IGFBP7 ratio in HFpEF

Abstract

The present invention relates to a method for differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF) in subjects suffering from heart failure, said method comprising the steps of: determining the amounts of IGFBP7 (insulin-like growth factor binding protein 7), BNP-type peptide and optionally CRP (C-reactive protein) in a sample from said subject, calculating (i) the ratio of the amount of said IGFBP7 to the amount of said BNP peptide or (ii) the ratio of the sum of the amounts of said IGFBP7 and said CRP to the amount of said BNP-type peptide, comparing the calculated ratio with a reference ratio, and differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF). The present invention also relates to a method for diagnosing HFpEF.

Description

[0001] The present invention relates to a method for differentiating between heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) in a subject suffering from heart failure, the method comprising the steps of: determining the amounts of IGFBP7 (insulin-like growth factor binding protein 7), BNP-type peptide and optionally CRP (C-reactive protein) in a sample from the subject, calculating (i) the ratio of the amount of the IGFBP7 to the amount of the BNP peptide or (ii) the ratio of the sum of the amounts of the IGFBP7 and the CRP to the amount of the BNP-type peptide, comparing the calculated ratio with a reference ratio, and differentiating between heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). The present invention also relates to a method for diagnosing HFpEF.

[0002] The use of biomarkers for the care of patients with heart failure (HF) has expanded significantly. Natriuretic peptides (NPs) (including B-type natriuretic peptide (BNP) and its amino-terminal cleavage equivalent (NT-proBNP)) are now widely used for the diagnosis, prognosis and management of affected patients. After BNP and NT-proBNP, a wide variety of new biomarkers are being investigated, each of which has the potential to provide complementary assessment of patients with complex HF pathophysiological situations. In this regard, considerable effort has been made to better understand the mechanistic link between cardiac biomarker concentrations and the underlying cardiovascular pathophysiological processes that release them.

[0003] Many prior art references describe the use of biomarker combinations in the assessment of heart failure, for example, in relation to heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). For example, Sanders evaluated whether biomarkers reflecting pathophysiological pathways differed between HFpEF and HfrEF and whether the prognostic value of the biomarkers differed in HFpEF versus HfrEF (Eur J Heart Fail. October 2015;17(10):1006-14. doi:10.1002 / ejhf.414. Epub October 16, 2015). The study described that patients with HFpEF exhibited higher soluble interleukin 1 receptor-like 1, high-sensitivity C-reactive protein and cystatin-C. In contrast, patients with HFrEF exhibited higher NT-proBNP, high-sensitivity troponin T and hemoglobin.

[0004] Sinning described the measurement of CRP, GDF-15, sST2 and NT-proBNP in patients with HFrEF and HFpEF (Int J Cardiol. 2017 Jan 15;227:272-277. doi: 10.1016 / j.ijcard.2016.11.110. Epub 2016 Nov 9). According to Sinning, the index ((CRP + GDF-15s + sST2) / NT-proBNP) was able to distinguish HFpEF and HFrEF.

[0005] Previously, the insulin-like growth factor axis has been found to be a predictor of HF outcomes (Watanabe et al., Insulin-like growth factor axis (insulin-like growth factor-i / insulin-like growth factor-binding protein-3) as a prognostic predictor of heart failure: Association with adiponectin, Eur J Heart Fail. 2010;12:1214-1222). IGFBP-7 (insulin-like growth factor-binding protein 7) is a 30-kDa modular glycoprotein known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono et al., Biochem Biophys Res Comm 202 (1994) 1490). IGFBP-7 has been described as being associated with cellular senescence, tissue aging, and obesity. IGFBP-7 has been proposed as a marker for diastolic dysfunction and HFpEF, a disease of the elderly and obese. A mechanistic link between IGFBP-7 and impaired diastolic function has been demonstrated in a mouse model (US2018 / 0127752). Elevated circulating levels of IGFBP-7 have been described as being associated with diastolic abnormalities in the PARAMOUNT study (Januzzi et al., Circ Heart Fail. (2018) 11).

[0006] WO 2008 / 089994 discloses the use of IGFBP7 in the assessment of heart failure. In addition to IGFBP7, NTproBNP and CRP can also be measured.

[0007] WO 2015 / 144767 describes IGFBP7 as a marker for diastolic dysfunction. In Example 2.2, patients with HFpEF were analyzed. In this example, IGFBP7 was combined with, for example, osteopontin, troponin T, and NTproBNP.

[0008] WO 2014 / 086833 describes IGFBP7 as a marker for selecting heart failure therapies.

[0009] The relationship between IGFBP7 and HFpEF and HFrEF was also analyzed. For example, Hage described that IGFBP-7 in HFpEF was lower than that in HFrEF (Am J Cardiol. June 15, 2018; 121(12):1558-1566. doi:10.1016 / j.amjcard.2018.02.041. Epub Mar 14, 2018).

[0010] Due to the normal ejection fraction, the diagnosis of HFpEF is challenging. It is mainly based on potentially vague symptoms and the exclusion of other causes, such as lung diseases. In addition, biomarker-based diagnosis of HFpEF is troublesome. So far, no biomarker has been established in clinical routine to assist in the diagnosis of HFpEF. NTproBNP shows a stronger association with systolic dysfunction than with diastolic dysfunction. It only moderately increases in HFpEF. Although IGFBP-7 shows a mechanistic link with diastole, it has not been described that IGFBP-7 helps to identify patients with preserved ejection fraction.

[0011] As commented, the manifestations and pathophysiology of HFpEF are diverse, and its management still poses challenges (Clin Res Cardiol. January 2018; 107(1):1-19. doi:10.1007 / s00392-017-1170-6. Epub Oct 10, 2017). Until now, there has been no therapy to improve the survival rate of HFpEF patients. Therefore, the goal of treatment is to relieve symptoms, improve quality of life and reduce cardiac decompensation by controlling fluid retention and managing risk factors and comorbidities. For example, angiotensin-aldosterone inhibitors, diuretics, calcium channel blockers (CBB) and β-blockers are currently used to treat HFpEF. However, it has not been proven in large randomized controlled trials that these drugs can reduce mortality. Recently, new targets for treating HFpEF have been identified, such as soluble guanylate cyclase stimulators, inorganic nitrates, angiotensin receptor neprilysin inhibitor LCZ 696 and SGLT2 inhibitors. Patients with HFpEF may particularly benefit from these new treatments.

[0012] There is a great need for biomarker-based methods that can be used to reliably evaluate HFpEF and distinguish between HFrEF and HFpEF. The technical problem of the present invention can be seen as providing means and methods to meet the above needs.

[0013] This technical problem is solved by the claims and the embodiments characterized hereinafter.

[0014] Advantageously, in the studies of the present invention, it has been found that, compared to each biomarker alone, the ratio of IGFBP-7 to a BNP-type peptide (such as NT-proBNP) and the ratio of the sum of the amounts of IGFBP-7 + CRP to the amount of the BNP-type peptide improve the unique identification of HFpEF in patients with heart failure. Thus, the above ratios allow for a reliable distinction between heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) in subjects with heart failure.

[0015] Accordingly, the present invention relates to a method for distinguishing heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) in a subject with heart failure, the method comprising the steps of:

[0016] (a) determining the amounts of IGFBP7 (insulin-like growth factor binding protein 7), a BNP-type peptide, and optionally CRP (C-reactive protein) in a sample from the subject,

[0017] (b) calculating (i) the ratio of the amount of the IGFBP7 to the amount of the BNP peptide or (ii) the ratio of the sum of the amounts of the IGFBP7 and the CRP to the amount of the BNP-type peptide,

[0018] (c) comparing the ratio calculated in step (b) with a reference ratio, and

[0019] (d) distinguishing heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF).

[0020] The present invention also relates to a method for diagnosing HFpEF in a subject suspected of having heart failure with preserved ejection fraction (HFpEF), the method comprising the steps of:

[0021] (a) determining the amounts of IGFBP7 (insulin-like growth factor binding protein 7), a BNP-type peptide, and optionally CRP (C-reactive protein) in a sample from the subject,

[0022] (b) calculating (i) the ratio of the amount of the IGFBP7 to the amount of the BNP peptide or (ii) the ratio of the sum of the amounts of the IGFBP7 and the CRP to the amount of the BNP-type peptide,

[0023] (c) comparing the ratio calculated in step (b) with a reference ratio, and

[0024] (d) diagnosing heart failure with preserved ejection fraction.

[0025] In one embodiment of the method of the present invention, the amounts of said IGFBP7, said BNP-type peptide and said CRP are determined in step (a), and the ratio of the sum of the amounts of said IGFBP7 and said CRP to the amount of said BNP-type peptide is calculated in step (b). Thus, steps (a) and (b) are as follows:

[0026] (a) Determine the amounts of IGFBP7, BNP-type peptide and CRP in a sample from said subject,

[0027] (b) Calculate the ratio of the sum of the amounts of said IGFBP7 and said CRP to the amount of said BNP-type peptide.

[0028] The method of the present invention is preferably an ex vivo method, or in particular an in vitro method. In addition, it may also include steps other than those explicitly mentioned above. For example, additional steps may involve sample pretreatment or evaluation of the results obtained by said method. The method may be performed manually or assisted by automation. Preferably, steps (a), (b), (c) and / or (d) may be assisted by automation in whole or in part, such as by suitable robotic and sensing devices for the measurement in step (a) or the computer-implemented calculation in step (b) or the computer-implemented comparison in step (c) and / or the computer-implemented discrimination / diagnosis based on the comparison in step (c).

[0029] As used herein, the term "diagnosis" means evaluating whether the subject mentioned herein has HFpEF. In one embodiment, the subject is diagnosed with HFpEF. In an alternative embodiment, the subject is diagnosed as not having HFpEF.

[0030] The diagnosis in step (d) or the discrimination in step (d) is based on comparing the results of step (c). Preferably, the diagnosis in step (d) is based on comparing the results of step (c). However, it should be understood that the actual diagnosis of whether the subject has AF may include additional steps, such as confirmation of the diagnosis. Therefore, the diagnosis mentioned herein should allow an assessment of the likelihood that the subject has HFpEF. As can be seen from the above, the diagnosis of HFpEF is understood to contribute to the diagnosis of HFpEF. Therefore, the term "diagnosis" in the context of the present invention also includes assisting the physician in evaluating whether the subject has HFpEF.

[0031] As used herein, the term "differentiate" means differentiating HFpEF from HFrEF in diseased subjects. This term as used herein preferably includes differential diagnosis of HFpEF and HFrEF in subjects with heart failure. Preferably, the differentiation in step (d) is based on comparing the results of step (c). Additionally, the methods of the present invention allow for the assessment of whether a subject with atrial fibrillation has paroxysmal atrial fibrillation or persistent atrial fibrillation. The actual differentiation may include additional steps, such as confirmation of the differentiation. Thus, the term "differentiate" in the context of the present invention also includes assisting a physician in differentiating HFpEF and HFrEF.

[0032] As will be understood by those skilled in the art, the assessments (i.e., differentiations or diagnoses) described herein are generally not intended to be correct for all (i.e., 100%) patients to be diagnosed / graded. In embodiments of the present invention, a statistically significant portion of patients (e.g., a cohort in a cohort study) can be identified. Those skilled in the art can readily determine whether a portion is statistically significant using various well-known statistical assessment tools (e.g., determination of confidence intervals, p-value determination, Student t-test, Mann-Whitney test, etc.). See Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983 for details. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005 or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the patients in a population can be correctly diagnosed / differentiated by the methods of the present invention.

[0033] The "subjects" referred to herein are preferably mammals. 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). Preferably, the subject is a human subject. The terms "subject", "patient" and "individual" are used interchangeably herein.

[0034] According to the method for differentiating HFpEF and HFrEF, the patient to be tested should have heart failure. The term "heart failure" is well known in the art. As used herein, this term preferably relates to impaired cardiac function with symptoms of heart failure as known to those skilled in the art. Thus, the patient preferably has symptomatic heart failure.

[0035] The ACC / AHA classification is a heart failure classification developed by the American College of Cardiology and the American Heart Association (see J. Am. Coll. Cardiol. 2001; 38; 2101-2113, updated in 2005, see J. Am. Coll. Cardiol. 2005; 46; e1-e82). Four stages A, B, C, and D are defined. Stages A and B are not HF (heart failure), but are considered helpful in early identification of patients before developing into "true" HF. Patients in stages A and B are preferably defined as those with risk factors for developing HF. For example, patients with coronary artery disease, hypertension, or diabetes who have not yet shown impaired left ventricular (LV) function, hypertrophy, or geometric ventricular deformation will be considered stage A, while asymptomatic patients who show LV hypertrophy (LVH, a phenomenon of thickening of the ventricular wall) and / or impaired LV function will be designated stage B. Stage C represents patients with current or past symptoms of HF related to underlying structural heart disease (most patients have HF), while stage D designates patients with true refractory HF.

[0036] In one embodiment of the method of the present invention, the patient to be tested should preferably have heart failure stage C or D according to the ACC / AHA classification (see citation above). At these stages, the patient shows symptoms of heart failure. The symptoms of heart failure are well known in the art and include dyspnea, fatigue, and fluid retention. Fluid retention may lead to pulmonary congestion and peripheral edema, and the typical physical examination signs are edema and rales. Therefore, the patient to be tested preferably shows symptoms of heart failure.

[0037] According to the present invention, heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) should be distinguished in subjects with heart failure.

[0038] Subjects with HFrEF have heart failure with reduced left ventricular ejection fraction (LVEF). The term "left ventricular ejection fraction" is well known in the art. Patients with reduced LVEF preferably have an LVEF of less than 50%, more preferably less than 45%, and most preferably less than 40%. In addition, it is contemplated that the patient has an LVEF of less than 30%.

[0039] Subjects with HFpEF have heart failure with preserved LVEF. Therefore, the term HFpEF preferably refers to heart failure with an LVEF equal to or greater than 50%. Patients with preserved LVEF may also have an LVEF greater than 55% or greater than 60%.

[0040] How to evaluate LVEF is well known in the art. In one embodiment, LVEF can be determined as described, for example, by McMurray et al. (European Heart Journal (2012) 33, 1787-1847, see, for example, page 1800 and below).

[0041] In one embodiment of the method for diagnosing HFpEF, the patient to be tested should be suspected of having heart failure, especially HFpEF. The subject suspected of having heart failure preferably exhibits symptoms of heart failure. In an alternative embodiment of the method for diagnosing HFpEF, the patient to be tested should have heart failure. Thus, it should be known that the patient has heart failure.

[0042] The patient to be tested according to the method for diagnosing HFpEF may have a normal ejection fraction, i.e., preserved LVEF.

[0043] According to the present invention, the subject to be tested can be elderly and / or overweight.

[0044] Preferably, the subject is greater than 50 years old, more preferably greater than 60 years old, or most preferably greater than 75 years old. Alternatively or additionally, the subject has a body mass index (BMI) of more than 25 kg / m 2 , especially more than 27.5 kg / m 2 In addition, the subject may have a BMI of more than 30.0 kg / m 2 .

[0045] The term "sample" refers to a sample of a body fluid, a sample of isolated cells, or a sample from a tissue or organ. Samples of body fluids can be obtained by well-known techniques and include samples of blood, plasma, serum, urine, lymph fluid, sputum, ascites, or any other bodily secretion or derivative thereof. Preferred body fluid samples are urine, blood, serum, or plasma. Tissue or organ samples can be obtained from any tissue or organ by, for example, biopsy. Isolated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. For example, cell, tissue, or organ samples can be obtained from those cells, tissues, or organs that express or produce a biomarker. The sample can be a frozen, fresh, fixed (e.g., formalin-fixed), centrifuged, and / or embedded (e.g., paraffin-embedded) sample, etc. Of course, various well-known post-collection preparation and storage techniques (e.g., nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) can be performed on cell samples before assessing the amount of the biomarker in the sample.

[0046] In addition, it is contemplated that the blood sample is a dried blood spot sample. The dried blood spot sample can be obtained by applying a blood drop to an absorbent filter paper. The blood is allowed to fully soak the paper and air-dried for several hours. The blood can be drawn from the subject to be tested (e.g., from a finger) using a lancet.

[0047] In a preferred embodiment, the sample is a blood (i.e., whole blood), serum or plasma sample. Serum is the liquid fraction obtained after blood clotting in whole blood. To obtain serum, the clot is removed by centrifugation and the supernatant is collected. Plasma is the cell-free liquid portion of blood. To obtain a plasma sample, whole blood is collected in an anticoagulant-treated tube (e.g., a citrate-treated tube or an EDTA-treated tube). The cells are removed from the sample by centrifugation and the supernatant (i.e., the plasma sample) is obtained.

[0048] According to the present invention, the amount of insulin-like growth factor binding protein 7 (= IGFBP-7) should be determined. Preferably, the amount of IGFBP-7 polypeptide is determined. IGFBP-7 is a 30-kDa modular glycoprotein known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono, Y. et al., Biochem Biophys Res Comm 202(1994)1490-1496). Preferably, the term "IGFBP-7" refers to human IGFBP-7. The sequence of this protein is well known in the art and can be obtained, for example, via Uni-Prot (Q16270, IBP7_HUMAN) or via GenBank (NP_001240764.1). A detailed definition of the biomarker IGFBP-7 is provided, for example, in WO2008 / 089994, the entire content of which is incorporated herein by reference. IGFBP-7 has two isoforms: isoform 1 and isoform 2, which are generated by alternative splicing. In an embodiment of the present invention, the total amount of the two isoforms is determined (for the sequences, see UniProt database entries (Q16270-1 and Q16270-2)).

[0049] IGF-binding protein 7 (= IGFBP7) is a 30-kDa modular glycoprotein known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono, Y. et al., Biochem Biophys Res Comm 202 (1994) 1490-1496). In that literature, the molecule was also named FSTL2; IBP 7; IGF-binding protein-related protein I; IGFBP 7; IGFBP7v; IGFBP rPl; IGFBP7; IGFBPRP1; insulin-like growth factor-binding protein 7; insulin-like growth factor-binding protein 7 precursor; MAC25; MAC25 protein; PGI2-stimulating factor; and PSF or prostacyclin-stimulating factor. Northern blot studies have revealed widespread expression of this gene in human tissues including heart, brain, placenta, liver, skeletal muscle, and pancreas (Oh, Y. et al., J. Biol. Chem. 271 (1996) 30322-30325).

[0050] IGFBP7 was initially identified as a gene differentially expressed in normal leptomeninges and mammary epithelial cells compared to their corresponding tumor cells and was named meningioma-associated cDNA (MAC25) (Burger, A.M. et al., Oncogene 16 (1998) 2459-2467). The expressed protein was independently purified as a tumor-derived adhesion factor (later renamed vasoregulin) (Sprenger, C.C. et al., Cancer Res 59 (1999) 2370-2375) and as a prostacyclin-stimulating factor (Akaogi, K., et al., Proc Natl Acad Sci USA 93 (1996) 8384-8389). It has also been reported as T1Al2, a gene downregulated in breast cancer (StCroix, B. et al., Science 289 (2000) 1197-1202).

[0051] Preferably, the term "IGFBP7" refers to human IGFBP7. The sequence of this protein is well known in the art and is available, for example, via GenBank (NP_001240764.1). IGFBP7 as used herein preferably also includes variants of the specific IGFBP7 polypeptide.

[0052] As used herein, the term "BNP-type peptide" includes pre-proBNP, proBNP, NT-proBNP, and BNP. The pre-propeptide (134 amino acids in the case of pre-proBN) contains a short signal peptide that is enzymatically cleaved to release the propeptide (108 amino acids in the case of proBNP). The propeptide is further cleaved into the N-terminal propeptide (NT-propeptide, 76 amino acids in the case of NT-proBNP) and the active hormone (32 amino acids in the case of BNP). Preferably, the BNP-type peptides according to the invention are NT-proBNP, BNP, and variants thereof. BNP is the active hormone and has a shorter half-life than the corresponding inactive counterpart NT-proBNP. BNP is metabolized in the blood, while NT-proBNP circulates in the blood as an intact molecule and is thus cleared by the kidneys. The in vivo half-life of NT-proBNP is 120 minutes longer than that of BNP, and the in vivo half-life of BNP is 20 minutes (Smith 2000, J Endocrinol. 167:239-46). The pre-analytical of NT-proBNP is more reliable, and samples can be easily transported 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 can be mailed or transported without loss of recovery. In contrast, storage of BNP at room temperature or 4 °C for 48 hours results in at least a 20% loss of concentration (Mueller loc.cit.; Wu 2004, Clin Chem 50:867-73). Thus, depending on the time course or nature of the purpose, it may be advantageous to measure the active or inactive form of the natriuretic peptide. The most preferred natriuretic peptide according to the invention is NT-proBNP or a variant thereof. As briefly discussed above, the human NT-proBNP referred to according to the invention is a polypeptide that preferably contains 76 amino acids corresponding to the N-terminal portion of the human NT-proBNP molecule. The structures of human BNP and NT-proBNP have been described in detail in the prior art (e.g., WO 02 / 089657, WO 02 / 083913, or Bonow loc.cit). Preferably, the human NT-proBNP as used herein is the human NT-proBNP disclosed in EP 0648228 B1. These prior art documents are incorporated herein by reference for the specific sequences of NT-proBNP and its variants disclosed therein.

[0053] C-reactive protein (CRP) is an acute-phase protein that was discovered over 75 years ago as a blood protein that binds to the C-polysaccharide of pneumococcus. CRP is known as a reactive inflammation marker and is produced by the distal organ, namely the liver, in response to chemokines or interleukins originating from the primary lesion site. CRP is known to consist of five monomeric subunits that are non-covalently linked and assembled into a cyclic pentamer with a molecular weight of approximately 110-140 kDa. Preferably, CRP as used herein refers to human CRP. The sequence of human CRP is well known and has been published, for example, by Woo et al. (J. Biol. Chem. 1985. 260(24), 13384-13388). CRP levels are usually low in normal individuals but can increase 100 to 200-fold or more due to inflammation, infection, or injury (Yeh (2004) Circulation. 2004; 109:11-11-11-14). CRP is known to be an independent factor for predicting cardiovascular risk. In particular, CRP has been shown to be suitable as a predictor for myocardial infarction, stroke, peripheral arterial disease, and sudden cardiac death. In addition, an elevated amount of CRP can also predict recurrent ischemia and death in subjects with acute coronary syndrome (ACS) and in subjects undergoing coronary intervention.

[0054] As used herein, the term "amount" includes the absolute amount of the biomarker mentioned herein, the relative amount or concentration of the biomarker, and any value or parameter related to or derivable from it. Such values or parameters include intensity signal values from all specific physical or chemical properties obtained from the peptide by direct measurement, such as intensity values in a mass spectrum or an NMR spectrum. In addition, included are all values or parameters obtained by indirect measurement specified elsewhere in this specification, such as the amount of response determined from a biological readout system in response to a peptide or an intensity signal obtained from a specifically bound ligand. It should be understood that values related to the above amounts or parameters can also be obtained by all standard mathematical operations.

[0055] In some embodiments of the present invention, the term "amount" refers to "mass concentration", which is defined as the mass of the biomarker divided by the sample volume. The SI unit of mass concentration is kg / m 3 (kilogram per cubic meter), which is the same as mg / mL and g / L. For the concentration of a biomarker, other units are usually used, such as "mg / ml" or "pg / ml". Thus, the amount of a biomarker can be the mass of the biomarker in 1 ml of the test sample.

[0056] The term "determining" the amount of a biomarker as mentioned herein refers to the quantification of the biomarker, for example, using a suitable detection method described elsewhere herein to determine the level of the biomarker in a sample.

[0057] In one embodiment, the amount of the biomarker is determined by contacting a sample with a reagent that specifically binds to the biomarker, thereby forming a complex between the reagent and the biomarker, detecting the amount of the formed complex, and thereby determining the amount of the biomarker.

[0058] The biomarkers mentioned herein can be detected using methods generally known in the art. Detection methods generally include methods for quantifying the amount of a biomarker in a sample (quantitative methods). Those skilled in the art generally know which of the following methods are suitable for the qualitative and / or quantitative detection of biomarkers. Commercially available Western and immunoassays (such as ELISA, RIA, fluorescence- and luminescence-based immunoassays) can be used to conveniently determine, for example, proteins in a sample. Other suitable methods for detecting biomarkers include determining the physical or chemical properties unique to a peptide or polypeptide, such as its exact molecular mass or NMR spectrum. Such methods include, for example, biosensors, optical devices coupled to immunoassays, biochips, analytical devices (such as mass spectrometers, NMR analyzers, or chromatographic devices). In addition, methods include microplate ELISA-based methods, fully automated or robotic immunoassays (such as those available on Elecsys TM analyzers), CBA (such as the enzymatic cobalt-binding assay available on Roche-Hitachi TM analyzers), and latex agglutination assays (such as those available on Roche-Hitachi TM analyzers).

[0059] For the detection of the biomarker proteins mentioned herein, various immunoassay techniques using such assay formats are available. See, for example, U.S. Patent Nos. 4,016,043, 4,424,279, and 4,018,653. These techniques include non-competitive types of single-site and two-site or "sandwich" assays, as well as traditional competitive binding assays. These assays also include the direct binding of a labeled antibody to the target biomarker. The sandwich assay is one of the most useful immunoassays.

[0060] Methods using electrochemiluminescent labels are well known. Such methods utilize the ability of special metal complexes to achieve an excited state by oxidation, and the special metal complexes decay from this excited state to the ground state, thereby emitting electrochemiluminescence. For a review, see Richter, M.M., Chem. Rev. 104 (2004) 3003-3036.

[0061] In one embodiment, the detection antibody (or antigen-binding fragment thereof) to be used for determining the amount of biomarker is ruthenated or iridiated. Thus, the antibody (or antigen-binding fragment thereof) should contain a ruthenium tag. In one embodiment, the ruthenium tag is a bipyridine-ruthenium(II) complex. Alternatively, the antibody (or antigen-binding fragment thereof) should contain an iridium tag. In one embodiment, the iridium tag is a complex as disclosed in WO 2012 / 107419.

[0062] Determining the amount of a polypeptide such as IGFBP-7 may preferably comprise the steps of: (a) contacting the polypeptide with a reagent that specifically binds to the polypeptide, (b) (optionally) removing unbound reagent, and (c) determining the amount of bound binder, i.e., the complex of the reagent formed in step (a). According to a preferred embodiment, the contacting, optionally removing, and determining steps may be performed by an analyzer unit. According to some embodiments, the steps may be performed by a single analyzer unit of the system or by more than one analyzer unit operably communicating with each other. For example, according to a particular embodiment, the system disclosed herein may include a first analyzer unit for performing the contacting and optionally removing steps; and a second analyzer unit for performing the determining step, the second analyzer unit being operably connected to the first analyzer unit via a transfer unit (e.g., a robotic arm).

[0063] Reagents that specifically bind to biomarkers (also referred to herein as "binders") can be covalently or non-covalently conjugated to a label, thereby allowing the detection and measurement of the bound reagent. Labeling can be performed by direct or indirect methods. Direct labeling involves the direct (covalent or non-covalent) conjugation of the label to the binder. Indirect labeling involves the binding (covalent or non-covalent) of a secondary binder to the first binder. The secondary binder should specifically bind to the first binder. The secondary binder can be conjugated to a suitable label and / or be the target (receptor) to which a tertiary binder binds to the secondary binder. Suitable secondary and higher-order binders can include antibodies, secondary antibodies, and well-known binding systems such as the streptavidin-biotin system (Vector Laboratories, Inc.). The binder or substrate can also be "labeled" with one or more labels known in the art. Such labels can then be targets for higher-order binders. Suitable labels include biotin, digoxin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose binding protein, etc. In the case of peptides or polypeptides, the label is preferably located at the N-terminus and / or C-terminus. A suitable label is any label that can be detected by an appropriate detection method. Typical labels include gold particles, latex beads, acridinium esters, luminol, ruthenium complexes, iridium complexes, enzyme activity labels, radioactive labels, magnetic labels (e.g., magnetic beads, including paramagnetic and superparamagnetic labels), and fluorescent labels. Enzyme activity labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and their derivatives. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, available as a ready-to-use stock solution from Roche Diagnostics), CDP-Star TM (Amersham Bio-sciences), ECF TM (Amersham Biosciences). Suitable enzyme-substrate combinations can produce a colored reaction product, fluorescence, or chemiluminescence, which can be assayed according to methods known in the art (e.g., using photographic film or a suitable imaging system). For assaying enzyme reactions, the standards given above apply similarly. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa 568). Additional fluorescent labels are available, for example, from Molecular Probes (Oregon). Similarly, the use of quantum dots as fluorescent labels is also contemplated. Radioactive labels can be detected by any known and appropriate method (e.g., photographic film or a phosphor imager).

[0064] The amount of the polypeptide can also be preferably determined as follows: (a) contacting a solid support having a binder containing the polypeptide as described elsewhere herein with a sample containing the peptide or polypeptide, and (b) determining the amount of the peptide or polypeptide bound to the support. Materials for making the support are well known in the art and include, in particular, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon wafers and surfaces, nitrocellulose strips, membranes, sheets, duracyte, the wells and walls of reaction plates, plastic tubes, etc.

[0065] In another aspect, the sample is removed from the complex formed between the binder and a biomarker before measuring the amount of the formed complex. Thus, in one aspect, the binder can be immobilized on a solid support. In another aspect, the sample can be removed from the complex formed on the solid support by applying a washing solution.

[0066] "Sandwich assay" is one of the most useful and commonly used assays, including many variants of sandwich assay techniques. Briefly, in a typical assay, an unlabeled (capture) binder is immobilized or can be immobilized on a solid substrate, and a sample to be tested is contacted with the capture binder. After a suitable incubation period, after a period sufficient to allow the formation of a binder - biomarker complex, then a second (detection) binder labeled with a reporter molecule capable of generating a detectable signal is added, and incubated for a time sufficient to form another complex of binder - biomarker - labeled binder. Optionally, any unreacted material can be washed away. The presence of the biomarker is determined by observing the signal generated by the reporter molecule bound to the detection binder. The results can be qualitative by simply observing the visible signal, or can be quantitative by comparison with a control sample containing a known amount of the biomarker.

[0067] The incubation steps of a typical sandwich assay can be varied as needed and when appropriate. Such variations include, for example, simultaneous incubation in which two or more binders and a biomarker are co - incubated. For example, both the sample to be analyzed and the labeled binder are added simultaneously to the immobilized capture binder. It is also possible to first incubate the sample to be analyzed and the labeled binder, and then add an antibody that binds to or is capable of binding to the solid.

[0068] The complex formed between a specific binder and a biomarker should be proportional to the amount of the biomarker present in the sample. It should be understood that the specificity and / or sensitivity of the binder to be applied defines the degree of proportion of at least one biomarker contained in the sample that can be specifically bound. Further details on how the measurement can be carried out can also be found elsewhere herein. The amount of the formed complex should be converted into the amount of the biomarker, so as to reflect the amount actually present in the sample.

[0069] The terms "binding agent", "specific binding agent", "analyte-specific binding agent", "detection agent", and "reagent that specifically binds to a biomarker" are used interchangeably herein. Preferably, it refers to a reagent comprising a binding moiety that specifically binds to a corresponding biomarker. Examples of a "binding agent" or "reagent" are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, peptide nucleic acids (PNAs), or compounds. Preferred reagents are antibodies or antigen-binding fragments thereof that specifically bind to the biomarker to be measured. The term "antibody" is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the required antigen-binding activity (i.e., their antigen-binding fragments). Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.

[0070] The term "specific binding" or "specifically binds" refers to a binding reaction in which binding pair molecules bind to each other under conditions where they do not bind significantly to other molecules. The term "specific binding" or "specifically binds" when referring to a protein or peptide as a biomarker means a binding reaction in which the binding agent binds to the corresponding biomarker with an affinity of at least 10 -7 M. The term "specific binding" or "specifically binds" preferably refers to an affinity for its target molecule of at least 10 -8 M or even more preferably at least 10 -9 M. The term "specific" or "specificity" is used to indicate that other molecules present in the sample do not bind significantly to the binding agent specific for the target molecule.

[0071] In step (b) of the present invention, (i) the ratio of the amount of IGFBP7 to the amount of BNP peptide. Alternatively, (ii) the ratio of the sum of the amounts of IGFBP7 and CRP to the amount of BNP-type peptide.

[0072] The term "calculate a ratio" as referred to herein involves calculating the ratio of the amount by dividing (i) the amount of IGFBP7 or (ii) the sum of the amounts of IGFBP7 and CRP by the amount of BNP-type peptide or by performing any other comparable mathematical calculation that relates the amount in (i) or (ii) to the amount of BNP-type peptide. Preferably, the amount in (i) or (ii) is divided by the amount of BNP-type peptide to calculate the ratio (thus, calculating the ratio of the amount in (i) or (ii) to the amount of BNP-type peptide).

[0073] In some embodiments, the sum of the amounts of IGFBP7 and CRP is determined by adding the amounts of IGFBP7 and CRP (e.g., by adding the mass concentration of IGFBP7 and the mass concentration of CRP). Generally, the same unit is used for the amounts of CRP and IGFBP7, such as "ng / mL". Then the sum of the determined amounts of IGFBP7 and CRP is divided by the amount of the BNP-type peptide. For the BNP-type peptide, the same unit as the amounts of CRP and IGFBP7 can be used. However, it is also conceivable to use amounts expressed in pg / ml for the calculation. In some embodiments, amounts expressed in ng / ml are used for CRP and IGFBP7, while amounts expressed in pg / ml are used for the BNP-type peptide.

[0074] If the amount in i) or ii) is divided by the amount of the BNP-type peptide to calculate a ratio, then the following ratios are calculated:

[0075] 1. IGFBP7 / BNP-type peptide (i.e., the ratio of the amount of IGFBP7 to the amount of the BNP-type peptide), or

[0076] 2. (IGFBP7 + CRP) / BNP-type peptide (i.e., the ratio of the sum of the amounts of IGFBP7 and CRP to the amount of the BNP-type peptide)

[0077] If the above ratios (1. or 2.) are determined, then the following applies to the diagnostic algorithm in the method for differentiating HFpEF and HFrEF.

[0078] Preferably, a ratio higher than the reference ratio indicates heart failure with preserved ejection fraction (HFpEF), while a ratio lower than the reference ratio indicates heart failure with reduced ejection fraction (HFrEF).

[0079] If the above ratios (1. or 2.) are determined, then the following applies to the diagnostic algorithm in the method for diagnosing HFpEF.

[0080] Preferably, a ratio higher than the reference ratio indicates a subject with heart failure with preserved ejection fraction (HFpEF), while a ratio lower than the reference ratio indicates a subject without HFpEF, i.e., a subject with HF or suspected of having HF but not having HFpEF.

[0081] Also preferably, the amount of the BNP-type peptide is divided by the amount in i) or ii) to calculate a ratio (thus, calculating the ratio of the amount of the BNP-type peptide to the amount in i) or ii)).

[0082] Therefore, the following ratios are calculated

[0083] 3. BNP-type peptide / IGFBP7, or

[0084] 4. BNP-type peptide / (IGFBP7 + CRP)

[0085] If the above ratio (3. or 4.) is determined, then the following applies to the diagnostic algorithm in the method for differentiating HFpEF and HFrEF.

[0086] Preferably, a ratio below the reference ratio indicates heart failure with preserved ejection fraction (HFpEF), while a ratio above the reference ratio indicates heart failure with reduced ejection fraction (HFrEF).

[0087] If the above ratio (3. or 4.) is determined, then the following applies to the diagnostic algorithm in the method for diagnosing HFpEF.

[0088] Preferably, a ratio below the reference ratio indicates a subject with heart failure with preserved ejection fraction (HFpEF), while a ratio above the reference ratio indicates a subject without HFpEF, i.e., a subject with HF or suspected of having HF but without HFpEF.

[0089] In step c) of the method of the present invention, the ratio calculated in step (b) is compared with the reference ratio.

[0090] As used herein, the term "comparing" preferably refers to comparing the ratio calculated according to the method of the present invention with the reference ratio (i.e., the appropriate reference ratio). It should be understood that comparing as used herein generally refers to comparing values. The comparison can be performed manually or in a computer-aided manner. Thus, the comparison can be performed by a computing device. The values of the calculated ratio and the reference ratio can be compared, for example, with each other, and the comparison can be automatically performed by a computer program that executes an algorithm for comparison. The computer program that performs the evaluation will provide the required evaluation in a suitable output format. For computer-aided comparison, the value of the calculated ratio can be compared with the value corresponding to the appropriate reference ratio, which is stored in a database by the computer program. The computer program can further evaluate the result of the comparison, i.e., automatically provide the required evaluation in a suitable output format. The result of the comparison can preferably be used as an aid in diagnosing HFpEF or differentiating HFpEF and HFrEF.

[0091] In the method for differentiating HFpEF and HFrEF, HFpEF and HfrEF in heart failure subjects can be differentiated based on the comparison of the calculated ratio with the reference ratio. Thus, the reference ratio in the method for differentiating HFpEF and HFrEF is selected such that the difference or similarity of the comparison values allows for the differentiation of HFpEF and HFrEF. Thus, depending on the difference between HFpEF and HfrEF, the term "reference ratio" refers to a value / ratio that allows for the differentiation of HFpEF and HFrEF. Thus, the reference ratio can be a threshold that separates these groups from each other.

[0092] In a method for diagnosing HFpEF, subjects with HFpEF can be distinguished from subjects without HFpEF based on a comparison of a calculated ratio with a reference ratio. Thus, the reference ratio is selected such that the difference or similarity of the comparison values allows for the discrimination between HFpEF and HFrEF. Accordingly, for the diagnosis of HFpEF, the term "reference ratio" refers to a value / ratio that allows for the discrimination between subjects with HFpEF and subjects without HFpEF.

[0093] The reference ratio applicable to an individual subject can vary according to various physiological parameters such as age, sex, or subgroup, as well as the means used to measure the polypeptides or peptides mentioned herein. A suitable reference ratio can be determined from a reference sample that is to be analyzed together with (i.e., simultaneously or subsequently to) the test sample.

[0094] In principle, a reference ratio for a cohort of subjects can be calculated based on the average ratio by applying standard statistical methods. In particular, the accuracy of a test (such as a method designed to diagnose the occurrence or non-occurrence of an event) is best described by its receiver operating characteristic (ROC) (see especially Zweig 1993, Clin.Chem. 39:561-577). An ROC curve is a curve of all pairs of sensitivity and specificity generated by continuously varying the decision threshold over the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign subjects to a certain classification or diagnosis. The ROC curve shows the overlap between the two distributions by plotting sensitivity against 1-specificity for the entire range of thresholds applicable to the classification. On the y-axis is sensitivity, or the true positive fraction, which is defined as the ratio of the number of true positive test results to the product of the number of true positive test results and the number of false negative test results. This is also called positive in the presence of a disease or disorder. It is calculated only from the affected subgroup. On the x-axis is the false positive fraction, or 1-specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negative results and the number of false positive results. It is an index of specificity and is calculated entirely from the unaffected subgroup. Since the true positive fraction and the false positive fraction are calculated completely separately, the ROC curve is independent of the prevalence of the event in the cohort by using test results from two different subgroups. Each point on the ROC curve represents a sensitivity / -specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap between the two outcome distributions) has an ROC curve that passes through the upper left corner, where the true positive fraction is 1.0 or 100% (perfect sensitivity), and the false positive fraction is 0 (perfect specificity). The theoretical curve for a test with no discrimination (identical outcome distributions for the two groups) is a 45-degree diagonal line from the lower left corner to the upper right corner. Most curves fall between these two extremes. If the ROC curve is completely below the 45-degree diagonal, it can be easily corrected by reversing the criterion for "positive" from "greater than" to "less than", and vice versa. Qualitatively, the closer the curve is to the upper left corner, the higher the overall accuracy of the test. Depending on the desired confidence interval, a threshold can be derived from the ROC curve, thus allowing the classification or diagnosis mentioned herein to be made at an appropriate balance of sensitivity and specificity, respectively. Thus, the reference (i.e., the threshold that allows discrimination between HFpEF or HFrEF or allows diagnosis of HFpEF) for the method of the present invention can preferably be generated by establishing an ROC for the cohort as described above and deriving a threshold ratio therefrom. Depending on the sensitivity and specificity required for the diagnostic method, the ROC curve allows the derivation of a suitable threshold.

[0095] The definitions and explanations given above apply, mutatis mutandis, to the following methods of the present invention.

[0096] The present invention also relates to a computer-implemented method for differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF) in subjects suffering from heart failure, said method comprising the steps of:

[0097] (a) receiving, at a processing unit, values of the amount of IGFBP7 (insulin-like growth factor binding protein 7), values of the amount of BNP-type peptide, and optionally values of the amount of CRP (C-reactive protein) in a sample from said subject,

[0098] (b) calculating, by said processing unit, (i) the ratio of the amount of said IGFBP7 to the amount of said BNP peptide or (ii) the ratio of the sum of the amounts of said IGFBP7 and said CRP to the amount of said BNP-type peptide,

[0099] (c) comparing, by said processing unit, the ratio calculated in step (b) with a reference ratio, and

[0100] (d) differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF).

[0101] The present invention also relates to a computer-implemented method for diagnosing HFpEF in a subject suspected of having heart failure with preserved ejection fraction (HFpEF), said method comprising the steps of:

[0102] (a) receiving, at a processing unit, values of the amount of IGFBP7 (insulin-like growth factor binding protein 7), values of the amount of BNP-type peptide, and optionally values of the amount of CRP (C-reactive protein) in a sample from said subject,

[0103] (b) calculating, by said processing unit, (i) the ratio of the amount of said IGFBP7 to the amount of said BNP peptide or (ii) the ratio of the sum of the amounts of said IGFBP7 and said CRP to the amount of said BNP-type peptide,

[0104] (c) comparing, by said processing unit, the ratio calculated in step (b) with a reference ratio, and

[0105] (d) diagnosing heart failure with preserved ejection fraction.

[0106] The above methods are computer-implemented methods. Preferably, all steps of the computer-implemented method are performed by one or more processing units of a computer (or computer network). Thus, the evaluation in step (d) (i.e., the diagnosis or differentiation) is implemented by the processing unit. Preferably, the evaluation is based on the result of step (c).

[0107] The values received in step (a) will be derived from the determination of the amounts of IGFBP-7, BNP-type peptides (and optionally CRP) in a sample from the subject as described elsewhere herein. Preferably, these values are values of the amounts of the markers. These values will typically be received by the processing unit by uploading or sending the values to the processing unit. Alternatively, the value can be received by the processing unit by entering the value via a user interface.

[0108] In an embodiment of the above method, a reference ratio as described in step (c), i.e., a value of the reference ratio, is established from the memory.

[0109] In an embodiment of the above computer-implemented method of the invention, the result of the assessment performed in step d) is provided via a display configured to present the result.

[0110] In an embodiment of the above computer-implemented method of the invention, the method may include an additional step of transmitting information about the assessment performed in step d) to a personal electronic medical record.

[0111] The invention also relates to a computer program comprising computer-executable instructions which, when the program is executed on a computer or a computer network, are for performing the steps of a computer-implemented method according to the invention for differentiating HFpEF and HFrEF or for diagnosing HFpEF. Generally, the computer program may specifically comprise 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.

[0112] The invention also relates to a method for differentiating heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) in a subject suffering from heart failure, the method comprising the steps of:

[0113] (a) receiving a sample from the subject,

[0114] (b) determining the amounts of IGFBP7 (insulin-like growth factor binding protein 7), BNP-type peptides and optionally CRP (C-reactive protein) in the sample, and

[0115] (c) providing the values of the amounts of the IGFBP7, the BNP-type peptides and optionally the CRP to the attending physician, thereby allowing differentiation of HFpEF and HFrEF.

[0116] The invention also relates to a method for diagnosing HFpEF in a subject suspected of having heart failure with preserved ejection fraction (HFpEF), the method comprising the following steps:

[0117] (a) Receive a sample from the subject,

[0118] (b) Determine the amounts of IGFBP7 (insulin-like growth factor binding protein 7), BNP-type peptide and optionally CRP (C-reactive protein) in the sample, and

[0119] (c) Provide the values of the amounts of the IGFBP7, the BNP-type peptide and optionally the CRP to the attending physician, thereby allowing the diagnosis of HFpEF.

[0120] The physician according to the foregoing method should be the physician who requests the determination of the biomarkers IGFBP-7, BNP-type peptide and optionally CRP for diagnosis or differentiation, i.e., the physician is the attending physician. The physician should treat the subject to be tested. The above method should respectively assist the attending physician in differentiation and diagnosis.

[0121] The step a) of receiving the sample does not include drawing the sample from the subject. Instead, a sample obtained from the subject is provided (e.g., under the supervision of the attending physician). For example, the sample can be provided by delivering it to a laboratory that determines the amounts of the biomarkers in the sample.

[0122] After determining these amounts, information on the values of the determined amounts or information on the calculated ratios is provided to the physician. Additionally, information on the values of the reference ratios can be provided. The information provided may also include an indication of whether the subject has HFrEF or HFpEF.

[0123] Instead of providing the values of the amounts of IGFBP7, BNP-type peptide and optionally CRP (as described in step c of the above method) to the attending physician, the values of the ratios as described herein can be provided to the attending physician, thereby allowing the differentiation of HFpEF and HfrEF (or the diagnosis of HFpEF).

[0124] The method for diagnosing HFpEF of the present invention may further include the step of treating the subject based on the result of the diagnosis. Preferably, the subject diagnosed with HFpEF by the method of the present invention is treated. Thus, the method may include the step of selecting a subject with HFpEF. The selection should be based on the result of the comparison step.

[0125] The treatment may include any treatment that allows the treatment of HFpEF. This term includes lifestyle changes, dietary regimens, physical interventions, and the administration of appropriate medications.

[0126] In some embodiments, treatment includes administering at least one drug that permits treatment of HFpEF. In some embodiments, treatment includes administering at least one drug selected from the group consisting of angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin II receptor blockers (also commonly referred to as angiotensin II receptor antagonists), β-adrenergic blockers (also referred to herein as β-blockers), aldosterone antagonists, and diuretics.

[0127] In one embodiment, an ACE inhibitor is administered, such as benazepril, captopril, cilazapril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, or trandolapril.

[0128] In one embodiment, a β-blocker is administered, such as acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bupranolol, carteolol, carteolol hydrochloride, carvedilol, celiprolol, metipranolol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, talinolol, or timolol.

[0129] In one embodiment, an angiotensin II receptor antagonist is administered, such as losartan, valsartan, irbesartan, candesartan, telmisartan, or eprosartan.

[0130] In one embodiment, a diuretic is administered, such as thiazide and thiazide-like diuretics or potassium-sparing diuretics.

[0131] In one embodiment, an aldosterone antagonist is administered, such as eplerenone, spironolactone, canrenone, mexrenone, or prorenone.

[0132] Other therapies for the treatment of HFpEF are described in et al. (Clin Res Cardiol. January 2018; 107(1):1-19. doi: 10.1007 / s00392-017-1170-6. Epub October 10, 2017), the entire contents of which are incorporated herein by reference. Preferably, treatment includes administering at least one drug selected from the group consisting of SGLT2 (sodium-glucose cotransporter-2) inhibitors, soluble guanylate cyclase stimulators, inorganic nitrates, and angiotensin receptor-neprilysin inhibitors (ARNi).

[0133] In one embodiment, the drug is an SGLT2 (sodium-glucose cotransporter-2) inhibitor. In another embodiment, the drug is a soluble guanylate cyclase stimulator. In another embodiment, the drug is an inorganic nitrate. In another embodiment, the drug is an angiotensin receptor-neprilysin inhibitor (ARNi), i.e., a combination drug comprising a neprilysin inhibitor (such as sacubitril) and an angiotensin receptor blocker (such as valsartan).

[0134] Furthermore, the present invention relates to the use of IGFBP7, BNP-type peptides and optionally CRP in a sample from a subject for differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF) or for diagnosing HFpEF.

[0135] The method of the present invention for differentiating HFpEF and HFrEF may further include the step of treating the subject based on the differentiation result. Generally, subjects diagnosed with HFpEF and subjects diagnosed with HFrEF are treated. Generally, subjects diagnosed with HFpEF are treated as described above in connection with the method for diagnosing HFpEF. In some embodiments, treating the subject includes administering at least one drug selected from SGLT2 (sodium-glucose cotransporter-2) inhibitors, soluble guanylate cyclase stimulators, inorganic nitrates and angiotensin receptor-neprilysin inhibitors (ARNi).

[0136] Subjects determined to have HFrEF are preferably treated according to guidelines. In some embodiments, treating HFrEF includes administering at least one drug selected from angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin II receptor blockers (also commonly referred to as angiotensin II receptor antagonists), β-adrenergic blockers (also referred to herein as β-blockers), aldosterone antagonists and diuretics.

[0137] Finally, the present invention relates to the use of at least one detection agent that binds to IGFBP7 in a sample from a subject, at least one detection agent that binds to BNP-type peptides in a sample from a subject and optionally at least one detection agent that binds to CRP in a sample from a subject for differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF) or for diagnosing HFpEF.

[0138] In one embodiment, the detection agent is an antibody or an antigen-binding fragment thereof.

[0139] Embodiments of the present invention

[0140] The following embodiments of the present invention can be used in combination with any other embodiments described herein. The definitions and explanations provided above apply, mutatis mutandis, to the following.

[0141] 1. A method for differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF) in a subject suffering from heart failure, the method comprising the steps of:

[0142] (a) determining the amounts of IGFBP7 (insulin-like growth factor binding protein 7), BNP-type peptide, and optionally CRP (C-reactive protein) in a sample from the subject,

[0143] (b) calculating (i) the ratio of the amount of the IGFBP7 to the amount of the BNP peptide or (ii) the ratio of the sum of the amounts of the IGFBP7 and the CRP to the amount of the BNP-type peptide,

[0144] (c) comparing the ratio calculated in step (b) with a reference ratio, and

[0145] (d) differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF).

[0146] 2. The method according to embodiment 1, wherein the subject is a human.

[0147] 3. The method according to embodiments 1 and 2, wherein the sample is a blood, serum or plasma sample.

[0148] 4. The method according to any one of embodiments 1 to 3, wherein the BNP-type peptide is BNP or NT-proBNP.

[0149] 5. The method according to any one of embodiments 1 to 4, wherein the ratio of the amount of the IGFBP7 to the amount of the BNP-type peptide is calculated.

[0150] 6. The method according to any one of embodiments 1 to 4, wherein the ratio of the sum of the amounts of the IGFBP7 and the CRP to the amount of the BNP-type peptide is calculated.

[0151] 7. The method according to embodiment 5 or 6, wherein a ratio higher than the reference ratio indicates heart failure with preserved ejection fraction (HFpEF), and / or wherein a ratio lower than the reference ratio indicates heart failure with reduced ejection fraction (HFrEF).

[0152] 8. A method for diagnosing HFpEF in a subject suspected of having heart failure with preserved ejection fraction (HFpEF), the method comprising the steps of:

[0153] (a) Determine the amounts of IGFBP7 (insulin-like growth factor binding protein 7), BNP-type peptide, and optionally CRP (C-reactive protein) in a sample from the subject,

[0154] (b) Calculate (i) the ratio of the amount of the IGFBP7 to the amount of the BNP peptide or (ii) the ratio of the sum of the amounts of the IGFBP7 and the CRP to the amount of the BNP-type peptide,

[0155] (c) Compare the ratio calculated in step (b) with a reference ratio, and

[0156] (d) Diagnose heart failure with preserved ejection fraction.

[0157] 9. A computer-implemented method for differentiating heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) in a subject with heart failure, the method comprising the steps of:

[0158] (a) Receiving, at a processing unit, values of the amount of IGFBP7 (insulin-like growth factor binding protein 7), the amount of BNP-type peptide, and optionally the amount of CRP (C-reactive protein) in a sample from the subject,

[0159] (b) Calculating, by the processing unit, (i) the ratio of the amount of the IGFBP7 to the amount of the BNP peptide or (ii) the ratio of the sum of the amounts of the IGFBP7 and the CRP to the amount of the BNP-type peptide,

[0160] (c) Comparing, by the processing unit, the ratio calculated in step (b) with a reference ratio, and

[0161] (d) Differentiating heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF).

[0162] 10. A computer-implemented method for diagnosing HFpEF in a subject suspected of having heart failure with preserved ejection fraction (HFpEF), the method comprising the steps of:

[0163] (a) Receiving, at a processing unit, values of the amount of IGFBP7 (insulin-like growth factor binding protein 7), the amount of BNP-type peptide, and optionally the amount of CRP (C-reactive protein) in a sample from the subject,

[0164] (b) Calculating, by the processing unit, (i) the ratio of the amount of the IGFBP7 to the amount of the BNP peptide or (ii) the ratio of the sum of the amounts of the IGFBP7 and the CRP to the amount of the BNP-type peptide,

[0165] (c) Comparing the ratio calculated in step (b) with a reference ratio by means of the processing unit, and

[0166] (d) Diagnosing heart failure with preserved ejection fraction.

[0167] 11. The method according to embodiments 9 and 10, wherein the reference ratio is established from a memory.

[0168] 12. A method for differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF) in a subject suffering from heart failure, the method comprising the steps of:

[0169] (a) Receiving a sample from the subject,

[0170] (b) Determining the amounts of IGFBP7 (insulin-like growth factor binding protein 7), BNP-type peptide and optionally CRP (C-reactive protein) in the sample, and

[0171] (c) Providing the values of the amounts of the IGFBP7, the BNP-type peptide and optionally the CRP to a attending physician, thereby allowing differentiation between HFpEF and HFrEF.

[0172] 13. A method for diagnosing HFpEF in a subject suspected of having heart failure with preserved ejection fraction (HFpEF), the method comprising the steps of:

[0173] (a) Receiving a sample from the subject,

[0174] (b) Determining the amounts of IGFBP7 (insulin-like growth factor binding protein 7), BNP-type peptide and optionally CRP (C-reactive protein) in the sample, and

[0175] (c) Providing the values of the amounts of the IGFBP7, the BNP-type peptide and optionally the CRP to a attending physician, thereby allowing diagnosis of HFpEF.

[0176] 14. Use of IGFBP7, BNP-type peptide and optionally CRP as biomarkers in a sample from a subject for differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF) or for diagnosing HFpEF.

[0177] Use of at least one detection agent that binds to IGFBP7 in a sample from a subject, at least one detection agent that binds to a BNP-type peptide in a sample from a subject, and optionally at least one detection agent that binds to CRP in a sample from a subject for differentiating heart failure with preserved ejection fraction (HFpEF) from heart failure with reduced ejection fraction (HFrEF) or for diagnosing HFpEF.

[0178] The drawings show:

[0179] Figure 1 Show: Biomarker measurements for diagnosing HFpEF

[0180] Figure 1a : Measurement of NT-proBNP in 499 HFrEF patient samples and 123 HFpEF patient samples for diagnosing HFpEF

[0181] Figure 1b : Measurement of CRP in 411 HFrEF patient samples and 107 HFpEF patient samples for diagnosing HFpEF

[0182] Figure 1c : Measurement of GDF15 in 451 HFrEF patient samples and 110 HFpEF patient samples for diagnosing HFpEF

[0183] Figure 1d : Measurement of IGFBP-7 in 366 HFrEF heart failure patient samples and 96 HFpEF patient samples for diagnosing HFpEF

[0184] Figure 2 Show: GDF15 biomarker ratio for diagnosing HFpEF

[0185] Figure 2a : Diagnosis of HFpEF and HFrEF in heart failure patients using the ratio CRP + GDF15 (not elevated in HFrEF relative to HFpEF) / NTproBNP (elevated in HFrEF relative to HFpEF) in samples from 410 patients with HFrEF and 107 patients with HFpEF

[0186] Figure 2b : Diagnosis of HFpEF and HFrEF in heart failure patients using the ratio CRP + GDF15 + ST2 (not elevated in HFrEF relative to HFpEF) / NTproBNP (elevated in HFrEF relative to HFpEF) in samples from 411 patients with HFrEF and 107 patients with HFpEF

[0187] Figure 3 Shown: IGFBP-7 biomarker ratio for diagnosing HFpEF

[0188] Figure 3a : Diagnosis of HFpEF and HFrEF in heart failure patients using the ratio CRP + IGFBP-7 (not elevated in HFrEF compared to HFpEF) / NTproBNP (elevated in HFrEF compared to HFpEF) in samples from 359 patients with HFrEF and 96 patients with HFpEF

[0189] Figure 3b : Diagnosis of HFpEF and HFrEF in heart failure patients using the ratio CRP + IGFBP-7 + ST2 (not elevated in HFrEF compared to HFpEF) / NTproBNP (elevated in HFrEF compared to HFpEF) in samples from 359 patients with HFrEF and 96 patients with HFpEF

[0190] Figure 3c : Diagnosis of HFpEF and HFrEF in heart failure patients using the ratio GDF-15 + IGFBP-7 (not elevated in HFrEF compared to HFpEF) / NTproBNP (elevated in HFrEF compared to HFpEF)

[0191] All references cited herein are incorporated herein by reference for their general disclosure or for the specific disclosure above.

[0192] The present invention will now be illustrated by the following examples, which are not intended to limit or define the scope of the invention. Examples

[0193] Example 1: Evaluation of HFpEF with circulating biomarkers

[0194] NTproBNP, GDF-15, ST2, CRP (hs CRP) and IGFBP-7 levels were determined in HF patients with reduced and preserved ejection fraction.

[0195] Results are shown in Figure 1. NTproBNP was significantly elevated in heart failure with reduced ejection fraction (HFrEF) patients compared to heart failure with preserved ejection fraction (HFpEF) patients (AUC 0.659), see Figure 1a . Figure 1b Shown, CRP was not significantly elevated in heart failure with reduced ejection fraction (HFrEF) patients compared to heart failure with preserved ejection fraction (HFpEF) patients (AUC 0.575). Figure 1cIt was shown that growth differentiation factor 15 (GDF15) was not significantly elevated in patients with heart failure with reduced ejection fraction (HFrEF) compared to patients with heart failure with preserved ejection fraction (HFpEF) (AUC 0.554). Figure 1d It was shown that insulin-like growth factor binding protein 7 (IGFBP-7) was not significantly elevated in patients with HFrEF compared to patients with HFpEF (AUC 0.494).

[0196] Table 1 shows the titers of circulating biomarkers in subgroups of patients with HFrEF and patients with HFpEF. Biomarker data for the full panel of biomarkers (NTproBNP, GDF15, ST2, CRP hs, and IGFBP7) were available for 366 HFrEF patient samples and 96 HFpEF patient samples.

[0197] Table 1: Titers of circulating biomarkers in HFpEF and HFrEF patients.

[0198]

[0199] Table 1 clearly shows that in univariate analysis, significantly different biomarker concentrations of NTproBNP were detected in the two subgroups (HFrEF population compared to the HFpEF population).

[0200] Compared to HFpEF patients, HFrEF patients showed higher NT-proBNP titers [median 4410 vs 2189 pg / mL, p<0.001]. In contrast, for GDF15, ST2, CRP, and IGFBP7, only slightly elevated biomarker concentrations were detected in the HFpEF patient subgroup compared to the HFrEF patient subgroup. However, in univariate analysis, the observed differences in biomarker concentrations between HFpEF and HFrEF were not significant for GDF15, ST2, CRP hs, IGFBP-7. Compared to HFrEF patients, HFpEF patients showed slightly higher titers of GDF15, ST2, CRP hs, IGFBP-7 [median 4304 vs 3939 pg / mL, 37.6 vs 35.7 pg / mL; 8.54 vs 6.66 ng / mL; 237.1 vs 242.1 ng / mL; p>0.001].

[0201] As described above, the differential diagnosis and identification of HFpEF patients are challenging. Only a significantly elevated circulating titer of NTproBNP was observed in patients with HFrEF and HFpEF. In contrast, no significant differences in biomarker levels were detected between HFpEF and HFrEF patients for CRP, GDF15, ST2, or IGFBP-7. Therefore, there is no clear indication that using a biomarker combination can improve the diagnosis of HFpEF compared to measuring NTproBNP.

[0202] Example 2: GDF15 ratio for diagnosing HFpEF

[0203] As shown in Table 1, elevated circulating NTproBNP levels were detected in HFrEF patients compared to HFpEF patients. In contrast, no significant differences in circulating levels were detected for CRP and GDF15 between HF patients with reduced or preserved ejection fraction.

[0204] Evaluate the ratio CRP + GDF15 (not elevated in HFrEF compared to HFpEF) / NTproBNP (elevated in HFrEF compared to HFpEF) to improve the diagnosis of HFpEF.

[0205] As Figure 2a shown, the AUC of the ratio (CRP + GDF15) / NTproBNP for detecting HFpEF was 0.671. As Figure 2b shown, the ratio (CRP + GDF15 + ST2) / NTproBNP did not further improve compared to the ratio (CRP + GDF15) / NTproBNP. The AUC of the ratio (CRP + GDF15 + ST2) / NTproBNP for detecting HFpEF was 0.671.

[0206] Based on these results, it can be concluded that the ratio (CRP + GDF15) / NTproBNP improved the diagnosis of HFpEF compared to the determination of a single biomarker of NTproBNP, GDF15, or CRP.

[0207] Example 3: IGFBP-7 biomarker ratio for diagnosing HFpEF

[0208] As shown in Table 1, elevated circulating NTproBNP levels can be detected in patients with HFrEF compared to patients with HFpEF. In contrast, for CRP and IGFBP-7, no significant differences in circulating levels were detected between HF patients with reduced or preserved ejection fraction. The ratio CRP + IGFBP-7 (not elevated in HFrEF compared to HFpEF) / NTproBNP (elevated in HFrEF compared to HFpEF) was evaluated to improve the diagnosis of HFpEF.

[0209] As Figure 3a shown, the AUC of the calculated index of the sum of biomarkers for detecting HFpEF (CRP + IGFBP-7) / NTproBNP was 0.699. In contrast, the AUC of the ratio IGFBP-7 / NTproBNP for detecting HFpEF was 0.680. As Figure 3c shown, the AUC of the calculated index of the sum of biomarkers for detecting HF (GDF-15 + IGFBP-7) / NTproBNP was 0.693. As Figure 3b shown, the ratio (CRP + IGFBP-7 + ST2) / NTproBNP was observed to have comparable performance compared to the ratio (CRP + IGFBP-7) / NTproBNP. The AUC of the ratio (CRP + IGFBP-7 + ST2):NTproBNP for detecting HFpEF was 0.703. Surprisingly, the calculated index of the sum of biomarkers (CRP + IGFBP-7):NTproBNP showed even better performance in diagnosing HFpEF than the ratio (CRP + GDF15) / NTproBNP. The ratio (CRP + IGFBP-7) / NTproBNP showed even better performance than the ratio (CRP + GDF15 + ST2) / NTproBNP, as described by Sinning et al. (AUC 0.699 vs. AUC 0.671).

[0210] Table 2 shows the fold changes of the calculated indices in patients with HFpEF and HFrEF.

[0211]

[0212] The fold change was calculated as ((median HFpEF) / (median HFrEF)) - 1 * 100.

[0213] Table 2 clearly shows that the calculated index of the sum of (IGFBP-7 + CRP) / NTproBNP has improved performance in the differential diagnosis of HFpEF versus HFrEF compared to the calculated index of the sum of (GDF15 + CRP + ST2) / NTproBNP (fold changes of 87.81% vs. 65.34%). A fold change of 74.97% was observed compared to the ratio IGFBP-7 / NTproBNP.

[0214] In summary, IGFBP-7 improves the identification of HFpEF compared to NTproBNP, GDF15, CRP-based scores without IGFBP-7.

[0215] The current score for IGFBP-7 shows a better effect size compared to the score published by Sinning et al.

[0216] It can be concluded that an algorithm including the ratio (CRP + IGFBP-7) / NTproBNP helps in the diagnosis of HFpEF patients. This algorithm can be used to diagnose HFpEF in patients with heart failure and to identify HFpEF in individuals suspected of having heart failure.

[0217] Example 4: Case study

[0218] A 76-year-old obese female patient with hypertension presented with shortness of breath. NTproBNP, IGFBP-7, and CRP were measured in a serum sample obtained from the patient (using Elecsys NTproBNP, Elecsys IGFBP-7, and Cobas CRP). Due to the presence of obesity, the NTproBNP value may be in the grey zone, and thus a differential diagnosis of heart failure was performed. The NTproBNP value was 476 pg / mL, the IGFBP-7 value was 190.8 ng / mL, and the CRP value was 5.8 ng / mL. The calculated index (CRP + IGFBP-7):NTproBNP was 0.41, which was elevated relative to the calculated reference ratio (0.09). The reference ratio was calculated as the median ratio in the reference cohort. The ratio obtained (0.41) indicated heart failure with preserved ejection fraction, and it was determined that the patient would benefit more from treatment with an HFpEF-regulating strategy (e.g., treatment with an SGLT2 inhibitor). The patient's therapy was adjusted accordingly.

[0219] An 81-year-old male patient with hypertension had a history of atrial fibrillation and shortness of breath. NTproBNP, IGFBP-7, and CRP were measured (using the above kit) in a serum sample obtained from the patient. The observed ejection fraction was in the grey area (50%). Due to the presence of atrial fibrillation, the NTproBNP value may also be elevated, so a differential diagnosis of heart failure was performed. The NTproBNP value was 3289 pg / mL, the IGFBP-7 value was 250.7 ng / mL, and the CRP value was 10.8 ng / mL. The calculated index (CRP + IGFBP-7):NTproBNP was 0.79, which was elevated relative to the calculated reference ratio (0.09). The reference ratio was calculated as the median ratio in the reference cohort. The obtained ratio (0.79) indicated heart failure with preserved ejection fraction, and it was determined that the patient would benefit more from treatment with an HFpEF modulation strategy (e.g., treatment with an SGLT2 inhibitor). Accordingly, the patient's therapy was adjusted.

[0220] A 68-year-old obese female with diabetes and hypertension presented with symptoms of exercise intolerance. Due to the similar symptoms of obesity and heart failure with preserved ejection fraction (e.g., exercise intolerance), a differential diagnosis of heart failure was performed. NTproBNP, IGFBP-7, and CRP were measured (using the above kit) in a serum sample obtained from the patient. The NTproBNP value was 4643 pg / mL, the IGFBP-7 value was 245.5 ng / mL, and the CRP value was 5.6 ng / mL. The calculated index (CRP + IGFBP-7):NTproBNP was 0.05, which was decreased relative to the reference ratio (0.09). This result indicated heart failure with reduced ejection fraction, and it was determined that the patient would not benefit more from treatment with an HFpEF modulation strategy (e.g., treatment with an SGLT2 inhibitor). Therefore, the patient's therapy was not adjusted accordingly.

Claims

1. A computer-readable data carrier storing computer-executable instructions for performing the steps of a method on a computer or computer network, the method for differentiating between heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) in subjects with heart failure, the method comprising the steps of: (a) receiving, at a processing unit, values of the amount of IGFBP7 (insulin-like growth factor binding protein 7), values of the amount of BNP-type peptide, and values of the amount of CRP (C-reactive protein) in a blood, serum, or plasma sample from the subject; (b) calculating, by the processing unit, the ratio of the sum of the amounts of IGFBP7 and CRP to the amount of the BNP-type peptide; (c) comparing, by the processing unit, the ratio calculated in step (b) with a reference ratio, wherein the reference ratio is calculated by applying standard statistical methods based on the mean ratio or median ratio of a cohort of subjects, and the calculated reference ratio is 0.09; and (d) differentiating between heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF).

2. A computer-readable data carrier storing computer-executable instructions for performing the steps of a method on a computer or computer network, the method for diagnosing HFpEF in a subject suspected of having heart failure with preserved ejection fraction (HFpEF), the method comprising the steps of: (a) receiving, at a processing unit, values of the amount of IGFBP7 (insulin-like growth factor binding protein 7), values of the amount of BNP-type peptide, and values of the amount of CRP (C-reactive protein) in a blood, serum, or plasma sample from the subject; (b) calculating, by the processing unit, the ratio of the sum of the amounts of IGFBP7 and CRP to the amount of the BNP-type peptide; (c) comparing, by the processing unit, the ratio calculated in step (b) with a reference ratio, wherein the reference ratio is calculated by applying standard statistical methods based on the mean ratio or median ratio of a cohort of subjects, and the calculated reference ratio is 0.09; and (d) diagnosing heart failure with preserved ejection fraction.

3. The computer-readable data carrier according to claim 1 or 2, wherein the reference ratio is established from a memory.

4. The computer-readable data carrier according to any one of claims 1 to 3, wherein the subject is a human.

5. The computer-readable data carrier according to any one of claims 1 to 3, wherein the BNP-type peptide is BNP or NT-proBNP.

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