Monitoring of cardiovascular markers in interstitial fluid and its application in assessment of heart failure

Abstract

The present invention relates to methods of assessing heart failure (HF), determining the risk of developing HF, monitoring HF, classifying HF, risk stratification and prognosis in patients with HF and determining the therapeutic effect of a treatment regimen for HF in a subject by determining the level of one or more cardiac related (poly)peptide biomarkers in the interstitial fluid (ISF) from the subject, and comparing the determined level to a reference value. Further this invention refers to the use of ISF in the methods described herein.

Description

[0001] Monitoring of cardiovascular markers in interstitial fluid and its application in assessment of heart failure

[0002] The present invention relates to methods of assessing heart failure (HF), determining the risk of developing HF, monitoring HF, classifying HF, risk stratification and prognosis in patients with HF and determining the therapeutic effect of a treatment regimen for HF in a subject by determining the level of one or more cardiac related (poly)peptide biomarkers in the interstitial fluid (ISF) from the subject, and comparing the determined level to a reference value. Further this invention refers to the use of ISF in the methods described herein.

[0003] Background of the Invention

[0004] Heart failure (HF), also known as congestive heart failure (CHF), is, according to the guidelines of the European Society of Cardiology, updated in 2021 : "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 due to a structural and / or functional abnormality of the heart that results in elevated intracardiac pressures and / or inadequate cardiac output at rest and / or during exercise.” The shortness of breath may occur with exertion or while lying down, and may wake people up during the night. Chest pain, including angina, is not usually caused by heart failure, but may occur if the heart failure was caused by a heart attack. The severity of heart failure is measured by signs or symptoms at rest or during exercise or work out. Other comorbid conditions that may have signs and symptoms similar to heart failure include obesity, kidney failure, liver and lung diseases, anemia, and thyroid disease.

[0005] Common causes of heart failure include coronary artery disease, heart attack, high blood pressure, atrial fibrillation, valvular heart disease, excessive alcohol consumption, infection, and cardiomyopathy. These cause heart failure by altering the structure or the function of the heart or in some cases both. There are different types of heart failure: right-sided heart failure, which affects the right part of the heart, left-sided heart failure, which affects the left part of the heart, and biventricular heart failure, which affects both sides of the heart. Left-sided heart failure may be present with a reduced ejection fraction or with a preserved ejection fraction. Heart failure is not the same as cardiac arrest, in which blood flow stops completely due to the failure of the heart to pump effectively. Heart failure is divided into distinct phenotypes based on the measurement of left ventricular ejection fraction: heart failure with reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF).

[0006] Diagnosis is typically based on risk factors, symptoms, physical findings, abnormal electrocardiogram (ECG) and echocardiography. Blood tests and a chest x-ray may be useful to determine the underlying cause.

[0007] Treatment depends on severity and case. For people with chronic, stable, mild heart failure, treatment usually consists of lifestyle changes, such as not smoking, physical exercise, and dietary changes, as well as medications. In heart failure due to left ventricular dysfunction, angiotensin-converting-enzyme inhibitors, angiotensin receptor blockers, or valsartan / sacubitril along with beta blockers and sodium-glucose co-transporter 2 inhibitors (SGLT2i) are recommended. In severe disease, mineralocorticoid receptor antagonists or hydralazine with a nitrate can be used. Diuretics may also be prescribed to prevent fluid retention and the resulting shortness of breath. Depending on the case, an implanted device such as a pacemaker or implantable cardiac defibrillator may sometimes be recommended. In some moderate or more severe cases, cardiac resynchronization therapy (CRT) or cardiac contractility modulation may be beneficial. In severe disease that persists despite all other measures, a cardiac assist device ventricular assist device (for the left, right, or both heart chambers), or, occasionally, heart transplantation may be recommended.

[0008] Heart failure is a common, costly, and potentially fatal condition, and is the leading cause of hospitalization and readmission in older adults. Heart failure often leads to more drastic health impairments than failure of other, similarly complex organs such as the kidneys or liver.

[0009] Acute heart failure (AHF) is a major contributor to the morbidity and mortality of patients with heart failure (HF) (1). Despite this increased risk, the vast majority of AHF patients are not closely followed and are substantially undertreated during the first few months after discharge from an AHF admission - a “vulnerable period” during which most of the adverse events occur (2-7). Some authors have retrospectively examined the association of more follow-up and quicker up-titration of medications after an AHF event, but with mixed results (8-14,16). Based on this lack of evidence from prospective randomized studies, recent European guidelines for the treatment of HF recommend some follow-up of patients after an AHF admission and initiation of recommended therapies, but the level of evidence for this recommendation is low (C) (17).

[0010] The Safety, Tolerability and efficacy of Rapid Optimization, helped by NT-proBNP testinG, of Heart Failure therapies (STRONG-HF) study was a randomized, prospective clinical trial, designed to assess the safety and efficacy of rapid up-titration of medical therapy including beta-blockers (BB)s; angiotensin converting enzyme inhibitors (ACEi), angiotensin receptor blockers (ARB), or angiotensin receptor neprilysin inhibitors (ARNi), and mineralocorticoid receptor antagonists (MRA) prior to discharge from an AHF admission and during the following weeks (ClinicalTrials.gov NCT03412201). The safety of up-titration was guided by physical examination and laboratory evaluations including NT -proBNP. Guideline- recommended oral HF medications for patients in the “high intensity care” arm were up-titrated to half optimal doses at discharge and to full optimal doses at 2 weeks post discharge with safety visits 1 week after any up-titration and follow-up visits at 6 weeks and 3 months. At each visit, patients were assessed by physical examination for congestion and blood tests including NT -proBNP measurements (18,19). The STRONG-HF study came to the conclusion that an intensive treatment strategy of rapid up-titration of guideline-directed medication and close follow-up after an acute heart failure admission was readily accepted by patients because it reduced symptoms, improved quality of life, and reduced the risk of 180-day all-cause death or heart failure readmission compared with usual care (20).

[0011] In summary, HF is a progressive disease with recurring episodes of acute worsening. Despite guideline-recommended treatment options, HF outcomes remain poor. Decompensation and re-hospitalization poses a major challenge in the management of heart failure patients.

[0012] Monitoring of other biomarkers like creatinine or potassium in addition to BNP -type peptides like NT -proBNP will enable physicians to monitor the patients’ condition, chose the most promising treatment and optimize doses of life-saving treatments in heart failure (21).

[0013] There is a high need for a non-invasive and quick assessment and monitoring of cardiac related biomarkers, which allows a reliable and continuous risk assessment and / or monitoring of patients exhibiting signs and symptoms of (acute) heart failure.

[0014] The present invention, therefore, provides methods complying with these needs.

[0015] Accordingly, the present invention, inter alia, relates to methods for assessing heart failure in patients by determining the level of cardiac related biomarkers, in particular BNP -type peptides in the ISF.

[0016] Summary of the Invention

[0017] In a first aspect, the present invention relates to a method for assessing heart failure or determining the risk of developing heart failure (HF) in a subject, the method comprising the steps of: a) determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid (ISF) from the subject; b) comparing the level of the one or more cardiac related (poly)peptide biomarker with the level of the same cardiac related (poly)peptide biomarker in a control sample or with a pre-determined reference level for the same cardiac related biomarker; and c) identifying a subject as having heart failure or as having an increased risk of developing heart failure based on the comparison in step b).

[0018] In a second aspect the present invention relates to a method for monitoring HF in a subject, the method comprising the steps of i. determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, optionally in accordance with method steps a) to b) of claim 1 ; ii. repeating step i. after a time interval; and iii. comparing the levels of the cardiac related (poly)peptide biomarker identified in i. with the levels identified in ii., wherein a change in the levels from i. to ii. is indicative of a change in HF in the subject.

[0019] In a third aspect, the present invention relates to a method for classifying the stage of HF in a subject, the method comprising the steps of a) determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) classifying the stage of HF in the subject if the comparison in step b) indicates that the subject has an increased or decreased level of the one or more cardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

[0020] In a fourth aspect the present invention relates to a method for determining the therapeutic effect of a treatment regimen for HF in a subject, the method comprising the steps of: i. determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, optionally in accordance with method steps a) to b) of claim 1; ii. repeating step i. after a time interval; and iii. comparing the levels of the cardiac related (poly)peptide biomarker identified in i. with the levels identified in ii., and identifying that the treatment regimen has a therapeutic effect if the level of the cardiac related (poly)peptide biomarker decreased after treatment.

[0021] In a fifth aspect the present invention relates to a use of ISF for assessing HF, determining the risk of developing heart failure, classifying the stage of heart failure, monitor heart failure and / or determine the therapeutic effect of a treatment in a subject by determining the level of one or more cardiac related (poly)peptide biomarker therein.

[0022] In a sixth aspect the invention relates to a computer-implemented method for assessing a subject with suspected heart failure comprising the steps of

[0023] (a) receiving a value for level of a first cardiac related (poly)peptide biomarker in the interstitial fluid of the subject, said first biomarker being a BNP -type peptide;

[0024] (b) receiving a value for the level of at least one additional cardiac related biomarker in the interstitial fluid of the subject, wherein said additional biomarker is selected from the group consisting of 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 Cluster of Differentiation 146 (or Cell surface glycoprotein MUC18, sCD146), bioAdrenomedullin (bio ADM), Midregional proadrenomedullin (MR-proADM), Soluble suppression of tumorigenesis-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),

[0025] (c) comparing the values for the levels of steps (a) - (b) to references for said biomarkers and / or calculating a score for assessing the subject with suspected heart failure based on the levels of the biomarkers; and

[0026] (d) assessing said subject based on the comparison and / or the calculation made in step (c).

[0027] In a further aspect the present invention refers to a method for treating heart failure in a subject, the method comprising: a) requesting a test providing the results of an analysis to determine the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject and comparing the level to a reference value, wherein an increase in the level as compared to the reference value is indicative of heart failure or of an increased risk of developing heart failure in a subject; and b) administering the subject that has been identified as having or being at risk of developing heart failure a treatment for heart failure, thereby treating the subject.

[0028] In a further aspect the invention relates to a method for determining a subject’s compliance with a prescribed treatment for heart failure, the method comprising: a) determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject before treatment; b) determining the level of the same one or more cardiac related (poly)peptide biomarker as in step a) in the ISF from the subject after treatment; and c) comparing the level obtained in step a) and step b), wherein a decrease in the level obtained in step b) as compared to the level in step a) indicates that the subject is / and or has complied with the prescribed treatment.

[0029] In a further aspect, the present invention relates to a method for risk stratification of progressing to severe disease of a subject with HF, the method comprising the steps of: a) determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) stratifying the risk of progressing to severe disease of the subject with HF if the comparison in step b) indicates that the subject has an increased or decreased level of the one or more cardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

[0030] In a further aspect, the present invention relates to a method for making a prognosis of future disease course of a subject with HF, the method comprising the steps of: a) determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) making a prognosis of future disease course of the subject with HF if the comparison in step b) indicates that the subject has an increased or decreased level of the one or more cardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

[0031] Suitably, the cardiac related (poly)peptide biomarker may be a BNP -type peptide.

[0032] Suitably, the cardiac related (poly)peptide biomarker may be N-terminal pro-B-type natriuretic peptide (NT-proBNP) or B-type natriuretic peptide (BNP). Suitably, additionally to the level of the one or more cardiac related (poly)peptide biomarker the level of one or more further cardiac related biomarkers selected from the group consisting of 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 Cluster of Differentiation 146 (or Cell surface glycoprotein MUC18, sCD146), bioAdrenomedullin (bio ADM), Midregional proadrenomedullin (MR-proADM), Soluble suppression of tumorigenesis-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) may be determined.

[0033] Suitably, HF may be selected from the group consisting of acute heart failure, heart failure due to reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF), heart failure with preserved ejection fraction (HFpEF), left-sided failure, rightsided failure, biventricular failure.

[0034] Suitably, selecting a treatment regimen for the subject based on the comparison of the level of the biomarker with the control sample or with the pre-determined reference level may be further comprised.

[0035] Suitably, administering the selected treatment regimen to the subject, optionally wherein the selected treatment regimen comprises drug-based therapy and / or surgery may be further comprised.

[0036] Suitably, the subject’s stage of heart failure may be classified as stage I, stage II, stage III or stage IV heart failure according to the New York Heart Association (NYHA) Functional Classification.

[0037] It will be appreciated that, except for where the context requires otherwise, the considerations set out in this disclosure should be considered to be applicable to all aspects of the invention.

[0038] Various aspects of the invention are described in further detail below.

[0039] Brief description of the Figures

[0040] Examples of the invention are further described hereinafter with reference to the accompanying drawings. Figure 1: Calibration curve of a Simoa® Assay for NT -proBNP. On the x-axis, the concentrations of the respective NT -proBNP calibrators (in pg / mL) are shown on a logarithmic scale. The y-axis shows the mean signals of the Simoa® Assay measurements for these calibrators (in AEB (Average number of Enzyme labels per Bead) units), also on a logarithmic scale. The error bars shown in Fig 1. represent the standard deviation. The calibration curve shown in Fig. 1 is based on a cubic fitting with 1 / y2weighting.

[0041] Figure 2: Comparison plot of corresponding NT -proBNP values in plasma (x-axis, in pg / mL) and the ones in ISF (y-axis, also in pg / mL) for samples of the same donor. The error bars shown in Fig 2. represent the standard deviation of the respective plasma or ISF measurements.

[0042] Detailed Description of the Invention

[0043] The invention provides a new and surprising approach for assessing heart failure, determining the risk of developing heart failure, classifying the stage of heart failure, monitor heart failure and / or determine the therapeutic effect of a treatment in a subject.

[0044] This new approach is based on the inventors’ unexpected finding that the ISF is reliable for the purpose of determining the levels of cardiac related (poly)peptide biomarkers. Due to varying accessibilities, concentrations of components (e.g. ions, peptides, proteins, metabolites, small molecules, etc.), sizes of components, equilibria, etc. the various body fluids (e.g., whole blood, blood plasma, tears, sweat, synovial fluid, ISF, urine, saliva, lymphatic fluid, vitreous humor, or pericardial fluid) contain different components in different concentrations. This reliability is due to the fact that the level of cardiac related (poly)peptide biomarkers in the ISF correlates to the level found in blood for that biomarker(s). Depending on the biomarker the correlation between the level of the cardiac related (poly)peptide biomarker in the ISF and in the blood can be either linear / proportional or can substantially be the same. In this context levels being “substantially the same” can be considered levels when considering a deviation of ± 20 % due to measurement and dilution errors. Currently, determining the biomarker levels in blood is the gold standard. The inventors’ finding allows the use of ISF to determine the levels of cardiac related (poly)peptide biomarkers instead of using blood samples that need to be obtained by medical doctors or trained medical assistants.

[0045] The inventors show for the first time that levels of NT -proBNP measured in ISF are comparable to the levels of NT-proBNP in blood. The present application shows a close to 1 : 1 correlation between NT -proBNP concentrations in serum and ISF. With this surprising finding the present application demonstrates that in general ISF has to be considered as a potential source for body fluids in order to determine levels of biomarkers, with ISF having the advantage of being easily accessible and obtainable.

[0046] There is an unmet medical need to determine and to regularly monitor cardiac related biomarkers in subjects with signs for a HF in a quick, reliable and simple way for several reasons:

[0047] • assessing cardiac decompensation

[0048] • diagnosing congestion or assessing or monitoring the extent of congestion in a subject

[0049] • assessing the decision on (re)hospitalization of a subject

[0050] • assessing the decision on hospital discharge of a subject

[0051] • optimization of (doses of) treatments

[0052] • optimization of doses of treatments

[0053] • assessment of decongestion or residual congestion in HF (including quantifying, diagnosing, determining, monitoring etc.), eventually followed by optimized and / or guided decongestion therapy

[0054] • predicting / assessing decongestion or residual congestion after therapy or intervention of congestion in a subject

[0055] • predicting or determining or monitoring need of therapy or intervention or predicting or determining or monitoring the success of a therapy or intervention of congestion or guiding a therapy or intervention in a subject, or

[0056] • determining a subject’ s compliance with a prescribed treatment for HF

[0057] • risk stratification of progressing to severe disease of a subject with HF

[0058] • prognosis of future disease course of a subject with HF

[0059] The use of ISF as body fluid within which the cardiac related biomarker levels are determined has the advantage of being easily accessible and allowing a continuous monitoring of cardiac related biomarkers in a simple and reliable way. No medical doctor or trained medical assistant is required in order to obtain an ISF sample. More advantageously, by its easy accessibility a continuous measurement of corresponding biomarkers can be carried out without the need of actually obtaining a sample of ISF but rather by direct measurement within the ISF in the body without requiring a medical intervention.

[0060] The inventors have investigated the levels of the cardiac related biomarker NT -proBNP in ISF and blood samples of donors. Surprisingly, they found that the levels of NT-proBNP correlate in ISF and blood of the donors. The data presented herein show that by determining the levels of cardiac related (poly)peptide biomarkers, in particular 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 a treatment regimen for HF can be determined. By this the need to be able to determine and to regularly monitor cardiac related biomarkers in subjects with signs for a HF in a quick, reliable and simple way is met and can be used for all the reasons and purposes listed above.

[0061] In general, the methods described herein are in vivo, ex vivo or in vitro methods.

[0062] Preferably the methods described herein are in vivo methods. Thereby the levels of cardiac related (poly)peptide biomarkers are (continuously) measured by a corresponding device, e.g. a needle type sensor (permanently) attached to the subject’s body in a way allowing to carry out the measurement in the ISF of the subject.

[0063] In case of in vitro methods they are performed using a sample that has already been 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). The methods may therefore include the step of providing a biological fluid sample from a subject.

[0064] The methods provided herein may comprise providing an ISF sample from a subject. Advantageously, methods for obtaining ISF samples from a subject are typically low-invasive or non-invasive.

[0065] The sample is an in vitro sample, it will be analyzed in vitro and not transferred back into the body.

[0066] Definitions

[0067] The word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0068] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

[0069] Concentrations, levels, amounts, and other numerical data may be expressed or presented herein in a “range” format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly recited values of 150 mg to 600 mg, but to also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 150, 160, 170, 180, 190, ... 580, 590, 600 mg and sub-ranges such as from 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges reciting only one numerical value. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0070] The term “about” when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5% smaller than the indicated numerical value and having an upper limit that is 5% larger than the indicated numerical value.

[0071] As used herein, “provide”, "obtain" or "obtaining" can be any means whereby one comes into possession of the sample by "direct" or "indirect" means. Directly obtaining a sample means performing a process (e.g., performing a physical method such as extraction) to obtain the sample. Indirectly obtaining a sample refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample).

[0072] Analysis of a sample may be accomplished on a visual or chemical basis. Chemical analysis includes but is not limited to the detection of the presence or absence of specific indicators or alterations in their amount, concentration or level.

[0073] In the context of present invention, the terms “biomarker”, “marker”, “cardiac related biomarker” and “cardiac related (poly)peptide biomarker” are used interchangeably and refer to a peptide or polypeptide (= (poly)peptide) within a biological system that is used as an indicator of a biological state of said system. In the art, the term „biomarker“ is sometimes also applied to means for the detection of said endogenous substances (e.g. antibodies, nucleic acid probes etc, imaging systems). In the context of present invention, the term “biomarker“ shall be only applied for the substance, not for the detection means. Thus, biomarkers can be any kind of (poly)peptide present in a living organism, such as a protein (cell surface receptor, cytosolic protein etc.), polypeptide, peptide, isomeric form thereof, immunologically detectable fragment thereof, which is differentially present in a subject / sample taken from a subject having HF compared with a subject not having HF. In case other biomarkers than (poly)peptide biomarkers are meant to be covered by these term in the context of this invention these biomarkers are explicitly indicated.

[0074] The term “(poly)peptide” as used herein refers to chains of amino acids linked by peptide bonds with a length of up to 150 amino acids including oligopeptides, dipeptides, tripeptides, and tetrapeptides.

[0075] Examples of “cardiac related (poly)peptide biomarkers are BNP-type peptides. As used herein, the term “BNP -type peptides” comprise pre-proBNP, proBNP, NT-proBNP, and BNP. The pre-pro peptide (134 amino acids in the case of pre-proBNP) comprises a short signal peptide, which is enzymatically cleaved off to release the pro peptide (108 amino acids in the case of proBNP). The pro peptide is further cleaved into an N-terminal pro peptide (NT- pro peptide, 76 amino acids in case of NT-proBNP) and the active hormone (32 amino acids in the case of BNP). Preferably, BNP -type peptides according to the present invention are NT- proBNP, BNP, and variants thereof. BNP is the active hormone and has a shorter half-life than the respective inactive counterpart NT-proBNP. BNP is metabolized in the blood, whereas NT- proBNP circulates in the blood as an intact molecule and as such is eliminated renally. The in- vivo half-life of NT-proBNP is 120 min longer than that of BNP, which is 20 min (Smith 2000, J Endocrinol. 167: 239-46.). Preanalytics are more robust with NT-proBNP allowing easy transportation of the sample to a central laboratory (Mueller 2004, Clin Chem Lab Med 42: 942-4.). Blood samples can be stored at room temperature for several days or may be mailed or shipped without recovery loss. In contrast, storage of BNP for 48 hours at room temperature or at 4° Celsius leads to a concentration loss of at least 20 % (Mueller loc.cit.; Wu 2004, Clin Chem 50: 867-73.). Therefore, depending on the time-course or properties of interest, either measurement of the active or the inactive forms of the natriuretic peptide can be advantageous. The most preferred natriuretic peptides according to the present invention are NT-proBNP or variants thereof. As briefly discussed above, the human NT-proBNP, as referred to in accordance with the present invention, is a polypeptide comprising, preferably, 76 amino acids in length corresponding to the N-terminal portion of the human NT-proBNP molecule. The structure of the human BNP and NT-proBNP has been described already in detail in the prior art, e.g., WO 02 / 089657, WO 02 / 083913 or Bonow loc. cit. Preferably, human NT-proBNP as used herein is human NT-proBNP as disclosed in EP 0 648 228 Bl. These prior art documents are herewith incorporated by reference with respect to the specific sequences of NT-proBNP and variants thereof disclosed therein.

[0076] In addition to the cardiac related (poly)peptide biomarkers the methods described herein can also comprise the determination of levels or one or more further cardiac related biomarkers that do not have to be (poly)peptides. Examples of such cardiac related biomarkers are 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 Cluster of Differentiation 146 (or Cell surface glycoprotein MUC18, sCD146), bioAdrenomedullin (bioADM), Midregional proadrenomedullin (MR- proADM), Soluble suppression of tumorigenesis-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). A biomarker is differentially present if the mean or median level of the biomarker in the different groups is calculated to be statistically relevant. Common tests for statistical significance include, among others, t-test (e.g., student t-test), ANOVA, Kruskal-Wallis, Wilcoxon, Mann- Whitney, Receiver Operating Characteristic (ROC curve), accuracy and odds ratio. Biomarkers, alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another.

[0077] Therefore, they are useful as markers for disease (diagnostics), therapeutic effectiveness of a drug and drug toxicity.

[0078] The methods provided herein refer to “determining” the level of one or more (poly)peptide biomarkers. As would be clear to a person of skill in the art, the level of one or more (poly)peptide biomarkers is typically “determined” by measuring the level of the biomarker in the sample / subject. The term “determining” can therefore be replaced with the term “measuring” or “determining by measuring” herein.

[0079] The terms “determining” or “assessing” as used herein also refer to assessing / determining whether a patient suffers from HF. Accordingly, assessing / determining as used herein includes diagnosing HF, assessing the risk that a subject suffers from HF, selecting for therapy of HF, monitoring a patient suffering from HF or being treated 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 reference. Typically, the assessment referred to in accordance with the present invention is the assessment of the presence and / or development of HF.

[0080] The term “assessing heart failure therapy” or “determining the therapeutic effect of a treatment regimen for HF” as used herein, preferably refers to the identification of patients who are eligible to change of heart failure therapy as described elsewhere herein. Thus, patients can be identified or selected which would benefit from a change of therapy.

[0081] The term "measurement", "measuring" or "determining" preferably comprises a qualitative, a semi-quantitative or a quantitative measurement.

[0082] Conventional in vitro "determining" methods may include sending a clinical sample(s) to a commercial laboratory for measurement of the biomarker levels in the biological fluid sample, or the use of commercially available assay kits for measuring the biomarker levels in the biological fluid sample. Exemplary kits and suppliers will be apparent to a person of skill in the art. In various examples, biomarkers may be determined, detected and / or quantified using ELISA assays or lateral flow devices, such as for point-of-care use, as well as spot check colorimetric tests. The level of biomarker present in the biological fluid (sample) may be determined by e.g. assaying the amount of biomarker present in the fluid (sample). Assays for measuring the amount of a specified protein are well known in the art and include direct or indirect measures. The level of protein biomarker in a fluid (sample) may also be determined by determining the level of protein biomarker activity in a fluid (sample). Accordingly, protein “level” encompasses both the amount of protein per se, or its level of activity.

[0083] The biomarkers as referred to herein can be detected using methods generally known in the art. Methods of detection generally encompass methods to quantify the level of a biomarker in the sample (quantitative method). It is generally known to the skilled artisan which of the following methods are suitable for qualitative and / or for quantitative detection of a biomarker. Samples can be conveniently assayed for, e.g., proteins using Westerns and immunoassays, like ELISAs, RIAs, fluorescence-based immunoassays, which are commercially available. Further suitable methods to detect biomarker include measuring a physical or chemical property specific for the peptide or polypeptide such as its precise molecular mass or NMR spectrum. Said methods comprise, e.g., biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass- spectrometers, NMR- analyzers, or chromatography devices. Further, methods include microplate ELISA-based methods, fully-automated or robotic immunoassays (available for example on Elecsys™ analyzers), CBA (an enzymatic Cobalt Binding Assay, available for example on Roche-Hitachi™ analyzers), and latex agglutination assays (available for example on Roche-Hitachi™ analyzers).

[0084] For the detection of (poly)peptide biomarkers or biomarker proteins as referred to herein a wide range of immunoassay techniques using such an assay format are available, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and 4,018,653. These include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target biomarker.

[0085] Sandwich assays are among the most useful and commonly used immunoassays.

[0086] Methods for measuring electro chemiluminescent phenomena are well-known. Such methods make use of the ability of special metal complexes to achieve, by means of oxidation, an excited state from which they decay to ground state, emitting electrochemiluminescence. For review see Richter, M.M., Chem. Rev. 104 (2004) 3003-3036.

[0087] Biomarkers can also be detected by generally known methods including magnetic resonance spectroscopy (NMR spectroscopy), Gas chromatography-mass spectrometry (GC-MS), Liquid chromatography-mass spectrometry (LC-MS), High and ultra-HPLC HPLC such as reverse phase HPLC, for example, ion-pairing HPLC with dual UV-wavelength detection, capillary electrophoresis with laser-induced fluorescence detection, anion exchange chromatography and fluorescent detection, thin layer chromatography.

[0088] Preferably, measuring the level of a biomarker as referred to herein comprises the steps of (a) contacting a cell capable of eliciting a cellular response the intensity of which is indicative of the level of the peptide or polypeptide with the said peptide or polypeptide for an adequate period of time, (b) measuring the cellular response. For measuring cellular responses, the sample or processed sample is, preferably, added to a cell culture and an 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, e.g. a peptide, polypeptide, or a small molecule. The expression or substance shall generate an intensity signal which correlates to the level of the peptide or polypeptide.

[0089] Also preferably, measuring the level of a peptide or polypeptide comprises the step of measuring a specific intensity signal obtainable from the peptide or polypeptide in the sample. As described above, such a signal may be the signal intensity observed at an m / z variable specific for the peptide or polypeptide observed in mass spectra or a NMR spectrum specific for the peptide or polypeptide.

[0090] Measuring the level of a peptide or polypeptide may, preferably, comprises the steps of (a) contacting the peptide with a specific binding agent, (b) (optionally) removing non-bound binding agent, (c) measuring the level of bound binding agent, i.e. the complex of the binding agent formed in step(a). According to a preferred embodiment, said steps of contacting, removing and measuring may be performed by an analyzer unit of the system disclosed herein. According to some embodiments, said steps may be performed by a single analyzer unit of said system or by more than one analyzer unit in operable communication with each other. For example, according to a specific embodiment, said system disclosed herein may include a first analyzer unit for performing said steps of contacting and removing and a second analyzer unit, operably connected to said first analyzer unit by a transport unit (for example, a robotic arm), which performs said step of measuring.

[0091] The bound binding agent, i.e. the binding agent or the binding agent / peptide complex, will generate an intensity signal. Binding according to the present invention includes both covalent and non-covalent binding. A binding agent according to the present invention can be any compound, e.g., a peptide, polypeptide, nucleic acid, or small molecule, binding to the peptide or polypeptide described herein. Preferred binding agents include antibodies, nucleic acids, peptides or polypeptides such as receptors or binding partners for the peptide or polypeptide and fragments thereof comprising the binding domains for the peptides, and aptamers, e.g. nucleic acid or peptide aptamers. Methods to prepare such binding agents are well-known in the art. For example, identification and production of suitable antibodies or aptamers is also offered by commercial suppliers. The person skilled in the art is familiar with methods to develop derivatives of such binding agents with higher affinity or specificity. For example, random mutations can be introduced into the 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. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab)2 fragments that are capable of binding the antigen. The present invention also includes single chain antibodies and humanized hybrid antibodies wherein amino acid sequences of a non-human donor antibody exhibiting a desired antigen- specificity are combined with sequences of a human acceptor antibody. The donor sequences will usually include at least the antigen-binding amino acid residues of the donor but may comprise other structurally and / or functionally relevant amino acid residues of the donor antibody as well. Such hybrids can be prepared by several methods well known in the art. Preferably, the binding agent or agent binds specifically to the pep-tide or polypeptide. Specific binding according to the present invention means that the ligand or agent should not bind substantially to (“cross-react” with) another peptide, polypeptide or substance present in the sample to be analyzed. Preferably, the specifically bound peptide or polypeptide should be bound with at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher affinity than any other relevant peptide or polypeptide. Non-specific binding may be tolerable, if it can still be distinguished and measured unequivocally, e.g. according to its size on a Western Blot, or by its relatively higher abundance in the sample. Binding of the binding agent can be measured by any method known in the art. Preferably, said method is semi-quantitative or quantitative. Further suitable techniques for the determination of a polypeptide or peptide are described in the following.

[0092] Binding of a binding agent may be measured directly, e.g. by NMR or surface plasmon resonance. Measurement of the binding of a binding agent, according to preferred embodiments, is performed by an analyzer unit of a system disclosed herein. Thereafter, a level of the measured binding may be calculated by a computing device of a system disclosed herein. If the binding agent also serves as a substrate of an enzymatic activity of the pep-tide or polypeptide of interest, an enzymatic reaction product may be measured (e.g. the level of a protease can be measured by measuring the level of cleaved substrate, e.g. on a Western Blot). Alternatively, the binding agent may exhibit enzymatic properties itself and the “binding agent / peptide or polypeptide” complex or the binding agent which was bound by the peptide or polypeptide, respectively, may be contacted with a suitable substrate allowing detection by the generation of an intensity signal. For measurement of enzymatic reaction products, preferably the level of substrate is saturating. The substrate may also be labeled with a detectable label prior to the reaction. Preferably, the sample is contacted with the substrate for an adequate period of time. An adequate period of time refers to the time necessary for a detectable, preferably measurable, level of product to be produced. Instead of measuring the level of product, the time necessary for appearance of a given (e.g. detectable) level of product can be measured. Third, the binding agent may be coupled covalently or non-covalently to a label allowing detection and measurement of the binding agent. Labeling may be done by direct or indirect methods. Direct labeling involves coupling of the label directly (covalently or non- covalently) to the binding agent. Indirect labeling involves binding (covalently or non- covalently) of a secondary binding agent to the first binding agent. The secondary binding agent should specifically bind to the first binding agent. Said secondary binding agent may be coupled with a suitable label and / or be the target (receptor) of tertiary binding agent binding to the secondary binding agent. The use of secondary, tertiary or even higher order binding agents is often used to increase the signal. Suitable secondary and higher order binding agents may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The binding agent or substrate may also be "tagged" with one or more tags as known in the art. Such tags may then be targets for higher order binding agents. Suitable tags include biotin, digoxygenin, His-Tag, Glutathion-S-Transferase, FLAG, GFP, myc-tag, influenza A virus haemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and / or C-terminus. Suitable labels are any labels detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluo-rescent labels. Enzymatically active labels include e.g. horseradish peroxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'- tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3- indolyl-phosphate, avail-able as ready-made stock solution from Roche Diagnostics), CDP- Star™ (Amersham Bio-sciences), ECF™ (Amersham Biosciences). A suitable enzymesubstrate combination may result in a colored reaction product, fluorescence or chemoluminescence, which can be measured according to methods known in the art (e.g. using a light-sensitive film or a suit-able camera system). As for measuring the enzymatic reaction, the criteria given above apply analogously. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes (e.g. Alexa 568). Further fluorescent labels are available e.g. from Molecular Probes (Oregon). Also the use of quantum dots as fluorescent labels is contemplated. A radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager.

[0093] The level of a peptide or polypeptide may be, also preferably, determined as follows: (a) contacting a solid support comprising a binding agent for the peptide or polypeptide as specified above with a sample comprising the peptide or polypeptide and (b) measuring the level peptide or polypeptide which is bound to the support. The binding agent, preferably chosen from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, is preferably present on a solid support in immobilized form. Materials for manufacturing solid supports are well known in the art and include, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc. The binding agent or agent may be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. Suitable methods for fixing / immobilizing said binding agent are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like. It is also contemplated to use “suspension arrays” as arrays according to the present invention (No-lan 2002, Trends Biotechnol. 20(l):9-12). In such suspension arrays, the carrier, e.g. a mi-crobead or microsphere, is present in suspension. The array consists of different microbeads or microspheres, possibly labeled, carrying different binding agents. Methods of producing such arrays, for example based on solid-phase chemistry and photo-labile protective groups, are generally known (US 5,744,305).

[0094] The term "detection agent" as used herein refers to an agent that is capable of specifically recognizing and binding to the biomarker present in a sample. The term is used interchangeably with the term "ligand" herein. Moreover, the said agent shall allow for direct or indirect detection of the complex formed by the said agent and the biomarker. Direct detection can be achieved by including into the agent a detectable label. Indirect labeling maybe achieved by a further agent that specifically binds to the complex comprising the biomarker and the detection agent wherein the said further agent is than capable of generating a detectable signal. Suitable compounds which can be used as detection agents are well known in the art. Preferably, the detection agent is an antibody, in particular a monoclonal antibody, or aptamer which specifically binds to the biomarker as referred to herein. The term “antibody” herein is used in the broadest sense and encompasses various anti-body structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigenbinding activity. Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.

[0095] The terms “level” or "amount" as used herein encompass the absolute amount of a biomarker as referred to herein, the relative amount or concentration of the said biomarker as well as any value or parameter which correlates thereto or can be derived therefrom. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said peptides by direct measurements, e.g., intensity values in mass spectra or NMR spectra. Moreover, encompassed are all values or parameters which are obtained by indirect measurements specified elsewhere in this description, e.g., response amounts measured from biological read out systems in response to the peptides or intensity signals obtained from specifically bound ligands. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations. "Signs" or "signals" of a disease include but are not limited to the change or alteration such as the presence, absence, increase or elevation, decrease or decline, of specific indicators such as biomarkers or molecular markers, or the development, presence, or worsening of symptoms.

[0096] The term "disease" and "disorder" are used interchangeably herein, referring to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a tissue, an organ or an individual is not able to efficiently fulfil its function anymore. Typically, but not necessarily, a disease is associated with specific symptoms or signs indicating the presence of such disease. The presence of such symptoms or signs may thus, be indicative for a tissue, an organ or an individual suffering from a disease. An alteration of these symptoms or signs may be indicative for the progression of such a disease. A progression of a disease is typically characterised by an increase or decrease of such symptoms or signs which may indicate a "worsening" or "bettering" of the disease. The "worsening" of a disease is characterised by a decreasing ability of a tissue, organ or organism to fulfil its function efficiently, whereas the "bettering" of a disease is typically characterised by an increase in the ability of a tissue, an organ or an individual to fulfil its function efficiently.

[0097] “Heart failure” (HF), also known as congestive heart failure (CHF), is a syndrome, a group of signs and symptoms caused by an impairment of the heart's blood pumping function. Symptoms typically include shortness of breath, excessive fatigue, and leg swelling. The shortness of breath may occur with exertion or while lying down, and may wake people up during the night. Chest pain, including angina, is not usually caused by heart failure, but may occur if the heart failure was caused by a heart attack. The severity of heart failure is measured by the signs or symptoms at rest or during exercise or work out. Other conditions that may have signs and symptoms similar to heart failure include obesity, kidney failure, liver disease, anemia, and thyroid disease.

[0098] The term “acute heart failure” is well known in the art and e.g. described in the 2021 Heart Failure ESC guidelines (Authors / Task Force Members, McDonagh et al., Reference 17). Typically, the term AHF refers to rapid or gradual onset of symptoms and / or signs of HF, severe enough for the patient to seek urgent medical attention leading to an unplanned hospital admission. Patients with AHF require urgent evaluation AHF is a leading cause of hospitalizations in subjects aged >65 years and is associated with high mortality and rehospitalization rates.

[0099] The subject may be referred to herein as a patient. The terms “subject”, “individual”, and “patient” are used herein interchangeably and, preferably, refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, 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, the patient is a human patient. The subject can be symptomatic (e.g., the subject presents symptoms associated with HF), or the subject can be asymptomatic (e.g., the subject does not present symptoms associated with HF). The subject may be diagnosed with, be at risk of developing or present with symptoms of HF. The subject may have, or be suspected of having (e.g., present with symptoms or a history indicative or suggestive of) HF.

[0100] Accordingly, in some examples, the subject has HF (and the method diagnoses, identifies, (or detects) that the subject has HF). In this context, the terms “diagnose” “identify”, and “detect” can be used interchangeably.

[0101] By detecting increased levels of a cardiac related (poly)peptide biomarker, in particular a BNP- type peptide, in a subject the suspicion that the subject suffers from HF would be confirmed and there is a high risk that the subject suffers from HF . In particular, in cases where the subject already exhibits clinical parameters, signs and / or symptoms of HF the determination of increased cardiac related (poly)peptide biomarker levels would confirm the presence of HF.

[0102] The term "comparing" as used herein refers to comparing the amount / level of the biomarker in the ISF (sample) from the subject with the reference amount or reference value of the biomarker specified elsewhere in this description. It is to be understood that comparing as used herein usually refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from the biomarker in a sample is compared to the same type of intensity signal obtained from a reference sample. The comparison may be carried out manually or computer assisted. Thus, the comparison may be carried out by a computing device. The value of the measured or detected amount of the biomarker in the sample from the subject and the reference amount can be, e.g., compared to each other and the said comparison can be automatically carried out by a computer program executing an algorithm for the comparison. The computer program carrying out the said evaluation will provide the desired assessment in a suitable output format. For a computer-assisted comparison, the value of the measured amount may be compared to values corresponding to suitable references which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format. For a computer-assisted comparison, the value of the measured amount may be compared to values corresponding to suitable references which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provides the desired assessment in a suitable output format.

[0103] The terms “(appropriate) reference value / level”, "reference sample" or "control (sample)" as used herein, refers to a sample which is analysed in a substantially identical manner as the sample of interest and whose information is compared to that of the sample of interest. A reference sample thereby provides a standard allowing for the evaluation of the information obtained from the sample of interest. A control sample may be derived from a healthy individual, thereby providing a standard of a healthy status. Differences between the status of the normal reference sample and the status of the sample of interest may be indicative of the presence or further progression of HF. Differences between the status of the normal or abnormal reference sample and the status of the sample of interest may be indicative of the absence or bettering of HF.

[0104] A reference sample / value / level may also be derived from the subject the sample / value of interest but has been taken at an earlier time point. Differences between the status of the earlier taken reference value / sample and the status of the value / sample of interest may be indicative of the progression of HF, i.e. a bettering or worsening of HF over time and by this may optionally allow to classify the stage of HF of being stage I, II, III or IV according to the New York Heart Association (NYHA) Functional Class in cases when classification is desired (see Table 4 of reference 17.). Stages of HF can also be described with the ACC / AHA stages that emphasize the development and progression of disease (see Figure 1 and Table 3 of reference 22.).

[0105] The determined value can be compared to more than one (appropriate) reference values, which can be of different kind. For example, the determined value can be compared to one or more values obtained from the same subject at earlier time points and in parallel it can be compared to one or more values obtained from other subjects (for example, with a known stage of HF).

[0106] The control sample may be an internal or an external control sample. An internal control sample is used, i.e. the biomarker level(s) is(are) assessed in the test sample as well as in one or more other sample(s) taken from the same subject to determine if there are any changes in the level(s) of said marker(s). Or measurements carried out at an earlier time point in the subject serve as control samples for measurements carried out at a later time point in the same subject. For an external control sample the presence or amount of a marker in a sample derived from the individual or measured in a subject is compared to its presence or amount in an individual known to suffer from, or known to be at risk of, a given condition; or an individual known to be free of a given condition, i.e., "normal individual".

[0107] It will be appreciated by the skilled artisan that such external control sample may be obtained from a single individual or may be obtained from a reference population that is age-matched and free of confounding diseases. Typically, samples from 100 well-characterized individuals from the appropriate reference population are used to establish a "reference value". However, reference population may also be chosen to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals represent a preferred reference population for establishing a control value.

[0108] For example, a marker concentration in a patient (sample) can be compared to a concentration known to be associated with a specific course of a certain disease. For example, it can be compared to a concentration known to be associated with a certain stage of HF. Usually, the (sample's) marker concentration is directly or indirectly correlated with a diagnosis and the marker concentration is e.g. used to determine whether an individual is at risk for a certain suffering from that disease. Alternatively, the marker concentration can be compared to marker concentrations obtained from the same subject at an earlier time point. Alternatively, the sample's marker concentration can e.g., be compared to a marker concentration known to be associated with a response to therapy in a certain disease, the diagnosis of a certain disease, the assessment of the severity of a certain disease, the guidance for selecting an appropriate drug to a certain disease, in judging the risk of disease progression, or in the follow-up of patients. Depending on the intended diagnostic use an appropriate control sample is chosen and a control or reference value for the marker established therein. As also clear to the skilled artisan, the absolute marker values established in a control sample will be dependent on the assay used.

[0109] The most common control samples and / or reference values derived therefrom for the methods described herein are obtained from but not limited to “healthy controls”. The corresponding subjects from which these samples are obtained are “healthy subjects” respectively.

[0110] “Healthy controls” refer to control samples of subjects that do not suffer from HF and do not show any symptoms or signs that could be associated with HF, like shortness of breath with activity or when lying down, fatigue and weakness, swelling in the legs, ankles and feet, rapid or irregular heartbeat, reduced ability to exercise, persistent cough or wheezing with white or pink blood-tinged mucus, swelling of the belly area (abdomen), very rapid weight gain from fluid buildup, nausea and lack of appetite, difficulty concentrating or decreased alertness, chest pain if heart failure is caused by a heart attack.

[0111] The control sample may be assayed at the same time, before or after, separately or simultaneously with the test sample / subject. The control value that is used in the comparison with the test sample / subject may be a value that is calculated as an average or median of more than one (e.g., two or more, five or more, ten or more, a group etc) of control samples. Alternatively, the control sample may be a sample that originated from (i.e., is a mix of) more than one (e.g., two or more, five or more, ten or more, a group etc) individual that is not suffering from HF.

[0112] For classification of the stage of HF a comparison of the test sample with several different control samples can or has to be carried out in order to be able to allocate the result to a certain stage. For example, a comparison to a healthy sample and to a NYHA class II sample. Or a comparison to a healthy sample, to a NYHA class II sample and to a NYHA class IV sample. This can also be combined with surgery or other interventions to confirm or determine a certain stage of HF. Alternatively, the level of (poly)peptide biomarker in the ISF may be compared to a predetermined reference level for the biomarker of interest. As used herein, a “predetermined reference level” refers to a biomarker level obtained from a reference database, which may be used to generate a pre-determined cut off value, i.e., a score that is statistically predictive of HF. In one example, the predetermined reference level is the average or median level of the biomarker in at least one individual not suffering from HF from the same species. The predetermined reference value may be calculated as the average or median, taken from a group or population of individuals that are not suffering from HF. The individual or the population of individuals can be the same age or in the same state or condition of health as the subject from which the test sample is obtained.

[0113] In one example, the pre-determined reference level is therefore the average level of the biomarker in a control subject that does not have HF.

[0114] Typically, in methods for diagnosing / assessing HF in a subject, the control sample or predetermined reference are obtained from an individual or group of individuals that are distinct from the subject that is being tested (i.e., the subject within which the biomarker level is determined or from which the test sample is obtained / provided). In such examples, the control or predetermined reference are used as a bench line to determine whether the tested subject has HF.

[0115] In an alternative example, the control or predetermined reference value may be obtained from the same individual as the test sample, but at an earlier time point. This is particularly relevant for the methods described herein that classify the stage of HF, that determine the progression in a subject, that determine the therapeutic effect of a treatment regimen for HF, and / or that determine a subject’s compliance or adherence with a prescribed treatment regimen for HF. For this the measurements in the ISF are carried out in the same subject or the ISF samples are taken from the same subject.

[0116] In such examples, the control sample or predetermined reference level is used to determine any changes in the level of the biomarker(s) over a time interval for the same subject. The predetermined reference level or control sample can therefore be from the same subject that the test sample is obtained from, for example obtained at an earlier time point. This earlier time point can be before they were diagnosed with HF.

[0117] A pre-determined level can be a single cut-off value, such as a median or a mean. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where the risk in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n-quantiles being subjects with the highest risk. Moreover, the reference could be a calculated reference, most preferably the average or median, for the relative or absolute amount of a biomarker of a population of individuals comprising the subject to be investigated. How to calculate a suitable reference value, preferably, the average or median, is well known in the art.

[0118] Thus, in some cases the level of the biomarker in a subject being greater than or equal to the level of the biomarker of the control sample or pre-determined reference level is indicative of a clinical status (e.g., indicative of HF). In other cases, the level of the biomarker in a subject being less than or equal to the level of biomarker of the control sample or predetermined reference level is indicative of a certain stage of HF.

[0119] Typically, but not necessarily, the greater than, or the less than, that is sufficient to distinguish a subject from a control subject is a statistically significantly greater than, or a statistically significant less than. In cases where the level of the biomarker in a subject being equal to the level of the biomarker in a control subject is indicative of a stage of HF, the "being equal" refers to being approximately equal (e.g., not statistically different).

[0120] The pre-determined value can depend upon a particular population of subjects (e.g., human subjects) selected. For example, an apparently healthy population will have a different 'normal' range of the biomarker than will a population of subjects which have, or are likely to have, HF. Accordingly, the pre- determined values selected may take into account the category (e.g., healthy, diseased, stage of disease) in which a subject (e.g., human subject) falls.

[0121] Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.

[0122] Suitably, the level of the specific biomarker detected in a subject / sample (e.g., a test sample, a control sample etc) may be normalized by adjusting the measured level (amount or activity) of the biomarker using the level of a reference (protein) in the same sample, wherein the reference (protein) is not a marker itself (it is e.g., a protein that is constitutively expressed). This normalization allows the comparison of the biomarker level in one sample to another sample, or between samples from different sources. This normalized level can then optionally be compared to 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, alternatively, it may be normalized against a known protein constitutively expressed in whole blood such as albumin, immunoglobulins or plasma protein concentration. For example, when measuring a protein biomarker in a serum (or plasma) sample the biomarker may be expressed as an absolute concentration or, alternatively, it may be normalized against a known protein constitutively expressed in serum (or plasma).

[0123] The biomarker level(s) in the test sample may be compared to the level of the same biomarker in a control sample or with a pre-determined reference level for the same biomarker to identify an increase or decrease in a level of the one or more biomarker in the sample of the subject.

[0124] In the methods described herein, the subject may be identified as having HF if the comparison (between biomarker level(s) in the control subject / sample / predetermined reference value and the test subject / sample of the subject) indicates that the subject has an increased level of the BNP -type peptide compared to the control sample or the pre- determined reference level.

[0125] As will be understood by those skilled in the art, the assessment made in accordance with the present invention, although preferred to be, may usually not be correct for 100% of the investigated subjects. The term, typically, requires that a statistically significant portion of subjects can be correctly assessed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistical evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc.. Details may be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically envisaged confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-values are, typically, 0.2, 0.1, 0.05.

[0126] The terms "lowered" or "decreased" level of an indicator refer to the level of such indicator in the sample being reduced in comparison to the reference (value) or reference sample. The terms "decrease", "decreased" "reduced", "reduction" or “down- regulated", “lower” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, "reduced", "reduction", "decreased" or "decrease" means a decrease by at least 10% as compared to a reference level / control, for example a decrease by 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 up to and including a 100% decrease (i.e. absent level as compared to a reference / control sample), or any decrease between 10-100% as compared to a reference level / control, or at least about a 0.5 -fold, or at least about a 1.0-fold, or at least about a 1.2-fold, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold decrease, or any decrease between 1.0-fold and 10-fold or greater as compared to a reference level / control.

[0127] The terms "elevated" or "increased" level of an indicator / (bio)marker refer to the level of such indicator in the sample being higher in comparison to the reference (value) or reference sample. E.g. a protein that is detectable in higher amounts in a fluid sample of one individual suffering from a given disease than in the same fluid sample of individuals not suffering from said disease, has an elevated level. The terms "increased", "increase" or "up-regulated", “higher” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased" or "increase" means an increase of at least 10% as compared to a reference level / control, for example an increase of 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 up to and including a 100% increase or any increase between 10-100% as compared to a reference level / control, or at least about a 0.5 -fold, or at least about a 1.0-fold, or at least about a 1.2-fold, or at least about a 1.5 -fold, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 1.0-fold and 10-fold or greater as compared to a reference level / control.

[0128] Embodiments

[0129] The inventors have surprisingly identified that the ISF is reliable for the purpose of determining the levels of cardiac related (poly)peptide biomarkers, in particular BNP -type peptides like NT- proBNP.

[0130] The ISF can be used for assessing heart failure, determining the risk of developing heart failure, classifying the stage of heart failure, monitor heart failure and / or determine the therapeutic effect of a treatment in a subject. Further, by determining the level of one or more cardiac related (poly)peptide biomarker in the ISF a quick, easy and reliable approach for assessing HF is provided.

[0131] ISF as the body fluid to determine cardiac related biomarker levels can advantageously be used in any of the methods, kits, or uses described herein.

[0132] Methods for assessing HF or determining the risk of developing HF in a subject

[0133] In one aspect the invention relates to a method for assessing heart failure (HF) or determining the risk of developing HF in a subject, the method comprising the steps of a) determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid (ISF) from the subject; b) comparing the level of the one or more cardiac related (poly)peptide biomarker with the level of the same cardiac related (poly)peptide biomarker in a control sample or with a pre-determined reference level for the same cardiac related biomarker; and c) identifying a subject as having heart failure or as having an increased risk of developing heart failure based on the comparison in step b).

[0134] The method can be in vivo, ex vivo or in vitro. Preferably the method is an in vivo method. As used herein, the terms "biological (fluid) sample", “test sample”, "sample" are used interchangeably, and variations thereof refer to a sample obtained or derived from a subject. For the purposes described herein, the sample is, or comprises, an ISF sample. Notably, since the methods described herein can also be in vivo methods these terms may also refer to a “sample” in the sense that it represents a value obtained by an in vivo measurement, determination without actually obtaining a sample that has been transferred out of the subject’s body.

[0135] The ISF can be any type of ISF, preferably subcutaneous, i.e. dermal ISF. Dermal ISF refers to ISF within the skin layers epidermis, dermis and hypodermis.

[0136] In particular embodiments the subject is a human.

[0137] The method can be carried out based on the suspicion that the subject might suffer from or might have an increased risk in developing HF. Alternatively, the cardiac related (poly)peptide biomarker levels are determined routinely as part of screening tests without any suspicion on HF.

[0138] Patients “at risk of developing HF” are not healthy but have comorbid conditions (stage A = risk factors) or cardiac abnormalities in absence of signs / symptoms of HF (stage B) that may lead to the development of HF. This corresponds to the stages A and B of the ACC / AHA classification (see Figure 1 and Table 3. of reference 22.)

[0139] Patients with diabetes are at risk of developing HF but are not healthy; same for arterial hypertension of all the risk factors associated with higher risk of HF. For a list of risk factors for the development of HF, see Table 10 of reference 17.

[0140] In embodiments, an elevated level of the one or more cardiac related (poly)peptide biomarker in the ISF of the subject compared to the control sample or to the pre-determined reference level is indicative for the subject having HF or has an increased risk of developing HF.

[0141] In embodiments, the level(s) of one, two, three, or four cardiac related (poly)peptide biomarkers are determined.

[0142] In particular, a level of cardiac related (poly)peptide biomarker in the ISF elevated by 25% or more, is indicative of the presence or the risk of developing HF. In particular, a level of cardiac related (poly)peptide biomarker in the ISF elevated by 30% or more, is indicative of the presence of HF. In particular, a level of cardiac related (poly)peptide biomarker in the ISF elevated by 50% or more, is indicative of the presence of HF. In particular, a level of cardiac related (poly)peptide biomarker in the ISF elevated by 100% or more, is indicative of the presence of HF. In particular, a level of cardiac related (poly)peptide biomarker in the ISF elevated by 150% or more, is indicative of the presence of HF. In particular, a level of cardiac related (poly)peptide biomarker in the ISF elevated by 200% or more, is indicative of HF.

[0143] Suitably, the cardiac related (poly)peptide biomarker may be a BNP -type peptide.

[0144] Suitably, 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.

[0145] The present application shows a close to 1 : 1 correlation between NT-proBNP in serum and ISF. This allows to consider NT-proBNP levels detected in the ISF as reliable biomarker for cardiac related disorders / diseases.

[0146] Due to the close to 1 : 1 correlation between NT-proBNP in serum and ISF, NT-proBNP not only qualifies as biomarker in ISF, but the correlation even allows to apply already known protocols, treatment regimens or other suitable measures as they are already known and established for the corresponding NT-proBNP levels in serum.

[0147] Suitably, additionally to the level of the one or more cardiac related (poly)peptide biomarker the level of one or more further cardiac related biomarkers selected from the group consisting of 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 Cluster of Differentiation 146 (or Cell surface glycoprotein MUC18, sCD146), bioAdrenomedullin (bio ADM), Midregional proadrenomedullin (MR-proADM), Soluble suppression of tumorigenesis-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) may be determined.

[0148] By determining the level(s) of one or more cardiac related biomarkers in addition to the BNP- type peptide biomarker(s) further information about the status of the subject, the status of HF / HF progression can be obtained. By this a more precise and reliable assessment may be possible.

[0149] In embodiments, the level(s) of one, two, three, or four cardiac related (poly)peptide biomarkers are determined together with one, two, three, four ore even more further biomarkers selected from the group consisting of 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 Cluster of Differentiation 146 (or Cell surface glycoprotein MUC18, sCD146), bioAdrenomedullin (bioADM), Midregional proadrenomedullin (MR-proADM), Soluble suppression of tumorigenesis-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) may be determined.

[0150] When determining the level(s) of one, two, three, or four cardiac related (poly)peptide biomarkers together with one, two, three, four ore even more further biomarkers, preferably the following combinations of the determinations can be made for improved diagnostic outcomes: a cardiac related (poly)peptide biomarker alone a cardiac related (poly)peptide biomarker in combination with sodium and / or potassium (e.g. to monitor HF progression and therapeutic response) a cardiac related (poly)peptide biomarker in combination with sodium and / or potassium and / or further biomarkers (e.g. glucose or creatinine) a cardiac related (poly)peptide biomarker in combination with sodium and / or potassium and / or further biomarkers (e.g. glucose or creatinine) and / or further clinical information of the subject (e.g. additional known diseases of the subject, age, gender, weight, size)

[0151] In one example the level of NT-proBNP and creatinine are determined.

[0152] In one example the level of NT-proBNP and potassium are determined.

[0153] In one example the level of NT-proBNP, creatinine and potassium are determined.

[0154] Preferred combinations for the monitoring of patients with HF include a cardiac related (poly)peptide biomarker and serum sodium and serum potassium.

[0155] Preferred combinations for the assessment of congestion include a cardiac related (poly)peptide biomarker with 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.

[0156] Suitably, the heart failure (HF) may be selected from the group consisting of acute heart failure, heart failure due to reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF), heart failure with preserved ejection fraction (HFpEF), left-sided failure, right-sided failure, biventricular failure.

[0157] Suitably, the method may further comprise selecting a treatment regimen for the subject based on the comparison of the level of the cardiac related (poly)peptide biomarker with the control sample or with the pre- determined reference level.

[0158] In particular embodiments, the method may further comprise administering the selected treatment regimen to the subject, optionally wherein the selected treatment regimen comprises drug-based therapy and / or surgery.

[0159] Treatment can include, for example, surgery and, in some cases, therapy, or combinations thereof. However, in some cases, immediate treatment may not be required, and the subject may be selected for active surveillance.

[0160] As used herein, the terms “active surveillance”, “monitoring” and “watchful waiting” are used interchangeably herein to mean closely monitoring a patient's condition without giving any treatment until symptoms appear or change.

[0161] As used herein, the terms “treat”, “treating” and "treatment" are taken to include an intervention performed with the intention of altering the pathology of a condition, disorder or symptom (i.e., in this case HF). Accordingly, "treatment" refers to therapeutic treatment, wherein the object is to slow down (lessen) the targeted condition, disorder or symptom. “Treatment” therefore encompasses a reduction, slowing or inhibition of the symptoms of HF, for example of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to the symptoms before treatment.

[0162] In the context of HF, appropriate treatment may include drug therapy and / or surgery.

[0163] As used herein, the term “surgery” applies to surgical methods, like, for example, angioplasty (also known as Percutaneous Coronary Interventions [PCI], Balloon Angioplasty and Coronary Artery Balloon Dilation), artificial heart valve surgery (also known as Heart Valve Replacement Surgery), atherectomy, bypass surgery (also known as CABG, Coronary Artery Bypass Graft done via Open-Heart Surgery), cardiomyoplasty, heart transplant, minimally invasive heart surgery (also known as Limited Access Coronary Artery Surgery and includes Port- Access Coronary Artery Bypass (PACAB or PortC AB) 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). As used herein, the term “therapy” may include drug-based therapy, radiation, hormonal therapy, cryosurgery, chemotherapy, immunotherapy, biologic therapy, and high-intensity focused ultrasound.

[0164] Drug-based therapy of HF can for example comprise the administration of P blockers, angiotensin converting enzyme (ACE) inhibitors (or angiotensin receptor blockers [ARBs] if the patient was intolerant to ACE inhibitors) or angiotensin receptor-neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists, sodium-glucose co-transporter 2 inhibitors, loop diuretics, If channel inhibitors, soluble guanylate cyclase receptor stimulators, hydralazine and isosorbide dinitrates, digoxin, ferric carboymaltose.

[0165] The type of treatment will vary depending on the particular form and / or stage of HF that the subject has, is suspected of having. Depending on whether the assessment rather hints to a severe disease stage or not the person skilled in the art would be well aware of how to select the most appropriate and promising treatment regimen.

[0166] Definitions for the cardiac related (poly)peptide biomarkers, combinations with further biomarkers, methods steps, subjects, types of HF, treatments, reference values, time intervals, etc. provided elsewhere herein equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0167] Methods for monitoring HF in a subject

[0168] In one aspect the invention relates to a method for monitoring heart failure in a subject, the method comprising the steps of i. determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid (ISF) from the subject, optionally in accordance with method steps a) to b) of claim 1 ; ii. repeating step i. after a time interval; and iii. comparing the levels of the cardiac related (poly)peptide biomarker identified in i. with the levels identified in ii., wherein a change in the levels from i. to ii. is indicative of a change in heart failure in the subject.

[0169] In particular, due to the easy accessibility of ISF continuous monitoring of subjects will be possible without requiring professional assistance. Measurements within the ISF can be carried out by the subject, for example, with a batch having a needle type sensor applied to the subject’s skin. Such batches are for example known for glucose measurements in diabetes patients. This allows to monitor the biomarker levels over a long time at desired time intervals without the need for the patient to see a medical doctor or professional assistance but rather during the daily life.

[0170] In embodiments, a patient suffering from HF is monitored to determine if the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject is changing over time. In particular, a patient suffering from HF is monitored to determine if the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject is increasing, decreasing or not changing over time. In embodiments, a patient suffering from HF is monitored if an elevated level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid (ISF) from the subject is determined.

[0171] The method may be used to monitor the progression of any kind of HF described herein.

[0172] Such monitoring methods may be performed on subjects that have not yet been treated for HF or on subjects that have already been treated for HF. Such methods may be performed on patients before, during or after (re-)hospitalization.

[0173] Monitoring the progression of HF in a subject over time assists in the earliest possible identification of HF progression (e.g., a worsening in disease status or disease symptoms). Such monitoring naturally involves the taking of repeated samples over time. The method may therefore be repeated at one or more time intervals for a particular subject and the results compared to monitor the development, progression or improvement in HF of that subject over time, wherein a change in the amount of level of the biomarker tested for in the ISF is indicative of a change in the progression of HF in the subject.

[0174] HF progression may be indicated by an increase in the level cardiac related (poly)peptide biomarker detected over time when the results of two or more time intervals are compared for the same subject.

[0175] In other words, if the method is performed a plurality of times, HF progression may be indicated when the level of cardiac related (poly)peptide biomarker detected at the later time interval(s) is higher than that detected at the earlier time interval(s). An “increase” in the level of cardiac related (poly)peptide biomarker encompasses detection of cardiac related (poly)peptide biomarker at a later time interval when no cardiac related (poly)peptide biomarker was detected (i.e., it was not present at detectable levels) when the method was performed previously (i.e., at an earlier time interval) on the same subject. This is particularly relevant when monitoring the progression of HF in subjects that based on other signs or symptoms are suspected to develop HF.

[0176] Suitable time intervals for monitoring HF progression can easily be identified by a person of skill in the art and will depend on the specific form of HF being monitored. As a non-limiting example, the method may be repeated at least every day, week, month, six months, or at least every year, or whenever clinically needed, i.e., in case of a significant change in HF symptoms. The frequency of monitoring HF progression will depend on the available health-care setting and severity of the disease. In some cases, such as patients hospitalized with acute HF and treated with intravenous diuretics, a reasonable frequency can be up to every 2-6 hours or every 2-12 hours.

[0177] For regular monitoring the monitoring may be performed at regular intervals. The term “at regular intervals” as used herein refers to the periodical determination of the cardiac related (poly)peptide biomarker levels in the same subject after a predetermined time. Since every subject is different the speed of development of HF may vary from subject to subject. Whereas in one subject HF progression can progress through (for example, from stage I to stage IV) within a few years it might stagnate at one stage for several years in another subject. Therefore, to obtain a continuous picture of the development of the cardiac related (poly)peptide biomarker levels in a subject and to be able to allocate the obtained cardiac related (poly)peptide biomarker levels to the corresponding disease stages I, II, III and IV the intervals should be chosen in a way that no stages of disease progression are skipped. Preferably the cardiac related (poly)peptide biomarker levels of a subject are determined every 2, 4, 6, 8, 10, 11, or 12 weeks.

[0178] The most important determinant of the frequency of monitoring will be when the subjects are rapidly decompensating and present signs and symptoms of acute HF (Heavy breathing, sensation like suffocating, struggling to breathe while lying down, tight chest, arrhythmia, chest pain, cough, fluid retention (edema) in the arms or legs) or after discharge from hospital for acute HF.

[0179] For such cases shorter intervals between the monitoring times should be chosen. A skilled person would be well aware to choose the appropriate frequency depending on every individual scenario.

[0180] In patients with stable HF, monitoring every week or month may be suffcicient.

[0181] Definitions for the cardiac related (poly)peptide biomarkers, combinations with further biomarkers, methods steps, subjects, types of HF, treatments, reference values, time intervals, etc. provided elsewhere herein equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0182] Methods for classifying the stage of HF in a subject In one aspect the invention relates to a method for classifying the stage of heart failure in a subject, the method comprising the steps of a) determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) classifying the stage of heart failure in the subject if the comparison in step b) indicates that the subject has an increased or decreased level of the one or more cardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

[0183] In embodiments, an elevated or reduced level or amount or concentration of the cardiac related (poly)peptide biomarker in the ISF of the subject can be indicative of the stage of HF in the subject. In particular, a level of the cardiac related (poly)peptide biomarker in the ISF of the subject is indicative of the stage of HF in the subject if the level of the cardiac related (poly)peptide biomarker in the ISF of the subject is higher or lower than the level of the cardiac related (polyjpeptide biomarker according to a reference value of which the stage of HF is known. If necessary, comparisons to several reference values are made in order to classify the stage of HF. In addition, this can be combined with surgery or other parameters used to determine the stage of HF to allow an even more precise classification.

[0184] In particular, if the cardiac related (poly)peptide biomarker is detectable in higher levels in the ISF of the subject assessed for the presence of HF than in the ISF of subjects not suffering from HF then this is indicative of any stage of HF. By further comparing the obtained value to reference values of which the stage of HF is known an allocation of the obtained value to a certain stage of HF is possible.

[0185] Suitably, the subject’s stage of heart failure may be classified as stage I, stage II, stage III or stage IV heart failure according to the New York Heart Association (NYHA) Functional Classification.

[0186] In a particular embodiment the control sample or pre-determined reference level is i. a level of the cardiac related (poly)peptide biomarker in a healthy subject or in a subject that has stage I, stage II, stage III or stage IV HF according to the New York Heart Association (NYHA) Functional Classification, or is ii. an average level of the cardiac related (poly)peptide biomarker in a group of healthy subjects or in a group of subjects that have stage I, stage II, stage III or stage IV HF according to the New York Heart Association (NYHA) Functional Classification, or wherein the at least one appropriate reference value is iii. a predetermined value of a level of the cardiac related (poly)peptide biomarker in a healthy subject or in a subject that has stage I, stage II, stage III or stage IV HF according to the New York Heart Association (NYHA) Functional Classification, or is iv. a predetermined average value of a level of the cardiac related (poly)peptide biomarker in a group of healthy subjects or in a group of subjects that have stage I, stage II, stage III or stage IV HF according to the New York Heart Association (NYHA) Functional Classification.

[0187] For example, a level of the cardiac related (poly)peptide biomarker obtained that is higher than in a healthy subject but lower than in a subject with stage II HF is indicative of stage I HF.

[0188] For example, a level of the cardiac related (poly)peptide biomarker obtained that is higher than in a subject with stage I HF and higher than in a subject with stage III HF is indicative of stage II HF.

[0189] Definitions for the cardiac related (poly)peptide biomarkers, combinations with further biomarkers, methods steps, subjects, types of HF, treatments, reference values, time intervals, etc. provided elsewhere herein equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0190] Methods for determining the therapeutic effect of a treatment regimen for HF in a subject In one aspect the invention relates to a method for determining the therapeutic effect of a treatment regimen for heart failure in a subject, the method comprising the steps of: i. determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid (ISF) from the subject, optionally in accordance with method steps a) to b) of claim 1; ii. repeating step i. after a time interval; and iii. comparing the levels of the cardiac related (poly)peptide biomarker identified in i. with the levels identified in ii., and identifying that the treatment regimen has a therapeutic effect if the level of the cardiac related (poly)peptide biomarker decreased after treatment.

[0191] In one example, the change in level of the cardiac related (poly)peptide biomarker that is indicative of a therapeutic effect is a decrease in the cardiac related (poly)peptide biomarker level in the ISF after treatment. An “decrease” in the level of the cardiac related (poly)peptide biomarker encompasses no detection of the cardiac related (poly)peptide biomarker (i.e., it is not present at detectable levels) at a later time interval when the cardiac related (poly)peptide biomarker was detected when the method was performed previously (i.e. at an earlier time interval) on the same subject.

[0192] Step i. may first be performed in accordance with the method at a time point before the treatment regimen for HF began. Alternatively, step i. may first be performed at the same time as commencing the treatment regimen, or at a time point after the treatment regimen for HF began. The method can therefore be used to determine the therapeutic effect of a treatment regimen for HF from the outset (i.e., from the start of the regimen) or from a time point after the treatment regimen has started (i.e., determining the therapeutic effect of a treatment regimen for HF during the treatment regimen itself).

[0193] In embodiments, an unaltered or increasing level of the cardiac related (poly)peptide biomarker in the ISF of the subject being treated for HF is indicative of the therapy being potentially insufficient, i.e., an unaltered or increasing level of the cardiac related (poly)peptide biomarker in the ISF of the subject being treated for HF may be indicative of persisting or recurring HF. In particular, the treatment for HF is ineffective if the level of the cardiac related (poly)peptide biomarker is increasing to 30% or more. In particular, the treatment for HF is ineffective if the level of the cardiac related (poly)peptide biomarker is increasing to 50% or more. In particular, the treatment for HF is ineffective if the level of the cardiac related (poly)peptide biomarker is increasing to 100% or more. In particular, the treatment for HF is ineffective if the level of the cardiac related (poly)peptide biomarker is increasing to 150% or more. In particular, the treatment for HF is ineffective if the level of the cardiac related (poly)peptide biomarker is increasing to 200% or more. Same applies vice versa to cardiac related (poly)peptide biomarker whose decrease is indicative of a deterioration of the HF condition.

[0194] Alternatively, an unaltered level of the cardiac related (poly)peptide biomarker could mean that the HF progression stagnates.

[0195] An improvement in disease status or symptoms (e.g., over a treatment period) may also be indicated by stabilised levels of the cardiac related (poly)peptide biomarker over time (compared to the level of the cardiac related (poly)peptide biomarker observed in the absence of treatment over the equivalent time period or compared to equivalent controls).

[0196] 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 development of symptoms (e.g., over a treatment period).

[0197] A treatment regimen may also be identified as having a therapeutic effect if it results in an improvement in disease status or symptoms (e.g., over a treatment period). Methods for determining if the treatment regimen has a therapeutic effect are well known in the art. A treatment period refers to a time interval over which treatment occurs (e.g., 1 month, 3 months, 6 months, 1 year, 2 years, or even lifelong).

[0198] As would be clear to a person of skill in the art, the direction of change in the cardiac related (poly)peptide biomarker levels that is indicative of a therapeutic effect may depend on the disease status of the subject prior to treatment and the control / reference used.

[0199] The change in level of the cardiac related (poly)peptide biomarker can also be indicative of compliance or adherence with the prescribed treatment after treatment.

[0200] The trends for identifying that the subject has complied or adhered with the prescribed treatment regimen are equivalent to those described in detail above in respect of determining the therapeutic effect of a treatment regimen for HF. This is because a “prescribed treatment regimen” is a recommended treatment regimen and therefore typically has a therapeutic effect (and thus, observation of the therapeutic effect on the biomarker levels is an indication of subject compliance or adherence with the prescribed treatment regimen).

[0201] In embodiments, the subject is monitored several times at different time points. In embodiments, the patient is monitored several times within a time frame of days, weeks, months, or years. In particular embodiments, a subject is monitored is once a month or once a year. In embodiments, a subject suffering from HF is monitored once a month or once a year after diagnosis of HF. In embodiments, a subject being treated for HF is monitored once after therapy, in particular once after surgical therapy. In particular, the subject being treated for HF is monitored once a month or once a year to determine the efficacy of treatment and / or the recurrence of HF.

[0202] The method can also be useful as a screening tool for determining if specific regimens or treatment modalities have a therapeutic effect on HF. The tested regimens or treatment modalities may be new regimens or treatment modalities, modified regimens or treatment modalities, or known regimens or treatment modalities that need further testing. In this context, a treatment modality is e.g., a drug or medicament that is useful or suspected to be useful in the treatment of HF.

[0203] Definitions for the cardiac related (poly)peptide biomarkers, combinations with further biomarkers, methods steps, subjects, types of HF, treatments, reference values, time intervals, etc. provided elsewhere herein equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0204] Risk stratification of progressing to severe disease In one aspect the invention relates to a method for risk stratification of progressing to severe disease of a subject with HF, the method comprising the steps of: a) determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) stratifying the risk of progressing to severe disease of the subject with HF if the comparison in step b) indicates that the subject has an increased or decreased level of the one or more cardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

[0205] As used herein the term “progressing to severe disease” means worsening of signs or symptoms of HF or of physical capacity of a subject with HF

[0206] When determining the level of one or more cardiac related (poly)peptide biomarker and realizing that the HF condition of the subject has deteriorated because the biomarker increased or decreased by at least 10% as compared to a reference level / control, for example by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90% or by at least about 100%.

[0207] Definitions for the cardiac related (poly)peptide biomarkers, combinations with further biomarkers, methods steps, subjects, types of HF, treatments, reference values, time intervals, etc. provided elsewhere herein equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0208] Makins a prognosis of future disease course

[0209] In another aspect the invention relates to a method for making a prognosis of future disease course of a subject with HF, the method comprising the steps of: a) determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) making a prognosis of future disease course of the subject with HF if the comparison in step b) indicates that the subject has an increased or decreased level of the one or more cardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

[0210] As used herein the term “making a prognosis of future disease course” refers to the evaluation of the future evolution of severity of the HF of a subject with HF. The future evolution of severity of HF can include both a worsening or an improvement of the severity of HF and may be directed to a future worsening or improvement of signs or symptoms of HF or of physical capacity of a subject with HF. The prognosis may also include future events like hospitalization of a subject with HF and / or the occurrence of fatal events of the subject with HF.

[0211] Definitions for the cardiac related (poly)peptide biomarkers, combinations with further biomarkers, methods steps, subjects, types of HF, treatments, reference values, time intervals, etc. provided elsewhere herein equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0212] Uses

[0213] In one aspect the present invention relates to the use of interstitial fluid (I SF) for assessing heart failure, determining the risk of developing heart failure, classifying the stage of heart failure, monitor heart failure and / or determine the therapeutic effect of a treatment in a subject by determining the level of one or more cardiac related (poly)peptide biomarker therein.

[0214] Definitions for the cardiac related (poly)peptide biomarkers, combinations with further biomarkers, methods steps, subjects, types of HF, treatments, reference values, time intervals, etc. provided elsewhere herein equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0215] Computer-implemented method for assessing a patient with HF

[0216] The definitions and explanations provided above preferably apply mutatis mutandis to the following. The methods of the present invention can be also carried out as computer-implemented methods. In computer-implemented methods, typically, all steps of the computer-implemented method of the present invention are performed by one or more processing units of a computer or a computer network. However, the computer-implemented method may comprise additional steps, such as the determination of the level of a biomarker, such as the amount of a BNP -type peptide.

[0217] Thus, the present invention further relates to a computer-implemented method for assessing a subject with suspected heart failure comprising the steps of

[0218] (a) receiving a value for level of a first cardiac related (poly)peptide biomarker in the interstitial fluid of the subject, said first biomarker being a BNP -type peptide;

[0219] (b) optionally, receiving a value for the level of at least one additional cardiac related biomarker in the interstitial fluid of the subject, wherein said additional biomarker is selected from the group consisting of 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 Cluster of Differentiation 146 (or Cell surface glycoprotein MUC18, sCD146), bioAdrenomedullin (bio ADM), Midregional proadrenomedullin (MR-proADM), Soluble suppression of tumorigene sis-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),

[0220] (c) comparing the values for the levels of steps (a) and optionally step (b) to appropriate references for said biomarkers and / or calculating a score for assessing the subject with suspected heart failure based on the levels of the biomarkers; and

[0221] (d) assessing said subject based on the comparison and / or the calculation made in step (c).

[0222] The term “computer-implemented” as used herein means that the method is carried out in an automated fashion on a data processing unit which is, typically, comprised in a computer or similar data processing device. The data processing unit shall receive values for the amount of the biomarkers. Such values can be the amounts, relative amounts or any other calculated value reflecting the amount as described elsewhere herein in detail. Accordingly, it is to be understood that the aforementioned method does not require the determination of amounts for the biomarkers but rather uses values for already predetermined amounts. The present invention further relates to a computer program including computer-executable instructions for performing the steps of the computer-implemented method according to the present invention, when the program is executed on a computer or computer network. Typically, the computer program specifically may contain computer-executable instructions for performing the steps of the method as disclosed herein. Specifically, the computer program may be stored on a computer-readable data carrier.

[0223] The present invention further relates to a computer program product with program code means stored on a machine-readable carrier, in order to perform the method according to the present invention, when the program is executed on a computer or computer network, such as one or more of the above-mentioned steps discussed in the context of the computer program. As used herein, a computer program product refers to the program as a tradable product. The product may generally exist in an arbitrary format, such as in a paper format, or on a computer-readable data carrier. Specifically, the computer program product may be distributed over a data network.

[0224] The present invention further relates to a computer or computer network comprising at least one processing unit, wherein the processing unit is adapted to perform all steps of the method according to the present invention, in particular steps a), b), c) and d).

[0225] The present invention also, in principle, contemplates a computer program, computer program product or computer readable storage medium having tangibly embedded said computer program, wherein the computer program comprises instructions when run on a data processing device or computer carry out the method of the present invention as specified above. Specifically, the present disclosure further encompasses:

[0226] A computer or computer network comprising at least one processor, wherein the processor is adapted to perform the method according to one of the embodiments described in this description, a computer loadable data structure that is adapted to perform the method according to one of the embodiments described in this description while the data structure is being executed on a computer, a computer script, wherein the computer program is adapted to perform the method according to one of the embodiments described in this description while the program is being executed on a computer, a computer program comprising program means for performing the method according to one of the embodiments described in this description while the computer program is being executed on a computer or on a computer network, a computer program comprising program means according to the preceding embodiment, wherein the program means are stored on a storage medium readable to a computer, a storage medium, wherein a data structure is stored on the storage medium and wherein the data structure is adapted to perform the method according to one of the embodiments described in this description after having been loaded into a main and / or working storage of a computer or of a computer network, a computer program product having program code means, wherein the program code means can be stored or are stored on a storage medium, for performing the method according to one of the embodiments described in this description, if the program code means are executed on a computer or on a computer network, a data stream signal, typically encrypted, comprising a data for the BNP -type peptide, and a data stream signal, typically encrypted, comprising the calculations by the methods of the present invention and, preferably, providing information of the assessment.

[0227] Definitions for the cardiac related (poly)peptide biomarkers, combinations with further biomarkers, methods steps, subjects, types of HF, treatments, reference values, time intervals, etc. provided elsewhere herein equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0228] Data storage aspects

[0229] Biomarker levels and / or reference levels may be stored in a suitable data storage medium (e.g., a database) and are, thus, also available for future diagnoses. This also allows efficiently diagnosing prevalence for a disease because suitable reference results can be identified in the database once it has been confirmed (in the future) that the subject from which the corresponding reference sample was obtained did have HF. As used herein a "database" comprises data collected (e.g., analyte and / or reference level information and / or patient information) on a suitable storage medium. Moreover, the database, may further comprise a database management system. The database management system is, preferably, a networkbased, hierarchical or object-oriented database management system. Furthermore, the database may be a federal or integrated database. More preferably, the database will be implemented as a distributed (federal) system, e.g., as a Client-Server-System. More preferably, the database is structured as to allow a search algorithm to compare a test data set with the data sets comprised by the data collection. Specifically, by using such an algorithm, the database can be searched for similar or identical data sets being indicative of HF (e.g., a query search). Thus, if an identical or similar data set can be identified in the data collection, the test data set will be associated with HF. Consequently, the information obtained from the data collection can be used to diagnose HF or based on a test data set obtained from a subject. More preferably, the data collection comprises characteristic values of all analytes comprised by any one of the groups recited above.

[0230] The methods described herein may further include communication of the results or diagnoses (or both) to technicians, physicians or patients, for example. In certain examples, computers will be used to communicate results or diagnoses (or both) to interested parties, e.g., physicians and their patients.

[0231] In some examples, the results or diagnoses (or both) are communicated to the subject as soon as possible after the diagnosis is obtained. The results or diagnoses (or both) may be communicated to the subject by the subject's treating physician. Alternatively, the results or diagnoses (or both) may be sent to a subject by email or communicated to the subject by phone. A computer may be used to communicate the results or diagnoses by email or phone. In certain examples, the message containing results or diagnoses may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications.

[0232] Companion diagnostic

[0233] The methods and uses provided herein may be used as part of a companion diagnostic e.g., as part of a medical device, often an in vitro device, which provides information that is essential for the safe and effective use of a corresponding drug or biological product (wherein the corresponding drug or biological product is for treating or preventing HF).

[0234] All patents, patent applications, and publications or public disclosures referred to or cited herein are incorporated by reference in their entirety.

[0235] The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

[0236] Examples a. Samples

[0237] Paired plasma and ISF samples from healthy donors were provided by Ascilon AB (Stockholm, Sweden).

[0238] Interstitial fluid (ISF) was collected from skin (upper arm). The ISF collection was performed by using "Asciiion's dISF sampling solution" based on microneedles made from monocrystalline silicon. Eight different samples were obtained from four different healthy donors (donor A - D). Type of sample (blood plasma or ISF) and volume are summarized in the table below. b. NT-proBNP in ISF and plasma determination

[0239] The determination of NT-proBNP in the ISF and plasma samples was performed by using a Simoa® Homebrew Assay. A detailed description of the assay is described below:

[0240] • Reagents

[0241] • Simoa® Homebrew Assay

[0242] Cadences 47-7 (35-5 min)

[0243] Reagent Volume [pL]

[0244] Beads 25

[0245] Detector 25

[0246] SBG 100

[0247] RGP 25

[0248] Calibrator / Sample 50

[0249] All calibrators and samples were measured in duplicates.

[0250] • Calibrators NT-proBNP(l-76)amid (Roche Diagnostics GmbH); Stock 5.2 pg / mL

[0251] O-Standard: Quanterix Homebrew Detector / Sample Diluent

[0252] “Control” sample: 1 :175 dilution of PreciControl Cardiac II (Roche Diagnostics GmbH) Control 1 target value: 0.883 pg / mL (Mean of 6 runs on SIMOA)

[0253] • Sample dilution:

[0254] Samples were diluted in the Sample Diluent

[0255] Results

[0256] • Calibration and Control

[0257] The run was calibrated using a cubic fitting with 1 / y2weighting. The fitting was checked by the recovery of the fitted concentration to the target concentration of the calibrators. This recovery should be between 85 % and 115 %. With recoveries between 86 % and 113 %, the fitting can be considered as successful.

[0258] To verify the run, a control with a defined target value was measured resulting in a concentration of 0.954 pg / mL. As the recovery to the target is within ± 21 %, the verification was successful.

[0259] • Samples

[0260] The samples were prepared as stated above and measured in duplicates.

[0261] The measured concentration should be above the determined Lower Detection and Quantification Limit of 0.205 pg / mL and the duplicates should have a coefficient of variation (CV) of concentration < 15 %.

[0262]

[0263] To minimize the influence of measurement errors, the diluted NT-proBNP concentration in the next table was determined based on the mean signal. NT -proBNP recovery in ISF samples compared to plasma is between 108 % to 145 %. Considering a deviation of ± 20 % due to measurement and dilution errors, the NT -proBNP concentration of Donor B and D can be considered as equal in plasma and ISF.

[0264] Results are represented in a comparison plot (Fig. 2). The observation of the results in the comparison plot indicates a linear correlation between the values of NT -proBNP measured in plasma and the ones measured in ISF within the same donor.

[0265] References

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Claims

Claims1. A method for assessing heart failure or determining the risk of developing heart failure (HF) in a subject, the method comprising the steps of: a) determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid (ISF) from the subject; b) comparing the level of the one or more cardiac related (poly)peptide biomarker with the level of the same cardiac related (poly)peptide biomarker in a control sample or with a pre-determined reference level for the same cardiac related (poly)peptide biomarker; and c) identifying a subject as having heart failure or as having an increased risk of developing heart failure based on the comparison in step b).

2. A method for monitoring heart failure in a subject, the method comprising the steps of i. determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid (ISF) from the subject, optionally in accordance with method steps a) to b) of claim 1 ; ii. repeating step i. after a time interval; and iii. comparing the levels of the cardiac related (poly)peptide biomarker identified in i. with the levels identified in ii., wherein a change in the levels from i. to ii. is indicative of a change in heart failure in the subject.

3. A method for classifying the stage of heart failure in a subject, the method comprising the steps of a) determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) classifying the stage of heart failure in the subject if the comparison in step b) indicates that the subject has an increased or decreased level of the one or morecardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

4. A method for determining the therapeutic effect of a treatment regimen for heart failure in a subject, the method comprising the steps of i. determining the level of one or more cardiac related (poly)peptide biomarker in the interstitial fluid (ISF) from the subject, optionally in accordance with method steps a) to b) of claim 1; ii. repeating step i. after a time interval; and iii. comparing the levels of the cardiac related (poly)peptide biomarker identified in i. with the levels identified in ii., and identifying that the treatment regimen has a therapeutic effect if the level of the cardiac related (poly)peptide biomarker decreased after treatment.

5. A method for risk stratification of progressing to severe disease of a subject with HF, the method comprising the steps of: a) determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) stratifying the risk of progressing to severe disease of the subject with HF if the comparison in step b) indicates that the subject has an increased or decreased level of the one or more cardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

6. A method for making a prognosis of future disease course of a subject with HF, the method comprising the steps of:a) determining the level of one or more cardiac related (poly)peptide biomarker in the ISF from the subject, b) comparing the level of the one or more cardiac related (poly)peptide biomarker to at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level, c) making a prognosis of future disease course of the subject with HF if the comparison in step b) indicates that the subject has an increased or decreased level of the one or more cardiac related (poly)peptide biomarker compared to the at least one appropriate reference value of the same one or more cardiac related (poly)peptide biomarker level.

7. Use of interstitial fluid (ISF) for assessing heart failure, determining the risk of developing heart failure, classifying the stage of heart failure, monitor heart failure and / or determine the therapeutic effect of a treatment in a subject by determining the level of one or more cardiac related (polyjpeptide biomarker therein.

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-type natriuretic peptide (NT-proBNP) or B-type natriuretic peptide (BNP).

10. The method according to claims 1 to 6 or the use according to claim 7, wherein additionally to the level of the one or more cardiac related (poly)peptide biomarker the level of one or more further cardiac related biomarkers selected from the group consisting of 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, CAI 25), Soluble Cluster of Differentiation 146 (or Cell surface glycoprotein MUC18,sCD146), bioAdrenomedullin (bio ADM), Midregional proadrenomedullin (MR- proADM), Soluble suppression of tumorigenesis-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 heart failure is selected from the group consisting of acute heart failure, heart failure due to reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF), heart failure with preserved ejection fraction (HFpEF), left-sided failure, right-sided failure, biventricular failure.

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

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

14. The method according to claim 3, wherein the subject’s stage of heart failure 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-implemented method for assessing a subject with suspected heart failure comprising the steps of(a) receiving a value for level of a first cardiac related (poly)peptide biomarker in the interstitial fluid of the subject, said first biomarker being a BNP -type peptide;(b) optionally, receiving a value for the level of at least one additional cardiac related biomarker in the interstitial fluid of the subject, wherein said additional biomarker is selected from the group consisting of 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 Cluster of Differentiation 146 (or Cell surface glycoprotein MUC18, sCD146), bioAdrenomedullin (bio ADM), Midregional proadrenomedullin (MR-proADM), Soluble suppression of tumorigene sis-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) comparing the values for the levels of steps (a) and optionally step (b) to appropriate references for said biomarkers and / or calculating a score for assessing the subject with suspected heart failure based on the levels of the biomarkers; and(d) assessing said subject based on the comparison and / or the calculation made in step (c).

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