HK40012159A - Biomarkers for risk assessment and treatment monitoring in heart failure patients guided by natriuretic peptides - Google Patents

Description

Biomarkers for risk assessment and therapy monitoring in heart failure patients guided by natriuretic peptides

The present application is a divisional application of chinese patent application 201580005871.5 "biomarker for risk assessment and therapy monitoring in heart failure patients guided by natriuretic peptides", filed on day 2015, 1, month 22.

Technical Field

The present invention relates to a method of identifying a patient suitable for intensive heart failure therapy. Furthermore, the present invention relates to methods of optimizing BNP-type peptide directed therapy for heart failure. The method is based on measuring the level of at least one marker in a sample from a patient having heart failure and receiving a BNP-type peptide directed heart failure therapy. Further contemplated by the invention are kits and devices suitable for carrying out the invention.

Background

Heart Failure (HF) is one of the leading causes of morbidity and mortality in many countries of the world. Although available treatment options can reduce morbidity and mortality in patients with HF, the relative number of suitable patients receiving these treatments remains unsatisfactorily low (O' Donoghue m. and Braunwald e., nat. rev. cardiol.2010;7: 13-20). Furthermore, in patients eligible for treatment, therapy has been primarily directed and regulated by signs and symptoms of HF to maximal tolerance of the drug (e.g., by NYHA stage, ACC/AHA stage, or congestion scores).

Measurement of natriuretic peptide markers such as B-type natriuretic peptide (BNP), or N-terminal proBNP of its amino-terminal fragment (NT-proBNP) has emerged as an important tool for diagnosis and risk stratification (stringification) of patients with HF. In addition, evidence is emerging that NT-proBNP may be useful for guiding medical therapy in heart Failure (Januzzi J, journal of Cardiac Failure, 2011; 17: 622-.

However, NT-proBNP-directed HF therapy does not identify all patients at risk for HF decompensation and adverse events. Thus, some patients are at risk even if they show a favorable response to therapy with respect to their NT-proBNP levels. Thus, not all patients will benefit from the intensification of heart failure therapy.

WO2008/015254 discloses a method for predicting the risk of death or a further cardiovascular event in a heart failure patient based on measuring NT-proBNP and GDF-15.

WO2010/0070411 discloses a method for monitoring a superficially stable patient suffering from heart failure based on GDF-15, NT-proANP, NT-proBNP and cardiac troponin detection. Furthermore, it discloses a method of diagnosing and/or deciding which therapy/medication is to be applied in a superficially stable patient suffering from heart failure and undergoing a change in its physiological state.

Böhm et al 2011 (Clin Res Cardiol, 100: 973-.

Miyata et al (j. of Cardiology 2012, 59, 352-. Half of the group was switched to the long acting diuretic azosemide while the other half remained on the short acting diuretic (furosemide). The authors found a significant reduction in the plasma levels of BNP and ANP after 3 months in the azosemide group. There were no significant differences in the changes in creatinine, BUN (═ blood urea nitrogen), sodium, potassium, and hematocrit in both groups.

Advantageously, it has been found in the studies carried out in the present invention that NT-proBNP or the combination of BNP with other markers and clinical parameters can be used for monitoring purposes and as therapy guidance in addition to the current standard-of-care therapy (standard-of-care) for the modulation and titration (titrate) of therapy in HF patients (chronic or acute HF after stabilization). These markers and parameters are creatinine, BUN (urea), glucose, HbA1c, hscRP, cystatin C, IL-6, prealbumin, sFlt-1, uric acid, GDF-15, sST2, galectin-3, endostatin, Mimecan, IGFBP-7, osteopontin, sodium, hemoglobin and hematocrit, as well as heart rate and QRS duration. In particular, the addition of these measurements to NT-proBNP or BNP, together with current standard therapies, enables further risk stratification of HF patients who have been guided by NT-proBNP but may need more intensive therapy and closer observation. The present invention thus optimizes heart failure therapy guidance beyond NT-proBNP by considering the combination of natriuretic peptides with other markers and/or clinical parameters.

Disclosure of Invention

In particular, it has been found in the studies of the present invention that the additional determination of the parameter markers as described above allows to identify a subgroup of patients exhibiting a level of the BNP-type peptide lower than a reference level of the BNP-type peptide indicative of an intensification of the heart failure therapy, but nevertheless suitable for an intensification of the heart failure therapy. Thanks to the present invention, it is possible to identify patients in need of an intensification of heart failure therapy, who would not receive an intensified heart failure therapy based on the measurement of the level of BNP type alone.

Accordingly, the present invention relates to a method for identifying or selecting a patient suitable for intensive heart failure therapy, said method comprising the steps of

(a) Measuring in a sample from a patient having heart failure and receiving a BNP-type peptide directed heart failure therapy, in particular NP-proBNP directed heart failure therapy or BNP directed heart failure therapy, the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFLt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin, and

(b) comparing the level(s) of marker(s) measured in (a) with a reference level(s).

In one embodiment, the method further comprises the step (c) of identifying or selecting a patient suitable for intensive heart failure therapy, i.e. a BNP-type peptide directed therapy.

Furthermore, the method may comprise the step (d) of strengthening heart failure therapy or recommending strengthening heart failure therapy if the patient has been identified as suitable for strengthening heart failure therapy. Accordingly, the present invention also contemplates a method of potentiating a heart failure therapy, said method comprising steps (a) to (d) as described above.

The methods of the invention are preferably ex vivo or in vitro methods. Further, it may comprise steps other than those explicitly mentioned above. For example, further steps may involve sample pre-treatment or evaluation of the results obtained by the method. The method can be carried out manually or with the aid of automation. Preferably, steps (a) and/or (b) may be wholly or partially assisted by automation, for example by suitable machinery and sensor means for the measurement in step (a) or computer-implemented authentication in step (c).

In one embodiment, the above method may additionally comprise assessing or providing QRS duration and comparing the QRS duration thus determined with a reference.

Furthermore, it is contemplated to evaluate or provide QRS duration instead of measuring the level of the at least one marker in step a) and to compare the QRS duration thus determined with a reference.

Thus, the present invention further contemplates a method for identifying or selecting a patient suitable for intensive heart failure therapy, said method comprising the steps of

(a) Assessing or providing QRS duration of a patient having heart failure and receiving a BNP-type peptide-directed heart failure therapy, and

(b) the QRS duration is compared to a reference,

wherein an increased QRS duration as compared to a reference indicates that the patient is suitable for intensive heart failure therapy and a decreased QRS duration as compared to the reference indicates that the patient is not suitable for intensive heart failure therapy.

As referred to herein, a "patient" is preferably a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human 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 terms "subject" and "patient" are used interchangeably herein.

The phrase "selecting a patient" or "identifying a patient" as used herein refers to identifying or selecting a patient as more or less likely to benefit from a heart failure-potentiating therapy using information or data generated regarding the level of at least one marker referred to in the context of the present invention in a patient sample. Preferably, a body adapted for said strengthening requires said strengthening, whereas a body not adapted for said strengthening does not require said strengthening.

It will be appreciated that subjects eligible for intensive heart failure therapy will benefit from the enrichment, while subjects not eligible for such enrichment may not benefit from the enrichment, e.g., may experience adverse side effects or victimization from the enrichment. In particular, a subject benefits from fortification if fortification reduces the risk of mortality and/or reduces the risk of hospitalization and/or cardiac decompensation of the subject (especially within a window period of 18 months or 3 years after a sample has been obtained). Preferably, the aforementioned risk is reduced by 5%, more preferably by 10%, even more preferably by 15%, and most preferably by 20%. Preferably, the hospitalization and death referred to herein should be due to heart failure.

Conversely, subjects who are not eligible for intensive heart failure therapy will not benefit from the enrichment (and in particular will not benefit significantly). In particular, a subject does not benefit from fortification if fortification does not reduce (in particular does not significantly reduce) the risk of death of the subject and/or does not reduce (in particular does not significantly reduce) the risk of hospitalization and/or cardiac decompensation of the subject in question and/or increases the risk of unwanted side effects (in particular within a window period of 18 months or 3 years after the sample has been obtained). In this case, if the therapy is not intensive, unnecessary healthcare costs can be avoided. In addition, adverse side effects that may result from fortification can be avoided.

Thus, by identifying a subject suitable for intensive heart failure therapy, it can be assessed whether the subject would benefit from intensive heart failure therapy. Accordingly, the present invention also relates to methods of identifying subjects that would benefit from intensive heart failure therapy based on the steps described elsewhere herein.

The information or data used or generated may be in any form, written, spoken or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof. In some embodiments, the communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof is performed by a computer device, an analyzer unit, or a combination thereof. In some further embodiments, the communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof is performed by a laboratory or medical professional. In some embodiments, the information or data comprises a comparison of the level of the at least one marker to a reference level.

As described in more detail herein below, subjects who are eligible for intensive heart failure therapy should also be monitored at short intervals, while subjects who are not eligible for intensive heart failure therapy (i.e., do not require intensive heart failure therapy) should be monitored at long intervals. Thus, in addition to determining whether heart failure treatment should be enhanced, it is also possible to assess whether a subject should be monitored at short or long time intervals.

As will be understood by those skilled in the art, the assessment by the method of the present invention is generally not intended to be 100% correct for the subject to be diagnosed. However, this term requires that the assessment be correct for a statistically significant portion of subjects (e.g., cohorts in a cohort study). Whether a moiety is statistically significant can be determined by one skilled in the art without further effort using various well-known statistical evaluation tools, such as determination of confidence intervals, p-value determination, student's t-test, Mann-Whitney test, and the like. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. 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.

It is envisaged in the context of the present invention that the subject suffers from Heart Failure (HF), in particular chronic heart failure. Further, the subject may suffer from stable acute heart failure.

The term "heart failure" as used herein relates to diastolic dysfunction or, in particular, systolic dysfunction of the heart, which is accompanied by obvious signs of heart failure as known to the person skilled in the art. Preferably, heart failure as referred to herein is chronic heart failure (which is preferably caused by systolic dysfunction). Heart failure according to the present invention includes overt heart failure and/or advanced heart failure. In overt heart failure, patients exhibit heart failure symptoms known to those skilled in the art.

HF can be classified to various degrees of severity.

According to the NYHA (New York Heart Association) classification, Heart failure patients may be classified as belonging to NYHA types I, II, III and IV. A patient with heart failure has experienced structural and functional changes in his pericardium, myocardium, coronary circulation, or heart valves. He will not be able to fully restore his health and will need therapeutic treatment. Patients of NYHA type I have no obvious symptoms of cardiovascular disease, but already have objective evidence of impaired function. Patients of NYHA type II have slight limitations of physical activity. Patients of NYHA type III show significant limitations of physical activity. Patients of NYHA type IV cannot perform any physical activity without discomfort. They showed symptoms of cardiac insufficiency at rest.

This functional classification is complemented by the newer classification of American College of medicine and the American HeartAssociation (see J.Am. Coll.Cardiol.2001;38; 2101. multidot. 2113, updated at 2005, see J.Am.Coll.Cardiol.2005;46; e1-e 82). 4 phases A, B, C and D are defined. Phases a and B are not HF but are believed to help identify patients earlier before progressing to "true" HF. Phase a and B patients are best defined as patients with risk factors for HF development. For example, a patient with coronary artery disease, hypertension, or diabetes who has not demonstrated impaired Left Ventricular (LV) function, hypertrophy, or geometric ventricular deformity will be considered stage a, while a patient who is asymptomatic but demonstrates LV hypertrophy and/or impaired LV function will be designated stage B. Then phase C represents patients with symptoms of HF currently or historically associated with an underlying structural heart disease (most patients with HF), and phase D designates patients with truly refractory HF.

As used herein, the term "heart failure" refers in particular to stages B and C of the ACC/AHA classification described above. At these stages, subjects show typical heart failure symptoms. Thus, the patient preferably has heart failure classified as stage B or C according to the ACC/AHA classification. Also preferably, the patient has heart failure according to class II or III of the NYHA classification.

Preferably, heart failure is due to impaired contractile function. Thus, patients with systolic heart failure are especially considered. Preferably, the patient has a Left Ventricular Ejection Fraction (LVEF) of less than 50%, more preferably less than 45%, and most preferably less than 40%.

The patient to be tested according to the method of the invention should receive a BNP-type peptide directed therapy, i.e. a BNP-type peptide directed therapy of heart failure. The terms "BNP-type peptide directed therapy" and "BNP-type peptide directed therapy of heart failure" are well known in the art. Therefore, the patient to be tested should receive a heart failure therapy directed by the BNP-type peptide (more precisely, at the time of obtaining the sample). Thus, it is considered that at least one decision regarding the heart failure therapy of the patient has been made in the past (and thus before obtaining the sample to be tested) based on the level of the BNP-type peptide in the patient, in particular based on the blood, serum or plasma level of the BNP-type peptide in the patient. Thus, the patient's level of BNP-type peptide may have been considered in the past decision on the treatment of heart failure. Further, it is the first decision to consider the level of BNP-type peptides that is considered to be a current decision regarding heart failure therapy. Thus, the patient receiving the BNP-type peptide-directed heart failure therapy may be a patient wherein the BNP-type peptide-directed heart failure therapy is initiated, in particular immediately after the test sample has been obtained. Nevertheless, the patient may have previously received a heart failure therapy that is not directed by BNP-type peptides.

Preferred BNP-type peptides are disclosed elsewhere herein. The BNP-type peptide-directed therapy may preferably be a BNP (brain natriuretic peptide) -directed therapy or especially a NT-proBNP (N-terminal brain natriuretic peptide precursor) -directed therapy (for the explanation of these markers see elsewhere).

In BNP-type peptide directed therapy, the level of BNP-type peptide is used to manage heart failure treatment. Based on this level, a decision is made on the heart failure therapy. In principle, patients with increased BNP-type peptide levels receive more intensive therapy than patients with decreased levels of this marker. BNP-type peptide-directed therapies are well known in the art and are described, for example, by Sanders-van Wijk et al Eur J Heart Fail (2013) 15 (8): 910-. Furthermore, BNP-type peptide-directed therapies are reviewed by Januzzi, see Archives of Cardiovascular diseases (2012), 105, 40-50. Both documents are incorporated herein by reference for their full disclosure.

In a preferred embodiment, the patient exhibits a level (in particular a blood, serum or plasma level) of the BNP-type peptide which is lower than a reference level of said BNP-type peptide, said reference level being indicative for an intensification of the heart failure therapy. Thus, the patient should be a patient having a BNP-type peptide level which, when taken alone (i.e. not in combination with at least one further marker as described in step (a) of the above method), is indicative for a patient who is not suitable for intensive heart failure therapy. Preferred reference levels of said BNP-type peptide indicative for an enhanced heart failure therapy to be applied in the context of the present invention are those described in the examples. Preferred reference levels are in the range of about 80-400 pg/ml for BNP, or specifically about 80-200pg/ml, or in the range of about 450-2200 pg/ml for NT-proBNP, or specifically about 800-1200 pg/ml. Further preferred reference levels are about 100 pg/ml or 400 pg/ml for BNP and about 1000pg/ml or 1200pg/ml for NT-proBNP. Thus, patients according to the invention may exhibit a level of NT-proBNP, in particular a blood, serum or plasma level of NP-proBNP, of less than 1000pg/ml or 1200 pg/ml.

Further, it is contemplated that patients exhibiting a level of a BNP-type peptide below a reference level of said BNP-type peptide indicative of an enhanced heart failure therapy have a BNP level (in particular a blood, serum or plasma level) in the range of about 80 to about 400 pg/ml, in particular in the range of about 80 to about 200 pg/ml. Moreover, a patient exhibiting a level of a BNP-type peptide lower than a reference level of said BNP-type peptide indicative of an intensive heart failure therapy may have a level of NT-proBNP (in particular a blood, serum or plasma level) in the range of 450 to 2200 pg/ml, in particular in the range of 800 to 1200 pg/ml.

Preferably, the term "about" as used herein encompasses a range of +/-20%, more preferably a range of +/-10%, even more preferably a range of +/-5%, and most preferably a range of +/-2% relative to the particular amount, e.g., an amount indicating "about 100" is meant to encompass amounts within a range of 80-120. Moreover, the term "about" refers to a precise amount. Preferably, the level is measured as described in the examples.

The term "heart failure therapy" (also referred to herein as "heart failure treatment") as used herein preferably refers to any treatment that allows for the treatment of heart failure. Preferably, the term encompasses lifestyle changes, dietary therapy, physical intervention as well as administration of suitable drugs, use of devices and/or organ transplantation for treating patients suffering from heart failure.

Lifestyle changes include smoking cessation, reduction of alcohol consumption, increased physical activity, weight loss, sodium (salt) restriction, weight management, and healthy diet (such as daily fish oil).

Preferred devices to be applied are pacemakers and resynchronization devices, defibrillators, intra-aortic capsule counterpulsators and left ventricular assist devices.

In a preferred embodiment, the heart failure therapy is a pharmaceutical heart failure therapy. Thus, heart failure therapy preferably encompasses administration of one or more drugs. The term "administration" as used herein is used in the broadest sense and includes oral, enteral, topical and "parenteral administration", among others. As used herein, "parenteral administration" and "parenterally administered" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal injection and infusion. In embodiments, the medicament is administered orally.

Drugs suitable for the treatment of Heart failure are well known in the art, see for example Heart Disease, 2008, eighth edition, edited by Braunwald, Elsevier Sounders, chapter 24 or ESC Guidelines for the diagnosis and treatment of acute and chronic Heart failure (European Heart Journal (2008) 29, 2388) 2442. preferably, Heart failure treatment comprises administration of at least one drug selected from the group consisting of angiotensin converting enzyme inhibitors (ACE inhibitors), angiotensin II receptor blockers (often also referred to as angiotensin II receptor antagonists), β adrenergic blockers (also referred to herein as β blockers), diuretics, aldosterone antagonists, adrenergic agonists, positive inotropic drugs, calcium antagonists, hydralazine, nitrates and aspirin.

Preferred ACE inhibitors include benazepril, captopril, cilazapril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, and trandolapril. A particularly preferred inhibitor is enalapril.

Preferred β blockers include acebutolol (cebutolol), alprenolol, atenolol, betaxolol, bisoprolol, blanalol, caraprolol, carteolol, carvedilol, celiprolol, metipranolol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, tarlinolol (tanilol), and timolol.A particularly preferred β blocker is atenolol, bisoprolol, carvedilol, or metoprolol.

Preferred angiotensin II receptor antagonists are losartan, valsartan, irbesartan, candesartan, telmisartan, and eprosartan. Particularly preferred antagonists are losartan or valsartan.

Preferred diuretics are loop diuretics, thiazine and thiazine-like diuretics, potassium sparing diuretics, mineralocorticoid receptor antagonists and vasopressin antagonists.

Preferred aldosterone antagonists are Eplerone, spironolactone, canrenone, Mexrenone, Prorenone; and statins (statins), in particular atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin. Particularly preferred antagonists are spironolactones.

Preferred inotropic agents are digoxin and digitoxin.

Preferred calcium antagonists are dihydropyridines, verapamil and diltiazem.

Preferred adrenergic agonists are dobutamine, dopamine, epinephrine, isoproterenol, norepinephrine, and phenylephrine.

In a preferred embodiment, however, the heart failure therapy comprises administration of at least one drug as described above, in an even more preferred embodiment, the heart failure therapy comprises administration of at least one drug selected from the group consisting of angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, β blockers, diuretics, and aldosterone antagonists.

According to the method of the present invention, it should be assessed whether the heart failure treatment of the patient to be tested should be intensive. Preferably, the intensive heart failure treatment comprises at least one of:

increasing the dose of the one or more drugs previously administered,

administering the additional or another drug(s), in particular administering the additional drug(s) having a different mode of action than the previously administered drug,

device therapy, in particular with pacemaker devices, Cardiac Resynchronization Therapy (CRT), implantable defibrillator devices (ICD) or Left Ventricular Assist Devices (LVAD), lifestyle changes, and

a combination thereof.

Preferably, potentiation comprises increasing the dose of one or more drugs previously administered, in particular increasing the dose of a drug selected from the group consisting of diuretics, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, aldosterone antagonists, and β blockers.

Also preferably, the fortification comprises administration of another drug(s), in particular, administration of another drug(s) having a different mode of action than the previously administered drug(s), or application of another device (i.e. application of a drug/device that was not administered/used before carrying out the method of the invention). Preferred additional agents include hydralazine, nitrates, inotropic agents (inotropic agents) and adrenergic agents. Preferred devices include pacemaker devices, Cardiac Resynchronization Therapy (CRT) and implantable defibrillator devices (ICD).

Moreover, the intensive heart failure treatment may further include monitoring the patient at short time intervals. Thus, by performing the methods of the invention, patients in need of closer monitoring, particularly with respect to heart failure therapy (and therefore closer observation), can be identified. By "more closely monitoring" is preferably meant that the level of the marker as referred to herein in connection with the method of the invention is measured in at least one further sample obtained from the patient after a short time interval after the sample referred to in step a) of the method of the invention. Preferred short time intervals are mentioned below.

Patients who do not require intensive heart failure treatment preferably can continue heart failure treatment without changing the treatment regimen. Thus, there is no need to modify the dose of the administered drug and/or change the drug.

The term "sample" refers to a sample of bodily fluid, refers to a sample of isolated cells or a sample from a tissue or organ. Samples of body fluids may be obtained by well-known techniques and include samples of blood, plasma, serum, urine, lymph, saliva, ascites, bronchial lavage or any other bodily secretion or derivative thereof. Tissue or organ samples may be obtained from any tissue or organ by, for example, biopsy. Isolated cells may be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. For example, a cell-, tissue-, or organ sample can be obtained from those cells, tissues, or organs that express or produce a biomarker. The sample may be frozen, fresh, fixed (e.g., formalin fixed), centrifuged, and/or embedded (e.g., paraffin embedded), and the like. The cell sample may, of course, be subjected to a variety of well-known post-collection preparation and storage techniques (e.g., nucleic acid and/or protein extraction, immobilization, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of marker in the sample. Likewise, biopsies can also undergo post-collection preparation and storage techniques, such as fixation.

In one embodiment, the sample is a blood, serum or, in particular, plasma sample.

The sample may be obtained from the patient at least one month, at least six months, or at least 12 months (in increasing order of preference) after initiation of heart failure therapy, in particular BNP-type peptide-guided therapy. Preferably, the therapy is a pharmaceutical heart failure therapy.

The level of a biomarker as referred to herein may be determined in the same sample from the patient or in different samples (i.e. in two or three different samples).

The term "measuring" a level of a marker as referred to herein designates an amount of the biomarker, e.g., to determine the level of the biomarker in a sample, using a suitable detection method as described elsewhere herein. In one embodiment, the level of at least one biomarker is measured by: contacting the sample with a detection reagent that specifically binds to each label, thereby forming a complex between the reagent and the label, detecting the level of the complex formed, and thereby measuring the level of the label. If the biomarker is uric acid, the level of said biomarker may be measured by contacting the sample with a detection reagent, in particular an enzyme or a compound, which allows the biomarker to be converted, for example allows uric acid to be oxidized. The enzyme may be uricase (EC 1.7.3.3) which catalyzes the oxidation of uric acid to 5-hydroxyisocuric acid (5-hydroxyisocourate). Furthermore, the enzyme may be a peroxidase. The compound may be phosphotungstic acid. If the label is urea, the detection reagent may be urease. If the label is glucose, the detection reagent may be hexokinase. If the label is creatinine, the detection reagent may be picric acid (which forms a complex with creatinine). The level of picric acid and creatinine complex can be measured.

The term "growth differentiation factor-15" or "GDF-15" relates to polypeptides that are members of the Transforming Growth Factor (TGF) cytokine superfamily. The terms polypeptide, peptide, and protein are used interchangeably throughout this specification. GDF-15 was originally cloned as macrophage-inhibitory cytokine 1 and was later identified as placental transforming growth factor-15, placental bone morphogenetic protein, NSAIAs, 1997 Biochim Biophys Acta 1354:40-44, Lawton 1997, Gene 203:17-26, Yokoyama-Kobayashi 1997, J Biochem (Tokyo), 122: 622-. Similar to other TGF-related cytokines, GDF-15 is synthesized as an inactive precursor protein that undergoes disulfide-linked homodimerization. After proteolytic cleavage of the N-terminal leader, GDF-15 is secreted as a dimeric protein of-28 kDa (Bauskin 2000, Embo J19: 2212-2220). The amino acid sequence of GDF-15 is disclosed in WO99/06445, WO00/70051, WO2005/113585, Bottner 1999, Gene 237: 105-. GDF-15 as used herein also encompasses variants of the specific GDF-15 polypeptides mentioned above. Such variants have at least the same basic biological and immunological properties as the particular GDF-15 polypeptide. In particular, they share the same basic biological and immunological properties if they can be detected by the same specific assays indicated in the present specification (e.g. by ELISA assays using polyclonal or monoclonal antibodies that specifically recognize the GDF-15 polypeptide). Preferred assays are described in the accompanying examples. Furthermore, it is to be understood that variants indicated according to the present invention should have different amino acid sequences, which differ due to at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of the variant also preferably has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98% or at least about 99% identity to a specific GDF-15 polypeptide amino acid sequence, preferably to a human GDF-15 amino acid sequence, more preferably over the entire length of a specific GDF-15, e.g., human GDF-15. The degree of identity between two amino acid sequences can be determined as described above. The variants referred to above may be allelic variants or specific homologues, orthologues or orthologues of any other species. Furthermore, variants referred to herein include fragments of the particular GDF-15 polypeptide or type of variant referred to above, provided that such fragments have the basic immunological and biological properties referred to above. Such fragments may be, for example, degradation products of a GDF-15 polypeptide. Further included are variants that differ due to post-translational modifications such as phosphorylation or myristylation.

The insulin-like growth factor binding protein (IGFBP) system plays an important role in cell growth and differentiation. It comprises two ligands, IGF-I and IGF-II, two receptor type 1 and type 2 IGF receptors, and is known as 1995 six IGF-binding proteins (IGFBPs), IGFBPs-1 to-6 (Jones, j.i., et al, endocr.rev. 16 (1995) 3-34). Recently, the IGFBP family has been expanded to include IGFBP-related proteins (IGFBP-rPs), which have significant structural similarity to IGFBP (Hwa, V., et al, endocr. Rev 20 (1999) 761-787). Thus, the IGFBP superfamily includes six conventional IGFBPs that have high affinity for IGFs, and at least 10 IGFBP-rP that not only have the conserved amino-terminal domains of IGFBPs but also show some degree of affinity for IGFs and insulin. IGFBP-rP is a group of cysteine-rich proteins that control various cellular functions such as cell growth, cell adhesion and migration, and extracellular matrix synthesis. In addition, these proteins may be involved in biological processes such as tissue proliferation and differentiation, regeneration, angiogenesis, injury repair, inflammation, fibrosis and tumorigenesis (Hwa, V., et al, Endocr. Rev 20 (1999) 761-787).

IGF binding protein 7 (= IGFBP 7) is a modular glycoprotein of 30-kDa 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 the literature, this molecule has also been named FSTL 2; IBP 7; IGF-binding protein-related protein I; IGFBP 7; IGFBP7 v; IGFBP rPl; IGFBP 7; IGFBPRP 1; insulin-like growth factor binding protein 7; insulin-like growth factor binding protein 7 precursor; a MAC 25; MAC25 protein; PGI2 stimulating factor; and PSF or prostacyclin stimulating factor. Northern blot studies revealed extensive expression of the gene in human tissues including heart, brain, placenta, liver, skeletal muscle and pancreas (Oh, Y., et al, J. biol. chem.271 (1996) 30322-30325).

IGFBP7 was originally identified as a gene differentially expressed in normal leptomeningeal and mammary epithelial cells compared to their corresponding tumor cells, and was named meningioma-associated cDNA (MAC25) (Burger, a.m., et al, Oncogene16 (1998) 2459-. The expressed proteins were independently purified as tumor-derived adhesion factor (later renamed angiomodulin) (Sprenger, C.C., et al, Cancer Res 59 (1999) 2370-. It has also been reported as T1Al2, a gene that is down-regulated in breast cancer (StCroix, B., et Al, Science 289 (2000) 1197-1202).

Differential expression of IGFBP7 mRNA was measured in patients with different diseases including heart disease, kidney disease, inflammatory disease (US 6,709,855 by scios inc.) and vascular graft disease (US 2006/0,003,338).

A number of different assays have been described and used to test the hormone binding properties of IGFBP 7. Low affinity IGF binding was analyzed by a competitive affinity cross-linking assay. The recombinant human mac25 protein specifically binds IGF-I and-II (Oh, Y., et al, J. biol. chem.271 (1996) 20322. sup. 20325; Kim, H.S., et al, Proc. Natl. Acad. Sci. USA 94 (1997) 12981. sup. 12986.). IGFBP activity can also be detected by measuring the ability of the protein to bind radiolabeled IGF in Western ligand blots.

Preferably, the term "IGFBP 7" refers to human IGFBP 7. The sequence of the protein is well known in the art and is accessible, for example, via GenBank (NP _ 001240764.1). IGFBP7 as used herein preferably also includes variants of a particular IGFBP7 polypeptide. For the interpretation of the term "variant" please see above.

Immunoassay cycling IGFBP7 was performed recently. Low levels of this analyte were detected in random human sera and increased serum levels were found to be associated with insulin resistance (Lopez-Bermejo, a., et al, j. clinical endoscopy and Metabolism 88 (2003) 3401-.

Endothelin, a marker, is well known in the art, it was originally isolated from murine vascular endotheliomas (O' Reilly, m.s. et al, Cell 88 (1997) 277 285) collagen, a family of extracellular matrix proteins with a characteristic triple helix conformation that forms supramolecular aggregates that play a dominant role in maintaining tissue structural integrity, excessive collagen deposition leads to fibrosis that disrupts normal function of surrounding tissues, collagen XVIII is a member of the Multiplexin family of collagens primarily in the basement membrane, with multiple discontinuities in the central triple helix domain and a distinct non-triple helix domain at the C-terminus, the sequence of a short isoform of the human α -1 type chain of collagen XVIII (SwissProt: P39060) is disclosed, for example, in WO2010/124821, the entire disclosure of which is incorporated herein by reference.

Endostatin is released from the α 1 chain of collagen XVIII by the action of various proteolytic enzymes (see Ortega, N. and Werb, Z., Journal of Cell Science 115 (2002) 4201-4214-the complete disclosure of which is incorporated herein by reference.) endostatin as used herein is represented by the fragment of collagen XVIII spanning from amino acid position 1337 to amino acid position 1519 of collagen XVIII as disclosed in WO2010/124821 the hinge region at the C-terminus of the α chain of collagen XVIII contains several protease sensitive sites, and many enzymes including neutrophil elastase, cathepsin and matrix metalloproteinase are known to produce endostatin by cleaving the collagen chain in this region.

Osteopontin (OPN), also known as bone sialoprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP 1), 2ar, and rickettsial resistance (Ric), are polypeptides that are highly negatively charged, extracellular matrix proteins that lack extensive secondary structure. It consists of about 300 amino acids (297 in mouse, 314 in human) and is expressed as a 33-kDa nascent protein; there are also functionally important cleavage sites. OPN can undergo post-translational modifications, which increase its apparent molecular weight to about 44 kDa. The sequence of osteopontin is well known in the art (human osteopontin: UniProt P10451, GenBank NP-000573.1), which is found in normal plasma, urine, milk and bile (US 6,414,219; US 5,695,761; Denhardt, D.T. and Guo, X., FASEB J. 7 (1993) 1475-1482; Oldberg, A., et al, PNAS 83 (1986) 8819-8823; Oldberg, A., et al, J.biol.chem.263 (1988) 33-19436; Giachelli, CM., et al, Trends Cardiovasc.Med.5 (1995) 88-95). Human OPN proteins and cDNAs have been isolated and sequenced (Kiefer M. C, et al, Nucl. acids Res.17 (1989) 3306). OPN plays a role in cell adhesion, chemotaxis, macrophage directed interleukin-10. OPN is known to interact with several integrin receptors. Increased OPN expression has been reported in several human cancers, and its cognate receptors have been identified (av-b 3, av-b5, and av-bl integrins and CD 44). In vitro studies by Irby, r.b., et al, clin. exp. metastasis 21 (2004) 515-523, indicated that both endogenous OPN expression (by stable transfection) and exogenous OPN (added to the culture medium) increased the mobility and invasiveness of human colon cancer cells in vitro.

Endostatin is a potent inhibitor of angiogenesis and vascular growth. The relationship between endothelin and the cytokine network has not been elucidated, but endothelin is known to alter the expression of a wide range of genes (Abdollahi, A. et al, MoI. cell 13 (2004) 649-.

Endothelin as used herein preferably also includes variants of a particular endothelin polypeptide. For the interpretation of the term "variant" please see above.

Mimecan is a small proteoglycan with leucine rich repeats and a precursor comprising 298 amino acids. Other names of mimecan are OGN, bone glycine, OG, OIF, SLRR 3A.

Mimecan is a member of the secreted small leucine-rich proteoglycan (SLRP) family with structurally related core proteins. All SLRPs share a common feature of tandem leucine-rich repeat (LRR) units in the C-terminal half of the core protein. However, in the N-terminal region, each class of SLRPs has a unique domain called LRR N-domain that contains cysteine clusters with conserved spacing. Class III SLRPs contain six carboxyl LRRs and include mimecan, epiphysin and optically active proteins (opticin).

Functional studies from mouse knockouts of class I and II members such as decorin, biglycan, lumecan and fibromodulin show that SLRPs lack a broad spectrum of defects that display a contribution to abnormal collagen fibrillogenesis, suggesting that these SLRPs play an important role in the establishment and maintenance of the collagen matrix (Ameye, l. and Young, m.f., Glycobiology12 (2002) 107R-116R). The absence of class III mimecan also causes abnormalities in collagen fibrils (Tasheva, E.S. et al, MoI.Vis. 8 (2002) 407-.

mimecan is a multifunctional component of the extracellular matrix. It binds to a variety of other proteins (IGF2, IKBK, IFNBl, INSR, CHUK, IKBKB, NFKBIA, ILl 5, Cd3, retinoic acid, APP, TNF, lipopolysaccharide, c-abl oncogene1, receptor tyrosine kinase, v-src sarcoma virus oncogene). These different binding activities can lead to the ability of mimecan to perform different functions in many tissues.

mimecan has been found to have altered expression patterns in cornea, bone, skin and other tissues in different case situations despite the increasing amount of data on the biological role of mimecan, its function is still unclear mimecan has been shown to be involved in the regulation of collagen fibrillogenesis, an essential process in development, tissue repair and metastasis (tashieva et al, moi. vis. 8 (2002) 407. cndot. 415.) it plays a role in bone formation associated with TGF- β -1 or TGF- β -2.

The sequence of human mimecan polypeptides is well known in the art and can be evaluated, for example, via GenBank accession number NP-054776.1 GI: 7661704. Further, the sequence is disclosed in WO 2011/012268. As used herein, mimecan preferably also includes variants of a particular mimecan polypeptide. For the interpretation of the term "variant" please see above. In the context of the present invention, mimecan is preferably determined as described in WO 2011/012268.

The term "soluble Flt-1" or "sFlt-1" as used herein refers to a polypeptide which is a soluble form of the VEGF receptor Flt 1. It was identified in conditioned medium of human umbilical vein endothelial cells. The endogenous soluble Flt1(sFlt1) receptor is chromatographically and immunologically similar to recombinant human sFlt1 and binds [125I ] VEGF with fairly high affinity. Human sFlt1 was shown to form a VEGF-stable complex in vitro with the extracellular domain of KDR/Flk-1. Preferably, sFlt1 refers to human sFlt 1. More preferably, human sFlt1 can be deduced from the amino acid sequence of Flt-1 shown in Genbank accession number P17948, GI: 125361. The amino acid sequence of mouse sFlt-1 is shown in Genbank accession No. BAA24499.1, GI: 2809071.

The term "sFlt-1" as used herein also encompasses variants of the specific sFlt-1 polypeptides mentioned above. Such variants have at least the same basic biological and immunological properties as the particular sFlt-1 polypeptide. In particular, they share the same basic biological and immunological properties if they can be detected by the same specific assay indicated in the present specification (e.g., by an ELISA assay using a polyclonal or monoclonal antibody that specifically recognizes the sFlt-1 polypeptide). For a more detailed explanation of the term "variant" please see above.

Galectin-3 (Gal-3) is a structurally distinct member of the β galactoside binding lectin family, expression of galectin-3 has been associated with epithelial and inflammatory cells including macrophages, neutrophils and mast cells galectins galectin-3 has been implicated in a number of biological processes important in heart failure including myofibroblast proliferation, fibrogenesis, tissue repair, cardiac remodeling and inflammation galectin-3 is about 30 and, like all galectins, contains a carbohydrate recognition binding domain (CRD) of about 130 amino acids capable of specifically binding β -galactosides, galectin-3 is encoded by a single gene ls3, which contains an N-terminal domain of a tandem repeat with a short amino acid segment (total of 110 amino acids) linked to a single C-terminal CRD of about 130 amino acids, expressed in nuclei, ay, lines, cell surfaces and extracellular spaces, which has been shown to be involved in cell failure, cell activation and adhesion failure (chemotaxis and cell growth and apoptosis, elevated levels of cell growth and extracellular space (see e.g. dietary supplement of heart failure, acute and acute phase R2009).

The protein sequence of galectin-3 is well known in the art, see, for example, uniprot accession No. P17931 (version 5, 2008/11/25), GenBank accession No. NP _002297.2 NM _ 002306.3.

ST2 is a member of the IL-1 receptor family, which is produced by cardiac fibroblasts and cardiomyocytes under mechanical stress conditions. ST2 is an interleukin-1 receptor family member and exists in both membrane-bound and soluble isoforms (sST 2). In the context of the present invention, the amount of soluble ST2 should be determined (see diepilger et al (clinical biochemistry, 43, 2010:1169 to 1170). ST2 is also known as interleukin 1 receptor-like 1 or IL1RL1, which is encoded by the IL1RL1 gene in humans the sequence of the human ST2 polypeptide is well known in the art and can be obtained, for example, by GenBank, see NP _003847.2 GI:27894328 soluble ST2 (sST 2) is believed to act as a decoy receptor by binding IL-33 and to abrogate the additional cardioprotective effect of IL-33 signaling by the cell membrane bound form of ST 2.

CRP, also referred to herein as C-reactive protein, is an acute phase protein that was discovered more than 75 years ago as a blood protein that binds the C-polysaccharide of pneumococci (pneumococci). CRP is known as a marker of reactive inflammation and is produced by remote organs (i.e., the liver) in response to or in response to chemokines or interleukins originating from major sites of pathology. CRP consists of five signal subunits, which are non-covalently linked and assembled as a circular pentamer with a molecular weight of approximately 110-140 kDa. Preferably, CRP as used herein relates to human CRP. The sequences of human CRP are well known and are disclosed, for example, in Woo et al (J.biol.chem.1985.260 (24), 13384-13388). CRP levels are usually lower in normal individuals, but may be increased by 100-fold or more due to inflammation, infection or injury (Yeh (2004) circulation.2004; 109: II-11-II-14). CRP is known to be an independent factor for cardiovascular risk prediction. In particular, CRP has been shown to be suitable as predictor for myocardial infarction, stroke, peripheral arterial disease and sudden cardiac death. In addition, elevated CRP levels can also predict recurrent ischemia and death in patients with Acute Coronary Syndrome (ACS) and patients undergoing coronary intervention. The determination of CRP in patients at risk for coronary Heart disease is recommended by the expert group (e.g., by the American Heart Association) (see also Pearson et al (2003) Markers of Inflammation and cardiovascular disease circulation, 107: 499-. The term CRP also relates to variants thereof.

Preferably, the amount of CRP in the patient sample is determined by using a CRP assay with high sensitivity. Determination of CRP by such an assay is also often referred to as high-sensitive CRP (hscrp). hsCRP assays, for example, are used to predict risk of heart disease. Suitable hsCRP assays are known in the art. A particularly preferred hscRP assay in the context of the present invention is the Roche/Hitachi CRP (Latex) HS test with a detection limit of 0.1 mg/l.

Interleukin 6 (abbreviated IL-6) is an interleukin secreted by T cells and macrophages to stimulate an immune response, for example during infection and after trauma, especially burns or other tissue injury leading to inflammation, it functions as a pro-inflammatory cytokine and an anti-inflammatory cytokine.in humans, it is encoded by the IL6 gene the sequence of human IL-6 can be evaluated via GenBank (polynucleotide sequence see NM-000600.3 and amino acid sequence see NP-000591.1). IL-6 signals via a cell surface type I cytokine receptor complex consisting of the ligand-binding IL-6R α chain (CD126) and the signal transduction component gp130 (also called CD 130). CD130 is a common signal transducer of several cytokines including Leukemia Inhibitory Factor (LIF), ciliary neurokine, oncostatin M, IL-11 and cardiotrophin-1 and is almost widely expressed in most tissues.conversely, the expression of CD126 is restricted to certain tissues because of the interaction of IL-6 with its receptors, thus initiating transcription of the protein kinase and protein kinase leads to the intracellular signal transduction complexes of the protein kinase and JAK receptor kinase activation of these JAK-6 receptor complexes.

The marker cystatin C is well known in the art. Cystatin C is encoded by the CST3 gene and is produced by all nucleated cells at a constant rate, and the rate of production in humans is significantly constant throughout life. Almost complete removal from circulation by glomerular filtration. For this reason, the serum concentration of cystatin C is independent of muscle mass and sex in the age range of 1-50 years. Therefore, cystatin C in plasma and serum has been proposed as a more sensitive marker of GFR. The sequence of the human cystatin C polypeptide can be evaluated by Genbank (see, e.g., accession number NP _ 000090.1). Biomarkers can be measured by particle-enhanced immunoturbidimetry assays. Human cystatin C was agglutinated with latex particles coated with anti-cystatin C antibody. The aggregates were subjected to turbidimetry.

The marker prealbumin is well known to the skilled person. It is a tryptophan-rich protein which is synthesized in hepatocytes and has a molar mass of 55000 daltons. Due to its greater rate of dispersion towards the anode, an electrophoretic band appeared < 2.5% relative amount before albumin at pH 8.6. Its function is to bind and transport low molecular weight retinol binding proteins (molar mass less than 21000 daltons), preventing their glomerular filtration. 30-50% of the circulating prealbumin forms a complex with retinol binding protein. In addition, it binds and transports thyroxine (T4), although its affinity for this hormone is less than that of thyroid-bound globulin. The sequence of the human prealbumin polypeptide may be assessed by Genbank (see, e.g., accession number NP _ 000362.1). Various methods are available for assaying prealbumin, such as Radioimmunodiffusion (RID), nephelometry and turbidimetry.

The marker "creatinine" is well known in the art, creatinine is endogenously synthesized from creatine and phosphocreatine in muscle metabolism, creatinine is excreted by glomeruli under normal renal function conditions, creatinine assays are performed for diagnosis and monitoring of acute and chronic renal disease and for monitoring of renal dialysis, creatinine concentration in urine is used as a reference for excretion of certain analytes (albumin, α -amylase). creatinine can be determined as described by Popper et al, (Popper H et al Biochem Z1937; 291:354), Seelig and W ü st (Seelig HP, W ü st H, Ärztl Labor 1969;15:34) or Bartels (Bartels H et al Clin Chim Acta 1972;37: Acta). for example, sodium hydroxide and picric acid can be added to a sample to initiate the formation of creatinine-picric acid complexes.

Uric acid is the end product of purine metabolism in the host organism. The IUPAC name is 7, 9-dihydro-3H-purine-2, 6, 8-trione. The compounds are often referred to as uric acid (urate), uric acid (lithoic acid), 2,6, 8-trioxypurine, 2,6, 8-trihydroxypurine, 2,6, 8-trioxypurine, 1H-purine-2, 6, 8-triol (formula C)₅H₄N₄O₃PubChem CID1175, CAS number 69-93-2).

Uric acid measurements are used to diagnose and treat a variety of renal and metabolic disorders, including renal failure, gout, leukemia, psoriasis, hunger or other wasting conditions, and patients receiving cytotoxic drugs. The oxidation of uric acid provides the basis for two methods for quantitatively determining this purine metabolism. One method is to reduce phosphotungstic acid to tungsten blue in an alkaline solution, which is measured by photometric measurements. The second method is described by Praetorius and Poulson, which uses the enzyme uricase to oxidize uric acid; the method eliminates the interference inherent to chemical oxidation (Praedius E, Poulsen H. Enzymatic Determination of Uricacid with defined orientations.Scandinav J Clin Lab interrogation 1953: 273-. Uricase may be used in methods involving UV measurement of uric acid consumption or in combination with other enzymes to provide a colorimetric assay. Another method is a colorimetric method developed by Town et al (Town MH, Gehm S, Hammer B, Ziegenhorn J. J ClinChem Clin Biochem 1985;23: 591). The sample is initially incubated with a reagent mixture containing ascorbate oxidase and a transparent system. It is important in this test system that any ascorbic acid present in the sample is eliminated in the initial reaction; this excludes any ascorbic acid interfering with the subsequent POD indicator reaction. After addition of the initial reagents, the oxidation of uric acid by uricase is started.

In the context of the present invention, uric acid may be determined by any method deemed suitable. Preferably, the biomarkers are determined by the methods mentioned above. More preferably, uric acid is determined by applying a slightly modified colorimetric method as described above. In this reaction, a peroxide is reacted in the presence of Peroxidase (POD), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS) and 4-aminopyrine to form a quinone diimine dye. The intensity of the red color formed is directly proportional to the uric acid concentration and is determined photometrically.

Urea is the major end product of protein nitrogen metabolism. Which has the chemical formula CO (NH)₂)₂And is synthesized from ammonia produced by deamination of amino acids through the urea cycle in the liver. Urea is mostly excreted through the kidneys, but small amounts are also excreted through sweat and degraded in the gut by bacterial action. The determination of blood urea nitrogen is the most widely used renal function screening test. Urea can be measured by an in vitro test that quantitatively determines urea/urea nitrogen in human serum, plasma and urine on the Roche/Hitachi cobas c system. The test was automated using different analyzers including cobas c311 and cobas c 501/502. The assay is a kinetic assay using urease and glutamate dehydrogenase. Urea is hydrolyzed by urease to form ammonium and carbonate salts. In a second reaction, 2-ketoglutarate reacts with ammonium in the presence of glutamate dehydrogenase (GLDH) and coenzyme NADH to produce L-glutamate. In this reaction, for each mole of urea hydrolyzed, 2 moles of NADH are oxidized to NAD⁺. The rate of decrease in NADH concentration is directly proportional to the urea concentration in the sample and is measured photometrically.

The marker glucose is well known in the art. As used herein, a label preferably refers to D-glucose. The level of the marker can be determined by well-known methods. For example, the marker may be phosphorylated to D-glucose-6-phosphate in the presence of the enzymes Hexokinase (HK) and adenosine 5 '-triphosphate (ATP), with the formation of adenosine 5' -diphosphate (ATP). In the presence of the enzyme glucose-6-phosphate dehydrogenase, D-glucose-6-phosphate is oxidized to D-gluconic acid phosphate by NADP, and reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is formed. The amount of NADPH formed in this reaction is stoichiometric with the amount of D-glucose. NADPH can be measured by light absorption.

The marker sodium is well known in the art. Sodium is the major extracellular cation and functions to maintain fluid distribution and osmotic pressure. Some causes of reduced sodium levels include prolonged vomiting or diarrhea, reduced reabsorption in the kidneys, and excessive fluid retention. Common causes of sodium increase include excessive fluid loss, high salt intake, and increased renal reabsorption. The level of the marker can be determined by applying an Ion Selective Electrode (ISE) that employs the unique properties of certain membrane materials to generate an electrical potential (electromotive force, EMF) for measuring ions in solution. The electrode has a selective membrane in contact with the test solution and the internal perfusion solution. The internal perfusion solution contains a fixed concentration of the test ion. Due to the specific nature of the membrane, the test ions will be intimately associated with the membrane on each side. The membrane EMF is determined by testing the difference in ion concentration in the test solution and the internal perfusion solution. EMF occurs according to the Nernst equation for a particular ion in solution.

The marker hemoglobin (Hb) is well known in the art. Hemoglobin comprises four protein subunits (each containing a heme moiety) and is a red-pigmented protein located in red blood cells. Its main function is to transport oxygen and carbon dioxide in the blood. Each Hb molecule is capable of binding four oxygen molecules. Hb consists of various subfractions and derivatives. The term "hemoglobin" as used herein preferably refers to total hemoglobin. Hemoglobin levels can be measured by well-known methods, for example, by hemoglobin from potassium (III) ferrocyanide (Fe)²⁺Fe³⁺) Oxidized to methemoglobin. Hemoglobin levels are proportional to color intensity and can be measured, for example, at a wavelength of 567 nm and at 37 ℃. The hemoglobin level may also beMeasured by contacting the sample with an antibody that specifically binds hemoglobin.

The marker HbA1c (glycated hemoglobin, glycohemoglobin) is well known in the art HbA1c is one of the glycated hemoglobin, linked to a subfraction formed by Hb molecules by various sugars HbA1c is formed in two steps by non-enzymatic reaction of glucose with the N-terminal amino group of the β chain of normal adult Hb (HbA). The first step is reversible and produces unstable HbA1c. this rearranges in the second reaction step to form stable HbA 1c. in red blood cells, the relative amount of HbA converted to stable HbA1c increases with the average concentration of glucose in the blood. the conversion to stable HbA1c is limited by the red cell lifetime of about 100-120 days.

Hematocrit (Ht or HCT), also known as hematocrit (PCV) or red blood cell volume fraction (EVF), is the volume percentage (%) of red blood cells in blood. As used, the term "hematocrit" preferably refers to the percentage of packed red blood cells in a volume of whole blood. Hematocrit can be determined by centrifuging heparinized blood in a capillary tube (also known as a microcytometer tube) for five minutes at 10,000 RPM. This stratifies the blood. The packed red blood cell volume divided by the total volume of the blood sample yields the PCV. Since a cuvette is used, this can be calculated by measuring the length of the layer. With modern laboratory devices, hematocrit is calculated by an automated analyzer and is not measured directly. It is determined by multiplying the red blood cell count by the average cell volume. The hematocrit is somewhat more accurate because the PCV includes a small amount of plasma trapped between the red cells. The estimated hematocrit as a percentage may be derived by multiplying the hemoglobin concentration in g/dL by three and removing units.

The term "QRS duration" is well known in the art. QRS duration is a standard measure in medicine and describes the duration of the QRS group on a surface Electrocardiogram (ECG), which indicates the duration of ventricular electrical excitation. Preferably, the QRS duration is measured by an ECG device.

The biomarkers referred to herein may be detected using methods generally known in the art. Detection methods generally encompass methods of quantifying the level of a biomarker in a sample (quantification methods). The following methods, which are suitable for the qualitative and/or quantitative detection of biomarkers, are generally known to the person skilled in the art. Samples can be conveniently assayed, for example using western blots and immunoassays for proteins, such as ELISA, RIA, fluorescence based immunoassays, which are commercially available. Further suitable methods of detecting biomarkers include measuring physical or chemical properties specific to the peptide or polypeptide, such as its exact molecular weight or NMR spectrum. The methods include, for example, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass spectrometers, NMR-analyzers, or chromatographic devices. Further, methods include microplate ELISA based methods, fully automated or robotic immunoassays (e.g., as available in Elecsys)^TMObtained from an analyzer), CBA (enzyme cobalt binding assay, e.g.as can be found in Roche-Hitachi^TMObtained on an analyzer) and latex agglutination assays (e.g., as may be found in Roche-Hitachi^TMObtained by an analyzer).

For biomarker protein detection as referred to herein, a wide range of immunoassay techniques using such assay formats are available, see, e.g., U.S. Pat. nos. 4,016,043, 4,424,279, and 4,018,653. These include single and double-site or "sandwich" assays of the non-competitive type, as well as traditional competitive binding assays. These assays may include direct binding of labeled antibodies to the target biomarkers.

Of these, the sandwich assay is the most useful and commonly used immunoassay.

Methods for measuring electrochemiluminescence phenomena are well known. Such methods use the ability of specific metal complexes to achieve excited states from which they decay from a ground state to emit electrochemiluminescence by oxidation. For a review see Richter, m.m., chem.rev. 104 (2004) 3003-3036.

Biomarkers can also be detected by commonly known methods including magnetic resonance spectroscopy (NMR spectroscopy), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), high and ultra-HPLC, such as reverse phase HPLC, e.g., ion-pairing HPLC with dual UV wavelength detection, capillary electrophoresis with laser induced fluorescence detection, anion exchange chromatography and fluorescence detection, thin layer chromatography.

Preferably, measuring the biomarker levels 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 said peptide or polypeptide for a sufficient time, (b) measuring the cellular response. For measuring cellular responses, the sample or treated sample is preferably added to a cell culture and either internal or external cellular responses are measured. The cellular response may include expression of a measurable reporter gene or secretion of a substance, such as a peptide, polypeptide, or small molecule. The expression or substance should generate an intensity signal which correlates with the level of the peptide or polypeptide.

Also preferably, measuring the level of the 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 specific for the peptide or polypeptide observed in the m/z variation, which is observed in a mass spectrum or NMR spectrum specific for the peptide or polypeptide.

Measuring the level of the peptide or polypeptide preferably comprises the steps of (a) contacting the peptide with a specific binding agent, (b) (optionally) removing unbound binding agent, (c) measuring the level of bound binding agent, i.e. the complex of 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 described herein. According to some embodiments, the steps may be performed by a single analyzer unit of the system or by more than one analyzer unit in operable communication with each other. For example, according to certain embodiments, the systems disclosed herein may include a first analyzer unit for performing the steps of contacting and removing, and a second analyzer unit operatively connected with the first analyzer unit via a transfer unit (e.g., robotic arm), the second analyzer unit performing the steps of measuring.

The bound binding agent, i.e. the binding agent or 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 invention may be any compound, such as a peptide, polypeptide, nucleic acid, or small molecule, which binds to a peptide or polypeptide described herein. Preferred binding agents include antibodies, nucleic acids, peptides or polypeptides, such as receptors or binding partners for peptides or polypeptides and fragments thereof comprising the peptide binding domain, and aptamers, e.g., nucleic acid or peptide aptamers. Methods of making such binders are well known in the art. For example, the identification and production of suitable antibodies or aptamers is also provided by commercial suppliers. The person skilled in the art is familiar with methods for developing derivatives of such binding agents with higher affinity or specificity. For example, random mutations can be introduced into a nucleic acid, peptide, or polypeptide. These derivatives can then be tested for binding according to screening procedures known in the art, such as 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, which are capable of binding to an antigen or hapten. The invention also includes single chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody that exhibits the desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence will typically include at least the antigen binding amino acid residues of the donor, but may also include other structurally and/or functionally relevant amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. Preferably, the binding agent or agents specifically bind to the peptide or polypeptide. Specific binding according to the present invention means that the ligand or agent should not substantially bind to (be "cross-reactive with") another peptide, polypeptide or substance present in the sample being analyzed. Preferably, the specific binding peptide or polypeptide should bind with at least 3-fold higher affinity than any other related peptide or polypeptide, more preferably at least 10-fold higher affinity and even more preferably at least 50-fold higher affinity. Non-specific binding may be tolerable if it can still be clearly distinguished and measured, for example, by 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, the method is semi-quantitative or quantitative. Further suitable techniques for determining the polypeptide or peptide are described below.

Binding of the binding agent can be measured directly by e.g. NMR or surface plasmon resonance the measurement of binding of the binding agent, according to preferred embodiments, is performed by the analyzer unit of the system disclosed herein, thereafter, the level of measured binding can be calculated by the computer device of the system disclosed herein, if the binding agent is also provided as a substance of the enzymatic activity of the peptide or polypeptide of interest, the enzyme reaction product can be measured (e.g., the level of protease is measured by measuring the level of cleaved substrate on a western blot), optionally, the binding agent can itself exhibit enzymatic properties, and the "binding agent/peptide or polypeptide" complex or binding agent bound respectively by the peptide or polypeptide can be contacted with a suitable substrate allowing detection by intensity signal generation, the substrate can also be labeled with a detectable label prior to the measurement of the enzyme reaction product, preferably a detectable label prior to the reaction using a detectable label for a sufficient period of time to contact the sample with the substrate for a sufficient period of time to be detectable, the level of the product to be produced, preferably measurable level of the appearance of the product, can be measured as a time required for the level of the measurement of the product, the measurement, the level of the presence of a given (e.g. a luciferase) can be measured by using a covalent binding of a secondary fluorescent label, or covalent binding of a secondary fluorescent label, e.g. a secondary fluorescent label, such as a fluorescent label, or fluorescent label, as a fluorescent label, or as a fluorescent label, can be obtained from a secondary label, or as a covalent binding agent, a secondary label, a kit, or as a kit, or a kit, or a kit, or kit, including a kit, a kit, a kit, a kit.

The level of peptide or polypeptide can also preferably be determined as follows: (a) contacting a solid support comprising a binding agent for a peptide or polypeptide as specified above with a sample comprising the peptide or polypeptide and (b) measuring the level of peptide or polypeptide bound to the support. A binding agent, preferably selected from the following: nucleic acids, peptides, polypeptides, antibodies and aptamers, preferably in immobilized form, on a solid support. Materials for preparing solid supports are well known in the art and include, inter alia: commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes, and the like. The binding agent or reagents may be bound to a variety of different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agarose, and magnetite. For the purposes of the present invention, the nature of the carrier may be soluble or insoluble. Suitable methods of immobilizing/immobilizing the binding agent are well known and include, but are not limited to, ionic interactions, hydrophobic interactions, covalent interactions, and the like. Also contemplated according to the invention is the use of "suspension arrays" as arrays (Nolan 2002, Trends Biotechnol.20(1): 9-12). In such a suspension array, a carrier such as a microbead or microsphere is suspended. The array consists of different microbeads or microspheres, which may be labeled, carrying different binders. Methods for producing such arrays, e.g. based on solid phase chemical or photolabile protecting groups, are generally known (US 5,744,305).

In an embodiment of the method of the invention, the level of the biomarker referred to herein is measured by using the assay described in the examples section.

In another embodiment of the method of the invention, the measurement in step a) may be carried out by an analyzer unit, in particular by an analyzer as defined elsewhere herein.

The term "binding agent" refers to a molecule comprising a binding moiety that specifically binds to the respective biomarker. Examples of "binding agents" are aptamers, antibodies, antibody fragments, peptides, Peptide Nucleic Acids (PNA) or compounds.

The term "specifically binds" or "specifically binds" refers to a binding reaction in which the binding pair molecules exhibit mutual binding under conditions in which they do not significantly bind to other molecules. The term "specifically binds" or "specifically binds" when referring to a protein or peptide as a biomarker refers to a binding reaction in which the binding agent binds to at least 10^-7The affinity of M binds to the corresponding biomarker. The term "specifically binds" or "specifically binds" preferably means at least 10 to its target molecule^-8M or even more preferably at least 10^-9Affinity of M. The term "specific" or "specifically" is used to indicate that other molecules present in the sample do not significantly bind to a binding agent specific for the target molecule. Preferably, the level of binding of molecules that are not target molecules results in a binding affinity that is only 10% or less, more preferably only 5% or less, of the affinity for the target molecules.

Examples of "binding agents" are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, Peptide Nucleic Acids (PNA) or compounds. Preferred binding agents are antibodies that specifically bind to the biomarker being measured. The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody 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 antigen-binding activity. Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.

In one aspect, another binding agent that may be used may be an aptamer that specifically binds to at least one biomarker in a sample. The term "specifically binds" or "specifically binds" when referring to a nucleic acid aptamer as a binding agent refers to a binding reaction in which the nucleic acid aptamer binds to the corresponding target molecule with an affinity in the low nM to pM range.

In yet another aspect, the sample is removed from complexes formed between the binding agent and the at least one label prior to measuring the level of the formed complexes. Thus, in one aspect, the binding agent may be immobilized on a solid support. In yet another aspect, the sample can be removed from the complex formed on the solid support by application of a wash solution. The complex formed should be proportional to the level of the at least one label present in the sample. It will be appreciated that the specificity and/or sensitivity of the binding agent to be applied defines the extent to which the proportion of the at least one label capable of being specifically bound is contained in the sample. Further details of how the determination is carried out may also be found elsewhere herein. The level of complex formed should translate into a level of at least one marker that reflects the level actually present in the sample. In one aspect, such a level may be substantially the amount present in the sample or may be, in another aspect, the amount of some portion thereof (due to the relationship between the amount present in the formed complex and the original sample).

The term "level" as used herein encompasses the absolute amount of a biomarker as indicated herein, the relative amount or concentration of the biomarker, and any value or parameter associated therewith or derivable therefrom. Such values or parameters include intensity signal values of all specific physical or chemical properties obtained from the peptide by direct measurement, for example intensity values in a mass spectrum or NMR spectrum. Furthermore, all values or parameters obtained from direct measurements as described elsewhere in the specification are covered, e.g. the amount of response determined from a biological readout system in response to the peptide or signal intensity obtained from a specific binding partner. It will be appreciated that values relating to the quantities or parameters mentioned above may also be obtained by standard mathematical operations.

The term "comparing" as used herein refers to comparing the level of a biomarker in a sample of an individual or patient to a reference level of a biomarker described elsewhere in this specification. It is to be understood that comparison as used herein generally refers to comparison of corresponding parameters or values, e.g., an absolute value is compared to an absolute reference amount, while a concentration is compared to a reference concentration or an intensity signal obtained from a sample biomarker is compared to the same type of intensity signal obtained from a reference sample. The comparison may be performed manually or with assistance from a computer. Thus, the comparison may be implemented by computer means (e.g. of the system disclosed herein). The value of the measured or detected level of the biomarker in the individual or patient sample and the reference level may be compared, for example, to each and the comparison may be automatically performed by a computer program executing an algorithm of comparison. A computer program implementing the evaluation will provide the desired assessment in a suitable output form. For a computer-aided comparison, the value of the determined quantity may be compared with a value corresponding to a suitable reference, which reference is 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 form. For a computer-aided comparison, the value of the determined quantity may be compared with a value corresponding to a suitable reference, which reference is 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 form.

In certain embodiments, the term "reference level" herein preferably refers to a predetermined value for each biomarker. In this context "level" encompasses absolute amounts, relative amounts or concentrations, as well as any value or parameter associated therewith or derivable therefrom. Preferably, the reference level is a level that allows assigning the patient to a group of patients suitable for intensive heart failure therapy, or to a group of patients not suitable for intensive heart failure therapy. Thus, the reference level should allow a distinction to be made between patients who are suitable for intensive heart failure therapy and patients who are not.

As will be appreciated by those skilled in the art, the reference levels are predetermined and are set to comply with conventional requirements, for example in specificity and/or sensitivity. These requirements may vary, for example, from one administrative department to another. It may be exemplified that the assay sensitivity or specificity, respectively, has to be set at certain limits, e.g. 80%, 90%, 95% or 98%, respectively. These requirements may also be defined as positive or negative predictive values. Nevertheless, based on the teachings given herein, it will always be possible for the skilled person to reach the reference values to meet those requirements. In one embodiment, the reference level is determined in a reference sample or sample from a patient (or group of patients) with heart failure and being suitable for intensive heart failure therapy, or in a reference sample or sample from a patient (or group of patients) with heart failure and being not suitable for intensive heart failure therapy. In one embodiment the reference level has been predetermined in a reference sample of the disease entity to which the patient belongs. In certain embodiments, the reference level may be set, for example, to any percentage between 25% and 75% of the overall distribution of values in the disease entity under study. In further embodiments, the reference level may for example be set to the median, the tertile, the quartile determined from the overall distribution of the median of the reference samples of the disease entity under investigation. In one embodiment, the reference level may be set as the median determined from the overall distribution of values for the disease entity under study. The reference level may vary depending on a number of physiological parameters, such as age, sex or subpopulation, and the method used to determine the biomarkers referred to herein. In one embodiment, the reference sample is derived from substantially the same type of cell, tissue, organ or bodily fluid source, such as a sample of an individual or patient undergoing the methods of the invention, e.g., if blood is used as a sample to determine biomarker levels in an individual according to the invention, the reference level is also determined in blood or a portion thereof.

In certain embodiments, the term "greater than a reference level" or "greater than a reference level" refers to a level of a biomarker in a sample of an individual or patient that is greater than the reference level or is increased by 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or more in total, as determined by the methods described herein compared to the reference level. In certain embodiments, the term increase refers to an increase in the level of a biomarker in a sample of an individual or patient, wherein the increase is at least about 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold greater compared to a reference level (e.g., predetermined from a reference sample).

In certain embodiments, the term "below a reference level" or "below" herein refers to a level of a biomarker in a sample of an individual or patient that is below a reference level or is reduced by 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more in total, as determined by the methods described herein compared to a reference level. In certain embodiments, the term decrease in the level of a biomarker in a sample of an individual or patient, wherein the level of decrease is at most about 0.9-, 0.8-, 0.7-, 0.6-, 0.5-, 0.4-, 0.3-, 0.2-, 0.1-, 0.05-or 0.01-fold or less of the reference level (e.g., predetermined from the reference sample).

If the at least one marker is selected from the group consisting of creatinine, urea, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, the following applies as a diagnostic algorithm:

preferably, a level(s) of the at least one marker in the sample from the patient that is higher than the reference level for the marker indicates that the patient is suitable for intensive heart failure therapy, and/or a level(s) of the at least one marker in the sample from the patient that is lower than the reference level for the marker indicates that the patient is not suitable for intensive heart failure therapy.

If the at least one marker is selected from sodium, hemoglobin, hematocrit and IGFBP-7, the following applies as a diagnostic algorithm:

preferably, a level(s) of the at least one marker in the sample from the patient that is lower than the reference level for the marker indicates that the patient is suitable for intensive heart failure therapy, and/or a level(s) of the at least one marker in the sample from the patient that is higher than the reference level for the marker indicates that the patient is not suitable for intensive heart failure therapy.

Table a below provides preferred ranges for reference levels for various markers (third column) and preferred specific reference levels (fourth column). The person skilled in the art is able to determine further reference levels without additional effort.

TABLE A

Markers/parameters	Unit of	Reference level within the range from	Reference level
				Creatinine	mg/dL	About 1.2 to 1.8	About 1.5
BUN (Urea)	mmol/L	About 10-12	About 11.1
				Glucose	mmol/L	About 10-13	About 11.6
HbA1c	%	About 0.05-0.07	About 0.06
				hsCRP	mg/mL	About 9 to 13	About 10.4
Cystatin C	mg/L	About 1.8 to 2.0	About 1.9
				IL-6	pg/mL	About 8 to 10	About 9.1
Prealbumin	g/L	About 0.14-0.18	About 0.16
				sFlt-1	pg/mL	About 85-100	About 87
Uric acid	mg/dL	About 9-10	About 9.1
				GFD-15	pg/mL	About 2500-	About 3210
sST2	ng/mL	About 38 to 47	About 41.5
				Galectin-3	ng/mL	About 24-30	About 25
Endostatin	ng/mL	About 230-	About 243
				Mimecan	ng/mL	About 44-50	About 45.2
IGFBP-7	ng/mL	About 70-77	About 71.4
				Osteopontin	ng/mL	About 110-	About 113.5
Hemoglobin	g/dL	About 7.5 to 8.5	About 8.04
				Hematocrit of blood	%	About 0.37-0.43	About 0.40
Duration of QRS	ms	About 140-	About 160 f
				Sodium salt	mmol/L	About 138-	About 141

With respect to QRS duration, the reference may be in the range of about 140 to about 180 ms. In an embodiment, the reference is about 160 ms.

In the context of the present invention, it is contemplated to measure the level of a single marker or a combination of multiple markers. Thus, it is contemplated to measure the levels of two, three, four or even more markers. Preferred embodiments are as follows:

for example, consider the following marker combinations:

creatinine and sodium

Hemoglobin and QRS duration

Urea and HbA1c

Hematocrit and creatinine.

For example, the marker may be used to assess whether treatment with a drug should be potentiated (e.g., whether the dose of drug administered should be increased). for example, if the marker to be measured is creatinine, the heart failure therapy to be potentiated is preferably treated with an β blocker.

The definitions given herein above apply mutatis mutandis to the following. Also, the steps performed in connection with the methods described herein above may be performed according to the following methods.

The invention also relates to a method, in particular an in vitro method, for identifying a patient suitable for intensive heart failure therapy, said method comprising the steps of

(a) Measuring the level of a BNP-type peptide in a sample from a patient having heart failure and receiving a BNP-type peptide directed therapy for heart failure;

(b) measuring the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin,

(c) comparing the level of the BNP-type peptide measured in (a) with one (or more) reference levels, and

(d) comparing the level(s) of the at least one marker measured in (b) with a reference level(s).

Identifying a patient suitable for intensive heart failure therapy by performing steps (c) or (d). In one embodiment, the method further comprises the step of (e) identifying or selecting a patient suitable for intensive heart failure therapy. In addition, the method may comprise the step (f) of strengthening heart failure therapy or recommending strengthening heart failure therapy if the patient is identified as suitable for strengthening heart failure therapy. Accordingly, the present invention also contemplates a method of potentiating a heart failure therapy, said method comprising steps (a) to (f) as described above.

In addition to the marker referred to in step a), or alternatively, QRS duration may be measured or provided and compared to a reference (as outlined elsewhere herein).

In addition to the above methods, the method further comprises the step of measuring the level of the BNP-type peptide.

As used herein, the term "BNP-type peptide" includes pre-proBNP, NT-proBNP and BNP. The pre-pro peptide (134 amino acids in case of pre-proBNP) comprises a short signal peptide which is enzymatically cleaved to release the pro peptide (108 amino acids in case of proBNP). The propeptide is further cleaved into an N-terminal propeptide (NT-pro peptide, 76 amino acids in the case of NT-proBNP) and an active hormone (32 amino acids in the case of BNP). Preferably, the BNP-type peptide according to the invention is NT-proBNP, BNP (brain natriuretic peptide) and variants thereof. BNP is an active hormone and has a shorter half-life than the respective inactive NT-proBNP. BNP is metabolized in the blood, whereas NT-proBNP circulates in the blood as a complete molecule and is thus excreted via the kidney. NT-proBNP has a 120 min longer half-life in vivo than BNP (which is 20 min) (Smith 2000, J Endocrinol.167: 239-46.). Pre-analysis with NT-proBNP (Preanalytics) is more robust, allowing samples to 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 weeks or can be mailed or transported without recovery losses. In contrast, BNP causes a loss of concentration of at least 20% when stored at room temperature or at 4 ℃ for 48 hours (Mueller loc. cit.; Wu 2004, Clin Chem 50: 867-73.). Thus, depending on the target time course or property, measurement of the active or inactive form of the natriuretic peptide can be advantageous. The most preferred BNP-type peptide according to the invention is NT-proBNP or a variant thereof. As briefly mentioned above, human NT-proBNP, as described according to the invention, is a polypeptide comprising a length of 76 amino acids preferably corresponding to the N-terminal part of the human NT-proBNP molecule. The structure of human BNP and NT-proBNP has been described in detail in the prior art, e.g. WO 02/089657, WO 02/083913 or Bonow low. cit. Preferably, the human NT-proBNP as used herein is human NT-proBNP as disclosed in EP 0648228B 1. These prior art documents are incorporated herein by reference with respect to the specific sequences of NT-proBNP and variants thereof disclosed therein. NT-proBNP referred to according to the invention further comprises alleles and other variants of the specific sequence of human NT-proBNP as discussed above. In particular, variant polypeptides are contemplated which are preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to human NT-proBNP at the amino acid level, preferably over the entire length of human NT-proBNP. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, wherein a fragment of an amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to a reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison can be carried out by the local homology algorithm of Smith and Waterman Add. Math.2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. mol. biol. 48:443 (1970), by the search similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, BLAST, PASTA and TFASTA in Wisconsin Genetics software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis), or by visual inspection. If two sequences have been identified for comparison, it is preferred to use GAP and BESTFIT to determine their optimal alignment and hence the degree of identity. Preferably, a default value of 5.00 is used for the gap weight and a default value of 0.30 is used for the gap weight length. The variants referred to above may be allelic variants or any other species-specific homologues, orthologues or orthologues. Substantially similar and also contemplated are proteolytic degradation products, which are still recognized by the diagnostic method or by ligands directed against the individual full-length peptides. Also included are variant polypeptides having amino acid deletions, substitutions, and/or additions compared to the amino acid sequence of human NT-proBNP, provided that the polypeptide has NT-proBNP properties. The NT-proBNP property as referred to herein is an immunological and/or biological property. Preferably, the variant NT-proBNP has immunological properties (i.e., epitope composition) comparable to those of human NT-proBNP. Thus, the variant should be recognizable by the above-described method or the ligand used to determine the amount of the natriuretic peptide. The biological and/or immunological NT-proBNP properties may be detected by assays described in: karl et al (Karl 1999, Scand J Clin Lab Invest 230: 177-. Furthermore, assays for the determination of NT-proBNP are described by Mueller T. et al, Clinica Chimica Acta 341 (2004) 41-48. In embodiments, NT-proBNP is performed as described in any one of the above-mentioned references. Variants also include post-translationally modified peptides, such as glycosylated peptides. Furthermore, a variant according to the invention is also a peptide or polypeptide which has been modified after collection of a sample, for example by covalent or non-covalent attachment of a label, preferably a radioactive or fluorescent label, to the peptide.

The term "reference level" has been defined above. The reference level of the BNP-type peptide should preferably be a level which, when taken alone (i.e. not in combination with the further marker referred to in the context of the present invention), is indicative for a patient who is not suitable for a therapy for heart failure. Preferred reference levels of said BNP-type peptide indicative for an enhanced heart failure therapy to be applied in the context of the present invention are those described in the examples. Preferred reference levels are in the range of about 80-400 pg/ml for BNP, or in particular about 80-200pg/ml, and in the range of about 450-2200 pg/ml for NT-proBNP, or in particular about 800-1200 pg/ml. Further preferred reference levels are about 100 pg/ml or 400 pg/ml for BNP and about 1000pg/ml or 1200pg/ml for NT-proBNP.

Preferred reference levels or ranges of reference levels for the markers creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high sensitivity CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and Osteopontin (OPN) are shown in Table A above.

If the at least one marker measured in step (b) is selected from the group consisting of creatinine, urea, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, the following applies as diagnostic algorithm:

(a) a level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of a BNP-type peptide which is higher than a reference level for said BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(b) a level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of the BNP-type peptide which is lower than the reference level for the BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(c) a level of at least one marker in a sample from the patient below a reference level for said marker and a level of said BNP-type peptide above a reference level for said BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy, and/or

(d) A level of the at least one marker in the sample from the patient that is lower than the reference level for the marker and a level of the BNP-type peptide that is lower than the reference level for the BNP-type peptide indicates that the patient is not suitable for intensive heart failure therapy.

Alternatively or additionally, if the at least one marker measured in step (b) is selected from sodium, hemoglobin, hematocrit and IGFBP-7, the following applies as a diagnostic algorithm:

(a) a level of the at least one marker in the sample from the patient below the reference level for the marker and a level of the BNP-type peptide above the reference level for the BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(b) a level of the at least one marker in the sample from the patient below the reference level for the marker and a level of the BNP-type peptide below the reference level for the BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(c) a level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of said BNP-type peptide which is higher than a reference level for said BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy, and/or

(d) A level of the at least one marker in the sample from the patient that is higher than the reference level for the marker and a level of the BNP-type peptide that is lower than the reference level for the BNP-type peptide indicates that the patient is not suitable for intensive heart failure therapy.

The patient to be tested according to the above-described method may exhibit any level of BNP-type peptide (in particular any blood, serum or plasma level).

Furthermore, the present invention relates to a method for optimizing a BNP-type peptide directed therapy for heart failure, said method comprising the steps of

(a) Measuring in a sample from a patient having heart failure and receiving a BNP-type peptide directed therapy the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin, and

(b) comparing the level(s) of marker(s) measured in (a) with a reference level(s), thereby optimizing BNP-type peptide directed therapy.

The patient according to the above method preferably exhibits a level (in particular a blood, serum or plasma level) of the BNP-type peptide which is lower than a reference level of said BNP-type peptide, said reference level being indicative of an intensification of the heart failure therapy.

The definitions given herein above apply mutatis mutandis to the following embodiments of the invention.

Method for predicting risk of cardiac decompensation, hospitalization and/or death

Furthermore, the present invention relates to a method, in particular an in vitro method, for predicting the risk of a patient suffering from cardiac decompensation, hospitalization and/or death (death) who has heart failure and receives a BNP-type peptide directed heart failure therapy, said method comprising the steps of

(a) Measuring in a sample from a patient having heart failure and receiving a BNP-type peptide directed heart failure therapy the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin, and

(b) comparing the level(s) of marker(s) measured in (a) with a reference level(s).

The method may further comprise a step (c) of predicting (or providing a prediction) the patient's risk of suffering from cardiac decompensation, hospitalization and/or death, in particular wherein a level(s) of the at least one marker being higher or lower than the reference level(s) indicates that the patient has an increased risk of suffering from cardiac decompensation, hospitalization and/or death, and wherein a level(s) of the at least one marker being higher or lower than the reference level(s) indicates that the patient has a reduced risk of suffering from cardiac decompensation, hospitalization and/or death.

In a preferred embodiment, the patient exhibits a level (in particular a blood, serum or plasma level) of the BNP-type peptide which is lower than a reference level of said BNP-type peptide, said reference level being indicative for an intensification of the heart failure therapy.

The following applications are diagnostic algorithms:

if the at least one marker is selected from the group consisting of creatinine, urea, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, the following applies as a diagnostic algorithm:

preferably, a level(s) of the at least one marker in the sample from the patient that is higher than the reference level of the marker indicates that the patient is at increased risk of suffering from cardiac decompensation, hospitalization and/or death, and/or wherein a level(s) of the at least one marker in the sample from the patient that is lower than the reference level of the marker indicates that the patient is at reduced risk of suffering from cardiac decompensation, hospitalization and/or death.

If the at least one marker is selected from sodium, hemoglobin, hematocrit and IGFBP-7, the following applies as a diagnostic algorithm:

preferably, a level(s) of the at least one marker in the sample from the patient that is lower than the reference level of the marker indicates that the patient is at increased risk of suffering from cardiac decompensation, hospitalization and/or death, and/or wherein a level(s) of the at least one marker in the sample from the patient that is higher than the reference level of the marker indicates that the patient is at reduced risk of suffering from cardiac decompensation, hospitalization and/or death.

Preferred reference levels or ranges of reference levels are disclosed elsewhere herein (see table a).

The phrase "providing a prediction" as used herein refers to using information or data generated regarding the level of at least one biomarker in a patient sample as referred to herein to predict the risk of the patient suffering from cardiac decompensation, hospitalization, and/or death. The information or data may be in any form, written, spoken or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, sending, transferring, providing, delivering, distributing, or a combination thereof. In some embodiments, communicating, presenting, reporting, storing, sending, transferring, providing, transferring, distributing, or a combination thereof, is performed by a computing device, an analyzer unit, or a combination thereof. In some further embodiments, communicating, presenting, reporting, storing, sending, transferring, providing, delivering, distributing, or a combination thereof is performed by a laboratory or medical professional. In some embodiments, the information or data comprises comparing the level of the at least one marker to a reference level. In some embodiments, the information or data comprises an indication that the patient is or is not at risk of suffering from cardiac decompensation, hospitalization, and/or death.

The invention also relates to a method, in particular an in vitro method, for predicting the risk of cardiac decompensation, hospitalization and/or death (death) of a patient having heart failure and receiving a BNP-type peptide directed heart failure therapy, said method comprising the steps of

(a) Measuring the level of a BNP-type peptide in a sample from a patient having heart failure and receiving a BNP-type peptide directed therapy for heart failure;

(b) measuring the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin,

(c) comparing the level of the BNP-type peptide measured in (a) with one (or more) reference levels, and

(d) comparing the level(s) of the at least one marker measured in (b) with a reference level(s).

The method may further comprise the step (f) of predicting (or providing a prediction) that the patient is at risk of suffering cardiac decompensation, hospitalization and/or death. The prediction is preferably based on the result of the comparing step.

The patient to be tested according to the above-described method may exhibit any level of BNP-type peptide (in particular any blood, serum or plasma level).

If the at least one marker measured in step (b) is selected from the group consisting of creatinine, urea, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, the following applies as diagnostic algorithm:

(a) a level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of a BNP-type peptide which is higher than a reference level for said BNP-type peptide indicates that the patient is at increased risk of suffering from cardiac decompensation, hospitalization and/or death,

(b) a level of the at least one marker in the sample from the patient which is higher than the reference level for the marker and a level of the BNP-type peptide which is lower than the reference level for the BNP-type peptide indicates that the patient is at increased risk of suffering from cardiac decompensation, hospitalization and/or death,

(c) a level of the at least one marker in the sample from the patient below the reference level for the marker and a level of the BNP-type peptide above the reference level for the BNP-type peptide indicates that the patient has an increased risk of suffering from cardiac decompensation, hospitalization, and/or death, and/or

(d) A level of the at least one marker in the sample from the patient that is lower than the reference level for the marker and a level of the BNP-type peptide that is lower than the reference level for the BNP-type peptide indicates that the patient is at reduced risk of suffering from cardiac decompensation, hospitalization and/or death.

Alternatively or additionally, if the at least one marker measured in step (b) is selected from sodium, hemoglobin, hematocrit and IGFBP-7, the following applies as a diagnostic algorithm:

(a) a level of the at least one marker in the sample from the patient below the reference level for the marker and a level of the BNP-type peptide above the reference level for the BNP-type peptide indicates that the patient is at increased risk of suffering from cardiac decompensation, hospitalization, and/or death,

(b) a level of the at least one marker in the sample from the patient which is lower than the reference level for the marker and a level of the BNP-type peptide which is lower than the reference level for the BNP-type peptide indicates that the patient is at increased risk of suffering from cardiac decompensation, hospitalization and/or death,

(c) a level of at least one marker in a sample from the patient that is higher than a reference level for the marker and a level of the BNP-type peptide that is higher than a reference level for the BNP-type peptide indicates that the patient has an increased risk of suffering from cardiac decompensation, hospitalization, and/or death, and/or

(d) A level of the at least one marker in the sample from the patient that is higher than the reference level for the marker and a level of the BNP-type peptide that is lower than the reference level for the BNP-type peptide indicates that the patient is at reduced risk of suffering from cardiac decompensation, hospitalization and/or death.

Preferred reference levels or ranges of reference levels are disclosed elsewhere herein (e.g., see table a).

The term "cardiac decompensation" is well known in the art. Preferably, the term refers to a condition of chronic heart failure in which the heart, without help, cannot ensure adequate cell perfusion in all parts of the body. Thus, the compensatory matrix of the body is no longer sufficient to maintain pump function.

The term "death" as used herein relates to any type of death, in particular death caused by cardiovascular complications. Preferably, the death is caused by heart failure.

The term "hospitalization" is well known in the art. As used herein, the term relates to hospitalization caused by cardiovascular complications. Preferably, the hospitalization is due to heart failure.

The term "predicting" as used herein refers to assessing the likelihood that a patient, as referred to herein, will suffer cardiac decompensation, hospitalization, and/or death in a defined time window (prediction window) in the future accordingly. The prediction window is the interval during which the patient will experience cardiac decompensation, will be hospitalized, and/or will die based on the predicted likelihood. The prediction window may be the entire remaining life of the patient after analysis by the method of the invention. Preferably, however, the prediction window is an interval of one, two, three, four, five, ten, fifteen or 20 years after the method of the invention has been carried out (more preferably and accurately, after the sample analysed by the method of the invention has been obtained). Most preferably, the prediction window is an interval of four or five years. As will be appreciated by those skilled in the art, such evaluations are generally not intended to be correct for 100% of the patients analyzed. However, this term requires that the assessment will be valid for a statistically significant portion of the patient being analyzed. Whether a moiety is statistically significant can be determined by one skilled in the art without further trouble using various well-known statistical evaluation tools, such as determination of confidence intervals, p-value determination, student's t-test, Mann-Whitney test, and the like. Details are found in Dowdy and Wearden, Statisticsfor Research, John Wiley & Sons, New York 1983. 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. Preferably, the possibilities envisaged by the present invention allow the prediction to be correct for at least 60%, at least 70%, at least 80% or at least 90% of the patients of a given population.

The expression "predicting the risk of cardiac decompensation, hospitalization or death" as used herein means that the patients analyzed by the method of the invention are assigned to a group of patients with an elevated risk population or a group with a reduced risk. The elevated risk referred to according to the present invention preferably means that the patient's risk of developing cardiac decompensation, risk of hospitalization or risk of death in a predetermined prediction window is significantly elevated (i.e. significantly increased) relative to the average risk of such events in a population of patients with heart failure and receiving BNP-type peptide-guided therapy. The reduced risk referred to according to the present invention preferably means that the risk of a patient to develop cardiac decompensation, hospitalization or death in a predetermined prediction window is significantly reduced relative to the average risk of such events in said patient population. In particular, a significant increase or decrease in risk is an increase or decrease in risk to the extent that it is considered to be meaningful for prognosis, in particular the increase or decrease is considered to be statistically significant. The terms "significant" and "statistically significant" are known to those skilled in the art. Thus, whether an increase or decrease in risk is significant or statistically significant can be determined without further trouble by one of skill in the art using a variety of well-known statistical evaluation tools.

Preferably, the increased risk is in the range of 3.0% -19.0%, more preferably in the range of 12.0% -17.0%, most preferably in the range of 8.0% -16.0% for a three year prediction window. Increased, and thus increased risk as used herein preferably relates to a risk of more than 3.0%, preferably more than 12.0%, more preferably more than 17%, even more preferably more than 20%, preferably with respect to a prediction window of three years. Reduced risk as used herein preferably relates to a risk of less than 8.0%, preferably less than 6%, even more preferably less than 4%, and most preferably in the range of 3.0% and 8.0%, preferably with respect to a prediction window of three years.

The invention also relates to at least one marker selected from the group consisting of creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, and/or said at least one marker (i.e. creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), Use of at least one detection agent for uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) or osteopontin), in a sample of a patient having heart failure and receiving BNP-type peptide-directed heart failure therapy, for identifying a patient suitable for intensive heart failure therapy, for predicting the risk of the patient suffering from cardiac decompensation, hospitalization and or death, or for optimizing BNP-type peptide-directed heart failure therapy.

The invention also relates to i) a QRS duration, optionally in combination with a BNP-type peptide, and/or a device for determining a QRS duration such as an ECG device (i.e. a device capable of generating an electrocardiogram), optionally in combination with at least one detection agent for a BNP-type peptide, for identifying the use of a patient suitable for intensive heart failure therapy, for predicting the risk of a patient suffering from cardiac decompensation, hospitalization and or death, or for optimizing BNP-type peptide-guided heart failure therapy. As outlined elsewhere herein, the patient should receive BNP-type peptide directed therapy for heart failure.

The invention also relates to a BNP-type peptide combination selected from creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, and/or a combination of detection agents specifically binding to a BNP-type peptide selected from creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), sFlt-1, Use of at least one detection agent for markers of the marker group of uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, in a sample of a patient having heart failure and receiving BNP-type peptide directed heart failure therapy, for identifying a patient suitable for intensive heart failure therapy or for predicting the risk of said patient suffering from cardiac decompensation, hospitalization or death.

The invention also relates to at least one marker selected from the group consisting of creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, IGFBP7, CRP (C-reactive protein, in particular high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, and/or said at least one marker (i.e. creatinine, urea, sodium, glucose, a1C (glycated hemoglobin), hemoglobin, hematocrit, IGFBP7, CRP (C-reactive protein, in particular high-sensitive CRP), cystatin C, IL-6 (interleukin 6), Use of at least one detection agent for prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) or osteopontin) for the preparation of a diagnostic composition for identifying a patient suitable for intensive heart failure therapy or for predicting the risk of a patient to suffer from cardiac decompensation, hospitalization or death (in particular, in a sample of a patient having heart failure and receiving BNP-type peptide-directed heart failure therapy).

The invention also relates to a BNP-type peptide combination selected from creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, IGFBP7, CRP (C-reactive protein, in particular high-sensitivity CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, and/or a combination of detection reagents specifically binding to a BNP-type peptide selected from creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, IGFBP7, CRP (C-reactive protein, in particular high-sensitivity CRP), cystatin C, and high-sensitivity CRP, Use of at least one detection agent for markers of the marker group of IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin for the preparation of a diagnostic composition for identifying a patient suitable for intensive heart failure therapy or for predicting the risk of a patient suffering from cardiac decompensation, hospitalization or death (in particular in a patient sample having heart failure and receiving BNP-type peptide-directed heart failure therapy).

If the marker is a polypeptide or peptide, in particular if the marker is HbA1C (glycated hemoglobin), CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, the detection reagent preferably binds specifically to said marker. In this case, the detection reagent is preferably a monoclonal or polyclonal antibody (for the definition of the term "antibody", see elsewhere herein). For the remaining labels, the detection reagent may be a reagent that forms a complex with the label, thereby allowing the level of label to be measured, or an enzyme that allows conversion of the label as described elsewhere herein.

If the label is creatinine, the detection reagent may be picric acid (which forms a complex with creatinine).

If the label is uric acid, the detection reagent may be uricase or peroxidase.

If the label is urea, the detection reagent may be urease.

If the label is glucose, the detection reagent may be hexokinase.

Thus, the present invention also preferably relates to a system for identifying a patient suitable for intensive heart failure therapy, the system comprising

a) An analyzer unit configured to contact a sample from a patient in vitro with a detection reagent (or reagents if the level of at least one marker is measured) for measuring the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin,

b) an analyzer unit configured to detect a signal from a patient sample aliquot contacted with the reagent,

c) a computing device having a processor and in operable communication with the analysis unit, an

d) A non-transitory machine-readable medium comprising a plurality of processor-executable instructions that, when executed, calculate a level of at least one marker and compare the level of the at least one marker to a reference level (or reference levels if levels of more than one marker are measured), thereby identifying a patient suitable for intensive heart failure therapy.

As mentioned above, the patient should have heart failure and should receive BNP-type peptide directed therapy for heart failure.

Furthermore, an apparatus adapted to carry out the method of the invention is provided, said apparatus comprising

a) An analyzer unit comprising one or more detection agents for measuring the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin, and

b) an analyzer unit for comparing the measured levels with reference levels, thereby identifying patients suitable for potentiation of a heart failure therapy antagonist, a diuretic and an inhibitor of the renin-angiotensin system, said unit comprising a database with reference levels and a computer-implemented algorithm for comparison.

Preferred reference levels and diagnostic algorithms are disclosed elsewhere herein.

Preferred embodiments of the present disclosure include systems for identifying a subject as suitable for administration of at least one drug selected from the group consisting of β blockers, aldosterone antagonists, diuretics, and inhibitors of the renin angiotensin system examples of systems include clinical chemistry analyzers, coagulation chemistry analyzers, immunochemical analyzers, urinalysis analyzers, nucleic acid analyzers for detectingThe outcome of or monitoring the progress of a chemical or biological reaction. More specifically, an exemplary system of the present disclosure may include Roche Elecsys^TMSystems and Cobas^®e immunoassay analyzer, Abbott Architect^TMAnd Axsym^TMAnalyzer, Siemens Centaur^TMAnd Immulite^TMAnalyzer and Beckman Coulter UniCel^TMAnd Access^TMAn analyzer, and the like.

Embodiments of the system may include one or more analyzer units for practicing the present disclosure. The analyzer unit of the system disclosed herein is in operable communication with the computing device disclosed herein via any known wired connection, bluetooth, LANS, or wireless signal. Additionally, according to the present disclosure, the analyzer unit comprises a stand-alone device, or a module in a larger instrument, which performs one or both assays, e.g. qualitative and/or quantitative evaluation of a sample for diagnostic purposes. For example, the analyzer unit may perform or assist with aspiration, dosing, mixing of the sample and/or reagents. The analyzer unit may comprise a reagent fixing unit for fixing a reagent to perform the assay. The reagents may be arranged, for example, in the form of containers or cassettes containing individual reagents or groups of reagents, arranged in suitable containers (receptacle) or locations in a storage compartment or conveyor. The detection reagent may also be in an immobilized form on a solid support, which is contacted with the sample. Further, the analyzer unit may comprise processing and/or detection components that may be optimized for a particular analysis.

According to some embodiments, the analyzer unit may be configured for optical detection of analytes, such as labels, with the sample. Exemplary analyzer units configured for optical detection include devices configured to convert electromagnetic energy into electrical signals, including single-element and multi-element or array optical detectors. In accordance with the present disclosure, an optical detector is capable of monitoring the photo-magnetic signal and providing an electrical output signal or a response signal relative to a baseline signal that is indicative of the presence and/or concentration of an analyte in a sample located in the optical path. Such devices may also include, for example, photodiodes, including avalanche photodiodes, phototransistors, photoconductive detectors, linear sensor arrays, CCD detectors, CMOS detectors, including CMOS array detectors, photomultiplier tubes, and photomultiplier tube arrays. According to certain embodiments, the optical detector, such as a photodiode or photomultiplier tube, may contain additional signal conditioning or processing electronics. For example, the optical detector may comprise at least one preamplifier, electronic filter or integrated circuit. Suitable preamplifiers include, for example, integrated, transimpedance and current gain (current mirror) preamplifiers.

Additionally, one or more analyzer units according to the present disclosure may include a light source for emitting light. For example, the light source of the analyzer unit may consist of at least one light emitting element (such as a light emitting diode, an electrically powered radiation source such as an incandescent lamp, an ionising light emitting lamp, a gas discharge lamp, a high intensity gas discharge lamp, a laser) for measuring the analyte concentration of the sample being tested or for enabling energy transfer (e.g. by fluorescence resonance energy transfer or a catalytic enzyme).

In addition, the analyzer unit of the system may include one or more incubation units (e.g., for maintaining the sample or reagent at a particular temperature or temperature range). In some embodiments, the analyzer unit may comprise a thermal cycler, including a real-time thermal cycler, for subjecting the sample to repeated temperature cycles and monitoring the sample for changes in the level of amplification products.

Additionally, the analyzer unit of the systems disclosed herein may include or be operatively connected to a reaction vessel or a cuvette feeding unit. Exemplary priming units include liquid handling units, such as pipetting units, to deliver samples and/or reagents to reaction vessels. The aspiration unit may comprise a reusable washable needle, e.g. a steel needle or a disposable tip. The analyzer unit may further comprise one or more mixing units, such as a vibrator to vibrate the cuvette containing the liquid, or a paddle to mix the liquid in the cuvette or reagent container.

Following the above, portions of some steps of the methods disclosed and described herein may be performed by a computing device, according to some embodiments of the present disclosure. The computing device may be, for example, a general purpose computer or a portable computing device. It should be understood that multiple computing devices may be used together, such as over a network or other data transmission method, for performing one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants ("PDAs"), such as BLACKBERRY brand devices, mobile devices, tablets, servers, and the like. Typically, a computing device includes a processor capable of executing various instructions (such as a software program).

The computing device accesses the memory. The memory is a computer-readable medium and may include a single storage device or multiple storage devices, which may be located locally to the computing device or may be connected to the computing device over a network, for example. Computer readable media can be any available media that can be accessed by the computing device and includes both volatile and nonvolatile media. Further, the computer readable medium may be one or both of a removable medium and a non-removable medium. By way of example, and not limitation, computer readable media may comprise computer storage media. Exemplary computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD-ROM, Digital Versatile Disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store a plurality of instructions that can be accessed by a computing device and executed by a processor of the computing device.

According to embodiments of the present disclosure, software may include instructions that, when executed by a processor of a computing device, may perform one or more steps of the methods disclosed herein. Some instructions may be adapted to generate signals that control the operation of other machines and may therefore be operated by those control signals to convert material remote from the computer itself. These descriptions and representations are the means used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art, for example.

The plurality of instructions may also include an algorithm that is generally conceived to be a self-consistent sequence of steps leading to a desired result. The steps are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic pulses or signals capable of being stored, transferred, transformed, combined, compared, and otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to these signals as values, features, display data, quantities, or the like, when referring to the physical items or representations in which such signals are embodied or expressed. It should be borne in mind, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities. According to some embodiments of the present disclosure, an algorithm for performing a comparison between a determined level of one or more markers disclosed herein and a suitable reference is embodied by executing instructions and performed. These results can be given as the output of raw data for parametric diagnosis or as absolute or relative levels. According to various embodiments of the systems disclosed herein, "diagnosis" may be provided by the computing device of the systems disclosed herein based on the comparison of the calculated "level" to a reference or threshold. For example, the computing device of the system may provide an indicator in the form of text, symbols, or numerical values that indicate a particular diagnosis.

The computing device may also access an output device. Exemplary output devices include, for example, facsimile machines, displays, printers, and documents. According to some embodiments of the present disclosure, a computing device may perform one or more steps of the methods disclosed herein, and then provide an output via an output device regarding the results, indications, proportions, or other factors of the methods.

Finally, the invention relates to a kit suitable for performing the method of the invention, said kit comprising at least one detection reagent (or a plurality of detection reagents, if the level of at least one marker is measured) for measuring the level of at least one marker selected from the group consisting of creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP7 (insulin growth factor binding protein 7), sST2 (soluble ST2), and osteopontin, reference is made to standards and instructions for carrying out the methods.

The term "kit" as used herein refers to a collection of the above-mentioned components, preferably provided separately or in a single container. The container also contains instructions for carrying out the method of the invention. These instructions may be provided in the form of a manual or may be provided by computer program code (which is capable of performing the comparisons relating to the methods of the invention and thus establishing a diagnosis when implemented in a computer or data processing apparatus). The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g. an optical disc), or directly on a computer or data processing device. Furthermore, the kit should comprise at least one standard for the above-defined reference, i.e. a solution with a predetermined level of the biomarker as mentioned herein representative of the reference level.

In some embodiments, the kits disclosed herein comprise at least one component or a combination of component packages for practicing the disclosed methods. By "packaged combination" is meant that the kit provides a single package containing a combination of one or more components, such as probes (e.g., antibodies), controls, buffers, instructions for reagents (e.g., conjugates and/or substrates), and the like, as disclosed herein. Kits containing a single container are also included in the definition of "packaged combination". In some embodiments, the kit comprises at least one probe, such as an antibody (with a particular affinity for an epitope of a biomarker disclosed herein). For example, the kit may include an antibody that is labeled with a fluorophore or an antibody that is a member of a fusion protein. In the kit, the probe may be immobilized, and may be immobilized in a specific conformation. For example, immobilized probes can be provided in a kit to specifically bind to a target protein, to detect the target protein in a sample, and/or to remove the target protein from a sample.

According to some embodiments, the kit comprises at least one probe, which may be immobilized, in at least one container. The kit may also include a plurality of probes in one or more containers, optionally immobilized. For example, multiple probes may be present in a single container, or in separate containers, e.g., where each container contains a single probe.

In some embodiments, a kit may include one or more non-immobilized probes and one or more solid supports, which may or may not include immobilized probes. Some such embodiments may include some or all of the reagents or supplies required for immobilization of one or more probes onto a solid support, or for binding of an immobilized probe to a particular protein in a sample.

In certain embodiments, a single probe (which includes multiple copies of the same probe) may be immobilized to a single solid support and provided in a single container. In other embodiments, two or more probes, each specific for a different target protein or a different form of a single target protein (such as a particular epitope), are provided in a single container. In some such embodiments, the immobilized probes can be provided in a plurality of different containers (e.g., single use format), or a plurality of immobilized probes can be provided in a plurality of different containers. In further embodiments, the probes may be immobilized on a plurality of different types of solid supports. Any combination of immobilized probes and containers is contemplated for the kits disclosed herein, and any combination thereof may be selected to achieve a suitable kit for a desired use.

The container of the kit can be any container suitable for packaging and/or containing one or more components disclosed herein, including, for example, probes (e.g., antibodies), controls, buffers, and reagents (e.g., conjugates and/or substrates). Suitable materials include, but are not limited to, glass, plastic, cardboard or other paper products, wood, metal, and any alloys thereof. In some embodiments, the container may completely surround the immobilized probe or may simply cover the probe to minimize contamination and exposure to light due to dust, oil, and the like. In some further embodiments, a kit may comprise a single container or a plurality of containers, and when a plurality of containers is present, each container may be the same as all other containers, different from other containers, or different from some but not all other containers.

Preferred embodiments of the invention

Hereinafter, preferred embodiments of the present invention are disclosed. The definitions and explanations given herein above and in the claims apply mutatis mutandis.

1. A method of identifying a patient suitable for intensive heart failure therapy, the method comprising the steps of

(a) Measuring in a sample from a patient having heart failure and receiving a BNP-type peptide directed heart failure therapy the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin, and

(b) comparing the level(s) of marker(s) measured in (a) with a reference level(s).

2. The method according to embodiment 1, further comprising the step (c) of identifying a patient as being suitable for intensive heart failure therapy.

3. The method of embodiment 1 or 2, wherein the patient exhibits a level of the BNP-type peptide that is lower than a reference level of the BNP-type peptide, which reference level is indicative of an intensification of the heart failure therapy.

4. The method of any of embodiments 1-4, wherein

i) At least one marker is selected from the group consisting of creatinine, urea, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, and wherein a level(s) of the at least one marker in the sample from the patient that is higher than the reference level of the marker indicates that the patient is suitable for intensive heart failure therapy, and/or wherein a level(s) of the at least one marker in the sample from the patient that is lower than the reference level of the marker indicates that the patient is suitable for intensive heart failure therapy, and/or

ii) the at least one marker is selected from the group consisting of sodium, hemoglobin, hematocrit and IGFBP-7, and wherein a level(s) of the at least one marker in the sample from the patient that is lower than the reference level for the marker indicates that the patient is suitable for intensive heart failure therapy, and/or wherein a level(s) of the at least one marker in the sample from the patient that is higher than the reference level for the marker indicates that the patient is not suitable for intensive heart failure therapy.

5. A method of identifying a patient suitable for intensive heart failure therapy, the method comprising the steps of

(a) Measuring the level of a BNP-type peptide in a sample from a patient having heart failure and receiving a BNP-type peptide directed therapy for heart failure;

(b) measuring the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin,

(c) comparing the level of the BNP-type peptide measured in (a) with one (or more) reference levels, and

(d) comparing the level(s) of the at least one marker measured in (b) with a reference level(s).

6. The method of embodiment 5, wherein

i) At least one marker is selected from the group consisting of creatinine, urea, glucose, HbA1C (glycated hemoglobin), CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, and wherein

(a) A level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of a BNP-type peptide which is higher than a reference level for said BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(b) a level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of the BNP-type peptide which is lower than the reference level for the BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(c) a level of at least one marker in a sample from the patient below a reference level for said marker and a level of said BNP-type peptide above a reference level for said BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy, and/or

(d) A level of the at least one marker in the sample from the patient which is lower than the reference level for the marker and a level of the BNP-type peptide which is lower than the reference level for the BNP-type peptide indicates that the patient is not suitable for intensive heart failure therapy, and/or

ii) at least one marker selected from sodium, hemoglobin, hematocrit and IGFBP-7,

(a) a level of the at least one marker in the sample from the patient below the reference level for the marker and a level of the BNP-type peptide above the reference level for the BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(b) a level of the at least one marker in the sample from the patient below the reference level for the marker and a level of the BNP-type peptide below the reference level for the BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(c) a level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of said BNP-type peptide which is higher than a reference level for said BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy, and/or

(d) A level of the at least one marker in the sample from the patient that is higher than the reference level for the marker and a level of the BNP-type peptide that is lower than the reference level for the BNP-type peptide indicates that the patient is not suitable for intensive heart failure therapy.

7. Method for optimizing a BNP-type peptide directed therapy for heart failure, said method comprising the steps of

(a) Measuring in a sample from a patient having heart failure and receiving a BNP-type peptide directed therapy the level of at least one marker selected from the group consisting of: creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP7 (insulin growth factor binding protein 7), sST2 (soluble ST2) and osteopontin, and

(b) comparing the level(s) of marker(s) measured in (a) with a reference level(s), thereby optimizing BNP-type peptide directed therapy.

8. The method according to any one of embodiments 1-7, wherein the patient is a human.

9. The method according to any one of embodiments 1-8, wherein said patient has heart failure classified according to the ACC/AHA classification as stage B or C, and/or wherein said patient has heart failure of class II or III according to the NYHA classification.

10. The method according to any one of embodiments 1-9, wherein the sample is a blood, serum or plasma sample.

11. The method according to any one of embodiments 1-10, wherein the heart failure therapy is a pharmaceutical heart failure therapy, in particular wherein the heart failure therapy comprises the administration of at least one drug selected from the group consisting of diuretics, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, β blockers and aldosterone antagonists.

12. The method of embodiment 11, wherein the heart failure therapy comprises combined administration of an β blocker and an ACE inhibitor.

13. The method according to any one of embodiments 1-12, wherein the intensive heart failure therapy comprises increasing a dose of a previously administered drug, administering an additional drug or drugs, particularly administering an additional drug or drugs having a different mode of action than the previously administered drug, device therapy, lifestyle modification, and combinations thereof.

14. i) at least one marker selected from the group consisting of creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, IGFBP7, CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, and/or ii) at least one detection reagent for said at least one marker, in a sample of a patient having heart failure and receiving BNP-type peptide-directed heart failure therapy, for identifying the patient as being suitable for intensive heart failure therapy, for predicting the patient to suffer from heart decompensation, insulin secretion, insulin, Use in hospitalization and/or risk of death, or for optimizing BNP-type peptide-directed therapy for heart failure.

15. The BNP-type peptide combination is selected from creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, IGFBP7, CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, IL-6 (interleukin 6), prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and at least one marker of osteopontin, and/or the combination of detection reagents specifically binding to the BNP-type peptide is selected from creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, blood cells, IGFBP7, CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, IL-6 (interleukin 6), Use of at least one detection agent for a marker of the marker group of prealbumin, sFlt-1 (soluble fms-like tyrosine kinase-1), uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, sST2 (soluble ST2) and osteopontin, in a sample of a patient having heart failure and receiving BNP-type peptide directed heart failure therapy, for identifying a patient suitable for intensive heart failure therapy or for predicting the risk of said patient suffering from heart decompensation, hospitalization or death.

All documents referred to above are incorporated herein by reference for their full disclosure as well as for the specific disclosure explicitly mentioned in the above description.

Detailed Description

Examples

The invention will now be illustrated by the following examples, which are not intended to limit or restrict the scope of the invention.

Example 1: patient's health

499 patients with HF (NYHA class II-IV contractile HF (LVEF ≦ 45%) were instructed according to NT-proBNP target or general care (Pfisterer m. et al jama.2009; 301: 383-92). overall, patients with NT-proBNP levels <1000 pg/mL (previously identified good outcome cut-off value) had significantly better outcomes than those with NT-proBNP levels that could not be reduced to these levels. These additional markers and parameters also provide additional important information for potential therapy guidance in the group with higher risk (i.e. NT-proBNP levels >1000pg/ml after 6 months).

The level of BNP and/or NT-proBNP is measured together with one or several markers and/or clinical parameters at regular intervals of up to every 6 months every few weeks. If intensive medical therapy is clinically necessary and/or indicated by BNP/NT-proBNP and/or one of these additional markers and/or parameters, these subjects are followed clinically every few weeks until optimal/maximal medical therapy is achieved, the BNP/NT-proBNP target goals of ≦ 100-200 pg/mL and ≦ 1,000 pg/mL, respectively, are achieved, and the following goals are achieved or the subjects require hospitalization.

Table 1 shows the cut-off values for additional markers and clinical parameters (based on the ROC optimized cut-off and the inflection point of the decile of the risk; both methods produce cut-offs identifying similar remaining risks; when both are used, a lower cut-off should be used):

table 1:

table 2 shows the Wald score, p-value and risk ratio (HR, with 95% confidence interval) of biomarkers and clinical parameters in patients guided by NT-proBNP. The Wald score and risk ratio indicate the remaining risk of cardiac decompensation, hospitalization or death in patients guided with BNP-type peptides.

All subjects with BNP/NT-proBNP concentrations >100-200 pg/mL and >1,000 pg/mL and marker/parameter levels above the cut-off values in the above table (or below the cut-off values for hemoglobin, hematocrit, IGFBP-7 and sodium), respectively, were considered drug therapy and/or device therapy potentiation regardless of symptom status, perceived stability, and carefully re-evaluated for the presence of "best" medical procedures. In addition, subjects with BNP/NT-proBNP concentrations <100-200 pg/mL and <1,000 pg/mL, respectively, and marker/parameter levels above the cut-off values shown above were considered drug therapy and/or device therapy potentiation. Management of HF patients for HF therapy according to such guided combination marker guided is the same as standard treatment and encompasses all medications, devices and treatment options as recommended by the practice guidelines. The therapy enhancement consisted of: increasing the dosage of previously prescribed medications or adding medications or device therapies of different modes of action, compliance with practical guidelines and exercise, diet, or combinations thereof consistent with optimal clinical practice. No special algorithms for drug titration or drug selection are used. Although loop diuretics may reduce NT-proBNP concentration, they are not generally considered "first-line" therapies for non-congestive (non-conflicted) patients with elevated NT-proBNP in view of the lack of mortality benefit of such agents in the chronic HF setting.

Once the drug adjustment results in achieving the target value or the subject is symptomatically stable, the subject is considered to be in the "optimal medical regimen for combined marker targeting" and is therefore removed from the follow-up loop for several weeks and seen at the next scheduled clinical visit (regular monitoring interval). The marker levels of the combination during further exploration or during near-patient combination marker measurement (using point of care assays) may be used to further guide HF therapy, i.e. to further increase or decrease the intensity of therapy in a similar exploration and BNP/NT-proBNP measurement loop as described above.

If the subject did not achieve the combined marker/parameter target goal but did reach a clear therapeutic limit, the subject would be removed from the week follow-up loop and would be seen in the next scheduled clinical visit. The subject re-assesses the combination marker levels and the opportunity to further titrate the drugs and adjust treatment options in the scheduled clinical visit. The invention according to additional stratification of other markers and parameters also provides benefits to patients with higher NT-proBNP targets, e.g., 3000 pg/mL. In particular, since not all patients can achieve NT-proBNP targets ≦ 1,000 pg/mL, higher target cutoff values can be used, and at these levels, other markers and clinical parameters can still provide additional risk stratification, monitoring, and therapy guidance benefits.

In contrast to the prior art and previous biomarker-directed HF methods, the present invention provides additional marker and parameter target levels outside the target range of BNP/NT-proBNP. The invention also improves the identification of patients that do not optimally benefit from BNP/NT-proBNP-directed HF therapy.

Furthermore, the association of marker levels, therapy changes and results indicate that the remaining risk reflected by the different parameters above can be changed using available therapies. This association indicates that different marker combinations may be applied to guide heart failure therapies other than NT-proBNP. An example of the association of these therapies is shown below:

NT-proBNP and creatinine to direct β blockers patients who were directed with NT-proBNP and high creatinine concentrations at 6 months experienced good results with either a high β blocker dose or an increased β blocker dose.

Example 2: measurement of

NT-proBNP was determined using the electrochemiluminescence ELISA sandwich assay of Roche, Elecsys proBNP II STAT (short turnaround time) assay. The test employs two monoclonal antibodies that recognize epitopes located in the N-terminal part (1-76) of proBNP (1-108).

IL-6 (interleukin-6) was measured by electrochemiluminescence immunoassay (ECLIA, Roche Diagnostics). The tests were performed using a Cobas E601 analyzer from Roche Diagnostics. The test is based on a first incubation with biotinylated monoclonal IL-6-specific antibody and a second incubation with monoclonal IL-6-specific antibody labeled with ruthenium complex and streptavidin-coated microparticles.

High sensitivity (hs) CRP was determined using a particle-enhanced immunoturbidimetry assay from Roche Diagnostics (Tina-quant cardiac C-reactive protein (latex) high sensitivity). In this test, latex particle-conjugated anti-CRP antibodies react with antigens in the sample to form antigen/antibody complexes. After agglutination, the complex is subjected to turbidimetric measurements.

To determine the concentration of GDF-15 in serum and plasma samples, an Elecsys prototype test was used using a polyclonal, GDF-15 affinity chromatography purified, goat anti-human GDF-15 IgG antibody from R & DSystems (AF 957). In each experiment, standard curves were generated using recombinant human GDF-15 from R & D Systems (957-GD/CF). Results with new batches or recombinant GDF-15 protein were tested in standard plasma samples and any deviation above 10% was corrected by introducing a regulatory factor to the assay. GDF-15 measurements in serum and plasma samples from the same patient yielded virtually identical results after correction for the final dilution factor. The detection limit of the assay was 200 pg/ml.

For detection of IGFBP7 in human serum or plasma, a sandwich ELISA was used. For capture and detection of antigen, aliquots of anti-IGFBP 7 polyclonal antibody from R & D Systems (Cat. No: AF 1334) were conjugated to biotin and digoxin, respectively.

Streptavidin-coated 96-well microtiter plates were incubated for 60 min with 100 pi biotinylated anti-IGFBP 7 polyclonal antibody at 1 pg/ml in 1 XPBS solution. After incubation, plates were washed three times with lx PBS + 0.02% Tween-20, blocked with PBS + 1% BSA (bovine serum albumin) and then washed three more times with lx PBS + 0.02% Tween-20. The wells were then incubated for 1.5 hours with serial dilutions of recombinant IGFBP7 as a standard antigen or with diluted serum or plasma samples (1: 50) from patients or control individuals, respectively. After IGFBP7 binding, the plates were washed three times with lx PBS + 0.02% Tween-20. For specific detection of bound IGFBP7, wells were incubated with 100 μ l of digoxin anti-IGFBP 7 polyclonal antibody at 1 μ g/ml in lxPBS + 1% BSA for 60 min. Subsequently, the plate was washed three times to remove unbound antibody. In the next step, the plates were incubated for 60 min with 75 mU/ml of anti-digoxin-POD conjugate (Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 1633716) in lxPBS + 1% BSA. The plate was then washed six times with the same buffer. For detection of antigen-antibody complexes, wells were incubated with 100 μ l ABTS solution (roche diagnostics GmbH, Mannheim, Germany, catalogue No. 11685767) and the Optical Density (OD) was measured after 15 min at 405 and 492 nm with an ELISA reader.

Gal-3 was determined by using a BGM Galectin-3 assay (BG medecine, Waltham, MA, USA). It quantitatively measures galectin-3 in serum or EDTA-plasma by enzyme linked immunosorbent assay (ELISA) on a microtiter plate platform. The assay utilized two monoclonal antibodies against galectin-3. One rat monoclonal anti-mouse galectin-3 antibody is coated on the surface of wells in a microtiter plate and used as a capture antibody to bind galectin-3 molecules in a sample, while the other mouse monoclonal anti-human galectin-3 antibody is provided in solution and functions as a tracer antibody for detecting galectin-3 molecules bound to the capture antibody.

For the detection of mimecan in human serum or plasma, a sandwich ELISA was used. For capture and detection of antigen, aliquots of anti-mimecan polyclonal antibody from R & D Systems (catalog # AF 2660) were conjugated to biotin and digoxin, respectively. Streptavidin-coated 96-well microtiter plates were incubated with 100 μ l of biotinylated anti-mimecan polyclonal antibody at 0.2[ mu ] g/ml in Ix PBS solution for 60 min. After incubation, plates were washed three times with lx PBS + 0.02% Tween-20, blocked with PBS + 2% BSA (bovine serum albumin) for 45 min and then washed three more times with Ix PBS + 0.02% Tween-20. The wells were then incubated with 100 μ l serial dilutions of recombinant mimecan as standard antigen or diluted serum or plasma samples (1: 5 in 1x PBS + 1% BSA) from patients or control individuals, respectively, for 1 hour. After binding of the mimecan, the plates were washed three times with Ix PBS + 0.02% Tween-20. For specific detection of bound mimecan, wells were incubated for 45 min with 100 μ l of digoxin anti-mimecan polyclonal antibody at 0.2 μ g/ml in Ix PBS + 1% BSA. Subsequently, the plate was washed three times to remove unbound antibody. In the next step, wells were incubated with 100 μ l of 75 mU/ml anti-digoxin-POD conjugate (Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 1633716) in lxPBS + 1% BSA for 30 min. The plate was then washed six times with the same wash buffer as above. For detection of antigen-antibody complexes, wells were incubated with 100 μ l ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, catalogue No. 11685767) and the Optical Density (OD) was measured after 15 min at 405 and 492 nm with an ELISA reader.

For measuring Endostatin in human serum or plasma, a commercially available sandwich ELISA (QuantikineHuman Endostatin Immunoassay, catalog No. DNSTO, R & D Systems) was used. Measurements were made according to the instructions provided by the manufacturer.

sST2 was determined using the Presage ™ ST2 assay from Critical Diagnostics (San Diego, Calif., USA). The assay is a quantitative sandwich monoclonal ELISA in 96-well plate format for measuring ST2 in serum or plasma. The diluted plasma was loaded into the appropriate wells of the anti-ST 2 antibody coated plate and incubated for the specified time. After a series of steps of washing the reagent from the plate, additional reagents are added and subsequently washed away, and finally the analyte is detected by addition of a colorimetric reagent and the resulting signal is measured spectrophotometrically at 450 nm.

The biomarker mimecan was determined as described in WO 2011/012268.

sFlt1 was detected using an ELECSYS immunoassay using two antibodies specific for sFlt1, respectively. The tests can be performed automatically using different Roche analyzers, including ELECSYS 2010 and cobra e411 and cobrae 601.

Uric acid was determined by applying an enzymatic colorimetric method. In this enzymatic reaction, peroxide is reacted in the presence of Peroxidase (POD), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), and 4-aminopyrine to form a quinone-diimine dye. The intensity of the red color formed is proportional to the uric acid concentration and is determined photometrically.

Urea measurement by in vitro assay for use on Roche/Hitachi cobalt c SystemFor the quantitative determination of urea/urea nitrogen in human serum, plasma and urine. The tests can be performed automatically using different analyzers, including cobas c311 and cobas c 501/502. The assay is a kinetic assay of urease and glutamate dehydrogenase. Urea is hydrolyzed by urease to form ammonium and carbonate. In a second reaction, 2-ketoglutarate reacts with ammonium in the presence of glutamate dehydrogenase (GLDH) and coenzyme NADH to produce L-glutamate. In this reaction, for every mole of urea hydrolyzed, 2 moles of NADH are oxidized to NAD⁺. The rate of decrease of NADH concentration is directly proportional to the urea concentration in the sample and is measured photometrically.

Creatinine in plasma samples is measured by the Jaffe Method adapted to the Rate blank (ratebranded) and compensated for by the Roche/Hitachi automatic analyzer (see also Foster-Swanson et al, Reference Interval Studies of the Rate-Blanked Creatinine/Jaff Method on BM/Hitachi Systems in Six U.S. laboratory. Clin Chem 1994; Abstract number 361). The assay is based on an in vitro test using kinetics that set rate blanks and compensate for the quantitative determination of creatinine in human serum, plasma and urine. Sodium hydroxide and picric acid were added to the sample to initiate formation of creatinine-picric acid complexes. In alkaline solution, creatinine and picric acid form a yellow-orange complex. Color intensity was measured photometrically and is proportional to the concentration of creatinine.

The measurement of D-Glucose in plasma samples was performed using an enzymatic assay from Roche/R-Biopharm (see also Schmidt, Die enzymatische Bestimung von Glucose and Fructose nebeinander, Klinische Wochenzeitschrift, 1961, 39, 1244-1247. the phosphorylation of the marker to D-Glucose-6-phosphate in the presence of the enzymes Hexokinase (HK) and adenosine 5' -triphosphate (ATP), with the formation of adenosine-5' -diphosphate (ATP) in the presence of the enzyme glucose-6-phosphate dehydrogenase, oxidation of D-glucose-6-phosphate to D-gluconic acid phosphate by NADP, and forming reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) the amount of NADPH formed in the reaction is stoichiometric for the amount of D-glucose NADPH is measured by light absorption.

Plasma sodium is measured by applying an Ion Selective Electrode (ISE) using a plasma sample through the ion selective electrode, which employs the unique properties of certain membrane materials to generate an electrical potential (electromotive force, EMF) for measuring ions in solution (COBAS Integr 400; Roche Diagnostics GmbH, Mannheim, Germany, Assay: "ISEindirect Na-K-Cl for Gen.2").

Hemoglobin (Hb) was measured using the Reflotron Hemoglobin assay. The test is based on the passage of potassium (III) ferrocyanide (Fe)²⁺To Fe³⁺) The oxidized hemoglobin is methemoglobin. Hemoglobin levels are proportional to color intensity and are measured at a wavelength of 567 nm at 37 ℃.

HbA1c (glycated hemoglobin, glycohemoglobin) was measured by using a Roche in vitro test which allows quantitative determination of HbA1c on a Roche/Hitachi cobas c system (Assay: "Tina-quant Hemoglobin A1c Gen.3", Roche Diagnostics GmbH, Mannheim, Germany).

Prealbumin was measured by using a Roche in vitro test that allowed quantitative determination of prealbumin in human samples on a Roche/Hitachi cobasc system (ACN 710, ACN 8710). The assay is an immunoturbidimetric assay. Human prealbumin was precipitated with specific antisera and assayed for turbidimetry.

Cystatin C was measured on a Roche automated clinical chemistry analyzer by using an immunoturbidimetric Assay for quantitative in vitro determination of Cystatin C in human serum and plasma (Assay: Tina-quant Cystatin C, Roche diagnostics GmbH, Mannheim, Germany). In this assay, human cystatin C agglutinates with latex particles coated with anti-cystatin C antibody. Aggregates were determined turbidimetrically at 546 nm.

And (4) conclusion:

NT-proBNP or a combination of BNP with other markers and clinical parameters may be used for monitoring purposes and as a guide to therapy beyond current standard treatments to modulate and titrate (titrate) therapy in HF patients (chronic or acute HF after stabilization), preferably in those patients where HF is due to impaired contractile function. These markers and parameters are preferably creatinine, eGFR (calculated from creatinine levels), BUN, glucose, HbA1c, hscRP, cystatin C, IL-6, prealbumin, sFlt-1, uric acid, GDF-15, sST2, galectin-3, endostatin, Mimecan, IGFBP-7, osteopontin, sodium, hemoglobin and hematocrit, as well as heart rate and QRS duration. In particular, the addition of these measurements to NT-proBNP or BNP, together with current standard therapies, enables further risk stratification of HF patients who have been guided by NT-proBNP but may need more intensive therapy and closer observation. The present invention thus optimizes heart failure therapy guidance beyond NT-proBNP by considering the combination of natriuretic peptides with other markers and/or clinical parameters.

Claims (15)

1. Use of a detection reagent for a marker selected from the group consisting of sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) in the manufacture of a kit for a method of identifying a patient suitable for intensive heart failure therapy, wherein said method comprises the steps of

(a) Measuring the level of at least one marker selected from sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) in a sample from a patient having heart failure and receiving a BNP-type peptide-directed therapy for heart failure, and

(b) comparing the level or levels of the marker or markers measured in (a) to one or more reference levels.

2. The use according to claim 1, wherein the method further comprises a step (c) of identifying a patient as being suitable for intensive heart failure therapy.

3. The use of claim 1 or 2, wherein the patient exhibits a level of the BNP-type peptide that is lower than a reference level of the BNP-type peptide, which reference level is indicative of an intensification of a heart failure therapy.

4. Use according to any one of claims 1 to 3, wherein

i) At least one marker is selected from the group consisting of sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, glucose and HbA1c (glycated hemoglobin), and wherein a level of one or more of the at least one marker in a sample from the patient that is higher than a reference level for the marker indicates that the patient is suitable for intensive heart failure therapy, and/or wherein a level of one or more of the at least one marker in a sample from the patient that is lower than a reference level for the marker indicates that the patient is not suitable for intensive heart failure therapy, and/or

ii) the at least one marker is selected from sodium, hemoglobin and hematocrit, and wherein a level or levels of the at least one marker in the sample from the patient that are below the reference level of the marker indicates that the patient is suitable for intensive heart failure therapy, and/or wherein a level or levels of the at least one marker in the sample from the patient that are above the reference level of the marker indicates that the patient is not suitable for intensive heart failure therapy.

Use of a detection agent for a BNP-type peptide and a detection agent for a marker selected from the group consisting of sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) in the preparation of a kit for a method of identifying a patient suitable for intensive heart failure therapy, and said method comprising the steps of

(a) Measuring the level of a BNP-type peptide in a sample from a patient having heart failure and receiving a BNP-type peptide directed therapy for heart failure;

(b) measuring the level of at least one marker selected from sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high-sensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) in a sample from said patient,

(c) comparing the level of the BNP-type peptide measured in (a) with one or more reference levels, and

(d) comparing the level or levels of the at least one marker measured in (b) to one or more reference levels.

6. The use of claim 5, wherein

i) At least one marker is selected from the group consisting of sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, glucose and HbA1c (glycated hemoglobin), and wherein

(a) A level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of a BNP-type peptide which is higher than a reference level for said BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(b) a level of at least one marker in a sample from the patient which is higher than a reference level for said marker and a level of the BNP-type peptide which is lower than the reference level for the BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy,

(c) a level of at least one marker in a sample from the patient below a reference level for said marker and a level of said BNP-type peptide above a reference level for said BNP-type peptide indicates that the patient is suitable for intensive heart failure therapy, and/or

(d) A level of the at least one marker in the sample from the patient that is lower than the reference level for the marker and a level of the BNP-type peptide that is lower than the reference level for the BNP-type peptide indicates that the patient is not suitable for intensive heart failure therapy.

7. Use of a detection agent for a marker selected from the group consisting of sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) in the manufacture of a kit for a method of optimizing a BNP-type peptide-directed therapy of heart failure, wherein said method comprises the steps of

(a) Measuring the level of at least one marker selected from sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) in a sample from a patient having heart failure and receiving BNP-type peptide directed therapy, and

(b) comparing the level or levels of the marker or markers measured in (a) with one or more reference levels, thereby optimizing BNP-type peptide-directed therapy.

8. Use of a detection reagent for a marker selected from the group consisting of sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimef, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) for the preparation of a kit for a method of predicting the risk of a patient suffering from heart decompensation, hospitalization and/or death in heart failure therapy with heart failure and receiving a BNP-type peptide, and the method comprises the steps of

(a) Measuring the level of at least one marker selected from sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially hypersensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) in a sample from a patient having heart failure and receiving a BNP-type peptide-directed therapy for heart failure, and

(b) comparing the level or levels of the marker or markers measured in (a) to one or more reference levels.

9. Use according to any one of claims 1 to 8, wherein the patient has heart failure classified according to the ACC/AHA classification as stage B or C, and/or wherein the patient has heart failure of class II or III according to the NYHA classification.

10. Use according to any one of claims 1 to 9, wherein the sample is a blood, serum or plasma sample, and/or wherein the patient is a human.

11. The use according to any one of claims 1 to 10, wherein the heart failure therapy is a pharmaceutical heart failure therapy, in particular wherein the heart failure therapy comprises the administration of at least one drug selected from the group consisting of diuretics, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, β blockers and aldosterone antagonists.

12. The use of claim 11, wherein the heart failure therapy comprises combined administration of an β blocker and an ACE inhibitor.

13. The use according to any of claims 1-12, wherein the intensive heart failure therapy comprises increasing a dose of a previously administered drug, administering an additional drug or drugs, in particular administering an additional drug or drugs having a different mode of action than the previously administered drug, device therapy, lifestyle modification, and combinations thereof.

14. Use of at least one detection reagent for a marker selected from the group of markers consisting of sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, especially high sensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) for the preparation of a kit for identifying a patient as being suitable for intensive heart failure therapy in a sample of a patient having heart failure and receiving BNP-type peptide-directed heart failure therapy, predicting the risk of the patient suffering from cardiac decompensation, hospitalization and/or death, or optimizing a BNP-type peptide-directed heart failure therapy.

15. Use of at least one detection agent which specifically binds to a BNP-type peptide in the manufacture of a kit of at least one detection agent selected from the group of markers consisting of sFlt-1 (soluble fms-like tyrosine kinase-1), IL-6 (interleukin 6), osteopontin, creatinine, urea, sodium, glucose, HbA1C (glycated hemoglobin), hemoglobin, hematocrit, CRP (C-reactive protein, in particular hypersensitive CRP), cystatin C, prealbumin, uric acid, GDF-15 (growth differentiation factor 15), galectin-3 (Gal-3), endostatin, Mimecan, IGFBP-7 (insulin growth factor binding protein 7) and sST2 (soluble ST2) for identifying a patient as being suitable for intensive heart failure therapy in a sample of a patient having heart failure and receiving a BNP-type peptide-directed heart failure therapy, or predicting the risk of the patient suffering from cardiac decompensation, hospitalization, or death.