a second biomarker, and optionally a third biomarker) to a standard for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker.

[0200] Therefore, the amounts of the first biomarker, the second biomarker, and optionally the third biomarker may be compared to the criteria for the first biomarker, the criteria for the second biomarker, and optionally the criteria for the third biomarker.

[0201] Alternatively, a score may be calculated based on the amounts of biomarkers, i.e., based on the amounts of a first biomarker, a second biomarker, and optionally a third biomarker. The score shall enable the assessment of myocardial infarction, for example, the distinction between type 1 and type 2 myocardial infarction. Optionally, the score may be compared to an appropriate baseline score.

[0202] As used herein, the term “compare” encompasses comparing a determined quantity of a biomarker referred to herein with a reference (or comparing a calculated score with a baseline score). It should be understood that as used herein, comparison refers to any type of comparison made between a quantity value and a reference. However, it should be understood that, preferably, values ​​of the same type are compared with one another, for example, if an absolute quantity is determined and compared in the method of the present invention, the baseline is also an absolute quantity; if a relative quantity is determined and compared in the method of the present invention, the baseline is also a relative quantity, and so on. Alternatively, as used herein, the term “compare” encompasses comparing a calculated score with an appropriate baseline score. Comparisons may be performed manually or with computer assistance. Quantity and baseline values ​​or scores may, for example, be compared with one another, and such comparisons may be performed automatically by a computer program that executes an algorithm for comparison. The computer program performing the evaluation provides the desired rating in an appropriate output format.

[0203] As described above, it is also conceivable to combine multiple biomarkers into a single score based on the amounts of the first and second biomarkers, or the first, second, or third biomarkers, and compare this score to a baseline score. This is how the score is calculated. Preferably, the score is based on the amounts of the first, second, and third biomarkers in the sample from the test subject, if the amount of the third biomarker is determined based on the amounts of the first and second biomarkers in the sample from the test subject. The score shall be based on the amounts of the biomarkers described above, but further biomarkers or further patient-specific parameters may contribute to the score.

[0204] The calculated score combines information about the quantities of at least two biomarkers. Furthermore, in the score, the biomarkers are preferably weighted according to their contribution to the establishment of the rating. Thus, the values ​​for individual markers are weighted, and the weighted values ​​are used to calculate the score. Appropriate coefficients (weights) can be determined even more easily by those skilled in the art. The score can also be calculated from a decision tree or a set of decision trees (ensemble) trained on at least two biomarkers. Depending on the combination of biomarkers applied in the method of the present invention, the weights of individual biomarkers and the structure of the decision trees may differ.

[0205] The score can be considered a classifier parameter for rating subjects as described herein. In particular, it allows for the provision of a rating based on a single score. The reference score is preferably a value, in particular a cutoff value that enables the rating of myocardial infarction as described herein. Preferably, the reference is a single value. Thus, it is not necessary to interpret all information regarding the amount of individual biomarkers. Using the scoring system described herein, advantageously, values ​​of different dimensions or units may be used for biomarkers, since the values ​​are mathematically converted into scores. Thus, for example, a value for absolute concentration may be combined with a peak area ratio in the score. The reference score to be applied may be selected based on the desired sensitivity or desired specificity. Methods for selecting an appropriate reference score are well known in the art.

[0206] The score is intended to enable the assessment of myocardial infarction, such as distinguishing between type 2 and type 1 myocardial infarction. Methods for calculating the score are well known in the art. Examples are provided in the Examples section (see Example 2, Example of using a combination of hsTnT and BMP10). Appropriate diagnostic algorithms can also be set up by those skilled in the art. For example, a calculated score higher than the baseline score indicates a subject with type 2 MI, and a calculated score lower than the baseline score indicates a subject with type 1 MI.

[0207] For the specific markers described herein, preferred changes ("directions") are found in Table A below. [Table A]

[0208] Therefore, cardiac troponin, MyBPC3, sFlt1, LDL, TRIGL (triglycerides), CHOL, ANG2, and BMP10 are increased in patients with type 1 MI (compared to the baseline) and decreased in patients with type 2 MI (compared to the baseline). The remaining markers shown in Table A are decreased in patients with type 1 MI (compared to the baseline) and increased in patients with type 2 MI (compared to the baseline).

[0209] Therefore, the method of the present invention may further include recommending or initiating appropriate therapeutic measures. Typically, such appropriate therapeutic measures depend on the results of the assessment described herein, namely whether the subject has type 1 or type 2 myocardial infarction. For example, it can be determined whether a patient will receive treatment aimed at treating type 2 myocardial infarction or type 1 myocardial infarction. A patient identified as having type 2 MI will receive treatment aimed at treating type 2 MI. A patient identified as having type 1 MI will receive treatment aimed at treating type 1 MI.

[0210] The method of the present invention can further guide the urgency and timing of treatment options according to various information obtained from biomarkers of AMI subtypes.

[0211] In one embodiment, the treatment measures recommended or initiated when a patient is assessed to have type 1 myocardial infarction include reperfusion strategies to address atherothrombotic plaque rupture with drugs (antiplatelet, anticoagulant) and emergency treatment using invasive revascularization (e.g., percutaneous coronary intervention [PCI], drug-eluting stent placement, or coronary artery bypass grafting [CABG]).

[0212] In one embodiment, the treatment measures recommended or initiated when a patient is assessed to have type 2 myocardial infarction include procedures to improve myocardial oxygen demand, such as addressing symptoms of decreased myocardial oxygen supply (hypotension, anemia, bradyarrhythmia, respiratory failure), and addressing symptoms of increased myocardial oxygen demand (tachycardia, tachyarrhythmia, severe hypertension, other comorbidities) with treatments and medications such as beta-blockers, vasodilators, anticoagulants, and diuretics. In one embodiment, the treatment measures recommended or initiated when a patient is assessed to have a high biomarker score can guide the appropriate timing of intervention (immediate, early, or delayed).

[0213] The definitions set forth in this specification above apply below, with necessary modifications.

[0214] The present invention also relates to a computer-implemented method for evaluating a target myocardial infarction, the method being: (a) A step of receiving a value for the amount of a first biomarker in the target sample, wherein the first biomarker is cardiac troponin, (b) A step of receiving a value for the amount of a second biomarker in the target sample, wherein the second biomarker is BMP10 type peptide, FGF23, BNP type peptide, cMyBPC, or ANG2, (c) A step of comparing the value for the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker, (d) assessing myocardial infarction based on the comparison and / or calculation performed in step (c), including:

[0215] When used herein, the term “computer implementation” means that the method is performed in an automated manner on a data processing unit, typically contained within a computer or similar data processing device. The data processing unit receives a value for the amount of the biomarker. Such a value may be an amount reflecting the amount described in detail elsewhere herein, a relative amount, or any other calculated value. Therefore, it should be understood that the method described above does not require the determination of an amount of the biomarker, but rather uses a value for an already predetermined amount.

[0216] Typically, in step (b) of the method, (i) If the value for the amount of BMP10 type peptide is received as the second biomarker, the method may further include receiving a value for the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP, or IL6, as a third biomarker, or (ii) If the value for the amount of FGF23 is received as the second biomarker, the method may further include receiving a value for the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2, or IGFBP7 as a third biomarker, or (iii) If the value for the amount of BNP-type peptide is received as the second biomarker, the method may further include receiving a value for the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, as a third biomarker, or (iv) If the value for the amount of ANG2 is received as the second biomarker, the method further includes receiving a value for the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7, as a third biomarker.

[0217] The present invention also, in principle, envisions a computer program, a computer program product, or a computer-readable storage medium in which such a computer program is tangibly incorporated, the computer program including instructions that, when executed on a data processing device or computer, perform the methods of the present invention as described above. Specifically, this disclosure further encompasses: - A computer or computer network comprising at least one processor, the processor configured to perform a method according to one of the embodiments described herein. - A computer-loadable data structure configured to perform a method according to one of the embodiments described herein when running on a computer, - A computer script, wherein a computer program is adapted to perform one of the embodiments described herein while the program is running on a computer. - A computer program comprising programming means for performing a method according to one of the embodiments described herein when running on a computer or on a computer network, - A computer program comprising the program means according to a prior embodiment, where the program means is stored on a storage medium readable by a computer. - A data structure is stored on a storage medium, and after the data structure is loaded into the main memory and / or working memory of a computer or computer network, the storage medium is adapted to perform the method according to one of the embodiments described herein. - A computer program product having program code means that can be stored or stored on a storage medium in order to perform a method according to one of the embodiments described in this specification when the program code means is executed on a computer or computer network. - Typically encrypted data stream signals, including data of parameters defined elsewhere in this specification, as well as - A typically encrypted data stream signal, including an evaluation provided by the method of the present invention.

[0218] The present invention relates to a device for assessing myocardial infarction in a subject having myocardial infarction, and the device is (a) A measurement unit for determining the amounts of a first biomarker, which is cardiac troponin, and a second biomarker in the target sample, wherein the second biomarker is BMP10 type peptide, FGF23, BNP type peptide, cMyBPC, or ANG2, and the measurement unit includes a detection system for the first biomarker and the second biomarker. (b) An evaluation unit operably connected to a measurement unit, comprising a database having stored references for a first biomarker and a second biomarker, preferably as described above, and a data processor including instructions for performing a comparison of the amounts of the first biomarker and the second biomarker with references, and / or for performing a score for evaluating myocardial infarction based on the amounts of biomarkers or a calculated score, and for evaluating myocardial infarction based on a reference amount (or reference score), wherein the evaluation unit can automatically receive values ​​for the amounts of biomarkers from the measurement unit.

[0219] When used herein, the term "device" refers to a system comprising the aforementioned units functionally linked together to enable the determination and evaluation of the amount of a biomarker by the method of the present invention, so that an assessment may be provided.

[0220] The analytical unit typically includes at least one reaction zone having a biomarker detector for first and second biomarkers and, preferably, a third biomarker, in a form immobilized on a solid support or carrier that comes into contact with the sample. Furthermore, conditions can be applied in the reaction zone to enable the specific binding of the detector(s) to the biomarkers contained in the sample.

[0221] The reaction zone may allow direct sample application, or it may be connected to a loading zone to which the sample is applied. In the latter case, the sample may be actively or passively transported to the reaction zone via a connection between the loading zone and the reaction zone. Furthermore, the reaction zone may also be connected to a detector. The connection may allow the detector to detect the binding of biomarkers to their respective detectors. The appropriate connection depends on the technique used to measure the presence or quantity of the biomarkers. For example, for optical detection, the propagation of light may be required between the detector and the reaction zone, and for electrochemical determination, a fluid connection may be required, for example, between the reaction zone and an electrode.

[0222] The detector is adapted to detect the determination of the amount of a biomarker. The determined amount can then be transmitted to an evaluation unit, which includes a data processing element such as a computer with an implemented algorithm for determining the amount present in the sample.

[0223] The processing units referred to in accordance with the methods of the present invention typically comprise a central processing unit (CPU) and / or one or more graphics processing units (GPUs) and / or one or more application-specific integrated circuits (ASICs) and / or one or more tensor processing units (TPUs) and / or one or more field-programmable gate arrays (FPGAs), etc. The data processing element may be, for example, a general-purpose computer or a portable computing device. It should also be understood that multiple computing devices may be used together, such as over a network or by other means of transferring data, to perform one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants ("PDAs"), cellular devices, smart or mobile devices, tablet computers, and servers, etc. Generally, the data processing element includes a processor capable of executing multiple instructions (e.g., software programs).

[0224] An evaluation unit typically includes or has access to memory. Memory is a computer-readable medium and may include, for example, a single or multiple storage devices located locally with the computing device or accessible to the computing device via a network. The computer-readable medium may be any available medium accessible by the computing device and may include both volatile and non-volatile media. Furthermore, the computer-readable medium may be either removable or non-removable media, or both. For example, non-limitingly, the computer-readable medium may include computer storage media. Exemplary computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory, or any other memory technology; CD-ROM, digital versatile disk (DVD), or other optical disk storage; magnetic cassette, magnetic tape, magnetic disk storage, or other magnetic storage devices; or any other medium accessible by the computing device and that can be used to store multiple instructions executable by the computing device's processor.

[0225] According to embodiments of this disclosure, the software may include instructions that, when executed by the processor of a computing device, can perform one or more steps of the methods disclosed herein. Some of the instructions may be adapted to generate signals that control the operation of other machines and thus can operate via those control signals to convert materials far away from the computer itself. These descriptions and expressions are means used by those skilled in the data processing technology to communicate, for example, the contents of the research to others skilled in the art in the most effective way.

[0226] Multiple instructions may also include algorithms that are generally considered to be a sequence of coherent steps leading to a desired result. These steps require the physical manipulation of physical quantities. While not always the case, these quantities typically take the form of electrical or magnetic pulses or signals that can be stored, transmitted, transformed, combined, compared, and otherwise manipulated. It has sometimes proven convenient, primarily for common-sense reasons, to refer to these signals as values, characters, display data, numbers, etc., as references to the physical items or representations in which such signals are embodied or represented. However, it should be noted that all of these and similar terms are used herein simply as convenient labels applied to and associated with appropriate physical quantities.

[0227] The evaluation unit may also include an output device or have access to an output device. Exemplary output devices include, for example, a fax machine, a display, a printer, and a file. According to some embodiments of the present disclosure, a computing device may perform one or more steps of a method disclosed herein and subsequently provide an output relating to the results of the method, an index, a ratio, or other factor via the output device.

[0228] Typically, the measurement unit includes a system for determining a third biomarker and detecting the third biomarker, the database includes stored criteria for the third biomarker, and the third biomarker is (i) If the BMP10 type peptide is the second biomarker, then it may be cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6. (ii) If FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2 or IGFBP7, or (iii) If the BNP-type peptide is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, or (iv) If ANG2 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7.

[0229] More typically, the detection system includes at least one detection agent that can specifically detect each of the biomarkers.

[0230] The present invention further envisions a device for assessing myocardial infarction in a subject, wherein the device comprises an evaluation unit including a database having stored criteria for a first biomarker which is cardiac troponin and a second biomarker which is a second biomarker which is a BMP10 type peptide, FGF23, BNP type peptide, cMyBPC, or ANG2, and a data processor which includes instructions for comparing the amounts of the first biomarker and the second biomarker with the criteria, preferably as described above, and assessing myocardial infarction based on the comparison, wherein the evaluation unit can receive values ​​for the determined amounts of the biomarkers in a sample of the subject.

[0231] Typically, the database contains stored criteria for a third biomarker, and the third biomarker is defined as follows: (i) If the BMP10 type peptide is the second biomarker, then it may be cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6, or (ii) If FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2, or IGFBP7, (iii) If the BNP-type peptide is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, (iv) If ANG2 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7.

[0232] The present invention also relates, in principle, to the use of a first biomarker, which is cardiac troponin, and a second biomarker, which is a BMP10 type peptide, FGF23, a BNP type peptide, cMyBPC, or ANG2, or i) at least one detection agent for the first biomarker and at least one detection agent for the second biomarker. Preferably, the biomarker or detection agent is used in the sample of the subject.

[0233] As used herein, the term “detector” refers to an agent that specifically detects a biomarker. In particular, the term typically refers to any agent that specifically binds to a biomarker, i.e., an agent that does not cross-react with other components present in the sample. Typically, a detector that specifically binds to a biomarker as referred herein may be an antibody, an antibody fragment or derivative, an aptamer, a ligand for a biomarker, a receptor for a biomarker, an enzyme known to bind to and / or convert a biomarker, or a small molecule known to specifically bind to a biomarker. For example, antibodies referred herein as detectors include both polyclonal and monoclonal antibodies, as well as their fragments, such as Fv, Fab, and F(ab)2 fragments that can bind to an antigen or hapten. The present invention also includes single-chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody exhibiting desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence typically includes at least the antigen-binding amino acid residues of the donor, but may also include other structurally and / or functionally valid amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. For example, the aptamer detection agent may be a nucleic acid or a peptide aptamer. Methods for preparing such aptamers are well known in the art. For example, random mutations can be introduced into the underlying nucleic acid or peptide for the aptamer. These derivatives can then be tested for binding according to screening procedures known in the art, such as phage display. Specific binding of the detection agent means that it does not substantially bind to, i.e., does not cross-react with, any other peptide, polypeptide, or substance present in the sample being analyzed. Preferably, the specifically bound biomarker should bind with an affinity at least 3 times higher, more preferably at least 10 times higher, and even more preferably at least 50 times higher than any other component of the sample.Nonspecific binding may be acceptable if it can still be clearly distinguished and measured, for example, according to its size on a Western blot or by its relatively higher abundance in the sample.

[0234] In a preferred embodiment, the detection agent is an antibody or its antigen-binding fragment that specifically binds to the marker.

[0235] Preferably, the biomarker or detection agent is used in the sample of interest.

[0236] The detection agent may be permanently or reversibly fused or ligated to a detectable label. Suitable labels are well known to those skilled in the art. A suitable detectable label is any label detectable by a suitable detection method. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g., magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels. Examples of enzymatically active labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, and their derivatives. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitrobluetetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, available as a ready-made storage solution from Roche Diagnostics), CDP-Star® (Amersham Biosciences), and ECF® (Amersham Biosciences). Appropriate enzyme-substrate combinations may produce colored reaction products, fluorescence, or chemiluminescence, which can be measured by methods known in the art (e.g., using photosensitive film or a suitable camera system). The diagnostic criteria described above apply similarly to the measurement of enzymatic reactions. Typical fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa 568). Further fluorescent labels are available, for example, from Molecular Probes (Oregon). Furthermore, the use of quantum dots as fluorescent labels is also considered. Typical radioactive labels include 35S, 125I, 32P, and 33P. Radioactive labels can be detected by any known and appropriate method, such as a photosensitive film or a phosphor imager.Appropriate labels may also include tags such as biotin, digoxygenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose-binding protein, etc., or may contain them.

[0237] The determination of lipid biomarkers by enzyme assays is described elsewhere in this specification. Preferred detection agents are also disclosed for these markers.

[0238] The determination of biomarkers as described herein may include mass spectrometry (MS) performed after a separation step (e.g., by LC or HPLC). When used herein, mass spectrometry encompasses all techniques that enable the determination of the molecular weight (i.e., mass) or the mass variable corresponding to the compound to be determined according to the invention, i.e., the biomarker. Preferably, when used herein, mass spectrometry relates to GC-MS, LC-MS, direct injection mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry, e.g., MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF, or any combination of approaches using the techniques described above. How these techniques are applied is well known to those skilled in the art. Furthermore, suitable devices are commercially available. More preferably, when used herein, mass spectrometry relates to LC-MS and / or HPLC-MS, i.e., mass spectrometry operably coupled to a prior liquid chromatography separation step. Preferably, mass spectrometry is tandem mass spectrometry (also known as MS / MS). Tandem mass spectrometry, also known as MS / MS, involves two or more mass spectrometry steps, with fragmentation occurring between the steps. In tandem mass spectrometry, two consecutive mass spectrometers are connected by a collision cell. The mass spectrometers are connected to a chromatography device. The sample separated by chromatography is sorted and weighed in the first mass spectrometer, then fragmented with an inert gas in the collision cell, and one or more fragments are sorted and weighed in the second mass spectrometer. The fragments are fractionated and weighed in the second mass spectrometer. Identification by MS / MS is more accurate.

[0239] In one embodiment, mass spectrometry, as used herein, includes quadrupole MS. Most preferably, the quadrupole MS is performed as follows: a) selection of the mass / charge ratio (m / z) of ions produced by ionization at a first analytical quadrupole of the mass spectrometer; b) fragmentation of the ions selected in step a) by applying an accelerating voltage at an additional subsequent quadrupole filled with collision gas and acting as a collision chamber; c) selection of the mass / charge ratio of ions produced by the fragmentation process in step b) at the additional subsequent quadrupole, wherein steps a) to c) of the method are performed at least once, and analysis of the mass / charge ratio of all ions present in the mixture of substances as a result of the ionization process is performed, the quadrupole is filled with collision gas, but no accelerating voltage is applied during the analysis. Further details regarding this most preferred mass spectrometry as used in accordance with the present invention can be found in International Publication No. 2003 / 073464.

[0240] More preferably, the mass spectrometry is liquid chromatography (LC) MS, for example, high-performance liquid chromatography (HPLC) MS, particularly HPLC-MS / MS. Liquid chromatography, as used herein, refers to all techniques that enable the separation of compounds (i.e., metabolites) in a liquid or supercritical phase.

[0241] For mass spectrometry, the analyte in the sample is ionized to generate charged molecules or molecular fragments. The mass-charge of the ionized analyte, particularly the ionized biomarker or its fragments, is then measured. Prior to ionization, the sample may be subjected to cleavage with a protease, such as trypsin. The protease cleaves the protein biomarker into smaller fragments.

[0242] Therefore, the mass spectrometry step preferably includes an ionization step in which the biomarker to be determined is ionized. Naturally, other compounds present in the sample / eluate are also ionized. Ionization of the biomarker can be carried out by any method deemed appropriate, in particular by electron impulse ionization, fast atomic collision, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption ionization (MALDI).

[0243] In a preferred embodiment, the ionization step (for mass spectrometry) is carried out by electrospray ionization (ESI). Therefore, mass spectrometry is preferably ESI-MS (or ESI-MS / MS if tandem MS is performed). Electrospray is a soft ionization method that results in ion formation without breaking chemical bonds.

[0244] More typically, a third biomarker or at least one detection agent for the third biomarker is additionally used, and the third biomarker is (i) If the BMP10 type peptide is the second biomarker, then it may be cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6. (ii) If FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2 or IGFBP7, or (iii) If the BNP-type peptide is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, or (iv) If ANG2 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7.

[0245] The present invention also relates to a kit for assessing myocardial infarction in a subject, wherein the kit comprises at least one detection agent for a first biomarker which is cardiac troponin, and at least one detection agent for a second biomarker which is BMP10 type peptide, FGF23, BNP type peptide, cMyBPC, or ANG2.

[0246] As used herein, the term “kit” typically refers to a collection of the components described above, provided separately or in a single container. The container also typically includes instructions for performing the methods of the present invention. These instructions may be in the form of a manual, or they may be provided by computer program code that can perform or support the determination of biomarkers referred to in the methods of the present invention when run on a computer or data processing device. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disk), or directly on a computer or data processing device, or in a download format, such as a link to an accessible server or cloud. Furthermore, the kit may typically include standards for reference amounts of biomarkers for softening purposes as described in detail elsewhere herein. The kit according to the present invention may also include further components necessary for performing the methods of the present invention, such as solvents, buffers, washing solutions, and / or reagents required for the detection of released second molecules. Furthermore, the kit may include the devices of the present invention in part or as a whole.

[0247] More typically, the kit further comprises at least one detection agent for a third biomarker, the third biomarker being (i) If the BMP10 type peptide is the second biomarker, then it may be cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6, or (ii) If FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2, or IGFBP7, (iii) If the BNP-type peptide is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, (iv) If ANG2 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7.

[0248] The definitions and explanations of the terms above apply below, with any necessary modifications.

[0249] Advantageously, some of the tested biomarkers, such as BMP10 peptide or FGF23, have been shown in the studies that form the basis of this invention to enable differentiation between type 1 and type 2 myocardial infarction when measured alone (see Table 1). Furthermore, the biomarker ANG2 enables differentiation between type 1 and type 2 myocardial infarction in diabetic patients (see Table 5).

[0250] Therefore, the present invention is a method for assessing myocardial infarction in a subject, (a) Determining the amount of a biomarker in the sample of the subject, wherein the biomarker is BMP10 peptide (bone morphogenesis protein type 10 peptide), FGF23 (fibroblast growth factor 23), or ANG2 (angiopoietin 2), (b) Comparing the amount of the biomarker with a standard for the biomarker, The present invention relates to a method comprising (c) assessing myocardial infarction based on the comparison performed in step (c).

[0251] In one embodiment of the above method, the sample is obtained from the subject during an examination in the emergency department. Preferably, the sample is a blood, serum, or plasma sample.

[0252] The direction of the biomarkers (increase vs. decrease) is disclosed in relation to Table A above.

[0253] If the biomarker is ANG2, the subjects are preferably those suffering from diabetes.

[0254] Furthermore, the present invention relates to a computer implementation method for evaluating a target myocardial infarction, and the method is: (a) A step of receiving a value for the amount of a biomarker in the target sample, wherein the biomarker is BMP10 type peptide, FGF23, or ANG2, (b) A step of comparing the value of the amount of the biomarker with a standard for the biomarker, (c) A step of assessing myocardial infarction based on the comparison performed in step (c), including the step of (c).

[0255] The term "assessment of myocardial infarction" is as described above. Therefore, this explanation applies. Preferably, the assessment of myocardial infarction is the distinction between type 1 myocardial infarction and type 2 myocardial infarction.

[0256] The present invention also relates to a device for assessing myocardial infarction in a subject having myocardial infarction, the device being (a) A measurement unit for determining the amount of a biomarker, which is a BMP10 type peptide, FGF23, or ANG2, in a target sample, wherein the measurement unit includes a biomarker detection system, An evaluation unit operably connected to a measurement unit comprising: (b) a database having one or more stored criteria for a biomarker; and a data processor comprising instructions for performing comparison of the amount of the biomarker with the criteria for evaluating myocardial infarction based on the comparison, wherein the evaluation unit can automatically receive from the measurement unit a value for the amount of the biomarker.

[0257] In one embodiment, the detection system comprises at least one detection agent for the biomarker.

[0258] The present invention further relates to a device for evaluating myocardial infarction in a subject, the device comprising: an evaluation unit comprising a database in which criteria for a biomarker are stored, the biomarker being a BMP10-type peptide, FGF23 or ANG2; and a data processor comprising instructions for performing comparison of the amount of the biomarker with the criteria and for evaluating myocardial infarction based on the comparison, wherein the evaluation unit can receive a value for the amount of the biomarker determined in a sample of the subject.

[0259] The present invention further relates to the use of i) a biomarker, which is a BMP10-type peptide, FGF23 or ANG2, or ii) at least one detection agent for the biomarker, for evaluating myocardial infarction in a subject.

[0260] Finally, the present invention relates to a kit for evaluating myocardial infarction in a subject, the kit comprising at least one detection agent for a biomarker, the biomarker being a BMP10-type peptide, FGF23 or ANG2.

[0261] It should be understood that the definitions and explanations of the terms executed above apply accordingly to all embodiments described in this specification and the appended claims. The following embodiments are specific embodiments contemplated in accordance with the present invention.

[0262] 1. A method for assessing myocardial infarction in a subject, comprising: (a) determining the amount of a first biomarker in a sample of the subject, wherein the first biomarker is a cardiac troponin such as cardiac troponin T or I; (b) determining the amount of a second biomarker in a sample of the subject, wherein the second biomarker is a BMP10-type peptide (bone morphogenetic protein 10-type peptide), FGF23 (fibroblast growth factor 23), a BNP-type peptide such as NT-proBNP or BNP, cMyBPC (cardiac myosin-binding protein C), or ANG2 (angiopoietin 2); (c) comparing the amount of the biomarker with a reference for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker; (d) assessing myocardial infarction based on the comparison and / or calculation performed in step (c).

[0263] 2. In step (b), (i) if the amount of the BMP10-type peptide is determined as the second biomarker, the method further comprises determining the amount of cMyBPC (cardiac myosin-binding protein C), at least one lipid biomarker such as cholesterol (CHOL), LDL (low density lipoprotein), TRIGLY (triglyceride), APOAT (apolipoprotein A-1), or HDL (high density lipoprotein), at least one vascular biomarker such as FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker such as hsCRP or IL6 as a third biomarker, or (ii) If the amount of FGF23 is determined to be the second biomarker, the method further comprises determining the amount of at least one lipid biomarker, e.g., cMyBPC, CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2, or IGFBP7, as a third biomarker, or (iii) If the amount of BNP-type peptide is determined to be the second biomarker, the method further comprises determining the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, as a third biomarker, or (iv) If the amount of ANG2 is determined to be the second biomarker, the method further comprises determining the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7, as a third biomarker.

[0264] 3. The method according to Embodiment 1 or 2, wherein the sample is obtained from the subject during an examination in the emergency department.

[0265] 4. The method according to any one of Embodiments 1 to 3, wherein the sample is a blood, serum, or plasma sample, and / or the subject is a human.

[0266] 5. Below: i. Cardiac troponin and BMP10 type peptide, ii. Cardiac troponin and FGF23, iii. Cardiac troponin and ANG2, iv. Cardiac troponin, BMP10 type peptide and CRP, v. Cardiac troponin, BMP10 type peptide, and at least one lipid biomarker, e.g., cholesterol (CHOL), LDL (low-density lipoprotein), TRIGLY (triglyceride), APOAT (apolipoprotein A-1), or HDL (high-density lipoprotein), vi. Cardiac troponin and FGF23, vii. Cardiac troponin and cMyBPC, viii. Cardiac troponin, FGF23, and at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL. ix. Cardiac troponin, ANG2 and cMyBPC, x. For example, in diabetic patients, cardiac troponin, ANG2 and LDL, or xi. Cardiac troponin, ANG2 and CHOL, The aforementioned amount of the marker is determined, The cardiac troponin is troponin T or I, for example, troponin T. The method according to any one of Embodiments 1 to 4, wherein the BMP10 type peptide is BMP10, proBMP10, or NT-proBMP10, for example, NT-proBMP10.

[0267] 6. A computer implementation method for assessing myocardial infarction in a subject, (a) A step of receiving a value for the amount of a first biomarker in the target sample, wherein the first biomarker is cardiac troponin, (b) A step of receiving a value for the amount of a second biomarker in the target sample, wherein the second biomarker is BMP10 type peptide, FGF23, BNP type peptide, cMyBPC, or ANG2, (c) A step of comparing the value for the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker, (d) A method comprising assessing myocardial infarction based on the comparison and / or calculation performed in step (c).

[0268] 7. In process (b), (i) If the value for the amount of BMP10 type peptide is received as the second biomarker, the method further includes receiving a value for the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP, or IL6, as a third biomarker, or (ii) If the value for the amount of FGF23 is received as the second biomarker, the method further includes receiving a value for the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2, or IGFBP7 as a third biomarker, or (iii) If the value for the amount of BNP-type peptide is received as the second biomarker, the method further includes receiving a value for the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, as a third biomarker, or (iv) If the value for the amount of ANG2 is received as the second biomarker, the method further comprises receiving a value for the amount of cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7, as a third biomarker.

[0269] 8. The method according to any one of Embodiments 1 to 7, wherein the assessment of myocardial infarction is a distinction between type 1 myocardial infarction and type 2 myocardial infarction.

[0270] 9. The method according to any one of Embodiments 1 to 7, wherein the assessment of myocardial infarction is a diagnosis of type 2 myocardial infarction.

[0271] 10. The method according to any one of Embodiments 1 to 7, wherein the assessment of myocardial infarction is guidance for the treatment of myocardial infarction.

[0272] 11. A device for assessing myocardial infarction in a subject with myocardial infarction, (a) A measurement unit for determining the amount of a first biomarker, which is cardiac troponin, and a second biomarker, which is a BMP10 type peptide, FGF23, BNP type peptide, cMyBPC, or ANG2, in the target sample, the measurement unit comprising a detection system for the first biomarker and the second biomarker, (b) An evaluation unit operably connected to the measurement unit, preferably having a database with stored criteria for the first biomarker and the second biomarker as described in any one of Embodiments 1 to 10, and preferably for performing a comparison of the amounts of the biomarker and the second biomarker with the criteria as described in any one of Embodiments 1 to 10, and / or for calculating a score for assessing myocardial infarction based on the amount of the biomarker or a score calculated based thereon, and for assessing myocardial infarction based on the comparison, and a data processor including instructions for assessing myocardial infarction based on the comparison, wherein the evaluation unit can automatically receive a value for the amount of the biomarker from the measurement unit, the evaluation unit.

[0273] 12. The measurement unit determines a third biomarker and includes a detection system for the third biomarker, the database includes stored criteria for the third biomarker, and the third biomarker is (i) When the BMP10-type peptide is the second biomarker, is cMyBPC, at least one lipid biomarker such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker such as hsCRP or IL6, (ii) When FGF23 is the second biomarker, is cMyBPC, at least one lipid biomarker such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker such as sFlt1, ANG2 or IGFBP7, (iii) When the BNP-type peptide is the second biomarker, is cMyBPC, at least one lipid biomarker such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker such as FGF23 or ANG2, or (iv) The device according to Embodiment 11, wherein ANG2 is a second biomarker, cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7.

[0274] 13. The device according to Embodiment 11 or 12, wherein the detection system comprises at least one detection agent for the first biomarker, at least one detection agent for the second biomarker, and optionally, at least one detection agent for the third biomarker.

[0275] 14. A device for assessing myocardial infarction in a subject, wherein the device comprises an evaluation unit including a database having stored criteria for a first biomarker which is cardiac troponin and a second biomarker, wherein the second biomarker is BMP10 type peptide, FGF23, BNP type peptide, cMyBPC, or ANG2, and a data processor which includes instructions for comparing the amounts of the first biomarker and the second biomarker with the criteria, preferably as described in any one of embodiments 1 to 10, and assessing myocardial infarction based on the comparison, wherein the evaluation unit can receive a value for the determined amount of the biomarker in a sample of the subject.

[0276] 15. The database includes stored criteria for a third biomarker, and the third biomarker is (i) If the BMP10 type peptide is the second biomarker, then it may be cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6. (ii) If FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2 or IGFBP7, or (iii) If the BNP-type peptide is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, or (iv) The device according to Embodiment 14, wherein ANG2 is a second biomarker, the second biomarker is cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7.

[0277] 16. Use of i) a first biomarker which is cardiac troponin and a second biomarker which is BMP10 type peptide, FGF23, BNP type peptide, cMyBPC or ANG2, or ii) at least one detection agent for the first biomarker and at least one detection agent for the second biomarker for assessment of myocardial infarction in a subject.

[0278] 17. A third biomarker or a detection agent for the third biomarker is further used, and the third biomarker is (i) If the BMP10 type peptide is the second biomarker, then it may be cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2, or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6. (ii) If FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2, or IGFBP7, (iii) If the BNP-type peptide is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, or (iv) The device according to Embodiment 16, wherein ANG2 is a second biomarker, the second biomarker is cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7.

[0279] 18. A kit for assessing myocardial infarction in a subject, comprising i) at least one detection agent for a first biomarker which is cardiac troponin, and ii) at least one detection agent for a second biomarker which the second biomarker is BMP10 type peptide, FGF23, BNP type peptide, cMyBPC, or ANG2.

[0280] 19. The kit further comprises a detection agent for a third biomarker, wherein the third biomarker is (i) If the BMP10 type peptide is the second biomarker, then it may be cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, e.g., FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, e.g., hsCRP or IL6, or (ii) If FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, e.g., sFlt1, ANG2 or IGFBP7, or (iii) If the BNP-type peptide is the second biomarker, then cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., FGF23 or ANG2, (iv) The kit according to Embodiment 18, wherein ANG2 is a second biomarker, the second biomarker is cMyBPC, at least one lipid biomarker, e.g., CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, e.g., ESM1 or IGFBP7.

[0281] 20. A method for assessing myocardial infarction in a subject, (a) Determining the amount of a biomarker in the sample of the subject, wherein the biomarker is BMP10 peptide (bone morphogenesis protein type 10 peptide), FGF23 (fibroblast growth factor 23), or ANG2 (angiopoietin 2), (b) Comparing the amount of the biomarker with a reference for the biomarker, A method for assessing myocardial infarction in a subject, comprising (c) assessing myocardial infarction based on the comparison performed in step (c).

[0282] 21. A computer implementation method for assessing myocardial infarction in a subject, (a) A step of receiving a value for the amount of a biomarker in the target sample, wherein the biomarker is BMP10 type peptide, FGF23, or ANG2, (b) A step of comparing the value of the amount of the biomarker with a standard for the biomarker, A computer implementation method for assessing myocardial infarction in a subject, comprising (c) assessing myocardial infarction based on the comparison performed in step (c).

[0283] 22. For assessing myocardial infarction in a subject, i) a biomarker which is a BMP10 type peptide, FGF23, or ANG2, or ii) at least one detection agent for the biomarker.

[0284] 23. A method, device, use, or kit according to any one of the prior embodiments, wherein the BMP10 type peptide is BMP10, proBMP10, or NT-proBMP10.

[0285] 24. The method, device, use, or kit according to any one of the prior embodiments, wherein the BNP-type peptide is NT-proBNP, proBNP, or BNP.

[0286] 25. The method, device, use, or kit according to any one of the prior embodiments, wherein the (second) biomarker is ANG2 and the subject is a subject suffering from diabetes.

[0287] 26. A method, device, use, or kit according to any one of the preceding embodiments, wherein the assessment of myocardial infarction is a distinction between type 1 and type 2 myocardial infarction.

[0288] All references referenced throughout this specification, in addition to the disclosures specifically mentioned above, are incorporated herein in their entirety. [Examples]

[0289] The examples provided are merely illustrative of the present invention and should not be construed as limiting its scope.

[0290] 1. Determination of biomarkers 1.1. Determination of biomarkers by sandwich assay on the cobas Elecsys® ECLIA platform (ECLIA assay manufactured by Roche Diagnostics, Germany) The following is a brief description of the Elecsys® electrochemiluminescence (ECL) technique and assay method for determining GDF-15. The concentration of GDF-15 was determined using a cobas e 801 analyzer. Detection of GDF-15 using the cobas e 801 analyzer is based on the Elecsys® electrochemiluminescence (ECL) technique. Briefly, biotin-labeled and ruthenium-labeled antibodies are combined with their respective amounts of undiluted sample and incubated in the analyzer. Subsequently, streptavidin-coated magnetic microparticles are added to promote the binding of the biotin-labeled immunocomplex and incubated in the instrument. After this incubation step, the reaction mixture is transferred to a measurement cell, where beads are magnetically trapped on the electrode surface. Next, ProCell M buffer containing tripropylamine (TPA) for the subsequent ECL reaction is introduced into the measurement cell to separate the bound immunoassay complex from the remaining free particles. Then, the induction of a voltage between the working electrode and the counter electrode initiates a reaction resulting in the emission of photons by the ruthenium complex and TPA. The resulting electrochemiluminescence signals are recorded by a photomultiplier tube and converted into numerical values ​​indicating the concentration levels of each analyte.

[0291] cTNTh (highly sensitive c-troponin T), NTpBNP (N-terminal prohormone of brain natriuretic peptide), sFLT-1 (soluble fms-like tyrosine kinase-1), GDF15 (growth / differentiation factor 15), IL6 (interleukin 6), cMyBPC (cardiac myosin-binding protein C), BMP10 (bone morphogenesis protein type 10 peptide), FGF23 (fibroblast growth factor 23), IGFBP7 (insulin-like growth factor-binding protein 7), ANG2 (angiopoietin 2), and ESM-1 (endothelial cell-specific molecule 1) were measured using a sandwich immunoassay.

[0292] cTNThs (highly sensitive c-troponin T), NTpBNP (N-terminal prohormone of brain natriuretic peptide), sFLT-1 (soluble fms-like tyrosine kinase-1), GDF15 (growth / differentiation factor 15), and IL6 (interleukin 6) were measured in EDTA plasma samples using a commercially available ECLIA assay developed for the cobas Elecsys® ECLIA platform (Roche Diagnostics, Germany) according to the manufacturer's instructions for the cobas Elecsys® ECLIA immunoassay platform (Roche Diagnostics, Germany).

[0293] cTNThs (troponin T hs Elecsys G5), electrochemiluminescence immunoassay. NTproBNP: (proBNP-II Elecsys), electrochemiluminescence immunoassay. GDF15 (GDF-15 Elecsys), electrochemiluminescence immunoassay. sFLT-1 (FLT1 Elecsys), electrochemiluminescence immunoassay. IL-6 (IL6 Elecsys), electrochemiluminescence immunoassay.

[0294] cMyBPC (cardiac myosin-binding protein C), BMP10 (bone morphogenesis protein type 10 peptide), FGF23 (fibroblast growth factor 23), IGFBP7 (insulin-like growth factor-binding protein 7), ANG2 (angiopoietin 2), and ESM-1 (endothelial cell-specific molecule 1) were measured using a robust prototype ECLIA assay. These sandwich immunoassays were developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assay manufactured by Roche Diagnostics, Germany). The assays include biotinylated and rutheniumized monoclonal antibodies that specifically bind to analytes from EDTA plasma samples and are measured using the cobas Elecsys® ECLIA immunoassay platform analyzer (Roche Diagnostics, Germany). For example, the assay was used for BMP10 using an antibody that binds to NT-proBMP10. Thus, this assay detects NT-proBMP10 and BMP10-type peptides containing the NT-proBMP10 sequence.

[0295] 1.2. Biomarker determination using the cobas® clinical chemistry analyzer platform (Roche Diagnostics, Germany) CRPh (high-sensitivity C-reactive protein), CysC2 (cystatin C), and lipid biomarkers CHOL (cholesterol), TRIGL (triglycerides), LDL (low-density lipoprotein), HDL (high-density lipoprotein), and APOAT (apolipoprotein A-1) were measured in EDTA plasma samples using commercially available assays (Roche Diagnostics, Germany) in accordance with the manufacturer's instructions for the cobas® clinical chemistry analyzer platform cobas c 501 (Roche Diagnostics, Germany).

[0296] The following assay was used: CRPhs: (Catalog number 04628918 190 Cardiac-C reactive protein (Latex) hs), particle-enhanced immunoturbidimetric assay APOAT: (Catalog number 03032566 Tina quant Apolipoprotein A-1 ver 2), immunoturbidimetric assay CHOL: (Catalog number 03039773 Cholesterol Gen.2), Enzyme colorimetric assay HDL: (Catalog number 07528566 HDL-C Gen), homogeneous enzyme colorimetric assay LDL: (Catalog number 07005717 LDL-C Gen3), homogeneous enzyme colorimetric assay TRIGL: (Catalog number 20767107 Triglycerides), Enzyme colorimetric assay

[0297] 2. Patient cohort, APACE study The favorable predictors for the Acute Coronary Syndrome Assessment (APACE) study are described in Nestelberger et al JAMA Cardiol.2021;6(7):771-780.doi:10.1001 / jamacardio.2021.0669. In short, this study is an international, multicenter, prospective cohort study enrolling unselected patients presenting with acute chest pain at rest within the past 12 hours in the emergency department (ED) and registering them in ClinicalTrials.gov (identifier: NCT00470587). The diagnosis of acute myocardial infarction (AMI) is made in patients consecutively enrolled against clinical criteria (final diagnosis) using central adjudication by two independent cardiologists according to the universal definition of AMI (Thygesen K et al, Eur Heart J.2019;40:226), using all clinical information including cardiac imaging and sequential measurements of high-sensitivity troponin.

[0298] AMI was diagnosed when there was evidence of myocardial necrosis in relation to a clinical context consistent with myocardial ischemia. Myocardial necrosis was diagnosed by at least one hs-cTnT / I value above the 99th percentile, along with significant elevation and / or decrease. Absolute changes in high-sensitivity troponin T and I (hs-cTnT / I) were used to determine significant changes based on the diagnostic superiority of absolute changes over relative changes. All other patients were categorized as having unstable angina, non-cardiac chest pain, cardiac but non-coronary artery disease (e.g., myocarditis, takotsubo cardiomyopathy, heart failure), and symptoms of unknown cause with normal hs-cTnT / I levels.

[0299] Definitions of type 1 and type 2 myocardial infarction: Type 1 AMI (T1MI) and Type 2 AMI (T2MI) were defined according to the universal definition of AMI (4th edition, Thygesen K et al, Eur Heart J. 2019;40:226). In short, both diagnoses (T1 and T2 AMI) require clinical evidence of acute myocardial ischemia, such as changes in cardiac troponin, but T1MI has an atherothrombotic origin, and T2MI has a non-atherothrombotic origin. More specifically, evidence of myocardial necrosis in a clinical setting consistent with Type 1 MI, which is acute myocardial ischemia, was defined as spontaneous MI associated with a primary atherothrombotic coronary event such as plaque erosion or plaque rupture, intraluminal coronary thrombosis, or distal microembolism. Type 2 MI was defined as secondary due to a mismatch between oxygen supply and demand. Symptoms reflecting an imbalance between myocardial oxygen supply and demand included bradycardic or tachyarrhythmias, hypoxemia, hypotension, hypertension, severe anemia, coronary artery spasm, dissection, and coronary artery embolism. Underlying coronary artery disease was possible but not necessary for the diagnosis of T2MI. Dynamic changes in hs-cTnT / I were required to qualify for T2MI, as was the case for T1MI. As recommended, documentation of clear triggers was essential for the diagnosis of T2MI. Coronary angiography was not essential for the diagnosis of T1MI to limit the potential impact of selection bias resulting from clinical inquiries to coronary angiography. No other subtypes of MI were reported.

[0300] Example 1: Patients in the APACE study In the APACE study, out of a total of 4,216 patients, N=874 (20.7%) were diagnosed with AMI. Of these, 708 patients were diagnosed with type 1 MI (564 with non-ST-elevation AMI and 144 with ST-elevation myocardial infarction), and 166 patients were diagnosed with type 2 MI (164 with non-ST-elevation myocardial infarction and 2 with ST-elevation myocardial infarction). • Cases (N=166): Patients with type 2 myocardial infarction (MI) secondary to ischemia caused by either increased oxygen demand or decreased oxygen supply. Of these, 58 patients were female and 108 were male. • Controls (N=708): Patients with spontaneous type 1 MI were associated with ischemia due to primary coronary events such as plaque erosion and / or plaque rupture, fissure formation, or dissection. Of these, 191 were female and 517 were male.

[0301] Statistical analysis: Biomarker values ​​were converted to base 2 logarithmically and mathematically combined using logistic regression. The "area under the receiver operating characteristic curve" (AUC) was used as a general measure of marker performance.

[0302] Biomarkers show different elevations in type 1 or type 2 myocardial infarction. [Table 1]

[0303] Example 2: Example of using a combination of hsTnT and BMP10 The following examples demonstrate how scores can actually be obtained from measured biomarker concentrations in patients with type 1 MI and type 2 MI, respectively. 1型 and BMP10 1型 These concentrations are derived from assays measuring hsTnT and BMP10 in type 1 MI patients, respectively. 2型 and hsTnT2型 Let them be the concentrations obtained from assays that measure hsTnT and BMP10 in type 2 MI patients, respectively.

[0304] Let β0 be the offset, and let β hsTnT and β BMP10 be the weight coefficients (coefficients) of the mathematical model used to combine the obtained concentrations.

[0305] Furthermore, let α be a predetermined cut-off that optimally separates type 1 and type 2 AMI patients.

[0306] Score 1型 The score of the biomarker concentration measured for type 1 AMI patients, shown as, is obtained by calculating the following. Score 1型 = β0 + β hsTnT log2(hsTnT 1型 ) + β BMP10 log2(BMP10 1型 )

[0307] Here, log2() represents the logarithmic function to base 2.

[0308] Score 1型 The score of the biomarker concentration measured for type 2 AMI patients, shown as, is obtained by calculating the following. Score 2型 = β0 + β hsTnT log2(hsTnT 2型 ) + β BMP10 log2(BMP10 2型 )

[0309] If the obtained score is greater than a predetermined value of the cut-off α, it is suggested that the patient has type 2 MI. If the obtained score is less than a predetermined value of the cut-off α, it is suggested that the patient has type 1 MI.

[0310] Table 2: The univariate performance of hsTnT(AUC), considering all patient subgroups and combinations of hsTnT with a second marker (bivariate marker combination) and a third marker (trivariate marker combination), shows an AUC improvement of at least 2.0 (collision AUC) compared to single-marker hsTnT. [Table 2-1] [Table 2-2]

[0311] Table 3: Univariate performance of hsTnT(AUC) considering all patient subgroups that did not have an improved AUC of at least 2.0 (clash AUC) compared to the single biomarker hsTnT, and combinations of hsTnT with a second marker (bivariate marker combination) and a third marker (trivariate marker combination). [Table 3]

[0312] As shown in Table 3, several biomarker combinations do not improve AUC values ​​beyond the single marker hsTnT for T1MI vs. T2MI rating.

[0313] Example 3: Diabetic patients from the APACE study • Cases (N=166): Patients with type 2 myocardial infarction (MI) secondary to ischemia caused by either increased oxygen demand or decreased oxygen supply. Of these, 37 patients had a history of diabetes, while 129 did not. • Controls (N=708): Patients with spontaneous type 1 MI were associated with ischemia due to primary coronary events such as plaque erosion and / or plaque rupture, fissure formation, or dissection. Of these, 196 patients had a history of diabetes, while 512 did not.

[0314] Table 5 provides the performance of marker combinations in patients with and without a history of diabetes. Table 5 shows the improved performance of hsTNT when combined with several biomarker panels. It is particularly interesting that all biomarker panels that improve the performance of hsTNT include ANG2. The hsTNT and ANG2 biomarker panels show improvement over hsTNT with a significant AUC delta change of 4.875 (AUC 66.870 ⇒ AUC 73.745). Adding a third biomarker, LDL, to hsTNT and ANG2 further improves performance compared to hsTNT in a subgroup of diabetic patients with an AUC delta change of 6.805 (AUC 68.870 ⇒ AUC 75.675).

[0315] As can be concluded from these results, the use of a biomarker combination of hsTNT and a second biomarker, ANG2, is beneficial in a subgroup of diabetic patients.

[0316] Table 4: Type 1 MI patients with or without a history of diabetes, and Type 2 MI patients with or without a history of diabetes. [Table 4]

[0317] Table 5: Univariate performance of hsTnT(AUC) for all patients with a history of diabetes, as well as combinations of hsTnT with a second marker (bivariate marker combination) and a third marker (trivariate marker combination). All bivariate and trivariate combinations show an improvement of at least 3.0 in AUC compared to patients without a history of diabetes. All bivariate and trivariate combinations showing an improvement of at least 3.0 in AUC compared to the univariate performance of hsTnT in both subgroups are reported for the markers used in the combinations for the criteria. [Table 5]

[0318] Table 6: Univariate performance of hsTnT(AUC) for all patients with a history of diabetes, and for combinations of hsTnT with a second marker (bivariate marker combination) and a third marker (trivariate marker combination). All bivariate and trivariate combinations that did not show at least a 3.0 improvement in AUC between patients with a history of diabetes and patients without a history of diabetes. Univariate performance in both subgroups is reported for the markers used in the combinations for the criteria. [Table 6]

Claims

1. A method for assessing myocardial infarction in a subject, (a) Determining the amount of the first biomarker in the sample of the subject, wherein the first biomarker is cardiac troponin such as cardiac troponin T or I, (b) Determining the amount of a second biomarker in the target sample, wherein the second biomarker is BMP10 type peptide (bone morphogenesis protein type 10 peptide), (c) Comparing the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker, (d) To provide an index for assessing myocardial infarction based on the comparison and / or calculation performed in step (c), The method, including the method described above.

2. In process (b), i) cMyBPC (cardiac myosin-binding protein C); ii) At least one lipid biomarker selected from the group consisting of cholesterol (CHOL), LDL (low-density lipoprotein), TRIGLY (triglycerides), APOAT (apolipoprotein A-1), and HDL (high-density lipoprotein); iii) At least one vascular biomarker selected from the group consisting of FGF23, sFlt1, GDF15, ESM1, ANG2, and IGFBP7; or, iv) At least one inflammatory biomarker selected from the group consisting of hsCRP and IL-6; This further includes determining the amount of a third biomarker. The method according to claim 1.

3. The method according to claim 1 or 2, wherein the sample is obtained from a subject during an examination in the emergency department.

4. The method according to claim 1 or 2, wherein the sample is a blood, serum, or plasma sample, and / or the subject is a human.

5. below: i. Cardiac troponin and BMP10 type peptide, ii. Cardiac troponin, BMP10 type peptide and CRP, or iii. Cardiac troponin, BMP10 type peptide, and at least one lipid biomarker selected from the group consisting of cholesterol (CHOL), LDL (low-density lipoprotein), TRIGLY (triglycerides), APOAT (apolipoprotein A-1), and HDL (high-density lipoprotein), The amount is determined, The cardiac troponin is troponin T or I, The BMP10 type peptide is BMP10, proBMP10, or NT-proBMP10. The method according to claim 1 or 2.

6. A computer implementation method for assessing myocardial infarction in a subject, (a) A step of receiving a value for the amount of a first biomarker in the target sample, wherein the first biomarker is cardiac troponin, (b) A step of receiving a value for the amount of a second biomarker in the target sample, wherein the second biomarker is a BMP10 type peptide, (c) A step of comparing the value for the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker, (d) A step of assessing myocardial infarction based on the comparison and / or calculation performed in step (c), including, Computer implementation method.

7. In process (b), i) cMyBPC (cardiac myosin-binding protein C); ii) At least one lipid biomarker selected from the group consisting of cholesterol (CHOL), LDL (low-density lipoprotein), TRIGLY (triglycerides), APOAT (apolipoprotein A-1), and HDL (high-density lipoprotein); iii) At least one vascular biomarker selected from the group consisting of FGF23, sFlt1, GDF15, ESM1, ANG2, and IGFBP7; or, iv) At least one inflammatory biomarker selected from the group consisting of hsCRP and IL-6; This further includes receiving a value for the amount of as a third biomarker. The computer implementation method according to claim 6.

8. A device for assessing myocardial infarction in a subject, wherein the device comprises an evaluation unit including a database having stored criteria for a first biomarker which is cardiac troponin and a second biomarker which is a BMP10 type peptide, and a data processor which includes instructions for comparing the amounts of the first biomarker and the second biomarker with the criteria and assessing myocardial infarction based on the comparison, wherein the evaluation unit can receive a value for the determined amount of the biomarker in the subject sample. A device for assessing myocardial infarction in a subject.

9. The database includes stored criteria for a third biomarker, and the third biomarker is i) cMyBPC (cardiac myosin-binding protein C); ii) At least one lipid biomarker selected from the group consisting of cholesterol (CHOL), LDL (low-density lipoprotein), TRIGLY (triglycerides), APOAT (apolipoprotein A-1), and HDL (high-density lipoprotein); iii) At least one vascular biomarker selected from the group consisting of FGF23, sFlt1, GDF15, ESM1, ANG2, and IGFBP7; or, iv) At least one inflammatory biomarker selected from the group consisting of hsCRP and IL-6; The device according to claim 8.

10. Use as a reference material for cardiac troponin, the first biomarker, and BMP10 type peptide, the second biomarker, to assess myocardial infarction in the target population.

11. A third biomarker was further used as a reference substance. The third biomarker is, i) cMyBPC (cardiac myosin-binding protein C); ii) At least one lipid biomarker selected from the group consisting of cholesterol (CHOL), LDL (low-density lipoprotein), TRIGLY (triglycerides), APOAT (apolipoprotein A-1), and HDL (high-density lipoprotein); iii) At least one vascular biomarker selected from the group consisting of FGF23, sFlt1, GDF15, ESM1, ANG2, and IGFBP7; or, iv) At least one inflammatory biomarker selected from the group consisting of hsCRP and IL-6; The use described in claim 10.

12. The use of at least one detector for a first biomarker, which is cardiac troponin, and at least one detector for a second biomarker, which is a BMP10 type peptide, to assess myocardial infarction in a subject.

13. A detection agent for a third biomarker is further used, and the third biomarker is i) cMyBPC (cardiac myosin-binding protein C); ii) At least one lipid biomarker selected from the group consisting of cholesterol (CHOL), LDL (low-density lipoprotein), TRIGLY (triglycerides), APOAT (apolipoprotein A-1), and HDL (high-density lipoprotein); iii) At least one vascular biomarker selected from the group consisting of FGF23, sFlt1, GDF15, ESM1, ANG2, and IGFBP7; or, iv) At least one inflammatory biomarker selected from the group consisting of hsCRP and IL-6; The use described in claim 12.

14. A kit for assessing myocardial infarction in a subject, comprising: i) at least one detection agent for a first biomarker which is cardiac troponin; and ii) at least one detection agent for a second biomarker which the second biomarker is a BMP10 type peptide. The aforementioned kit.

15. The kit further comprises a detection agent for a third biomarker, The third biomarker is, i) cMyBPC (cardiac myosin-binding protein C); ii) At least one lipid biomarker selected from the group consisting of cholesterol (CHOL), LDL (low-density lipoprotein), TRIGLY (triglycerides), APOAT (apolipoprotein A-1), and HDL (high-density lipoprotein); iii) At least one vascular biomarker selected from the group consisting of FGF23, sFlt1, GDF15, ESM1, ANG2, and IGFBP7; or, iv) At least one inflammatory biomarker selected from the group consisting of hsCRP and IL-6; The kit according to claim 14.

16. The BMP10 type peptide is BMP10, proBMP10, or NT-proBMP10. The method according to claim 1 or 2.

17. If the subject has diabetes, the third biomarker is ANG2. The method according to claim 2.

18. The aforementioned assessment of myocardial infarction, a) Distinguishing between type 1 myocardial infarction and type 2 myocardial infarction, b) Diagnosis of type 2 myocardial infarction, or c) Guidance on the treatment of myocardial infarction, The method according to claim 1 or 2.

19. The BMP10 type peptide is BMP10, proBMP10, or NT-proBMP10. The device according to claim 8 or 9.

20. If the subject has diabetes, the third biomarker is ANG2. The device according to claim 9.

21. The aforementioned assessment of myocardial infarction, a) Distinguishing between type 1 myocardial infarction and type 2 myocardial infarction, b) Diagnosis of type 2 myocardial infarction, or c) Guidance on the treatment of myocardial infarction, The device according to claim 8 or 9.

22. The BMP10 type peptide is BMP10, proBMP10, or NT-proBMP10. The use according to any one of claims 10 to 13.

23. If the subject has diabetes, the third biomarker is ANG2. The use according to claim 11 or 13.

24. The aforementioned assessment of myocardial infarction, a) Distinguishing between type 1 myocardial infarction and type 2 myocardial infarction, b) Diagnosis of type 2 myocardial infarction, or c) Guidance on the treatment of myocardial infarction, The use according to any one of claims 10 to 13.

25. The BMP10 type peptide is BMP10, proBMP10, or NT-proBMP10. The kit according to claim 14 or 15.

26. If the subject has diabetes, the third biomarker is ANG2. The kit according to claim 15.

27. The aforementioned assessment of myocardial infarction, a) Distinguishing between type 1 myocardial infarction and type 2 myocardial infarction, b) Diagnosis of type 2 myocardial infarction, or c) Guidance on the treatment of myocardial infarction, The kit according to claim 14 or 15.

28. The kit according to claim 14 or 15, wherein the detection agent is an antibody or an antigen-binding fragment thereof that specifically binds to the biomarker.

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