Its application in monitoring cardiovascular markers in interstitial fluid and evaluating heart failure.

Abstract

The present invention relates to a method for evaluating heart failure (HF), determining the risk of developing HF, monitoring HF, classifying HF, stratifying the risk of patients with HF, predicting prognosis, and determining the therapeutic effect of HF treatment regimens in a subject, by determining the level of one or more cardiac-related (poly)peptide biomarkers in interstitial fluid (ISF) from a subject and comparing the determined levels to reference values.

Description

Technical Field

[0001] The present invention relates to a method for evaluating heart failure (HF), determining the risk of developing HF, monitoring HF, classifying HF, stratifying the risk of patients with HF, predicting prognosis, and determining the therapeutic effect of an HF treatment regimen in a subject by determining the level of one or more heart-related (poly)peptide biomarkers in interstitial fluid (ISF) from the subject and comparing the determined level with a reference value. Furthermore, the present invention relates to the use of ISF in the methods described herein.

Background Art

[0002] Heart failure (HF), also known as congestive heart failure (CHF), was updated in 2021 in accordance with the guidelines of the European Society of Cardiology as: "It is not a single pathological diagnosis, but a clinical syndrome consisting of cardinal symptoms (e.g., breathlessness, ankle swelling, and fatigue) that may be accompanied by signs (e.g., elevated jugular venous pressure, pulmonary crackles, and peripheral edema). It is caused by structural and / or functional abnormality of the heart that results in elevated intracardiac pressure and / or insufficient cardiac output at rest and / or during exercise." Breathlessness can occur during exercise or while lying down, and may wake a person up at night. Chest pain including angina pectoris is not usually caused by heart failure, but may occur when heart failure is caused by a heart attack. The severity of heart failure is measured by signs or symptoms at rest or during exercise or training. Other comorbid conditions that can have signs and symptoms similar to those of heart failure include obesity, renal failure, liver and lung disease, anemia, and thyroid disease.

[0003] Common causes of heart failure include coronary artery disease, heart attack, hypertension, atrial fibrillation, valvular heart disease, excessive alcohol consumption, infections, and cardiomyopathy. These cause heart failure by altering the structure and / or function of the heart. There are various types of heart failure, including right heart failure, which affects the right part of the heart; left heart failure, which affects the left part of the heart; and biventricular heart failure, which affects both sides of the heart. Left heart failure can be recognized as reduced ejection fraction or preserved ejection fraction. Heart failure is not the same as cardiac arrest, in which blood flow completely stops due to the heart's inability to effectively pump blood. Heart failure is classified into different phenotypes based on the measurement of left ventricular ejection fraction: reduced ejection fraction heart failure (HFrEF), mild reduced ejection fraction heart failure (HFmrEF), and preserved ejection fraction heart failure (HFpEF).

[0004] Diagnosis is typically based on risk factors, symptoms, physical findings, abnormal electrocardiogram (ECG), and echocardiogram. Blood tests and chest X-rays may be useful in determining the underlying cause.

[0005] Treatment depends on the severity and the individual case. For people with chronic, stable, mild heart failure, treatment usually consists of lifestyle changes such as smoking cessation, physical exercise, and dietary changes, as well as pharmacotherapy. In heart failure due to left ventricular dysfunction, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, or valsartan / sacubitril are recommended, along with beta-blockers and sodium-glucose cotransporter 2 inhibitors (SGLT2i). In severe cases, mineralocorticoid receptor antagonists or hydralazine in combination with nitrates may be used. Diuretics may also be prescribed to prevent fluid retention and the resulting shortness of breath. In some cases, implantable devices such as pacemakers or implantable cardioverter-defibrillators may be recommended. In some moderate to severe cases, cardiac resynchronization therapy (CRT) or myocardial contractility adjustment may be beneficial. In severe cases that persist despite all other measures, cardiac assist devices such as ventricular assist devices (for the left, right, or both chambers), or in some cases, heart transplantation, may be recommended.

[0006] Heart failure is a common, costly, and potentially fatal condition, and is a leading cause of hospitalization and readmission in older adults. Heart failure often results in more dramatic health problems than damage to other similarly complex organs such as the kidneys or liver.

[0007] Acute heart failure (AHF) is a leading contributor to morbidity and mortality in patients with heart failure (HF) (1). Despite this increased risk, the majority of AHF patients are not closely followed and are substantially under-cared during the first few months after discharge from AHF hospitalization, which is a “vulnerable period” in which most adverse events occur (2–7). Some retrospective studies have investigated the association between increased follow-up after an AHF event and rapid dose escalation of medications, but the results have been mixed (8–14, 16). Based on this lack of evidence from prospective randomized studies, recent European guidelines for the management of HF recommend some follow-up of patients after AHF hospitalization and initiation of recommended therapy, but the level of evidence for this recommendation is low (C)(17).

[0008] The Safety, Tolerability, and Efficacy of Rapid Optimization of Heart Failure Treatment with NT-proBNP Measurement Support (STRONG-HF) study was a randomized prospective clinical trial designed to evaluate the safety and efficacy of rapid dose escalation of pharmacotherapy, including beta-blockers (BBs), angiotensin-converting enzyme inhibitors (ACEi), angiotensin receptor blockers (ARBs) or angiotensin receptor neprilysin inhibitors (ARNis), and mineralocorticoid receptor antagonists (MRAs), in the pre-discharge and subsequent weeks following AHF hospitalization (ClinicalTrials.gov NCT03412201). Safety of escalation was guided by physical examination and laboratory assessments, including NT-proBNP. Guideline-recommended oral HF pharmacotherapy for patients in the "high-intensity care" group was escalated to half of the optimal dose at discharge and to the full optimal dose two weeks post-discharge. A safety assessment visit was conducted one week after any escalation, and follow-up visits were conducted at six weeks and three months. At each visit, patients were evaluated by physical examination for congestion and blood tests including NT-proBNP measurement (18, 19). The STRONG-HF study concluded that an intensive care strategy consisting of rapid escalation of guideline-based medication and close follow-up after admission to acute heart failure was readily accepted by patients because it reduced symptoms, improved quality of life, and lowered the risk of all-cause mortality or rehospitalization for heart failure at 180 days compared to usual care (20).

[0009] In summary, heart failure (HF) is a progressive disease characterized by recurrent episodes of acute exacerbation. Despite the treatment options recommended by guidelines, the outcomes for HF remain poor. Decompensation and readmissions pose significant challenges in managing patients with heart failure.

[0010] Monitoring BNP-type peptides such as NT-proBNP, in addition to other biomarkers such as creatinine or potassium, allows physicians to monitor the patient's condition, select the most promising treatment, and optimize the dosage of life-saving treatment for heart failure (21).

[0011] There is a strong need for non-invasive and rapid assessment and monitoring of cardiac-related biomarkers, which would enable reliable and continuous risk assessment and / or monitoring of patients exhibiting signs and symptoms of (acute) heart failure.

[0012] Therefore, the present invention provides a method that meets these needs.

[0013] Therefore, the present invention relates, in particular, to a method for evaluating a patient's heart failure by determining the levels of cardiac-related biomarkers, especially BNP-type peptides, in ISF. [Overview of the project]

[0014] In a first aspect, the present invention relates to a method for evaluating heart failure in a subject or for determining the risk of developing heart failure (HF), wherein the method is a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the interstitial fluid (ISF) of a subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers with the level of the same cardiac-related (poly)peptide biomarkers in a control sample, or with a predetermined reference level of the same cardiac-related biomarkers, c) A step in which, based on the comparison in step b), subjects are identified as having heart failure or having an increased risk of developing heart failure. Includes.

[0015] In a second aspect, the present invention relates to a method for monitoring a target HF, wherein the method is i. A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in ISF from a subject according to optional method steps a) to b) of claim 1, ii. A process in which process i is repeated after a certain time interval, iii. A step of comparing the level of the cardiac-related (poly)peptide biomarker identified in i with the level identified in ii, wherein the change in level from i to ii indicates a change in HF in the subject. Includes.

[0016] In a third embodiment, the present invention relates to a method for classifying the disease stage of HF in a subject, wherein the method is a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in ISF from a subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has elevated or decreased levels of one or more cardiac-related (poly)peptide biomarkers compared to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, then the step of classifying the stage of HF in the subject and Includes.

[0017] In a fourth aspect, the present invention relates to a method for determining the therapeutic effect of a treatment regimen for a target HF, wherein the method is i. A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in ISF from a subject according to optional method steps a) to b) of claim 1, ii. A process in which process i is repeated after a certain time interval, iii. A step of comparing the levels of cardiac-related (poly)peptide biomarkers identified in i with the levels identified in ii, and identifying that the treatment regimen is effective if the levels of cardiac-related (poly)peptide biomarkers decrease after treatment. Includes.

[0018] In a fifth aspect, the present invention relates to the use of ISF for evaluating HF, determining the risk of developing heart failure, classifying the stage of heart failure, monitoring heart failure, and / or determining the therapeutic effect of a treatment in a subject, by determining the level of one or more cardiac-related (poly)peptide biomarkers in the subject.

[0019] In a sixth aspect, the present invention relates to a computer implementation method for evaluating a subject suspected of having heart failure, wherein the computer implementation method is (a) A step of receiving a value relating to the level of a first cardiac-related (poly)peptide biomarker in the interstitial fluid of a subject, wherein the first biomarker is a BNP-type peptide, (b) A step of receiving a value relating to the level of at least one additional cardiac-related biomarker in the interstitial fluid of the subject, wherein the additional cardiac-related biomarker is glucose, creatinine, potassium, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, hsCRP (high-sensitivity C-reactive protein), cancer A receiving step in which a substance is selected from the group consisting of antigen 125 (or carbohydrate antigen 125, CA125), soluble differentiation cluster 146 (or cell surface glycoprotein MUC18, sCD146), bioadrenomedullin (bioADM), central region proadrenomedullin (MR-proADM), soluble tumorigenesis inhibitor 2 (sST2), angiopoietin 2 (Ang-2), fibroblast growth factor 23 (FGF-23), BMP-10 (bone morphogenetic protein 10), ESM-1 (endothelial cell-specific molecule 1), and insulin-like growth factor binding protein 7 (IGFBP7), (c) A step of comparing the values ​​related to the levels of steps (a) to (b) with criteria related to biomarkers and / or calculating a score for evaluating subjects suspected of having heart failure based on the levels of biomarkers, (d) evaluating the subject based on the comparison and / or calculation performed in step (c); and comprising.

[0020] In a further aspect, the present invention relates to a method for treating heart failure in a subject, the method comprising: a) requesting a test that provides the result of an assay for determining the level of one or more cardiac-associated (poly)peptide biomarkers in ISF from the subject, and comparing the level to a reference level, wherein an increased level as compared to the reference level is indicative of heart failure or an increased risk of developing heart failure in the subject; b) administering a treatment for heart failure to the subject identified as having heart failure or being at risk of developing heart failure, thereby treating the subject; and comprising.

[0021] In a further aspect, the present invention relates to a method for determining compliance of a subject with a prescribed treatment for heart failure, the method comprising: a) determining the level of one or more cardiac-associated (poly)peptide biomarkers in ISF from the subject prior to treatment; b) determining the level of the same one or more cardiac-associated (poly)peptide biomarkers as in step (a) in ISF from the subject after treatment; c) comparing the levels obtained in step (a) and step (b), wherein a reduction in the level obtained in step (b) as compared to the level in step (a) indicates that the subject is and / or has been compliant with the prescribed treatment; and comprising.

[0022] In a further aspect, the present invention relates to a method for risk stratification of progression to severe disease in a subject having HF, the method comprising: a) determining the level of one or more cardiac-associated (poly)peptide biomarkers in ISF from the subject; b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has elevated or decreased levels of one or more cardiac-related (poly)peptide biomarkers compared to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, then the step of stratifying the risk of progression to serious disease for subjects with HF and Includes.

[0023] In a further embodiment, the present invention relates to a method for predicting the prognosis of the future disease course of a subject having HF, wherein the method is a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in ISF from a subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has elevated or decreased levels of one or more cardiac-related (poly)peptide biomarkers compared to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, then the step of predicting the future disease course of the subject having HF. Includes.

[0024] Ideally, cardiac-related (poly)peptide biomarkers can be BNP-type peptides.

[0025] Appropriately, cardiac-related (poly)peptide biomarkers may be N-terminal pro-B-type natriuretic peptide (NT-proBNP) or B-type natriuretic peptide (BNP).

[0026] Appropriately, in addition to the levels of one or more cardiac-related (poly)peptide biomarkers, glucose, creatinine, potassium, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, hsCRP (high-sensitivity C-reactive protein), cancer antigen 125 (or carbohydrate antigen 125, CA125), soluble The levels of one or more additional cardiac-related biomarkers selected from the group consisting of differentiation cluster 146 (or cell surface glycoprotein MUC18, sCD146), bioadrenomedullin (bioADM), central region proadrenomedullin (MR-proADM), soluble tumorigenesis inhibitor 2 (sST2), angiopoietin 2 (Ang-2), fibroblast growth factor 23 (FGF-23), BMP-10 (bone morphogenetic protein 10), ESM-1 (endothelial cell-specific molecule 1), and insulin-like growth factor-binding protein 7 (IGFBP7) can be determined.

[0027] Appropriately, HF may be selected from the group consisting of acute heart failure, reduced ejection fraction heart failure (HFrEF), mild reduced ejection fraction heart failure (HFmrEF), preserved ejection fraction heart failure (HFpEF), left heart failure, right heart failure, and biventricular failure.

[0028] Appropriately, the process may further include selecting a treatment regimen for the subject based on a comparison of the biomarker level with a control sample or a predetermined reference level.

[0029] Appropriately, the procedure may further include steps performed on the selected treatment regimen, and optionally, the selected treatment regimen may include drug-based treatments and / or surgery.

[0030] Appropriately, the stage of the heart failure in question may be classified as Stage I, Stage II, Stage III, or Stage IV heart failure according to the New York Cardiology Association (NYHA) functional classification.

[0031] Unless the context requires otherwise, it will be understood that the considerations described herein should be considered applicable to all aspects of the invention.

[0032] Various aspects of the present invention will be described in further detail below. [Brief explanation of the drawing]

[0033] The embodiments of the present invention will be further described below with reference to the attached drawings.

[0034] [Figure 1] Calibration curve for the Simoa® assay for NT-proBNP. The x-axis shows the concentration (pg / mL) of each NT-proBNP calibrator on a logarithmic scale. The y-axis shows the average signal (in AEB (average number of enzyme labels per bead)) of the Simoa® assay measurements for these calibrators, also on a logarithmic scale. The error bars shown in Figure 1 represent the standard deviation. The calibration curve shown in Figure 1 is based on a cubic fitting with 1 / y² weighting. [Figure 2] A comparative plot of corresponding NT-proBNP values ​​in plasma (x-axis, pg / mL) and ISF (y-axis, also in pg / mL) from samples of the same donor. The error bars shown in Figure 2 represent the standard deviation of each plasma or ISF measurement. [Modes for carrying out the invention]

[0035] The present invention provides a novel and remarkable approach to assessing heart failure, determining the risk of developing heart failure, classifying the stage of heart failure, monitoring heart failure, and / or determining the therapeutic effect of treatment in a subject.

[0036] This novel approach is based on the inventors' unexpected discovery that intravascular fluid (ISF) is reliable for determining levels of cardiac-related (poly)peptide biomarkers. Due to fluctuating accessibility, component concentrations (e.g., ions, peptides, proteins, metabolites, small molecules, etc.), component sizes, equilibrium, etc., various body fluids (e.g., whole blood, plasma, tears, sweat, synovial fluid, ISF, urine, saliva, lymph, vitreous fluid, or pericardial fluid) contain different components at different concentrations. This reliability stems from the fact that the level of cardiac-related (poly)peptide biomarkers in ISF correlates with the level of that biomarker found in the blood. Depending on the biomarker, the correlation between the levels of cardiac-related (poly)peptide biomarkers in ISF and blood can be linear / proportional or substantially the same. In this context, "substantially the same" levels can be considered levels when considering a ±20% deviation due to measurement and dilution errors. Currently, determining biomarker levels in blood is the gold standard. Our discovery makes it possible to determine the levels of cardiac-related (poly)peptide biomarkers using ISF, instead of using blood samples that would otherwise need to be obtained by a physician or trained medical assistant.

[0037] The inventors have shown for the first time that the level of NT-proBNP measured in ISF is comparable to the level of NT-proBNP in blood.

[0038] This application demonstrates a near 1:1 correlation between serum NT-proBNP concentrations and ISF concentrations. This remarkable finding demonstrates that ISF should generally be considered a potential source of bodily fluids for determining biomarker levels, and that ISF has the advantage of being readily accessible and available.

[0039] For several reasons, there is an unmet medical need to determine and regularly monitor cardiac-related biomarkers in subjects exhibiting signs of HF in a rapid, reliable, and simple manner: • Assessment of cardiac decompensation • To diagnose congestion, or to evaluate or monitor the degree of congestion in the subject. • Evaluate decisions regarding the patient's (read)hospitalization. • Evaluate the decision regarding the patient's discharge. • Optimization of treatment (dosage) • Optimization of treatment dosage • Assessment of decongestant or residual decongestant in HF (including quantification, diagnosis, assessment, monitoring, etc.), and ultimately, the subsequent optimized and / or induced decongestant therapy. • Prediction / assessment of congestion reduction or residual congestion after treatment or intervention for congestion in the target population. • Predicting, determining, or monitoring the need for treatment or intervention, or predicting, determining, or monitoring the success of treatment or intervention for congestion, or inducing treatment or intervention in a subject, • Determining the subject's compliance with prescribed treatments for HF. • Stratification of the risk of progression to severe disease in individuals with HF • Prognosis of the future disease course in patients with HF

[0040] The use of ISF as the body fluid in which cardiac-related biomarker levels are determined has the advantage of being easily accessible and enabling continuous monitoring of cardiac-related biomarkers in a simple and reliable manner. No physician or trained medical assistant is required to obtain ISF samples. Even more advantageously, its easy accessibility allows for the continuous measurement of corresponding biomarkers by directly measuring them in the ISF within the body, without actually obtaining ISF samples, or rather, without requiring medical intervention.

[0041] The inventors investigated the levels of the cardiac biomarker NT-proBNP in donor ISF and blood samples. Surprisingly, the inventors found that NT-proBNP levels correlated with those in donor ISF and blood.

[0042] The data presented herein demonstrate that by determining the levels of cardiac-related (poly)peptide biomarkers, particularly BNP-type peptides, HF can be assessed, the risk of developing HF can be determined, HF can be monitored, the stage of HF can be classified, and the therapeutic effect of HF treatment regimens can be determined. This satisfies the need for a rapid, reliable, and simple method to determine and regularly monitor cardiac-related biomarkers in subjects exhibiting signs of HF, and can be used for all the reasons and purposes listed above.

[0043] Generally, the methods described herein are in vivo, ex vivo, or in vitro methods.

[0044] Preferably, the method described herein is an in vivo method, in which the level of a cardiac-related (poly)peptide biomarker is measured (continuously) by a needle-type sensor (permanently) attached to the subject's body, enabling the measurement to be performed in the subject's ISF.

[0045] In the case of in vitro methods, these methods are performed using samples already obtained from the subject (i.e., the sample is provided for the method, and the steps taken to obtain the sample from the subject are not included as part of the method). Therefore, the method may include the step of providing a biological fluid sample from the subject.

[0046] Methods provided herein may include providing ISF samples from a subject. Advantageously, methods for obtaining ISF samples from a subject are typically minimally invasive or non-invasive.

[0047] The sample is an in vitro sample and will be analyzed in vitro; it will not be returned to the body.

[0048] definition It should be understood that the word "comprise," as well as variations such as "comprises" and "comprising," means the inclusion of the listed component or process, or group of components or processes, but not the exclusion of any other component or process, or group of components or processes.

[0049] As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple subjects unless otherwise explicitly indicated.

[0050] Concentration, level, quantity, and other numerical data may be expressed or presented in the form of “ranges” as used herein. It should be understood that such range forms are used merely for convenience and conciseness, and therefore should be flexibly interpreted to include not only the numbers explicitly listed as boundaries of the range, but also all individual numbers or subranges contained within that range, as if each number and subrange were explicitly listed. For example, the numerical range “150mg to 600mg” should be interpreted to include not only the explicitly listed values ​​of 150mg to 600mg, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 150, 160, 170, 180, 190, ... 580, 590, 600mg, and subranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600. The same principle applies to ranges listing only a single number. Furthermore, such interpretations should apply regardless of the scope or characteristics described.

[0051] When used in relation to a number, the term "approximately" means to encompass a range of numbers that have a lower limit 5% less than the given number and an upper limit 5% greater than the given number.

[0052] As used herein, “to provide,” “to obtain,” or “to acquire” in relation to a sample may be any means by which one comes into possession of the sample, whether directly or indirectly. Obtaining a sample directly means performing a process to obtain the sample (e.g., performing a physical method such as extraction). Obtaining a sample indirectly means receiving a sample from another entity or source (e.g., a third-party laboratory that directly obtained the sample).

[0053] The analysis of a sample may be achieved visually or chemically. Chemical analysis includes, but is not limited to, the detection of the presence or absence of specific indicators or changes in their quantity, concentration, or level.

[0054] In the context of the present invention, the terms “biomarker,” “marker,” “cardiac-related biomarker,” and “cardiac-related (poly)peptide biomarker” are used interchangeably and refer to peptides or polypeptides (=(poly)peptides) within a biological system that are used as indicators of the biological state of the biological system. In the art, the term “biomarker” may also be applied to means of detecting endogenous substances (e.g., antibodies, nucleic acid probes, imaging systems). In the context of the present invention, the term “biomarker” shall apply only to substances and not to means of detection. Accordingly, a biomarker may be any type of (poly)peptide present in a living organism, such as proteins (cell surface receptors, cytosolic proteins, etc.), polypeptides, peptides, their isomers, or immunodetectable fragments that are differentially present in a subject / sample taken from a subject having HF compared to a subject without HF. If other biomarkers other than (poly)peptide biomarkers are intended to be included in these terms in the context of the present invention, these biomarkers shall be explicitly indicated.

[0055] As used herein, the term "(poly)peptide" refers to a chain of amino acids linked by peptide bonds, having a length of up to 150 amino acids, including oligopeptides, dipeptides, tripeptides, and tetrapeptides.

[0056] An example of a "cardiac-related (poly)peptide biomarker" is a BNP-type peptide.

[0057] As used herein, the term “BNP-type peptide” includes pre-proBNP, proBNP, NT-proBNP, and BNP. The pre-propeptide (134 amino acids in the case of pre-proBNP) contains a short signal peptide, which is enzymatically cleaved to release the propeptide (108 amino acids in the case of proBNP). The propeptide is further cleaved into the N-terminal propeptide (NT-propeptide, 76 amino acids in the case of NT-proBNP) and the active hormone (32 amino acids in the case of BNP). Preferably, the BNP-type peptides according to the present invention are NT-proBNP, BNP, and their variants. BNP is the active hormone and has a shorter half-life than its inactive counterpart, NT-proBNP. BNP is metabolized in the blood, while NT-proBNP circulates in the blood as an unimpaired molecule and is released from the kidneys as such. The in vivo half-life of NT-proBNP is 120 minutes longer than that of BNP, which has a half-life of 20 minutes (Smith 2000, J Endocrinol. 167:239-46). Pre-analysis is more robust with NT-proBNP and allows for easy transport of samples to the central laboratory (Mueller 2004, Clin Chem Lab Med 42:942-4). Blood samples can be stored at room temperature for several days or shipped or transported without recovery loss. In contrast, storage of BNP at room temperature or 4°C for 48 hours results in at least a 20% concentration loss (Mueller loc.cit.; Wu 2004, Clin Chem 50:867-73). Therefore, depending on the time course or characteristics of the subject of interest, it may be advantageous to measure either the active or inactive form of the natriuretic peptide. The most preferred natriuretic peptide according to the present invention is NT-proBNP or its variants. As briefly discussed earlier, the human NT-proBNP referred to in this invention is preferably a polypeptide containing 76 amino acids in length, corresponding to the N-terminal portion of the human NT-proBNP molecule.The structures of human BNP and NT-proBNP have already been described in detail in the prior art, for example, International Publication No. 02 / 089657, International Publication No. 02 / 083913, or Bonow loc.cit. Preferably, the human NT-proBNP used herein is the human NT-proBNP disclosed in European Patent No. 0648228. These prior art documents are incorporated herein by reference with respect to the specific sequences of the NT-proBNP and its variants disclosed herein.

[0058] In addition to cardiac-related (poly)peptide biomarkers, the methods described herein may also include determining the levels of one or more further cardiac-related biomarkers that do not need to be (poly)peptides. Examples of such cardiac-related biomarkers include glucose, creatinine, potassium, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, hsCRP (high-sensitivity C-reactive protein), cancer antigen 125 (or carbohydrate antigen 125) These include CA125), soluble differentiation cluster 146 (or cell surface glycoprotein MUC18, sCD146), bioadrenomedullin (bioADM), central region proadrenomedullin (MR-proADM), soluble tumorigenesis inhibitor 2 (sST2), angiopoietin 2 (Ang-2), fibroblast growth factor 23 (FGF-23), BMP-10 (bone morphogenetic protein 10), ESM-1 (endothelial cell-specific molecule 1), and insulin-like growth factor-binding protein 7 (IGFBP7).

[0059] Biomarkers are differentially present when the mean or median levels of biomarkers in different groups are calculated to be statistically related. Common tests for statistical significance include, among others, t-tests (e.g., Student's t-test), ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney, receiver operating characteristics (ROC curves), precision, and odds ratios. Biomarkers, either alone or in combination, provide a measure of the relative risk of a subject belonging to one phenotypic state or another.

[0060] Therefore, these are useful as markers for disease (diagnosis), drug therapeutic efficacy, and drug toxicity.

[0061] The methods provided herein refer to “determining” the levels of one or more (poly)peptide biomarkers. As will be apparent to those skilled in the art, the levels of one or more (poly)peptide biomarkers are typically “determined” by measuring the levels of biomarkers in a sample / subject. Thus, the term “determine” may be replaced herein by the terms “measure” or “determine by measurement.”

[0062] As used herein, the terms “determine” or “evaluate” also refer to evaluating / determining whether a patient has HF. Therefore, as used herein, evaluation / determining includes diagnosing HF, assessing a subject’s risk of developing HF, selecting a treatment for HF, and monitoring patients who have HF or are receiving treatment for HF, by determining the level, amount, or concentration of one or more (poly)peptide biomarkers and comparing the determined level, amount, or concentration to a baseline. Typically, evaluations referred to in accordance with the present invention are evaluations of the presence and / or onset of HF.

[0063] As used herein, the terms “evaluate heart failure treatment” or “determine the therapeutic effect of a treatment regimen for HF” preferably refer to identifying patients who are eligible for a change in heart failure treatment, as described elsewhere herein. Thus, patients who would benefit from a change in treatment may be identified or selected.

[0064] The terms “measure,” “measure,” or “determine” preferably include qualitative, semi-quantitative, or quantitative measurement.

[0065] Conventional in vitro "determination" methods may include sending clinical samples to private laboratories to measure the levels of biomarkers in biological fluid samples, or using commercially available assay kits to measure the levels of biomarkers in biological fluid samples. Exemplary kits and suppliers will be obvious to those skilled in the art. In various examples, biomarkers can be determined, detected, and / or quantified using ELISA assays or lateral flow devices for point-of-care use, as well as spot-check colorimetric tests.

[0066] The level of biomarkers present in a biological fluid (sample) can be determined, for example, by assaying the amount of biomarkers present in the fluid (sample). Assays for measuring the amount of specific proteins are well known in the art and include direct or indirect measurements. The level of biomarkers of proteins in a fluid (sample) can also be determined by determining the level of biomarker activity of the proteins in the fluid (sample). Thus, the "level" of a protein encompasses both the amount of the protein itself or its activity level.

[0067] Biomarkers as used herein can be detected using methods generally known in the art. Detection methods generally include methods for quantifying the level of a biomarker in a sample (quantitative methods). It is generally known to those skilled in the art which of the following methods is suitable for the qualitative and / or quantitative detection of biomarkers. Samples can be conveniently assayed for proteins using commercially available immunoassays such as Western blotting and ELISA, RIAs, and fluorescence-based immunoassays. More suitable methods for detecting biomarkers include measuring physical or chemical properties specific to the peptide or polypeptide, such as its exact molecular weight or NMR spectrum. Such methods include analytical instruments such as biosensors, optical devices associated with immunoassays, biochips, mass spectrometers, NMR analyzers, or chromatography devices. Furthermore, the methods include microplate ELISA-based methods, fully automated or robotic immunoassays (available with Elecsys® analyzers), CBA (enzymatic Cobalt Binding Assay, e.g., available with Roche-Hitachi® analyzers), and latex agglutination assays (e.g., available with Roche-Hitachi® analyzers).

[0068] For the detection of (poly)peptide biomarkers or biomarker proteins as used herein, a wide range of immunological assay techniques using such assay formats are available; see, for example, U.S. Patents 4,016,043, 4,424,279, and 4,018,653. These include both conventional competitive binding assays and non-competitive one-site and two-site or “sandwich” assays. These assays also include direct binding of labeled antibodies to target biomarkers.

[0069] The sandwich assay is one of the most useful and commonly used immunoassays.

[0070] Methods for measuring electrochemiluminescence are well known. These methods utilize the properties of specific metal complexes, which are excited by oxidation. The metal complex then relaxes from the excited state to the ground state, emitting electrochemiluminescence. For a review, see Richter, MM, Chem. Rev. 104 (2004) 3003-3036.

[0071] Biomarkers can also be detected by commonly known methods including magnetic resonance spectroscopy (NMR spectroscopy), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), HPLC and ultraHPLC such as reversed-phase HPLC, ion-pair HPLC with dual UV wavelength detection, capillary electrophoresis with laser-induced fluorescence detection, anion exchange chromatography and fluorescence detection, and thin-layer chromatography.

[0072] Preferably, measuring the level of a biomarker as defined herein includes (a) contacting a cell capable of eliciting a cellular response whose intensity indicates the level of the peptide or polypeptide with the peptide or polypeptide for an appropriate period of time, and (b) measuring the cellular response. To measure the cellular response, the sample or treated sample is preferably added to a cell culture and the internal or external cellular response is measured. The cellular response may include the measurable expression of a reporter gene or the secretion of a substance, such as a peptide, polypeptide, or small molecule. The expression or substance generates an intensity signal that correlates with the level of the peptide or polypeptide.

[0073] Preferably, determining the level of the peptide or polypeptide involves measuring a specific intensity signal obtained from the peptide or polypeptide in the sample. As described above, such a signal may be the signal intensity observed in a peptide or polypeptide-specific m / z variable observed in a peptide or polypeptide-specific mass spectrum or NMR spectrum.

[0074] Measuring the level of a peptide or polypeptide may preferably include the steps of (a) contacting the peptide with a specific binder, (b) (optionally) removing the unbound binder, and (c) measuring the level of the bound binder, i.e., the binder complex formed in step (a). According to a preferred embodiment, the contacting, removal, and measuring steps may be performed by an analytical instrument unit of the system described herein. According to some embodiments, the steps may be performed by a single analytical instrument unit of the system or by a plurality of analytical instrument units in an operable communication state with each other. For example, according to a specific embodiment, the system disclosed herein may comprise a first analytical instrument unit for performing the contacting and removal steps, and a second analytical instrument unit operably connected to the first analytical instrument unit by a transport unit (e.g., a robotic arm) for performing the measuring step.

[0075] The bound conjugate, i.e., the conjugate or conjugate / peptide complex, generates an intensity signal. The binding according to the present invention includes both covalent and non-covalent bonds. The conjugate according to the present invention may be any compound that binds to the peptide or polypeptide described herein, e.g., peptides, polypeptides, nucleic acids, or small molecules. Preferred conjugates include antibodies, nucleic acids, peptides, or polypeptides, e.g., receptors or binding partners for the peptide or polypeptide, and fragments thereof containing a binding domain to the peptide, as well as aptamers, e.g., nucleic acid or peptide aptamers. Methods for preparing such conjugates are well known in the art. For example, the identification and production of suitable antibodies or aptamers are also provided by commercial suppliers. Those skilled in the art are familiar with methods for developing derivatives of such conjugates having higher affinity or specificity. For example, random mutations can be introduced into nucleic acids, peptides, or polypeptides. These derivatives can then be tested for binding according to screening procedures known in the art, e.g., phage display. The antibodies referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab, and F(ab)2 fragments capable of binding to antigens. 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 antigen-binding amino acid residues of the donor, but may also include other structurally and / or functionally relevant amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. Preferably, the binder or drug specifically binds to a peptide or polypeptide. Specific binding according to the present invention means that the ligand or drug does not substantially bind ("cross-react") to another peptide, polypeptide, or substance present in the sample being analyzed.Preferably, the specifically bound peptide or polypeptide should be bound 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 relevant peptide or polypeptide in the sample. Non-specific binding may be acceptable if it can still be clearly distinguished and measured, for example, by its size on a Western blot or by its relatively high abundance in the sample. The binding of the binder can be measured by any method known in the art. Preferably, the above method is semi-quantitative or quantitative. More preferred techniques for determining the polypeptide or peptide are described below.

[0076] The binding of the binder may be measured directly, for example, by NMR or surface plasmon resonance. According to a preferred embodiment, the measurement of binder binding is performed by an analytical instrument unit of the system disclosed herein. The measured binding level can then be calculated by a computing device of the system disclosed herein. If the binder also functions as a substrate for the enzymatic activity of the peptide or polypeptide of interest, the enzymatic reaction product may be measured (e.g., the level of protease can be measured by measuring the level of the cleaved substrate, for example, by Western blotting). Alternatively, the binder itself may exhibit enzymatic properties, and a "binder / peptide or polypeptide" complex or a binder to which a peptide or polypeptide is bound may be contacted with a suitable substrate and made detectable by generating an intensity signal. Preferably, the substrate level is saturated for the measurement of the enzymatic reaction product. The substrate may also be labeled with a detectable label before the reaction. Preferably, the sample is in contact with the substrate for an appropriate period of time. An appropriate period refers to the time required for a detectable, preferably measurable, level of product to be produced. Instead of measuring the level of the product, the time required for the appearance of a given (e.g., detectable) level of product may be measured. Thirdly, the conjugate may be covalently or acovalently bound to a label that enables the detection and measurement of the conjugate. Labeling may be carried out directly or indirectly. Direct labeling is carried out by directly (covalently or acovalently) binding the label to the conjugate. Indirect labeling is carried out by binding a second conjugate to the first conjugate (covalently or acovalently). The second conjugate shall bind specifically to the first conjugate. The second conjugate may be a target (receptor) for a third conjugate that binds to a suitable label and / or to the second conjugate. To enhance the signal, secondary, tertiary, or higher-order conjugates are often used. Suitable secondary and higher-order conjugates may include antibodies, secondary antibodies, and well-known streptavidin-biotin systems (Vector Laboratories, Inc.).The binder or substrate may also be “tagged” with one or more tags known in the art, thereby allowing such tags to become targets for higher-order binders. Preferred tags include biotin, digoxygenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose-binding protein, etc. In the case of peptides or polypeptides, the tag is preferably located at the N-terminus and / or C-terminus. A preferred 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. Enzymatically active labels include, for example, horseradish peroxidase, alkaline phosphatase, β-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 Bio-sciences), and ECF® (Amersham Bio-sciences). Depending on the enzyme-substrate combination, a colored reaction product, fluorescence, or chemiluminescence may be produced, which can be measured by methods known in the art (e.g., using photosensitive film or a suitable camera system). The criteria described above also apply to measuring 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 being considered.Radioactive labels can be detected by any known and appropriate method, such as a photosensitive film or a phosphor imager.

[0077] The level of peptides or polypeptides can also preferably be determined as follows: (a) contacting a sample containing a peptide or polypeptide with a solid support containing a binder for peptides or polypeptides as specified above, and (b) measuring the level of peptides or polypeptides bound to the support. Preferably, the binder, selected from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, is present on the solid support, preferably in an immobilized form. Materials for producing supports are well known in the art and include, among others, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracyte, reaction tray wells and walls, plastic tubes, etc. Binders or agents can be bound to many different supports. Examples of well known supports include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The properties of the support may be either soluble or insoluble for the purposes of the present invention. Suitable methods for immobilizing / fixing the above-mentioned binder are well known and not limited to, but include ionic, hydrophobic, and covalent interactions. It is also intended to use a "suspension array" as the array according to the present invention (No-lan 2002, Trends Biotechnol. 20(1):9-12). In such a suspension array, a support, such as microbeads or microspheres, is present in the suspension. The array consists of different microbeads or microspheres, which are optionally labeled and carry different binders. Methods for producing such arrays based, for example, on solid-phase chemistry and photodegradable protecting groups are generally known (U.S. Patent No. 5,744,305).

[0078] As used herein, the term “detector” refers to a drug capable of specifically recognizing and binding to a biomarker present in a sample. This term is interchangeable with the term “ligand” as used herein. Furthermore, the drug enables direct or indirect detection of a complex formed by the drug and the biomarker. Direct detection can be achieved by including a detectable label in the drug. Indirect labeling can be achieved by a further drug that specifically binds to the complex containing the biomarker and the detector, in which case the further drug can generate a detectable signal. Suitable compounds that can be used as detectors are also well known in the art. Preferably, the detector is an antibody, particularly a monoclonal antibody or aptamer, that specifically binds to the biomarker referred to herein. As used herein, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit the desired antigen-binding activity. Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.

[0079] Where used herein, the terms “level” or “quantity” encompass the absolute quantity of a biomarker referred to herein, the relative quantity or concentration of such biomarker, and any values ​​or parameters that correlate 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 peptide by direct measurement, e.g., intensity values ​​in mass spectra or NMR spectra. Furthermore, they encompass all values ​​or parameters obtained by indirect measurements as explicitly stated elsewhere herein, e.g., response quantities measured by a biological readout system in response to a peptide, or intensity signals obtained from a specifically bound ligand. It should be understood that values ​​correlated with the quantities or parameters described above can also be obtained by all standard mathematical operations.

[0080] The “signs” or “signals” of a disease include, but are not limited to, the presence or absence, increase or rise, decrease or decline, or other change or alteration of specific indicators such as biomarkers or molecular markers, or the onset, presence or worsening of symptoms.

[0081] The terms “disease” and “disorder” are used interchangeably herein and refer to an abnormal medical condition, particularly a disease or injury, in which a tissue, organ, or organism is no longer able to perform its function efficiently. Typically, though not necessarily, a disease is associated with certain symptoms or signs that indicate the presence of such a disease. The presence of such symptoms or signs may indicate a tissue, organ, or organism that is diseased. Changes in these symptoms or signs may indicate the progression of such a disease. The progression of a disease is typically characterized by an increase or decrease in such symptoms or signs, which may indicate a “worsening” or “improvement” of the disease. A “worsening” of a disease is characterized by a decrease in the ability of a tissue, organ, or organism to perform its function efficiently, while a “improvement” of a disease is typically characterized by an increase in the ability of a tissue, organ, or organism to perform its function efficiently.

[0082] Heart failure (HF), also known as congestive heart failure (CHF), is a syndrome characterized by a group of signs and symptoms caused by impaired blood pumping function of the heart. Symptoms typically include shortness of breath, excessive fatigue, and leg swelling. Shortness of breath can occur during exercise or while lying down and may wake a person at night. Chest pain, including angina, is not usually caused by heart failure but can occur if heart failure is caused by a heart attack. The severity of heart failure is measured by signs or symptoms at rest or during exercise or training. Other conditions that may have signs and symptoms similar to heart failure include obesity, renal failure, liver disease, anemia, and thyroid disease.

[0083] The term “acute heart failure” is well known in the art and is described, for example, in the 2021 Heart Failure ESC Guidelines (Authors / Task Force Members, McDonagh et al., Reference 17). Typically, the term AHF refers to the rapid or progressive onset of symptoms and / or signs of HF that are severe enough to cause a patient to seek emergency medical attention leading to unplanned hospitalization. Patients with AHF require emergency evaluation, and AHF is a leading cause of hospitalization in subjects aged over 65 years and is associated with high mortality and readmission rates.

[0084] Subjects may be referred to as patients herein. The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, a patient is a human patient. A subject may be symptomatic (e.g., a subject exhibits symptoms associated with HF) or asymptomatic (e.g., a subject does not exhibit symptoms associated with HF). A subject may be diagnosed with HF, at risk of developing HF, or exhibit symptoms of HF. A subject may have or be suspected of having HF (e.g., exhibit symptoms or a history that suggests or indicates HF).

[0085] Therefore, in some examples, the subject has HF (and the method diagnoses, identifies (or detects) that the subject has HF). In this regard, the terms “diagnose,” “identify,” and “detect” can be used interchangeably.

[0086] Detecting elevated levels of cardiac-related (poly)peptide biomarkers, particularly BNP-type peptides, in a subject confirms suspicion of HF and indicates a high risk of HF. Especially if the subject already exhibits clinical parameters, signs, and / or symptoms of HF, determining elevated levels of cardiac-related (poly)peptide biomarkers confirms the presence of HF.

[0087] The term "compare," as used herein, refers to comparing the amount / level of a biomarker in an ISF (sample) from a subject with a reference amount or reference value of the biomarker as specified elsewhere herein. It should be understood that, as used herein, comparison typically refers to a comparison of corresponding parameters or values; for example, an absolute amount is compared with an absolute reference amount, while a concentration is compared with a reference concentration, or an intensity signal obtained from a biomarker in a sample is compared with an intensity signal of the same type obtained from a reference sample. Comparisons may be performed manually or with computer assistance. Therefore, comparisons may be performed by a computing device. The measured or detected amount of a biomarker in a sample from a subject, and the value for the reference amount, can, for example, be compared with each other, and this comparison may be performed automatically by a computer program that executes an algorithm for comparison. The computer program performing the evaluation provides the desired evaluation in an appropriate output format. In a computer-assisted comparison, the measured value may be compared by the computer program with a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the comparison results, i.e., automatically provide the desired evaluation in a suitable output format. In computer-assisted comparisons, the value of a measured quantity may be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the comparison results, that is, it may automatically provide a desired evaluation in a suitable output format.

[0088] The terms “(appropriate) reference value / reference level,” “reference sample,” or “control (sample),” as used herein, refer to a sample that is analyzed in substantially the same manner as the sample of interest, and whose information is compared to the information of the sample of interest. The reference sample provides a standard that enables the evaluation of the information obtained from the sample of interest. A control sample may originate from a healthy individual and thereby provide a standard of healthy condition. Differences between the state of a normal reference sample and the state of the sample of interest may indicate the presence or further progression of HF. Differences between the state of a normal or abnormal reference sample and the state of the sample of interest may indicate the absence or improvement of HF.

[0089] Reference samples / values / levels may also originate from the same subject as the sample / value of interest, but are collected at an earlier point in time. The difference between the state of the earlier reference value / sample and the state of the value / sample of interest may indicate the progression of HF, i.e., improvement or deterioration of HF over time, which may optionally allow for the classification of the stage of HF as stage I, II, III, or IV according to the New York Cardiology Association (NYHA) functional classification, if classification is desired (see Table 4 in Reference 17). The stage of HF may also be described by the ACC / AHA staging classification, which emphasizes the onset and progression of the disease (see Figure 1 and Table 3 in Reference 22).

[0090] The determined value can be compared to multiple (appropriate) reference values, which may be of different types. For example, the determined value can be compared to one or more values ​​obtained from the same subject at an earlier point in time, and in parallel, to one or more values ​​obtained from other subjects (e.g., those with HF at a known stage).

[0091] Control samples may be internal or external control samples. Using internal control samples, i.e., evaluating biomarker levels in the test sample and one or more other samples taken from the same subject to determine if there is a change in the marker level. Alternatively, a measurement taken at an earlier point in time in the subject may serve as a control sample for a measurement taken at a later point in time in the same subject. For external control samples, the presence or amount of the marker in a sample originating from an individual or measured in the subject is compared to the presence or amount of the marker in individuals known to have or be at risk of having a given condition, or individuals known to be free from the given condition, i.e., "normal individuals."

[0092] Those skilled in the art will understand that such external control samples may be obtained from a single individual or from a reference population that is age-matched and free from confounding diseases. Typically, a “reference value” is established using samples from 100 well-characterized individuals from a suitable reference population. However, the reference population may also be selected to consist of 20, 30, 50, 200, 500, or 1000 individuals. Healthy individuals represent a preferred reference population for establishing control values.

[0093] For example, the marker concentration in a patient sample may be compared to a concentration known to be associated with a specific course of a particular disease. For instance, it may be compared to a concentration known to be associated with a specific stage of HF. Typically, the marker concentration in a sample correlates directly or indirectly with the diagnosis, and is used, for example, to determine whether an individual is at risk of developing the disease. Alternatively, the marker concentration may be compared to the concentration of a marker obtained from the same subject at an earlier point in time. Or, the marker concentration in a sample may be compared to a marker concentration known to be relevant, for example, in the response to treatment in a particular disease, in the diagnosis of a particular disease, in the assessment of the severity of a particular disease, in providing guidance for selecting appropriate drugs for a particular disease, in determining the risk of disease progression, or in patient follow-up. Depending on the intended diagnostic application, an appropriate control sample is selected, in which control or reference values ​​for the marker are established. As will be apparent to those skilled in the art, the absolute marker values ​​established in the control sample depend on the assay used.

[0094] The most common control samples and / or reference values ​​derived therefrom for the methods described herein are obtained from “healthy controls,” but are not limited to these. The corresponding subjects from which these samples are obtained are, each, “healthy subjects.”

[0095] A “healthy control” refers to a control sample of a subject that does not have HF and does not exhibit any symptoms or signs that may be associated with HF, such as active or supine dyspnea, fatigue and weakness, swelling of the legs, ankles and feet, rapid or irregular heartbeat, decreased exercise capacity, persistent cough or wheezing with white or pink bloody mucus, abdominal swelling, very rapid weight gain due to fluid retention, nausea and loss of appetite, difficulty concentrating or decreased alertness, or chest pain if heart failure is caused by a heart attack.

[0096] Control samples can be assayed simultaneously with the test sample / subject, before or after, separately or simultaneously. The control value used for comparison with the test sample / subject may be calculated as the mean or median of multiple (e.g., 2 or more, 5 or more, 10 or more, groups, etc.) control samples. Alternatively, the control sample may be a sample derived from multiple (e.g., 2 or more, 5 or more, 10 or more, groups, etc.) individuals that are not affected by HF (i.e., a mixture).

[0097] For the classification of HF stages, comparisons between test samples and several different control samples may or should be made so that results can be assigned to a specific stage. For example, a comparison between healthy samples and NYHA class II samples, or a comparison between healthy samples and NYHA class II and NYHA class IV samples. This can also be combined with surgery or other interventions to confirm or determine a specific stage of HF.

[0098] Alternatively, the level of a (poly)peptide biomarker in ISF may be compared to a predetermined reference level of the biomarker of interest. As used herein, “predetermined reference level” refers to a level of the biomarker obtained from a reference database that can be used to generate a predetermined cutoff value, i.e., a score for statistically predicting HF. In one example, the predetermined reference level is the mean or median level of the biomarker in at least one HF-free individual of the same species. The predetermined reference value may be calculated as the mean or median obtained from a group or population of HF-free individuals. The individuals or population of individuals may be of the same age or the same health status or conditions as those from which the test sample is obtained.

[0099] Therefore, in one example, the given reference level is the average level of the biomarker in a control group that does not have HF.

[0100] Typically, in a method for diagnosing / evaluating HF in a subject, the control sample or designated reference is obtained from a different individual or population than the subject being tested (i.e., the subject from which biomarker levels are determined or from which test samples are obtained / provided). In such cases, the control or designated reference is used as a bench line to determine whether the subject being tested has HF.

[0101] In alternative examples, controls or specified reference values ​​may be obtained from the same individual as the test sample, but at an earlier point in time. This is particularly relevant to the methods described herein for classifying the stage of HF, determining progression in a subject, determining the therapeutic effect of a treatment regimen for HF, and / or determining the compliance or adherence of a subject to a prescribed treatment regimen for HF. For this purpose, measurements in ISF are performed in the same subject, or ISF samples are taken from the same subject.

[0102] In such cases, a control sample or a predetermined baseline level is used to determine any change in the level of a biomarker over a time interval in the same subject. Thus, the predetermined baseline level or control sample can be obtained from the same subject from which the test sample was obtained, for example, at an earlier point in time. This earlier point in time may be before HF was diagnosed.

[0103] The predetermined level may be a single cutoff value, such as the median or mean. This can be a range of cutoff (or threshold) values, such as a confidence interval. This can be established based on a comparison group, such as when the risk of one defined group is twice as high or low as the risk of another defined group (e.g., about twice, four times, eight times, sixteen times, or more). It may be a range where, for example, the population of subjects (e.g., control subjects) is divided equally (or not equally) into groups such as low-risk, medium-risk, and high-risk groups, or into quartiles, with the lowest quartile being the lowest-risk subjects and the highest quartile being the highest-risk subjects, or into n-quartiles (i.e., n equally spaced intervals), with the lowest n-quartile being the lowest-risk subjects and the highest n-quartile being the highest-risk subjects. Furthermore, the reference may be a calculated reference, most preferably the mean or median, for the relative or absolute amount of a biomarker in a population of individuals containing the subjects under investigation. How to calculate a suitable reference value, preferably the mean or median, is well known in the art.

[0104] Therefore, in some cases, a biomarker level in a subject that is above the level of the biomarker in a control sample or a predetermined reference level indicates a clinical condition (e.g., HF). In other cases, a biomarker level in a subject that is below the level of the biomarker in a control sample or a predetermined reference level indicates a specific stage of HF.

[0105] Typically, but not always, being large or small enough to distinguish a subject from a control subject means being statistically significant in magnitude greater or statistically significant less. When the level of a biomarker in a subject being equal to the level of a biomarker in a control subject indicates the stage of HF, "equal" means approximately equal (e.g., no statistical difference).

[0106] The predetermined values ​​may depend on the specific population of the selected subjects (e.g., human subjects). For example, a population that appears healthy will have a different range of "normal" biomarkers than a population of subjects that have or are likely to have HF. Therefore, the selected predetermined values ​​may take into account the category (e.g., healthy, diseased, disease stage) to which the subjects (e.g., human subjects) belong.

[0107] The appropriate range and category can be selected solely through routine experiments by those skilled in the art.

[0108] Appropriately, the level of a specific biomarker detected in a subject / sample (e.g., test sample, control sample) can be normalized by adjusting the measured level (quantity or activity) of the biomarker using the level of a reference protein in the same sample, where the reference protein is not the marker itself (it is, for example, a constitutively expressed protein). This normalization allows for comparison of the level of a biomarker in one sample with that of another sample, or between samples from different sources. This normalized level can then be optionally compared with a reference value or control. For example, when measuring a protein biomarker in a whole blood sample, the biomarker may be expressed as an absolute concentration, or it may be normalized against a known protein constitutively expressed in whole blood, such as albumin, immunoglobulin, or plasma protein concentration.

[0109] For example, when measuring a protein biomarker in a serum (or plasma) sample, the biomarker may be expressed as an absolute concentration, or it may be normalized to a known protein constitutively expressed in serum (or plasma).

[0110] To identify an increase or decrease in the level of one or more biomarkers in a sample, the level of a biomarker in the test sample can be compared to the level of the same biomarker or a predetermined baseline level for the same biomarker in a control sample.

[0111] In the methods described herein, a subject may be identified as having HF if a comparison (comparison between the level of a biomarker at a control subject / sample / predetermined reference value and the test subject / sample of the subject) shows that the subject has an increased level of BNP-type peptide compared to the control sample or the predetermined reference level.

[0112] As will be understood by those skilled in the art, evaluations performed according to the present invention are preferable, but may not be accurate for 100% of the subjects investigated. This term typically requires that the statistically significant portion of the subjects can be accurately evaluated. Whether a portion is statistically significant can be further easily determined by those skilled in the art using various well-known statistical evaluation 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 and 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.

[0113] The term “reduced” or “decreased” level of an indicator refers to the level of such indicator in a sample that is reduced compared to a reference (value) or reference sample. The terms “reduced,” “decreased,” “reduced,” “reduced,” or “down-controlled” or “lower” are all used herein to generally mean a reduction of a statistically significant amount. However, to avoid misunderstanding, “reduced,” “decrease,” or “decrease” means a decrease of at least 10% compared to the baseline / control level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a decrease of 100% or less (i.e., a level that does not exist compared to the reference / control sample), or any decrease between 10% and 100% compared to the baseline / control level, or a decrease of at least about 0.5 times, or at least about 1.0 times, or at least about 1.2 times, or at least about 1.5 times, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any decrease of 1.0 times to 10 times or more compared to the baseline / control level.

[0114] The term "elevated" or "increased" level of an indicator / (bio)marker refers to a higher level of such indicator in a sample compared to a reference value or reference sample. For example, a protein that is detectable in a higher amount in a fluid sample from an individual with a given disease than in the same fluid sample from an individual without the disease has an elevated level. The terms “increased,” “boosted,” “upgraded,” or “higher” are all used herein to generally mean an increase of a statistically significant amount, and to avoid any misunderstanding, the terms “increased,” or “boosted” mean an increase of at least 10% compared to the baseline / control, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of 100% or less, or any increase between 10% and 100% compared to the baseline / control, or an increase of at least about 0.5 times, or at least about 1.0 times, or at least about 1.2 times, or at least about 1.5 times, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase of 1.0 times to 10 times or more compared to the baseline / control.

[0115] Embodiment To our surprise, we have identified ISF as a reliable tool for determining the levels of cardiac-related (poly)peptide biomarkers, particularly BNP-type peptides such as NT-proBNP.

[0116] ISF can be used to assess heart failure, determine the risk of developing heart failure, classify the stage of heart failure, monitor heart failure, and / or determine the therapeutic effect of treatment in a subject. Furthermore, determining the levels of one or more cardiac-related (poly)peptide biomarkers in ISF provides a rapid, easy, and reliable approach to assessing HF.

[0117] ISF as a body fluid for determining cardiac-related biomarker levels may, advantageously, be used in any of the methods, kits, or uses described herein.

[0118] A method for evaluating HF in a subject or determining the risk of developing HF. In one embodiment, the present invention relates to a method for evaluating heart failure (HF) in a subject or determining the risk of developing HF, wherein the method is a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the interstitial fluid (ISF) of a subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers with the level of the same cardiac-related (poly)peptide biomarkers in a control sample, or with a predetermined reference level of the same cardiac-related biomarkers, c) A step of identifying whether the subject has heart failure or has an increased risk of developing heart failure, based on the comparison in step b) Includes.

[0119] This method may be in vivo, ex vivo, or in vitro. Preferably, this method is an in vivo method.

[0120] As used herein, the terms “biological (fluid) sample,” “test sample,” and “sample” are interchangeable, and variations thereof refer to a sample obtained from or derived from a subject. For the purposes described herein, a sample is an ISF sample or includes an ISF sample. In particular, since the methods described herein may also be in vivo methods, these terms may also refer to “sample” in the sense that it represents a value obtained by in vivo measurement, determination, without actually obtaining a sample transferred from the body of the subject.

[0121] ISF can be any type of ISF, preferably subcutaneous ISF, i.e., cutaneous ISF. Cutaneous ISF refers to ISF within the skin layers of the epidermis, dermis, and subcutaneous tissue.

[0122] In certain embodiments, the subject is a human being.

[0123] This method may be performed based on the suspicion that a subject may have HF or may be at increased risk of developing HF. Alternatively, cardiac-related (poly)peptide biomarker levels may be routinely determined as part of a screening test without any suspicion of HF.

[0124] Patients at risk of developing HF are not healthy and have comorbid conditions (Stage A = risk factors) or cardiac abnormalities without signs / symptoms of HF that could lead to the development of HF (Stage B). This corresponds to Stages A and B of the ACC / AHA classification (see Figure 1 and Table 3 in Reference 22).

[0125] Patients with diabetes are at risk of developing HF, but are not healthy. The same applies to all risk factors associated with a higher risk of arterial hypertension and HF. For a list of risk factors for developing HF, see Table 10 in Reference 17.

[0126] In the embodiment, elevated levels of one or more cardiac-related (poly)peptide biomarkers in the subject's ISF, compared to a control sample or a predetermined reference level, indicate a subject with HF or an increased risk of developing HF.

[0127] In the embodiment, the levels of one, two, three, or four cardiac-related (poly)peptide biomarkers are determined.

[0128] In particular, an increase of 25% or more in the level of cardiac-related (poly)peptide biomarkers in ISF indicates the presence of HF or the risk of developing HF. In particular, an increase of 30% or more in the level of cardiac-related (poly)peptide biomarkers in ISF indicates the presence of HF. In particular, an increase of 50% or more in the level of cardiac-related (poly)peptide biomarkers in ISF indicates the presence of HF. In particular, an increase of 100% or more in the level of cardiac-related (poly)peptide biomarkers in ISF indicates the presence of HF. In particular, an increase of 150% or more in the level of cardiac-related (poly)peptide biomarkers in ISF indicates the presence of HF. In particular, an increase of 200% or more in the level of cardiac-related (poly)peptide biomarkers in ISF indicates HF.

[0129] Ideally, cardiac-related (poly)peptide biomarkers can be BNP-type peptides.

[0130] Suitablely, the cardiac-related (poly)peptide biomarker may be pre-proBNP, proBNP, NT-proBNP, and / or BNP. Preferably, the biomarker is NT-proBNP and / or BNP. More preferably, the biomarker is NT-proBNP.

[0131] This application demonstrates a nearly 1:1 correlation between NT-proBNP in serum and NT-proBNP in intravascular filtration (ISF). This makes it possible to consider NT-proBNP levels detected in ISF as a reliable biomarker for cardiac-related disorders / diseases.

[0132] Due to the near 1:1 correlation between NT-proBNP in serum and NT-proBNP in ISF, NT-proBNP is not only a suitable biomarker for ISF, but this correlation even allows for the application of already known protocols, treatment regimens, or other appropriate measures for corresponding NT-proBNP levels in serum, while remaining known and established.

[0133] Appropriately, in addition to the levels of one or more cardiac-related (poly)peptide biomarkers, glucose, creatinine, potassium, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, hsCRP (high-sensitivity C-reactive protein), cancer antigen 125 (or carbohydrate antigen 125, CA125), soluble The levels of one or more additional cardiac-related biomarkers selected from the group consisting of differentiation cluster 146 (or cell surface glycoprotein MUC18, sCD146), bioadrenomedullin (bioADM), central region proadrenomedullin (MR-proADM), soluble tumorigenesis inhibitor 2 (sST2), angiopoietin 2 (Ang-2), fibroblast growth factor 23 (FGF-23), BMP-10 (bone morphogenetic protein 10), ESM-1 (endothelial cell-specific molecule 1), and insulin-like growth factor-binding protein 7 (IGFBP7) can be determined.

[0134] In addition to BNP-type peptide biomarkers, determining the levels of one or more cardiac-related biomarkers can provide further information about the subject's condition and the state of HF / HF progression. This can enable more accurate and reliable assessments.

[0135] In multiple embodiments, the levels of one, two, three, or four cardiac-related (poly)peptide biomarkers include glucose, creatinine, potassium, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, hsCRP (high-sensitivity C-reactive protein), cancer antigen 125 (or carbohydrate antigen 125, CA125), and soluble This is determined along with one, two, three, four, or more additional biomarkers selected from the group consisting of differentiation cluster 146 (or cell surface glycoprotein MUC18, sCD146), bioadrenomedullin (bioADM), central region proadrenomedullin (MR-proADM), soluble tumorigenesis inhibitor 2 (sST2), angiopoietin 2 (Ang-2), fibroblast growth factor 23 (FGF-23), BMP-10 (bone morphogenetic protein 10), ESM-1 (endothelial cell-specific molecule 1), and insulin-like growth factor-binding protein 7 (IGFBP7).

[0136] When determining the levels of one, two, three, or four cardiac-related (poly)peptide biomarkers along with one, two, three, four, or even more additional biomarkers, the following combinations of determinations may be made, preferably to improve the diagnostic outcome: - Cardiac-related (poly)peptide biomarkers alone - Cardiac-related (poly)peptide biomarkers in combination with sodium and / or potassium (e.g., for monitoring the progression and treatment response of HF) - Cardiac-related (poly)peptide biomarkers combined with sodium and / or potassium and / or additional biomarkers (e.g., glucose or creatinine) - Cardiac-related (poly)peptide biomarkers combined with sodium and / or potassium and / or further biomarkers (e.g., glucose or creatinine) and / or further clinical information of the subject (e.g., further known diseases, age, sex, weight, size of the subject).

[0137] In one example, NT-proBNP and creatinine levels are determined.

[0138] In one example, NT-proBNP and potassium levels are determined.

[0139] In one example, NT-proBNP, creatinine, and potassium levels are determined.

[0140] A preferred combination for monitoring patients with HF includes cardiac-related (poly)peptide biomarkers as well as serum sodium and serum potassium.

[0141] A preferred combination for assessing congestion includes cardiac-related (poly)peptide biomarkers having glucose, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, sST2 (soluble ST2), hsCRP (high-sensitivity C-reactive protein), cancer antigen 125 (or carbohydrate antigen 125), angiopoietin 2, BMP-10 (bone morphogenetic protein 10), FGF-23 (fibroblast growth factor 23), ESM-1 (endothelial cell-specific molecule 1), and / or IGFBP7.

[0142] Appropriately, heart failure (HF) may be selected from the group consisting of acute heart failure, reduced ejection fraction heart failure (HFrEF), mild reduced ejection fraction heart failure (HFmrEF), preserved ejection fraction heart failure (HFpEF), left heart failure, right heart failure, and biventricular failure.

[0143] Appropriately, the method may further include a step of selecting a treatment regimen for a subject based on a comparison of the level of a cardiac-related (poly)peptide biomarker with a control sample or a predetermined reference level.

[0144] In certain embodiments, the method further comprises administering a selected treatment regimen, which optionally includes drug-based therapies and / or surgeries.

[0145] Procedures may include, for example, surgery and, in some cases, treatment, or a combination thereof. However, in some cases, immediate action may not be necessary, and the subject may be selected for active monitoring.

[0146] As used herein, the terms “active monitoring,” “monitoring,” and “careful observation” are interchangeable herein and mean closely monitoring the patient’s condition without taking any action until symptoms appear or change.

[0147] As used herein, the terms “to treat,” “to treat,” and “treatment” are interpreted to include interventions made with the intention of altering the condition, disorder, or symptom (i.e., in this case, HF). Thus, “treatment” refers to a therapeutic treatment aimed at slowing (reducing) the targeted condition, disorder, or symptom. Thus, “treatment” includes a reduction, delay, or inhibition of the symptoms of HF compared to the symptoms before treatment, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0148] In the context of HF, appropriate treatment may include drug therapy and / or surgery.

[0149] As used herein, the term “surgery” applies to surgical procedures such as, for example, angioplasty (also known as percutaneous coronary intervention [PCI], balloon angioplasty, and balloon coronary artery dilation), artificial heart valve surgery (also known as heart valve replacement surgery), atherectomy, bypass surgery (CABG performed via open heart surgery, i.e., also known as coronary artery bypass graft), cardiac reconstruction, heart transplantation, minimally invasive cardiac surgery (also known as limited-access coronary artery surgery, including port-access coronary artery bypass (PACAB or PortCAB) and minimally invasive coronary artery bypass graft (MIDCAB or minimally invasive CABG)), radiofrequency ablation (also known as catheter ablation), stent placement, and transmyocardial revascularization (TMR).

[0150] As used herein, the term “treatment” may include drug-based therapies, radiation, hormone therapy, cryosurgery, chemotherapy, immunotherapy, biological therapies, and high-intensity focused ultrasound.

[0151] Drug-based therapies for HF may include, for example, the administration of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors (or angiotensin receptor blockers [ARBs] if the patient is intolerant to ACE inhibitors) or angiotensin receptor-neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists, sodium-glucose cotransporter 2 inhibitors, loop diuretics, If channel inhibitors, soluble guanylate cyclase receptor agonists, hydralazine and isosorbide dinitrate, digoxin, and ferric carboxymaltose.

[0152] The type of treatment varies depending on the specific form and / or stage of HF that the subject has or is suspected to have. Depending on whether the assessment suggests a rather severe disease stage, those skilled in the art are well aware of how to select the most appropriate and promising treatment regimen.

[0153] Definitions of cardiac-related (poly)peptide biomarkers, combinations with further biomarkers, methods, subjects, types of HF, treatments, reference values, time intervals, etc., provided elsewhere in this specification, apply equally here. Definitions, embodiments, examples, etc., provided herein for one aspect of the present invention apply equally to all other aspects of the present invention. Unless otherwise apparent from the context, each embodiment listed above may be applicable to use in any aspect of the present invention.

[0154] Methods for monitoring HF in a target area In one aspect, the present invention relates to a method for monitoring heart failure in a subject, wherein the method is i. Optionally, a step of determining the level of one or more cardiac-related (poly)peptide biomarkers in interstitial fluid (ISF) from a subject according to steps a) to b) of the method of claim 1, ii. A process in which process i is repeated after a certain time interval, iii. A step of comparing the level of the cardiac-related (poly)peptide biomarker identified in i with the level identified in ii, wherein the change in level from i to ii indicates a change in heart failure in the subject. Includes.

[0155] In particular, the easy accessibility of the ISF allows for continuous monitoring of subjects without the need for specialized assistance. Measurements within the ISF can be performed by the subject, for example, using a batch with a needle-type sensor applied to the subject's skin. Such batches are known, for example, for glucose measurement in diabetic patients. This allows patients to monitor biomarker levels over a long period at desired time intervals, rather than requiring assistance from a doctor or specialist, during their daily lives.

[0156] In various embodiments, patients with HF are monitored to determine whether the levels of one or more cardiac-related (poly)peptide biomarkers in interstitial fluid (ISF) from the subject have changed over time. In particular, patients with HF are monitored to determine whether the levels of one or more cardiac-related (poly)peptide biomarkers in the ISF from the subject have increased, decreased, or remained unchanged over time. In embodiments, patients with HF are monitored if elevated levels of one or more cardiac-related (poly)peptide biomarkers in the interstitial fluid (ISF) from the subject are detected.

[0157] This method may be used to monitor the progression of any type of HF described herein.

[0158] Such monitoring methods may be implemented for subjects who have not yet received HF treatment or for subjects who have already received HF treatment. Such methods may be implemented for patients before, during, or after (re)hospitalization.

[0159] Monitoring the progression of a subject's HF over time helps identify the progression of HF (e.g., worsening of disease status or symptoms) as early as possible. Such monitoring naturally involves repeated sampling over time. Thus, the method can be repeated for a particular subject at one or more time intervals, and the results can be compared to monitor the onset, progression, or improvement of HF in that subject over time, and changes in the levels of biomarkers tested in ISF indicate changes in the progression of the subject's HF.

[0160] The progression of HF may be indicated by an increase in the levels of cardiac-related (poly)peptide biomarkers detected over time when comparing results from two or more time intervals for the same subject.

[0161] In other words, if the method is performed multiple times, progression of HF may be indicated when the levels of cardiac-related (poly)peptide biomarkers detected at later time intervals are higher than those detected at earlier time intervals. An "increase" in cardiac-related (poly)peptide biomarker levels includes the detection of cardiac-related (poly)peptide biomarkers at time intervals after the method was previously performed on the same subject (i.e., at earlier time intervals) in which the cardiac-related (poly)peptide biomarkers were not detected (i.e., were not present at detectable levels). This is particularly relevant when monitoring the progression of HF in subjects suspected of developing HF based on other signs or symptoms.

[0162] Appropriate time intervals for monitoring the progression of HF can be readily determined by those skilled in the art and depend on the specific form of HF being monitored. As a non-limiting example, the method can be repeated at least daily, weekly, monthly, every six months, or at least annually, or whenever clinically necessary, i.e., in the event of a significant change in the symptoms of HF.

[0163] The frequency of monitoring the progression of HF depends on the available medical environment and the severity of the disease. In some cases, such as patients hospitalized with acute HF and treated with intravenous diuretics, a reasonable frequency may be every 2–6 hours or every 2–12 hours.

[0164] For regular monitoring, monitoring may be performed at regular intervals. As used herein, the term “at regular intervals” refers to the periodic determination of cardiac-related (poly)peptide biomarker levels in the same subject after a predetermined time interval. Since all subjects are different, the rate of HF onset can vary from subject to subject. In some subjects, HF progression may progress within a few years (e.g., from stage I to stage IV), while in others, it may stagnate in one stage for several years. Therefore, intervals should be selected so as not to skip stages of disease progression, so that a sequence of cardiac-related (poly)peptide biomarker levels in a subject can be obtained and the obtained cardiac-related (poly)peptide biomarker levels can be assigned to the corresponding disease stages I, II, III, and IV. Preferably, cardiac-related (poly)peptide biomarker levels in a subject are determined every 2, 4, 6, 8, 10, 11, or 12 weeks.

[0165] The most important determinants of monitoring frequency are when the subject rapidly becomes decompensated and presents with signs and symptoms of acute HF (rapid breathing, feeling of choking, difficulty breathing while lying down, chest tightness, arrhythmia, chest pain, cough, fluid accumulation in the arms or legs (edema)), or after discharge for acute HF.

[0166] In such cases, shorter intervals between monitoring times should be selected. Those skilled in the art will be well aware that the appropriate frequency should be chosen according to the individual scenario.

[0167] In patients with stable HF, weekly or monthly monitoring may be sufficient.

[0168] Definitions of cardiac-related (poly)peptide biomarkers, combinations with further biomarkers, methods, subjects, types of HF, treatments, reference values, time intervals, etc., provided elsewhere in this specification, apply equally here. Definitions, embodiments, examples, etc., provided herein for one aspect of the present invention apply equally to all other aspects of the present invention. Unless otherwise apparent from the context, each embodiment listed above may be applicable to use in any aspect of the present invention.

[0169] A method for classifying the stage of HF in the subject. In one embodiment, the present invention relates to a method for classifying the stage of heart failure in a subject, wherein the method is a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the interstitial fluid of a subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has elevated or decreased levels of one or more cardiac-related (poly)peptide biomarkers compared to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, then the step of classifying the stage of heart failure in the subject and Includes.

[0170] In embodiments, an increase or decrease in the level, amount, or concentration of a cardiac-related (poly)peptide biomarker in the target ISF can indicate the stage of the target HF. In particular, if the level of the cardiac-related (poly)peptide biomarker in the target ISF is higher or lower than the level of the cardiac-related (poly)peptide biomarker based on known reference values ​​for the stage of HF, then the level of the cardiac-related (poly)peptide biomarker in the target ISF indicates the stage of the target HF. If necessary, comparisons with several reference values ​​are made to classify the stage of HF. Furthermore, this can be combined with surgery or other parameters used to determine the stage of HF to enable a much more accurate classification.

[0171] In particular, if cardiac-related (poly)peptide biomarkers are detectable at higher levels in ISF of subjects evaluated for the presence of HF than in ISF of subjects without HF, this indicates any stage of HF. By further comparing the obtained values ​​with reference values ​​for known stages of HF, the obtained values ​​can be assigned to a specific stage of HF.

[0172] Appropriately, the stage of the heart failure in question may be classified as Stage I, Stage II, Stage III, or Stage IV heart failure according to the New York Cardiology Association (NYHA) functional classification.

[0173] In certain embodiments, the control sample or a predetermined reference level is i. Levels of cardiac-related (poly)peptide biomarkers in healthy subjects or subjects with HF of stage I, stage II, stage III, or stage IV according to the New York Cardiology Association (NYHA) functional classification, or ii. The mean level of cardiac (poly)peptide biomarkers in a group of healthy subjects or in a group of subjects with HF of stage I, stage II, stage III, or stage IV according to the New York Cardiology Association (NYHA) functional classification. Alternatively, at least one appropriate reference value, iii. A specified level of cardiac-related (poly)peptide biomarker in a healthy subject or a subject with HF of stage I, stage II, stage III, or stage IV according to the New York Cardiology Association (NYHA) functional classification, or iv. A predetermined mean level of cardiac-related (poly)peptide biomarkers in a group of healthy subjects or in a group of subjects with HF of stage I, stage II, stage III, or stage IV according to the New York Cardiology Association (NYHA) functional classification.

[0174] For example, levels of cardiac-related (poly)peptide biomarkers obtained that are higher than those of healthy subjects but lower than those of subjects with stage II HF indicate stage I HF.

[0175] For example, a level of cardiac-related (poly)peptide biomarker obtained that is higher than that of subjects with stage I HF and higher than that of subjects with stage III HF indicates stage II HF.

[0176] Definitions of cardiac-related (poly)peptide biomarkers, combinations with further biomarkers, methods, subjects, types of HF, treatments, reference values, time intervals, etc., provided elsewhere in this specification, apply equally here. Definitions, embodiments, examples, etc., provided herein for one aspect of the present invention apply equally to all other aspects of the present invention. Unless otherwise apparent from the context, each embodiment listed above may be applicable to use in any aspect of the present invention.

[0177] Method for determining the therapeutic effect of treatment regimens for HF in a subject In one aspect, the present invention relates to a method for determining the therapeutic effect of a treatment regimen for a target heart failure, wherein the method is i. Optionally, a step of determining the level of one or more cardiac-related (poly)peptide biomarkers in interstitial fluid (ISF) from a subject according to steps a) to b) of the method of claim 1, ii. A process in which process i is repeated after a certain time interval, iii. A step of comparing the levels of cardiac-related (poly)peptide biomarkers identified in i with the levels identified in ii, and identifying that the treatment regimen is effective if the levels of cardiac-related (poly)peptide biomarkers decrease after treatment. Includes.

[0178] For example, a change in the level of cardiac-related (poly)peptide biomarkers indicating therapeutic effect is a decrease in the level of cardiac-related (poly)peptide biomarkers in ISF after treatment. A “decrease” in the level of cardiac-related (poly)peptide biomarkers includes the fact that if cardiac-related (poly)peptide biomarkers were detected when the method was previously performed on the same subject (i.e., at an earlier time interval), then they are not detected (i.e., not present at a detectable level) in subsequent time intervals.

[0179] Step i may be performed first according to this method before the treatment regimen for HF is initiated. Alternatively, step i may be performed first at the same time as the initiation of the treatment regimen, or at a point after the initiation of the treatment regimen for HF. Thus, the method can be used to determine the therapeutic effect of a treatment regimen for HF from the beginning (i.e., from the start of the regimen) or from a point after the initiation of the treatment regimen (i.e., determining the therapeutic effect of the treatment regimen for HF during the treatment regimen itself).

[0180] In embodiments, unchanged or increased levels of cardiac-related (poly)peptide biomarkers in the ISF of a subject being treated for HF may indicate inadequate treatment; that is, unchanged or increased levels of cardiac-related (poly)peptide biomarkers in the ISF of a subject being treated for HF may indicate that HF ​​will persist or recur. In particular, if the level of cardiac-related (poly)peptide biomarkers increases by 30% or more, treatment for HF is ineffective. In particular, if the level of cardiac-related (poly)peptide biomarkers increases by 50% or more, treatment for HF is ineffective. In particular, if the level of cardiac-related (poly)peptide biomarkers increases by 100% or more, treatment for HF is ineffective. In particular, if the level of cardiac-related (poly)peptide biomarkers increases by 150% or more, treatment for HF is ineffective. In particular, if the level of cardiac-related (poly)peptide biomarkers increases by 200% or more, treatment for HF is ineffective. Conversely, the same applies to cardiac-related (poly)peptide biomarkers whose decrease indicates worsening of the HF condition.

[0181] Alternatively, unchanged levels of cardiac-related (poly)peptide biomarkers may indicate a stagnation in HF progression.

[0182] Improvement in disease status or symptoms (for example, over the treatment period) may also be indicated by stabilized levels of cardiac-related (poly)peptide biomarkers over time (compared to levels of cardiac-related (poly)peptide biomarkers observed in the absence of treatment over a comparable period, or compared to a comparable control).

[0183] A treatment regimen may be identified as having a therapeutic effect if it results in a delay in disease progression or a delay in the onset of symptoms (for example, over the treatment period).

[0184] A treatment regimen can also be identified as having a therapeutic effect if it results in an improvement in the disease state or symptoms (for example, over the treatment period). Methods for determining whether a treatment regimen is therapeutic are well known in the art.

[0185] The treatment period refers to the time interval at which treatment is performed (for example, one month, three months, six months, one year, two years, or even a lifetime).

[0186] As will be apparent to those skilled in the art, the direction of changes in cardiac-related (poly)peptide biomarker levels that indicate therapeutic effects may depend on the state of the disease in the subject before treatment and the control / reference used.

[0187] Changes in the levels of cardiac-related (poly)peptide biomarkers can also indicate compliance or adherence to prescribed treatments after intervention.

[0188] The tendencies for identifying whether a subject complied with or adhered to a prescribed treatment regimen are equivalent to those described in detail above regarding determining the therapeutic effect of HF treatment regimens. This is because the “prescribed treatment regimen” is the recommended treatment regimen and therefore typically has a therapeutic effect (therefore, the presence of a therapeutic effect at the biomarker level is an indicator of the subject’s compliance with or adherence to the prescribed treatment regimen).

[0189] In embodiments, subjects are monitored several times at different points in time. In embodiments, patients are monitored several times within a time frame of days, weeks, months, or years. In certain embodiments, subjects are monitored once a month or once a year. In embodiments, subjects suffering from HF are monitored once a month or once a year after diagnosis of HF. In embodiments, subjects being treated for HF are monitored once after treatment, especially once after surgical treatment. In particular, subjects being treated for HF are monitored once a month or once a year to determine the effectiveness of treatment and / or recurrence of HF.

[0190] The method may also be useful as a screening tool to determine whether a particular regimen or treatment pattern has therapeutic effect on HF. The regimen or treatment pattern tested may be a new regimen or treatment pattern, a modified regimen or treatment pattern, or a known regimen or treatment pattern that requires further testing. In this regard, a treatment pattern may be, for example, a drug or medicine that is useful or is thought to be useful in the treatment of HF.

[0191] Definitions of cardiac-related (poly)peptide biomarkers, combinations with further biomarkers, methods, subjects, types of HF, treatments, reference values, time intervals, etc., provided elsewhere in this specification, apply equally here. Definitions, embodiments, examples, etc., provided herein for one aspect of the present invention apply equally to all other aspects of the present invention. Unless otherwise apparent from the context, each embodiment listed above may be applicable to use in any aspect of the present invention.

[0192] Stratification of the risk of progression to serious illness In one aspect, the present invention relates to a method for stratifying the risk of progression to a serious disease in subjects having HF, wherein the method is a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in ISF from a subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has elevated or decreased levels of one or more cardiac-related (poly)peptide biomarkers compared to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, then the step of stratifying the risk of progression to serious disease for subjects with HF and Includes.

[0193] As used herein, the term “progresses to a serious illness” means the signs or symptoms of HF or a deterioration in the physical ability of a person having HF.

[0194] This applies when determining the level of one or more cardiac-related (poly)peptide biomarkers and recognizing that the subject's HF condition has worsened because the biomarker has increased or decreased by at least 10%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the reference level / control.

[0195] Definitions of cardiac-related (poly)peptide biomarkers, combinations with further biomarkers, methods, subjects, types of HF, treatments, reference values, time intervals, etc., provided elsewhere in this specification, apply equally here. Definitions, embodiments, examples, etc., provided herein for one aspect of the present invention apply equally to all other aspects of the present invention. Unless otherwise apparent from the context, each embodiment listed above may be applicable to use in any aspect of the present invention.

[0196] Predicting the future course of the disease. In another aspect, the present invention relates to a method for predicting the prognosis of the future disease course of a subject having HF, wherein the method is a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in ISF from a subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has elevated or decreased levels of one or more cardiac-related (poly)peptide biomarkers compared to at least one appropriate reference value for the level of the same one or more cardiac-related (poly)peptide biomarkers, then the step of predicting the future disease course of the subject having HF. Includes.

[0197] As used herein, the term “predicting the prognosis of the future course of the disease” refers to an assessment of the future progression of the severity of HF in a subject with HF. Future progression of HF severity may include both worsening or improvement of HF severity and may include future deterioration or improvement of signs or symptoms of HF or the physical capacity of a subject with HF. Prognosis may also include future events such as hospitalization of a subject with HF and / or the occurrence of a fatal event in a subject with HF.

[0198] Definitions of cardiac-related (poly)peptide biomarkers, combinations with further biomarkers, methods, subjects, types of HF, treatments, reference values, time intervals, etc., provided elsewhere in this specification, apply equally here. Definitions, embodiments, examples, etc., provided herein for one aspect of the present invention apply equally to all other aspects of the present invention. Unless otherwise apparent from the context, each embodiment listed above may be applicable to use in any aspect of the present invention.

[0199] use In one aspect, the present invention relates to the use of interstitial fluid (ISF) for assessing heart failure, determining the risk of developing heart failure, classifying the stage of heart failure, monitoring heart failure, and / or determining the therapeutic effect of a treatment in a subject, by determining the level of one or more cardiac-related (poly)peptide biomarkers in the subject.

[0200] Definitions of cardiac-related (poly)peptide biomarkers, combinations with further biomarkers, methods, subjects, types of HF, treatments, reference values, time intervals, etc., provided elsewhere in this specification, apply equally here. Definitions, embodiments, examples, etc., provided herein for one aspect of the present invention apply equally to all other aspects of the present invention. Unless otherwise apparent from the context, each embodiment listed above may be applicable to use in any aspect of the present invention.

[0201] Computerized method for evaluating patients with HF The above definitions and explanations shall preferably apply mutatis mutandis to the following:

[0202] The method of the present invention may also be implemented as a computer implementation method. In a computer implementation method, typically all steps of the computer implementation method of the present invention are performed by one or more processing units of a computer or computer network. However, the computer implementation method may include additional steps, such as determining the level of a biomarker, such as the amount of a BNP-type peptide.

[0203] Therefore, the present invention further relates to a computer implementation method for evaluating a subject suspected of having heart failure, wherein the method is (a) A step of receiving a value relating to the level of a first cardiac-related (poly)peptide biomarker in the interstitial fluid of a subject, wherein the first biomarker is a BNP-type peptide, (b) Optionally, a step of receiving a value relating to the level of at least one additional cardiac-related biomarker in the interstitial fluid of the subject, wherein the additional cardiac-related biomarker is glucose, creatinine, potassium, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, hsCRP (high-sensitivity C-reactive protein), The process involves receiving a sample selected from the group consisting of: an antigen 125 (or carbohydrate antigen 125, CA125), soluble differentiation cluster 146 (or cell surface glycoprotein MUC18, sCD146), bioadrenomedullin (bioADM), central region proadrenomedullin (MR-proADM), soluble tumorigenesis inhibitor 2 (sST2), angiopoietin 2 (Ang-2), fibroblast growth factor 23 (FGF-23), BMP-10 (bone morphogenetic protein 10), ESM-1 (endothelial cell-specific molecule 1), and insulin-like growth factor binding protein 7 (IGFBP7). (c) A step of comparing the values ​​for the level in step (a) and optionally in step (b) with appropriate criteria for biomarkers and / or calculating a score for evaluating a subject suspected of having heart failure based on the level of the biomarkers, (d) A step of evaluating the object based on the comparison and / or calculation performed in step (c) Includes.

[0204] As used herein, the term “computer implementation” means that the method is performed in an automated manner on a data processing unit contained within a computer or similar data processing device. The data processing unit receives a value for the quantity of the biomarker. Such a value may be a quantity, a relative quantity, or any other calculated value reflecting a quantity as described in detail elsewhere herein. It should be understood that the method described above does not require the determination of the quantity of the biomarker, but rather uses a value for a quantity that has already been determined.

[0205] The present invention further relates to a computer program that includes computer-executable instructions for performing steps of a computer implementation method according to the present invention when the program is executed on a computer or computer network. Typically, the computer program may specifically include computer-executable instructions for performing steps of the method disclosed herein. Specifically, the computer program may be stored in a computer-readable data carrier.

[0206] The present invention further relates to a computer program product having program code means stored in a machine-readable carrier when the program is executed on a computer or computer network, such as one or more of the above-described steps considered in relation to a computer program for carrying out the method according to the present invention. As used herein, a computer program product refers to a program as a tradable product. The product may generally exist in any form, such as in the form of paper, or it may exist on a computer-readable data carrier. Specifically, the computer program product may be distributed over a data network.

[0207] The present invention further relates to a computer or computer network comprising at least one processing unit, wherein the processing unit is adapted to carry out all steps of the method according to the present invention, in particular steps a), b), c), and d).

[0208] The present invention also, in principle, envisions a computer program, a computer program product, or a computer-readable storage medium in which a computer program is tangibly incorporated, wherein the computer program includes 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 includes: - A computer or computer network having at least one processor, wherein the processor is adapted to carry out a method according to one of the embodiments described herein, - A computer-loadable data structure adapted to perform a method according to one of the embodiments described herein while the data structure is being executed on a computer. - A computer script, which is adapted so that the computer program performs a method according to 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 while the computer program is running on a computer or on a computer network, - A computer program comprising the program means described in a prior embodiment, stored on a storage medium readable by a computer, - A data structure is stored in 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 herein when the program code means is executed on a computer or computer network. - Typically encrypted data stream signals containing BNP-type peptide data, as well as - A typically encrypted data stream signal, which includes calculations by the method of the present invention and preferably provides evaluation information.

[0209] Definitions of cardiac-related (poly)peptide biomarkers, combinations with further biomarkers, methods, subjects, types of HF, treatments, reference values, time intervals, etc., provided elsewhere in this specification, apply equally here. Definitions, embodiments, examples, etc., provided herein for one aspect of the present invention apply equally to all other aspects of the present invention. Unless otherwise apparent from the context, each embodiment listed above may be applicable to use in any aspect of the present invention.

[0210] Data storage methods The levels of biomarkers and / or reference levels can be stored in a suitable data storage medium (e.g., a database) and thus available for future diagnoses. This also enables efficient diagnosis of disease prevalence, as appropriate reference results can be identified in the database when it is (future) confirmed that the subject from which the corresponding reference sample was obtained had HF. As used herein, “database” includes data (e.g., analytes and / or reference level information and / or patient information) collected in a suitable storage medium. Furthermore, the database may further comprise a database management system. The database management system is preferably a network-based hierarchical or object-oriented database management system. Furthermore, the database may be a federated or integrated database. More preferably, the database may be implemented as a distributed (federated) system, such as a client-server system. More preferably, the database is configured so that a search algorithm can compare test datasets with datasets contained in the data set. Specifically, by using such an algorithm, the database can be searched for similar or identical datasets that indicate HF (e.g., a query search). Thus, if identical or similar datasets can be identified in the data set, the test dataset is associated with HF. Therefore, the information obtained from the data set can be used to diagnose HF or based on test datasets obtained from the subject. More preferably, the data set includes characteristic values ​​for all analytes included in any one of the above groups.

[0211] The methods described herein may further include, for example, communicating results or diagnoses (or both) to technicians, physicians, or patients. In certain examples, a computer may be used to communicate results or diagnoses (or both) to relevant parties, such as physicians and their patients.

[0212] In some cases, after a diagnosis is made, the result or diagnosis (or both) is promptly communicated to the subject. The result or diagnosis (or both) may be communicated to the subject by the treating physician. Alternatively, the result or diagnosis (or both) may be sent to the subject by email or communicated by telephone. The result or diagnosis can be communicated by email or telephone using a computer. In certain cases, the message containing the result or diagnosis may be automatically generated and delivered to the subject using a combination of computer hardware and software well known to those skilled in the art of telecommunications.

[0213] Companion Diagnosis The methods and uses provided herein may be used, for example, as part of a medical device, often an in vitro device, as part of a companion diagnostic that provides information essential for the safe and effective use of the corresponding drug or biological substance (the corresponding drug or biological substance is intended to treat or prevent HF).

[0214] All patents, patent applications, and publications or disclosures mentioned or cited herein are incorporated in their entirety by reference.

[0215] The present invention will be further described with reference to the examples described herein. However, it should be understood that the present invention is not limited to such examples. [Examples]

[0216] a. Sample Paired plasma and ISF samples from healthy donors were provided by Ascilon AB (Stockholm, Sweden).

[0217] Interstitial fluid (ISF) was collected from the skin (upper arm). ISF collection was performed using "Ascilion's dISF sampling solution," which is based on microneedles made from single-crystal silicon.

[0218] Eight different samples were obtained from four different healthy donors (Donors A-D). The sample type (plasma or ISF) and volume are summarized in the table below. [Table 1]

[0219] b. Determination of ISF and NT-proBNP in plasma NT-proBNP levels in ISF and plasma samples were determined using the Simoa® Homebrew assay. A detailed description of the assay is provided below: ·reagent [Table 2] • Simoa (registered trademark) Homebrew Assay [Table 3] All calibrators and samples were measured in a dual-cycle setup. • Calibrator NT-proBNP(1-76)amid (Roche Diagnostics GmbH); Stock 5.2 μg / mL Zero standard: Quanterix Homebrew detector / sample diluent [Table 4] Control sample: 1:175 dilution of PreciControl Cardiac II (Roche Diagnostics GmbH) Control 1 target value: 0.883 pg / mL (average of 6 runs in SIMOA) • Sample dilution: The sample was diluted with the sample diluent.

[0220] result • Calibration and control Run is 1 / y 2Calibration was performed using cubic fitting with the specified weighting. The fitting was checked by the recovery rate of the fitted concentration to the target concentration of the calibrator. This recovery rate should be between 85% and 115%. A recovery rate of 86% to 113% can be considered a successful fitting.

[0221] To validate the run, a control with a specified target value was measured, and a concentration of 0.954 pg / mL was obtained. Since the recovery rate against the target was within ±21%, the validation was successful. [Table 5]

[0222] ·sample The sample was prepared as described above and measured using a double-barreled system.

[0223] The measured concentration should be above the determined detection and quantification limit of 0.205 pg / mL, and the dual measurement should have a coefficient of variation (CV) of ≤15% concentration. [Table 6]

[0224] To minimize the impact of measurement errors, the diluted NT-proBNP concentrations in the following table were determined based on the average signal. [Table 7]

[0225] The NT-proBNP recovery rate in ISF samples compared to plasma is 108%–145%. Considering a ±20% deviation due to measurement and dilution errors, the NT-proBNP concentrations of donors B and D can be considered equivalent in plasma and ISF.

[0226] The results are shown in the comparative plot (Figure 2). Observations in the comparative plot show a linear correlation between NT-proBNP values ​​measured in plasma from the same donor and values ​​measured in ISF.

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Claims

1. A method for evaluating heart failure in a subject or determining the risk of developing heart failure (HF), a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the interstitial fluid (ISF) from the subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers with the level of the same cardiac-related (poly)peptide biomarkers in a control sample, or with a predetermined reference level of the same cardiac-related (poly)peptide biomarkers, c) A step of identifying a subject as having heart failure or having an increased risk of developing heart failure, based on the comparison in step b) Methods that include...

2. A method for monitoring heart failure in subjects, i. A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the interstitial fluid (ISF) from the subject, optionally according to steps a) to b) of the method of claim 1, ii. A process that repeats process i after a certain time interval, iii. A step of comparing the level of a cardiac-related (poly)peptide biomarker identified in i with the level identified in ii, wherein the change in the level from i to ii indicates a change in heart failure in the subject. Methods that include...

3. A method for classifying the stage of heart failure, a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the interstitial fluid from the subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers with at least one appropriate reference value of the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has an increased or decreased level of one or more cardiac-related (poly)peptide biomarkers compared to the level of at least one appropriate reference value of the same one or more cardiac-related (poly)peptide biomarkers, then the step of classifying the stage of heart failure in the subject and Methods that include...

4. A method for determining the therapeutic effect of a treatment regimen for heart failure, i. A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the interstitial fluid (ISF) from the subject, optionally according to steps a) to b) of the method of claim 1, ii. A process that repeats process i after a certain time interval, iii. A step of comparing the level of the cardiac-related (poly)peptide biomarker identified in i with the level identified in ii, and determining that the treatment regimen has a therapeutic effect if the level of the cardiac-related (poly)peptide biomarker decreases after treatment. Methods that include...

5. A method for stratifying the risk of progression to serious diseases in subjects with HF, a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the ISF from the subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers with at least one appropriate reference value of the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has an increased or decreased level of one or more cardiac-related (poly)peptide biomarkers compared to the level of one or more of the same cardiac-related (poly)peptide biomarkers, then the step of stratifying the risk of progression to a serious disease for the subject having HF. Methods that include...

6. A method for predicting the future disease course of a subject with HF, a) A step of determining the level of one or more cardiac-related (poly)peptide biomarkers in the ISF from the subject, b) A step of comparing the level of one or more cardiac-related (poly)peptide biomarkers with at least one appropriate reference value of the level of the same one or more cardiac-related (poly)peptide biomarkers, c) If the comparison in step b) indicates that the subject has an increased or decreased level of one or more cardiac-related (poly)peptide biomarkers compared to the level of at least one appropriate reference value of the same one or more cardiac-related (poly)peptide biomarkers, then the step of predicting the future disease course of the subject having HF. Methods that include...

7. The use of interstitial fluid (ISF) to assess heart failure, determine the risk of developing heart failure, classify the stage of heart failure, monitor heart failure, and / or determine the therapeutic effect of a treatment in the subject, by determining the level of one or more cardiac-related (poly)peptide biomarkers in the subject.

8. The method according to claims 1 to 6 or the use according to claim 7, wherein the cardiac-related (poly)peptide biomarker is a BNP-type peptide.

9. The method according to claims 1 to 6 or the use according to claim 7, wherein the cardiac-related (poly)peptide biomarker is N-terminal pro-B natriuretic peptide (NT-proBNP) or B natriuretic peptide (BNP).

10. In addition to the levels of one or more cardiac-related (poly)peptide biomarkers, glucose, creatinine, potassium, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth and differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, hsCRP (high-sensitivity C-reactive protein), cancer antigen 125 (or carbohydrate antigen 125, CA125), soluble differentiation cluster 146 (or micro The method according to claims 1 to 6 or the use according to claim 7, wherein the level of one or more further cardiac-related biomarkers selected from the group consisting of cellular surface glycoproteins MUC18, sCD146), bioadrenomedullin (bioADM), central region proadrenomedullin (MR-proADM), soluble tumorigenesis inhibitor 2 (sST2), angiopoietin 2 (Ang-2), fibroblast growth factor 23 (FGF-23), BMP-10 (bone morphogenetic protein 10), ESM-1 (endothelial cell-specific molecule 1), and insulin-like growth factor-binding protein 7 (IGFBP7) is determined.

11. The method according to claims 1 to 6 or the use according to claim 7, wherein the heart failure is selected from the group consisting of acute heart failure, reduced ejection fraction heart failure (HFrEF), mild reduced ejection fraction heart failure (HFmrEF), fraction-preserving heart failure (HFpEF), left heart failure, right heart failure, and biventricular failure.

12. The method according to any one of claims 1 to 6 or the use according to claim 7, further comprising selecting a treatment regimen for the subject based on the comparison between the level of the cardiac-related (poly)peptide biomarker and the control sample or the predetermined reference level.

13. The method of claim 12, further comprising administering the selected treatment regimen to the subject, wherein the selected treatment regimen optionally includes a drug-based treatment and / or surgery.

14. The method according to claim 3, wherein the stage of the heart failure in the subject is classified as stage I, stage II, stage III, or stage IV heart failure according to the New York Heart Association (NYHA) functional classification.

15. A computer implementation method for evaluating individuals suspected of having heart failure, (a) A step of receiving a value relating to the level of a first cardiac-related (poly)peptide biomarker in the interstitial fluid of the subject, wherein the first biomarker is a BNP-type peptide, (b) Optionally, a step of receiving a value relating to the level of at least one additional cardiac-related biomarker in the interstitial fluid of the subject, wherein the additional cardiac-related biomarker is glucose, creatinine, potassium, sodium, urea, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), GDF-15 (growth differentiation factor 15), SHBG (sex hormone-binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, prealbumin, ferritin, osteopontin, hsCRP (high-sensitivity C-reactive protein). A receiving step in which a substance is selected from the group consisting of cancer antigen 125 (or carbohydrate antigen 125, CA125), soluble differentiation cluster 146 (or cell surface glycoprotein MUC18, sCD146), bioadrenomedullin (bioADM), central region proadrenomedullin (MR-proADM), soluble tumorigenesis inhibitor 2 (sST2), angiopoietin 2 (Ang-2), fibroblast growth factor 23 (FGF-23), BMP-10 (bone morphogenetic protein 10), ESM-1 (endothelial cell-specific molecule 1), and insulin-like growth factor binding protein 7 (IGFBP7), (c) A step of comparing the value for the level of step (a) and optionally step (b) with an appropriate standard for the biomarker and / or calculating a score for evaluating the subject suspected of having heart failure based on the level of the biomarker, (d) A step of evaluating the object based on the comparison and / or calculation performed in step (c) Computer implementation methods, including those mentioned above.

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