HK1213324B - Biomarkers in the selection of therapy of heart failure - Google Patents

Description

Biomarkers in the selection of heart failure therapies

The present invention relates to a method for identifying subjects suitable for administration of at least one drug selected from the group consisting of beta-adrenergic receptor blockers (beta-blockers), aldosterone antagonists, diuretics, and angiotensin-converting enzyme (ACE) or angiotensin receptor blocker (ARB) inhibitors, the latter two being collectively referred to as inhibitors of the renin-angiotensin system. The method is based on measuring the amount of at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF-binding protein 7), cardiac troponin, BNP-type peptide, uric acid, galectin-3 (Gal-3), soluble ST2 (sST2), PlGF, sFlt-1, PlNP, cystatin C, prealbumin, and transferrin in a sample from a subject suffering from heart failure. Furthermore, the method includes a step of comparing the measured amount to a reference amount. The present invention further relates to kits and devices suitable for performing the methods of the present invention. The present invention also relates to systems for identifying subjects suitable for administration of at least one agent as disclosed herein, as well as reagents and kits for performing the methods as disclosed herein.

The goal of modern medicine is to provide personalized or individualized treatment plans. These are treatment plans that take into account a patient's individual needs or risks. Personalized or individualized treatment plans should even include the measurements needed to make decisions about potential treatment options.

Heart failure (HF) is a significant and growing public health problem. It is estimated that approximately 5 million patients in the United States suffer from HF, more than 500,000 patients are diagnosed with HF for the first time each year, and more than 250,000 patients die from HF as the leading cause of death in the United States each year. Heart failure (HF) is one of the leading causes of morbidity and mortality in developed countries. Due to an aging population and the longer lifespan of patients with cardiovascular disease, the incidence and prevalence of HF are increasing.

Heart failure is a complex clinical syndrome that can arise from any structural or functional cardiac disorder that impairs the ability of the ventricles to fill or eject blood and ensure the body's metabolic needs for blood supply/oxygen supply. In such cases, the body attempts to compensate for the lack of supply through structural changes in the myocardium (e.g., hypertrophy, ultimately leading to fibrosis, apoptosis, necrosis) and neurotransmitter stimulation (activation of the sympathetic nervous system and the renin-angiotensin-aldosterone system) aimed at maintaining the required supply. HF is classified into different degrees of severity.

One classification is the so-called NYHA (New York Heart Association) classification. Patients with heart failure are classified into NYHA I, II, III, and IV classes, or American College of Cardiology and the American Heart Association (ACC/AHA) stages A, B, C, and D. They cannot fully recover their health and require treatment. Patients in NYHA I class do not have obvious symptoms of cardiovascular disease, but have objective evidence of functional impairment. Patients in NYHA II class have slight limitations in physical activity. Patients in NYHA III class show significant limitations in physical activity. Patients in NYHA IV class are unable to perform any physical activity without discomfort. They show symptoms of cardiac insufficiency when at rest.

This functional classification has been supplemented by more recent classifications from the American College of Cardiology and the American Heart Association (see Am. Coll. Cardiol. 2001;38;2101-2113, updated in 2005, see J. Am. Coll. Cardiol. 2005;46;el-e82). Four stages are defined: A, B, C, and D. A patient with heart failure stage B, C, or D has experienced structural and functional changes in the heart. He or she will not fully recover and requires treatment.

Guidelines for the treatment of acute and chronic heart failure and medications are described in the ESC's guidelines for the diagnosis and treatment of acute and chronic heart failure (European Heart Journal (2008) 29, 2388–2442). Although available treatment options can reduce morbidity and mortality in patients with heart failure (HF), the relative number of patients who are suitable for these treatments remains unsatisfactorily low (O'Donoghue M. & Braunwald E., Nat. Rev. Cardiol. 2010; 7: 13-20). Furthermore, among patients who are suitable for treatment, therapy has been primarily guided and tailored by the signs and symptoms of the underlying disease and HF's maximum drug tolerance (e.g., by NYHA stage, ACC/AHA stage, or congestion fraction).

Measurement of natriuretic peptide markers, such as B-type natriuretic peptide (BNP) or its amino-terminal fragment, N-terminal proBNP (NT-proBNP), has emerged as an important tool for guiding therapy in patients with systolic HF (O'Donoghue M. & Braunwald E., Nat. Rev. Cardiol. 2010;7:13–20). BNP/NT-proBNP-guided HF therapy can identify patients who require therapy intensification and indicate the timing of such intensification. However, BNP/NT-proBNP-guided HF therapy does not indicate which drug therapy a patient may benefit from.

For example, the beneficial effects of spironolactone on cardiovascular disease in patients with severe symptomatic HF of NYHA class II, III, or IV are well known, however, these drugs are also known for serious side effects such as hyperkalemia, arrhythmias, sudden death, hypotension, impotence, muscle weakness, gynecomastia, and gastritis (Williams et al. 2006 Clin. Cardiol. 29, 149-153). In particular, the risk of developing hyperkalemia is a threat (D.N. Juurlink et al. NEJM 2004; 351 543).

In other words, underlying disease, clinical judgment, and BNP/NT-proBNP-guided HF therapy alone may not provide sufficient information regarding the selection of one or more treatments or intensification of therapy. There is a need for new biomarkers, and in particular those that can predict response to therapy or aid in selecting appropriate therapy, intensifying a particular therapy, or conversely avoiding a particular therapy or its intensification.

Therefore, a need exists for biomarkers that allow identification of subjects who will benefit from, or conversely will experience harm from, certain heart failure therapies.

The IGFBP system plays an important role in cell growth and differentiation. IGF binding protein 7 (=IGFBP-7) is a 30-kDa modular glycoprotein known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts and epithelial cells (Ono, Y. et al., Biochem Biophys Res Comm 202 (1994) 1490-1496). In the literature, this molecule has also been named FSTL2; IBP 7; IGF binding protein-related protein 1; IGFBP 7; IGFBP 7v; IGFBP rP1; IGFBP7; IGFBPRP1; insulin-like growth factor binding protein 7; insulin-like growth factor binding protein 7 precursor; MAC25; MAC25 protein; PGI2 stimulating factor; and PSF or prostacyclin stimulating factor. Low levels of IGFB-7 have been detected in random human serum, and increased serum levels have been associated with insulin resistance (Lopez-Bermejo, A. et al., J. Clinical Endocrinology and Metabolism 88 (2003) 3401-3408, Lopez-Bermejo, A. et al., Diabetes 55 (2006) 2333-2339).

US20100285491 discloses the use of IGFBP-7 in the assessment of heart failure. It further discloses that the marker IGFBP-7 can be used to select a treatment regimen for patients with heart failure. Furthermore, it claims that elevated levels of IGFBP-7 during follow-up of patients with heart failure are a positive indicator of effective treatment for heart failure.

WO2008/015254 discloses a method for identifying subjects suitable for heart failure therapy based on GDF-15 detection, preferably drug-based therapy using agents such as aldosterone antagonists, including spironolactone. Preferably, TnT or NT-proBNP is also measured. Furthermore, preferably, a GDF-15 level greater than a reference amount (preferably 1200 pg/ml) indicates that the subject is suitable for heart failure therapy. However, the document does not disclose which drug therapy should be considered, or whether medication adjustments or therapy boosting should be considered.

Mimecan (also known as osteoglycin) is a multifunctional component of the extracellular matrix. Mimecan has been shown to be involved in regulating collagen fibrillogenesis, a process essential for development, tissue repair, and metastasis (Tasheva et al., MoI. Vis. 8 (2002) 407-415). It plays a role in bone formation in combination with TGF-β-1 or TGF-β-2. Transcriptome analysis in rat and human heart tissue revealed a high association of mimecan with left ventricular mass and extracellular remodeling in dilated cardiomyopathy (Petretto, E. et al., Nature Genetics 40 (2008) 546-552).

WO2011/012268 discloses the use of mimecan in a body fluid sample derived from an individual for the assessment of heart failure. It further relates to the use of the marker Mimecan in selecting a treatment regimen for a patient suffering from heart failure. Furthermore, it discloses that elevated levels of Mimecan during follow-up of a patient with heart failure are a positive indicator of effective treatment for heart failure. However, the document does not disclose which medications should be considered, or whether medication adjustments or intensification of therapy should be considered.

Endostatin was initially isolated from rat hemangioendothelioma as a 20 kDA proteolytic fragment of type XVIII collagen (O'Reilly, M.S. et al., Cell 88 (1997) 277-285). Collagen represents a family of extracellular matrix proteins that form supramolecular aggregates with a characteristic triple helical conformation, which plays a leading role in maintaining the integrity of tissue structure. Excessive collagen deposition leads to fibrosis that destroys the normal function of peripheral tissues. Endostatin is released from the α1 chain of collagen XVIII by the action of multiple proteolytic enzymes (Ortega, N. and Werb, Z., Journal of Cell Science 115 (2002) 4201-4214). Endostatin is a powerful inhibitor of angiogenesis and blood vessel growth. However, the document does not disclose what drug therapy should be considered, or whether drug adjustment or therapy enhancement should be considered.

WO2010/124821 discloses measuring endostatin for the assessment of heart failure. It further relates to the use of the marker endostatin in selecting a treatment regimen for patients suffering from HF. In addition, it claims that in the follow-up of HF patients, elevated levels of endostatin are a positive indicator of effective treatment of HF.

Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor beta cytokine superfamily and was first identified as macrophage inhibitory cytokine 1 (MIC-1) and later also named placental transforming growth factor beta (Bootcov 1997, Proc Natl Acad Sci 94:11514-11519; Tan 2000, Proc Natl Acad Sci 97:109-114). GDF-15 has also been described as a strong predictor of cardiovascular events and an indicator of cardiovascular complications (Brown, D.A. et al., 2002 The Lancet, 359:2159-2163; US2003/0232385; Kempf 2006, Circ Res 98:351-360). Kempf et al. showed that circulating levels of GDF-15 correlate with the severity of CHF and predict the risk of death in patients with chronic heart failure (Clinical Chemistry 53:2; 284–291 (2007); Am Coll Cardiol, 2007: 50:1054-1060).

WO2012/025355 discloses not only methods for diagnosing or monitoring functional and/or structural abnormalities of the heart preceding HF, but also guidelines for selecting appropriate drugs to treat such conditions. The marker may be IGFBP7.

WO2010/0070411 discloses a method for monitoring apparently stable subjects with heart failure based on the detection of GDF-15, NT-proANP, NT-proBNP, and cardiac troponin. Furthermore, it discloses methods for diagnosing and/or deciding which therapy/drug treatment to use in apparently stable subjects with heart failure who are experiencing a change in their physiological state.

WO2009/047283 describes a method for determining which treatment or combination of treatments should be used in the remodeling process of a subject after myocardial infarction by measuring BNP, cTnT, and at least one inflammatory marker: osteopontin (OPN), GDF-15, or CRP. It describes osteopontin levels >/= 500 pg/ml as an indicator for ACE inhibitors, angiotensin receptor antagonists, and aldosterone antagonists.

Kubo et al. 2011 (Circulation J, 75, 919-926) reported on the risk prediction of clinical worsening in patients with hypertrophic cardiomyopathy based on the detection of BNP and troponin. Many of the patients evaluated had heart failure. It is speculated that the combined measurement of these two markers could be used to monitor patients with hypertrophic cardiomyopathy.

Fonarow et al. 2008 (Am J Cardiol, 101, 231-237) disclose mortality risk prediction in patients with heart failure based on BNP and troponin measurements.

Mentz et al. 2011 (Circ J, 75(9):2031-7) disclose a method based on NTproBNP detection for therapy guidance and monitoring in heart failure patients, wherein the therapy is selected from treatment with diuretics, ACE inhibitors, beta blockers, spironolactone, nitrates or digoxin.

Böhm et al. 2011 (Clin Res Cardiol, 100:973-981) reviewed methods based on NTproBNP detection for therapy guidance and monitoring in patients with heart failure. They also mentioned the possibility of combining BNP with troponin for guiding heart failure therapy.

In the context of the research underlying the present invention, it has been advantageously shown that the measurement of GDF-15, endostatin, mimecan, IGFBP7, cardiac troponin, BNP-type peptides, uric acid, galectin-3, soluble ST2, PlGF, sFlt-1, PlNP, cystatin C, prealbumin and/or transferrin allows the identification of subjects who will benefit from certain heart failure therapies. It has been shown that only certain groups of patients will benefit from the administration of, for example, beta blockers and aldosterone antagonists, while other groups will not benefit from such administration. By measuring the amounts of the above biomarkers, it is possible to distinguish subjects who will benefit from therapy from subjects who will not benefit from therapy, or even subjects who will experience side effects or harm from therapy due to unnecessary boosting/uptitration in the absence of a beneficial effect.

The technical problem underlying the present invention can be seen as providing means and methods that comply with the above-mentioned need.

The technical problem is solved by the embodiments characterized in the claims and herein below.

Accordingly, the present invention relates to a method for identifying a subject suitable for administration of at least one agent for heart failure therapy, comprising:

a) determining the amount of at least one biomarker in a sample from a subject suffering from heart failure, and

b) comparing one or more amounts of the at least one biomarker determined in step a) with one or more reference amounts, thereby identifying a subject suitable for administration of the at least one agent.

Preferably, the at least one pharmaceutical agent is selected from the group consisting of beta blockers, aldosterone antagonists, diuretics and inhibitors of the renin-angiotensin system.

Preferably, the at least one biomarker is selected from the group consisting of IGFBP7 (IGF-binding protein 7), mimecan, osteopontin, endostatin, sFlt-1 (soluble fms-like casein kinase 1), PlGF (placental growth factor), cardiac troponin, BNP-type peptide, uric acid, GDF-15 (growth differentiation factor 15), sST2 (soluble ST2), Gal-3 (galectin-3), P1NP (procollagen type 1 N-terminal propeptide), cystatin C, prealbumin and transferrin.

Accordingly, the present invention relates to a method for identifying a subject suitable for administration of at least one agent selected from the group consisting of a beta blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system, the method comprising:

a) determining the amount of at least one biomarker selected from the group consisting of IGFBP7 (IGF binding protein 7), mimecan, osteopontin, endostatin, sFlt-1 (soluble fms-like casein kinase 1), PlGF (placental growth factor), cardiac troponin, BNP-type peptide, uric acid, GDF-15 (growth differentiation factor 15), sST2 (soluble ST2), Gal-3 (galectin-3), PINP (procollagen type 1 N-terminal propeptide), cystatin C, prealbumin, and transferrin in a sample from a subject with heart failure, and

b) comparing the one or more amounts determined in step a) with one or more reference amounts, thereby identifying a subject suitable for administration of the at least one pharmaceutical agent.

In a preferred embodiment of the methods of the present invention, at least one agent is an aldosterone antagonist. In this case, the at least one biomarker is selected from IGFBP7, endostatin, uric acid, a BNP-type peptide, cardiac troponin, sFlt-1, PlGF, sST2, Gal-3, and cystatin C. Preferably, the amount of only one of the above biomarkers is measured. However, measuring the amount of more than one biomarker in combination is also contemplated. Preferred combinations are s-Flt-1 and IGFBP7; s-Flt-1 and cardiac troponin; s-Flt-1 and Gal-3; cardiac troponin and Gal-3; cardiac troponin and IGFBP7; IGPBP7 and Gal-3; and combinations of these markers with a BNP-type peptide. A particularly preferred combination is IGFBP7 and mimecan. A further preferred combination is PlGF and sFlt-1. Furthermore, it is contemplated to calculate the ratio of the amount of PlGF to the amount of sFlt-1 (or the ratio of sFlt-1 to the amount of PlGF). In this case, the calculated ratio is compared with a reference ratio.

In another preferred embodiment of the methods of the present invention, the at least one agent is a beta-blocker. In this case, the at least one biomarker is preferably selected from IGFBP7, GDF-15, endostatin, mimecan, a BNP-type peptide, cardiac troponin, osteopontin, sFlt-1, and PlNP. Preferably, the amount of only one biomarker is measured. However, measuring the amount of more than one biomarker in combination is also contemplated. Preferred combinations are GDF-15 and endostatin; GDF-15 and cardiac troponin; GDF-15 and mimecan; GDF-15 and sST2; endostatin and cardiac troponin; endostatin and mimecan; endostatin and IGFBP7; mimecan and cardiac troponin; mimecan and IGFBP7; and cardiac troponin and IGFBP7. A particularly preferred combination is endostatin and sFlt-1. A further preferred combination is endostatin and PlGF.

In another preferred embodiment of the method of the present invention, at least one agent is an inhibitor of the renin-angiotensin system. In this case, the at least one biomarker is preferably osteopontin, GDF-15, a BNP-type peptide, sFlt-1, cardiac troponin, Gal-3, uric acid, or IGFBP7. Preferably, the amount of one biomarker is measured. However, it is also contemplated to measure the amount of more than one biomarker in combination. Preferred combinations are cardiac troponin and Gal-3, cardiac troponin and uric acid, and Gal-3 and uric acid. Further preferred combinations are osteopontin and sFlt-1, sFlt-1 and cardiac troponin, and osteopontin and cardiac troponin.

In another preferred embodiment of the method of the present invention, at least one agent is a diuretic. In this case, the at least one biomarker is preferably selected from GDF-15, endostatin, mimecan, a BNP-type peptide, uric acid, osteopontin, IGFBP-7, Gal-3, transferrin, or prealbumin. Preferably, the amount of only one biomarker is measured. However, it is also contemplated to measure the amount of more than one biomarker in combination. Preferred combinations are GDF-15 and endostatin; GDF-15 and a BNP-type peptide; GDF-15 and mimecan; GDF-15 and Gal-3; endostatin and a BNP-type peptide; endostatin and mimecan; endostatin and Gal-3; mimecan and a BNP-type peptide; mimecan and Gal-3; a BNP-type peptide and Gal-3; a BNP-type peptide and uric acid; a BNP-type peptide and IGFBP-7; a BNP-type peptide and osteopontin. In particular, a combination of uric acid and GDF-15 is contemplated.

Accordingly, the present invention particularly contemplates the following method:

Contemplated are methods for identifying a subject suitable for administration of an aldosterone antagonist (i.e., suitable for initial administration or boosting, particularly at a higher dose) comprising:

a) determining the amount of at least one biomarker selected from the group consisting of IGFBP7, endostatin, uric acid, BNP-type peptide, cardiac troponin, sFlt-1, PlGF, sST-2, Gal-3, and cystatin C in a sample from a subject suffering from heart failure, and

b) comparing the one or more amounts determined in step a) with one or more reference amounts, thereby identifying a subject suitable for administration of said aldosterone antagonist.

In one embodiment, the amounts of PlGF and sFlt-1 are determined in step a), the ratio of the amount of PlGF to the amount of sFlt-1 (or the ratio of the amount of sFlt-1 to PlGF) is calculated in a further step a1), and the calculated ratio is then compared with a reference ratio in step b).

Accordingly, the method may comprise the steps of:

a) determining the amount of PlGF and sFlt-1 in a sample from a subject suffering from heart failure,

a1) calculating the ratio of the amount of PlGF to the amount of sFlt-1 (or vice versa), and

b) comparing the ratio calculated in step a1) with a reference ratio, thereby identifying a subject suitable for administration of said aldosterone antagonist.

Also contemplated are methods for identifying a subject suitable for administration of a beta-blocker (i.e., suitable for initial administration or for a boost in administration, particularly at a higher dose), comprising:

a) determining the amount of at least one biomarker selected from the group consisting of IGFBP7, GDF-15, endostatin, mimecan, BNP-type peptide, cardiac troponin, osteopontin, sFlt-1, and P1NP in a sample from a subject suffering from heart failure, and

b) comparing the one or more amounts determined in step a) with one or more reference amounts, thereby identifying a subject suitable for administration of said beta blocker.

Further envisioned are methods for identifying a subject suitable for administration of a renin-angiotensin system inhibitor (i.e., suitable for initial administration or for a boost in administration, particularly at a higher dose), comprising:

a) determining the amount of a biomarker selected from the group consisting of osteopontin, GDF-15, BNP-type peptide, sFlt-1, cardiac troponin, Gal-3, uric acid, and IGFBP7 in a sample from a subject suffering from heart failure, and

b) comparing the one or more amounts determined in step a) with one or more reference amounts, thereby identifying a subject suitable for administration of said renin-angiotensin system inhibitor.

The present invention also contemplates a method for identifying a subject suitable for diuretic administration (i.e., suitable for initial administration or administration boost), comprising:

a) determining the amount of at least one biomarker selected from the group consisting of IGFBP7, cardiac troponin, endostatin, mimecan, BNP-type peptide, uric acid, osteopontin, Gal-3, prealbumin, and transferrin in a sample from a subject suffering from heart failure, and

b) comparing the one or more amounts determined in step a) with one or more reference amounts, thereby identifying a subject suitable for administration of said diuretic.

Preferably, the subject is identified as suitable for administration of said at least one medicament by performing the further step c) of identifying a subject suitable for administration of said at least one medicament.

In one embodiment, by making sample contact with the reagent of specific binding respective label, thus form the complex between reagent and described label, detect the amount of formed complex, and thus measure the amount of described label, measure the amount of at least one biomarker.If biomarker is uric acid, then the level of described biomarker can be measured by making sample contact with enzyme or compound, and described enzyme or compound allow the conversion of described biomarker, for example, allow the oxidation of uric acid.Enzyme can be the urease (EC 1.7.3.3) that catalyzes the oxidation of uric acid to 5-hydroxyisouric acid.Compound can be phosphotungstic acid.

The following biomarker-agent combinations are the most preferred combinations:

In a preferred embodiment of the methods, uses, kits, systems and devices of the present invention, the biomarker is IGFBP-7 and the pharmaceutical agent is a beta blocker.

In another preferred embodiment, the biomarker is mimecan and the pharmaceutical agent is a beta blocker.

In another preferred embodiment, the biomarker is osteopontin and the agent is an inhibitor of the renin-angiotensin system. It is also envisaged that the agent is a beta blocker.

In another preferred embodiment, the biomarker is endostatin and the agent is an aldosterone antagonist.

In another preferred embodiment, the biomarker is sFlt-1 and the agent is an aldosterone antagonist. Also preferred is that the biomarker is sFlt-1 and the agent is an inhibitor of the renin-angiotensin system. Further, it is contemplated that the agent is a beta blocker.

In another preferred embodiment, the biomarker is PlGF and the agent is an aldosterone antagonist.

In another preferred embodiment, the biomarker is cardiac troponin and the agent is an inhibitor of the renin-angiotensin system.

In another preferred embodiment, the biomarker is a BNP-type peptide and the pharmaceutical agent is an inhibitor of the renin-angiotensin system. It is also preferred that the biomarker is a BNP-type peptide and the pharmaceutical agent is a beta-blocker.

In another preferred embodiment, the biomarker is uric acid and the pharmaceutical agent is a diuretic. Also preferred is that the biomarker is uric acid and the pharmaceutical agent is an inhibitor of the renin-angiotensin system.

In another preferred embodiment, the biomarker is GDF-15 and the pharmaceutical agent is a diuretic. Also preferred is that the biomarker is GDF-15 and the pharmaceutical agent is an inhibitor of the renin-angiotensin system.

In another preferred embodiment, the biomarker is sST2 and the pharmaceutical agent is an aldosterone antagonist. Also preferred is that the biomarker is sST2 and the pharmaceutical agent is a beta blocker.

In another preferred embodiment, the biomarker is PINP and the pharmaceutical agent is a beta blocker.

In another preferred embodiment, the biomarker is cystatin C and the agent is an aldosterone antagonist.

In another preferred embodiment, the biomarker is prealbumin and the agent is a diuretic, such as a loop diuretic.

In another preferred embodiment, the biomarker is IGFBP7 and the agent is an inhibitor of the renin-angiotensin system.

Further preferred biomarker/agent combinations are disclosed in the Examples.

Method of the present invention is preferably ex vivo or in vitro method. In addition, it may include steps other than those clearly mentioned above. For example, a further step may relate to sample pretreatment or evaluate the result obtained by the method. The method can be manually carried out or assisted by automation. Preferably, step (a) and/or (b) can be assisted in whole or in part by automation, for example, by suitable machinery and sensing equipment for the mensuration in step (a) or based on the comparison and/or evaluation by computer implemented in step (b) compared.

Accordingly, the present invention also preferably relates to a system for identifying a subject suitable for administration of at least one agent selected from the group consisting of a beta blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system, the system comprising:

a) an analyzer unit configured to contact in vitro a portion of a sample from a subject with a ligand (or ligands if the amount of more than one biomarker is being determined), the ligand comprising a specific binding affinity for at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, a BNP-type peptide, uric acid, galectin-3 (Gal-3), soluble ST2 (sST2), PlGF, sFlt-1, PlNP, cystatin C, prealbumin, and transferrin,

b) an analyzer unit configured to detect a signal from a portion of the sample from the subject that has come into contact with the ligand (or ligands),

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

d) a non-transitory computer-readable medium comprising a plurality of instructions executable by a processor that, when executed, calculate an amount of at least one biomarker and compare the amount of at least one biomarker to a reference amount (or multiple reference amounts if the amount of more than one biomarker is determined), thereby identifying a subject suitable for administration of at least one pharmaceutical agent.

As used herein, the term "identification" means evaluating whether a subject is suitable for the administration of the at least one medicament. It should be understood that a subject suitable for the administration of the at least one medicament will benefit from the administration of the at least one medicament, while a subject not suitable for the administration of the at least one medicament may experience adverse side effects or injuries from the administration of the at least one medicament. In particular, if, particularly within a window period of 18 months or 3 years after the sample has been obtained, the administration of the at least one medicament reduces the mortality risk of the subject and/or reduces the hospitalization risk of the subject, the subject benefits from the administration of the at least one medicament. Preferably, the risk (or risks) is reduced by 5%, more preferably by 10%, even more preferably by 15%, and most preferably by 20%. Preferably, the hospitalization mentioned herein should be due to heart failure.

In contrast, the object that is not suitable for the administration of at least one medicament will not benefit from (particularly will not significantly benefit from) the administration of at least one medicament.Especially, if particularly in the window period of 18 months or 3 years after sample has been obtained, the administration of described at least one medicament does not reduce the mortality risk of described object (particularly not significantly reduce) and/or does not reduce the hospitalization risk of described object (particularly not significantly reduce) and/or increases unwanted effect risk, then object does not benefit from the administration of described at least one medicament.In this case, if medicament is not used, unnecessary health care costs can be avoided.Further, the adverse side effects that can be caused by administration can be avoided.

Thus, by identifying a subject suitable for administration of the at least one pharmaceutical agent, it is possible to assess whether the subject will benefit from administration (i.e., benefit from an initial administration or a boost in administration, particularly at a higher dose). Accordingly, the present invention also relates to methods for identifying a subject who will benefit from administration of the at least one pharmaceutical agent, based on the steps described elsewhere herein.

As will be understood by those skilled in the art, the evaluation of whether an object is suitable for the administration of the at least one agent is not generally expected to be 100% correct for the object to be evaluated. However, the term requires that the evaluation be correct for a statistically significant portion of the objects (e.g., a cohort in a cohort study). Whether a portion is statistically significant can be determined by those skilled in the art using a variety of well-known statistical evaluation tools without further complication, such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferably, the confidence interval is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80%, or at least 90% of the objects in a population can be appropriately identified by the method of the present invention.

In the context of the present invention, a subject should be identified as suitable for administration of at least one agent selected from the group consisting of beta blockers, aldosterone antagonists, diuretics, and inhibitors of the renin-angiotensin system.

"Administering" in the context of the methods of the invention means that (i) administration of the at least one agent should be initiated (and thus should be added to therapy), or (ii) administration of the at least one agent should be administered at a higher dose (and thus should be up-regulated).

As used herein, the term "dose" preferably refers to a daily dose.

In the first case (i), the subject should not have previously taken the at least one pharmaceutical agent. Thus, the at least one pharmaceutical agent should not have been administered to the subject before the sample to be tested has been obtained. Preferably, the at least one pharmaceutical agent has not been administered to the subject for at least three months, more preferably at least six months, before the sample to be tested has been obtained.

In the latter case (ii), the subject should have been treated with the at least one pharmaceutical agent (before obtaining the sample). Preferably, the subject has been treated with the at least one pharmaceutical agent for at least two weeks, more preferably at least two months, even more preferably at least six months, and most preferably at least one year before obtaining the sample to be tested. Preferably, the dosage of the at least one pharmaceutical agent is not changed during this period.

Particularly preferably, the subject to be tested has already been treated with the at least one pharmaceutical agent, and the subject is evaluated for suitability for administration of the at least one pharmaceutical agent at a higher dose, i.e., whether the therapy should be continued with a higher dose of the at least one drug. Thus, it can be evaluated whether the dose of the at least one pharmaceutical agent should be increased.

In a word, the object that is suitable for the administration of described at least one medicament is suitable for starting the administration of described at least one medicament, or with higher dosage of administration described at least one medicament, and the object that is not suitable for the administration of described at least one medicament is not suitable for starting the administration of described at least one medicament (before obtaining sample to be tested, when object is not treated with described medicament), or with higher dosage of administration described at least one medicament (before obtaining sample to be tested, when object is treated with described medicament). Accordingly, if object is suitable for the administration of medicament, then the administration of described medicament can be started, or the dosage of described medicament can be increased. However, if object is not suitable for the administration of medicament, then the administration of described medicament should not be started or should not be adjusted to higher dosage. Accordingly, if object is treated with described medicament before obtaining sample to be tested, then if object is not suitable for the administration of described medicament, then therapy should continue without changing dosage, particularly without increasing dosage. Also preferably, the object that is not suitable for the administration of described medicament can accept lower dosage of described medicament.

The agents mentioned in the context of the present invention are well known in the art, see for example Heart Disease, 2008, 8th edition, ed. Braunwald, Elsevier Sounders, Chapter 24 or the ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure (European Heart Journal (2008) 29, 2388-2442).

Beta blockers (also commonly referred to as beta adrenergic blockers) block the effects of the endogenous catecholamines adrenaline (epinephrine) and norepinephrine (norepinephrine), particularly beta adrenergic receptors. Preferably, beta blockers are selected from cebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bupranolol, carazolol, carteolol, carvedilol, celiprolol, metipranolol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, tanilolol and timolol. The most preferred beta blockers are atenolol, carvedilol, metoprolol and bisoprolol.

Aldosterone antagonists belong to a class of diuretic drugs that antagonize the effect of aldosterone on mineralocorticoid receptors. They are usually used as adjuvant therapy for the management of chronic heart failure in combination with other drugs. Preferably, aldosterone antagonists are selected from eplerenone, spironolactone, canrenone, hexenone, acerenone. Particularly preferred aldosterone antagonists are spironolactone (7α-acetylthio-3-oxo-17α-pregnane-4-ene 21,17-carboxylactone) or eplerenone.

Diuretics are diuretics that increase the elimination of sodium and water via urine. Particularly preferred diuretics are loop diuretics or thiazide diuretics.

Preferably, loop diuretics are used due to their efficacy and rapid onset of action. They act on the ascending loop of Henle in the kidney. Preferably, the loop diuretic is selected from the group consisting of furosemide, azosemide, bumetanide, piretanide, torsemide, edenic acid, and etosazoline. In particular, the loop diuretic is furosemide.

Preferably, the thiazide diuretic is selected from the group consisting of: hydrochlorothiazide and chlortaildon. A particularly preferred thiazide diuretic is hydrochlorothiazide.

Inhibitors of the renin-angiotensin system (RAS) include angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs, angiotensin II receptor antagonists) and direct renin inhibitors. Preferably, the inhibitor is an ACE inhibitor. Also preferably, the inhibitor is an angiotensin II receptor blocker.

Preferably, the ACE inhibitor is selected from benazepril, captopril, cilazapril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril and trandolapril. Particularly preferred ACE inhibitors are captopril, enalapril, lisinopril, ramipril or trandolapril.

Preferably, the angiotensin II receptor antagonist is selected from losartan, valsartan, irbesartan, candesartan, olmesartan, telmisartan and eprosartan. Particularly preferred ARBs are valsartan, losartan, candesartan or telmisartan.

A preferred direct renin inhibitor is aliskiren.

As described above herein, when a sample to be tested is obtained, the subject to be tested may have been treated with the at least one agent. Therefore, by carrying out the method of the present invention, identification is suitable for a subject to be treated with a higher dose of the at least one agent, the higher dose being a higher dose than the dose of the agent used before obtaining the sample to be tested. Preferably, the daily dose should be higher. In the context of the present invention, it is contemplated that the dosage is increased by up to 500% or more. Preferably, the dosage should be at least 30% higher than the dosage used before obtaining the sample to be tested, more preferably at least 50%, even more preferably at least 100%, or most preferably at least 200%.

As used herein in the context of the above methods, the term "subject" refers to an animal, preferably a mammal, and more preferably a human. In the context of the present invention it is envisaged that the subject suffers from heart failure (HF).

As used herein, the term "heart failure" refers to impaired systolic and/or diastolic function of the heart, accompanied by obvious signs of heart failure as known to those skilled in the art. Preferably, the heart failure referred to herein is also chronic heart failure. Heart failure according to the present invention includes overt and/or advanced heart failure. In overt heart failure, the subject shows symptoms of heart failure as known to those skilled in the art.

HF can be classified into different degrees of severity.

According to the NYHA (New York Heart Association) classification, patients with heart failure are classified as belonging to NYHA classes I, II, III, and IV. Patients suffering from heart failure have experienced structural and functional changes in their pericardium, myocardium, coronary circulation, or heart valves. They cannot fully recover their health and require treatment. Patients in NYHA class I do not have obvious symptoms of cardiovascular disease, but have objective evidence of functional impairment. Patients in NYHA class II have slight limitations in physical activity. Patients in NYHA class III show significant limitations in physical activity. Patients in NYHA class IV are unable to perform any physical activity without discomfort. They show symptoms of cardiac insufficiency when at rest.

This functional classification has been supplemented by more recent classifications by the American College of Cardiology and the American Heart Association (see Am. Coll. Cardiol. 2001;38;2101-2113, updated in 2005, see J. Am. Coll. Cardiol. 2005;46;el-e82). Four stages are defined: A, B, C, and D. Stages A and B are not HF but are considered to help identify patients early, before they develop true "HF." Stage A and B patients are best defined as those with risk factors for the development of HF. For example, patients with coronary artery disease, hypertension, or diabetes who do not have demonstrated impaired left ventricular (LV) function, hypertrophy, or geometric chamber distortion would be considered Stage A, while patients who are asymptomatic but have demonstrated LV hypertrophy and/or impaired LV function would be designated Stage B. Stage C designates patients with current or past HF symptoms related to underlying structural heart disease (the majority of patients with HF), and Stage D designates patients with true refractory HF.

As used herein, the term "heart failure" refers in particular to stages B, C and D of the ACC/AHA classification mentioned above. In these stages, subjects show typical symptoms of heart failure. Accordingly, subjects suffering from heart failure suffer from heart failure stage B, C or D according to the ACC/AHA classification, in particular stage C or D. Additionally preferably, the classifiable subject can be classified as belonging to NYHA class II; III or IV, in particular class III or IV.

Preferably, it is envisaged that the subject in the context of the present invention does not have impaired renal function. Preferably, the subject should not suffer from renal failure, in particular, the subject should not suffer from acute, chronic and/or end-stage renal failure. Further, the subject preferably should not suffer from renal hypertension. How to evaluate whether a subject shows impaired renal function is well known in the art. Renal disorders can be diagnosed by any means known and deemed appropriate. In particular, renal function can be evaluated by means of glomerular filtration rate (GFR). For example, GFR can be calculated by Cockgroft-Gault or MDRD formula (Levey 1999, Annals of Internal Medicine, 461-470). GFR is the volume of fluid filtered from the glomerular capillaries of the kidney into Bowman's capsule per unit time. Clinically, this is commonly used to measure renal function. All calculations derived from formulas such as the Cockgroft-Gault formula of the MDRD formula provide estimates, rather than the "true" GFR obtained by injecting insulin into the plasma). Because insulin is not reabsorbed by the kidney after glomerular filtration, its excretion rate is directly proportional to the filtration rate of water and solutes across glomerular filtration. However, in clinical practice, creatinine clearance is used to measure GFR. Creatinine is an endogenous molecule, synthesized in the body, which is freely filtered by the glomerulus (but also secreted in very small amounts by the renal tubules). Creatinine clearance (CrCl) is therefore a close approximation of GFR. GFR is usually recorded in milliliters per minute (mL/ minute). The normal GFR range for men is 97 - 137 mL/ minutes, and the normal GFR range for women is 88 - 128 ml/ minutes. Therefore, it is particularly contemplated that the GFR of an object with no impaired renal function is within this range. In addition, the object preferably has a blood creatinine level (particularly serum creatinine level) lower than 0.9 mg/dl, more preferably lower than 1.1 mg/dl and most preferably lower than 1.3 mg/dl.

Preferably, the subject does not have ACS (Acute Coronary Syndrome). As used herein, the term "ACS" includes STEMI (ST-segment elevation myocardial infarction); NSTEMI (non-ST-segment elevation myocardial infarction) and unstable angina. It is further contemplated that the subject to be tested does not have a history of ACS. In particular, the subject should not have had ACS within one month prior to performing the method of the present invention (more precisely, within one month prior to obtaining the sample).

As mentioned above, the object to be tested can be treated with the medicament shown in the context of the inventive method. Preferably, the object is treated with a diuretic, a beta blocker, an aldosterone antagonist and/or an inhibitor of the renin-angiotensin system. In this case, the method of the present invention allows identification of an object suitable for administering a medicament at a higher dose, or is used to identify an object that is not suitable for administering a medicament at a higher dose (which preferably can continue therapy without changing dosage).

The term "sample" refers to a body fluid sample, a sample of isolated cells or a sample from a tissue or organ. Body fluid samples can be obtained by well-known techniques and preferably include blood, plasma, serum or urine samples, more preferably blood, plasma or serum samples. Tissue or organ samples can be obtained from any tissue or organ by, for example, biopsy. Isolated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. Preferably, cell, tissue or organ samples are derived from those cells, tissues or organs that express or produce the peptides mentioned herein.

As used herein, the term "BNP-type peptide" encompasses pre-proBNP, proBNP, NT-proBNP, and BNP. The prepropeptide (134 amino acids in the case of pre-proBNP) contains a short signal peptide that is enzymatically cleaved to release the propeptide (108 amino acids in the case of proBNP). The propeptide is further cleaved into the N-terminal propeptide (NT propeptide, 76 amino acids in the case of NT-proBNP) and the active hormone (32 amino acids in the case of BNP). Preferably, the BNP-type peptides according to the present invention are NT-proBNP, BNP, and variants thereof. BNP is the active hormone and has a shorter half-life than its respective inactive counterpart, NT-proBNP. BNP is metabolized into the blood, while NT-proBNP circulates in the blood as an intact molecule and is thus cleared by the kidneys. The in vivo half-life of NT-proBNP is 120 minutes longer than that of BNP, which has a half-life of 20 minutes (Smith 2000, J Endocrinol. 167:239-46). Preanalytics are more robust for NT-proBNP, allowing for easy transport of samples to a central laboratory (Mueller 2004, Clin Chem Lab Med 42:942-4). Blood samples can be stored at room temperature for several days or mailed or shipped without recovery losses. In contrast, BNP storage at room temperature or at 4°C for 48 hours results in a loss of at least 20% of its concentration (Mueller, loc. cit.; Wu 2004, Clin Chem 50:867-73). Therefore, depending on the time course or properties of interest, either the active or inactive form of the natriuretic peptide may be advantageous. The most preferred natriuretic peptide according to the present invention is NT-proBNP or a variant thereof. As briefly discussed above, human NT-proBNP as referred to in accordance with the present invention is a polypeptide preferably comprising 76 amino acids, a length corresponding to the N-terminal portion of the human NT-proBNP molecule. The structures of human BNP and NT-proBNP are described in detail in the prior art, for example in WO 02/089657, WO 02/083913, or Bonow, loc. cit. Preferably, as used herein, human NT-proBNP is human NT-proBNP as disclosed in EP 0 648 228 B1. These prior art documents are hereby incorporated by reference with respect to the specific sequences of NT-proBNP and variants thereof disclosed herein. NT-proBNP as referred to in accordance with the present invention further encompasses alleles and other variants of the specific sequences of human NT-proBNP discussed above. Specifically, variant polypeptides are contemplated that 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 as described above. Also contemplated 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 possesses the properties of NT-proBNP. As referred to herein, the properties of NT-proBNP are immunological and/or biological properties. Preferably, the NT-proBNP variant has immunological properties (i.e., epitope composition) comparable to those of human NT-proBNP. Thus, the variant should be recognizable by the aforementioned means or ligands for determining the amount of natriuretic peptide. Biological and/or immunological NT-proBNP properties can be measured by the assays described in Karl et al. (Karl 1999, Scand J Clin Lab Invest 230: 177-181), Yeo et al. (Yeo 2003, Clinica Chimica Acta 338: 107-115).

The term "growth differentiation factor 15" or "GDF-15" refers to a polypeptide that is a member of the transforming growth factor (TGF) cytokine superfamily. The terms polypeptide, peptide, and protein are used interchangeably throughout this specification. GDF-15 was originally cloned as macrophage inhibitory cytokine 1 and has since been identified as placental transforming growth factor 15, placental bone morphogenetic protein, nonsteroidal anti-inflammatory drug-activated gene 1, and prostate-derived factor (Bootcov, supra; Hromas, 1997 Biochim Biophys Acta 1354:40-44; Lawton 1997, Gene 203:17-26; Yokoyama-Kobayashi 1997, J Biochem (Tokyo), 122:622-626; Paralkar 1998, J Biol Chem 273:13760-13767). Similar to other TGF-related cytokines, GDF-15 is synthesized as an inactive precursor protein that undergoes disulfide-bonded homodimerization. Following proteolytic cleavage of the N-terminal propeptide, GDF-15 is secreted as a ~28 kDa dimeric protein (Bauskin 2000, Embo J 19:2212-2220). The amino acid sequence of GDF-15 is disclosed in WO99/06445, WO00/70051, WO2005/113585, Bottner 1999, Gene 237:105-111, Bootcov loc. cit, Tan supra, Baek 2001, Mol Pharmacol 59:901-908, Hromas supra, Paralkar supra, Morrish 1996, Placenta 17:431-441, or Yokoyama-Kobayashi supra. As used herein, GDF-15 also encompasses variants of the aforementioned specific GDF-15 polypeptides. Such variants possess at least the same basic biological and immunological properties as the specific GDF-15 polypeptide. In particular, they share the same basic biological and immunological properties if they are detectable by the same specific assays described herein, such as by an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize the GDF-15 polypeptide. Preferred assays are described in the accompanying Examples. Furthermore, it should be understood that variants, as described herein, may have an amino acid sequence that differs due to at least one amino acid substitution, deletion, and/or addition, wherein the amino acid sequence of the variant is still preferably 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% identical to the amino acid sequence of the specific GDF-15 polypeptide, preferably to the amino acid sequence of human GDF-15, more preferably over the entire length of the 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 mentioned above can be allelic variants or any other species-specific homologs, paralogs, or orthologs. In addition, the variants mentioned herein include fragments of a specific GDF-15 polypeptide or variants of the above types, so long as these fragments possess the essential immunological and biological properties as mentioned above. Such fragments can be, for example, degradation products of the 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 receptors, type 1 and type 2 IGF receptors, and by 1995, six IGF binding proteins (IGFBPs), IGFBP-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-rP), which have significant structural similarity to IGFBP (Hwa, V. et al., Endocr. Rev 20 (1999) 761-787). Therefore, the IGFBP superfamily includes six conventional IGFBPs with high affinity for IGF, and at least 10 IGFBP-rPs, which not only share the conserved amino terminal domain of IGFBP, but also show a certain degree of affinity for IGF 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 the synthesis of the extracellular matrix. In addition, these proteins may be involved in biological processes such as tissue proliferation and differentiation, reproduction, angiogenesis, wound repair, inflammation, fibrosis, and tumorigenesis (Hwa, V. et al., Endocr. Rev 20 (1999) 761-787).

IGF binding protein 7 (= IGFBP7) is a 30-kDa modular glycoprotein known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono, Y. et al., Biochem Biophys Res Comm 202 (1994) 1490-1496). In the literature, this molecule has been designated as FSTL2; IBP 7; IGF binding protein-related protein 1; IGFBP 7; IGFBP 7v; IGFBP rP1; IGFBP7; IGFBPRP1; insulin-like growth factor binding protein 7; insulin-like growth factor binding protein 7 precursor; MAC25; MAC25 protein; PGI2 stimulating factor; and PSF or prostacyclin stimulating factor. Western blot analysis revealed widespread expression of this gene in human tissues including heart, brain, placenta, liver, skeletal muscle and pancreas (Oh, Y. et al., J. Biol. Chem. 271 (1996) 30322-30325).

IGFBP7 was originally identified as a gene differentially expressed in normal leptomeningeal and breast epithelial cells compared to their paired tumor cells and designated meningioma-associated cDNA (MAC25) (Burger, A.M. et al., Oncogene 16 (1998) 2459-2467). The expressed protein was independently purified as a tumor-derived adhesion molecule (later renamed angiomodulin) (Sprenger, C.C. et al., Cancer Res 59 (1999) 2370-2375) and a prostacyclin stimulator (Akaogi, K. et al., Proc Natl Acad Sci USA 93 (1996) 8384-8389). It has also been reported as T1A12, a gene downregulated in breast cancer (St Croix, B. et al., Science 289 (2000) 1197-1202).

Differential expression of IGFBP7 mRNA has been measured in patients with a variety of diseases including cardiac disease, renal disease, inflammatory disease (US 6,709,855 to Scios Inc.), and vascular transplant disease (US 2006/0,003,338).

Many different assays have been described and used to test the hormone binding protein properties of IGFBP7. Low-affinity IGF binding is analyzed via competitive affinity cross-linking assays. Recombinant human mac25 protein specifically binds IGF-I and -II (Oh, Y. et al., J. Biol. Chem. 271 (1996) 20322-20325; Kim, H.S. et al., Proc. Natl. Acad. Sci USA 94 (1997) 12981-12986). IGFBP activity can also be detected by measuring the ability of the protein to bind radiolabeled IGF in a protein ligand blot.

Preferably, the term "IGFBP7" refers to human IGFBP7. The protein sequence is well known in the art and can be obtained, for example, via GenBank (NP_001240764.1). As used herein, IGFBP7 preferably also encompasses variants of specific IGFBP7 polypeptides. For a description of the term "variant," see above.

Recently, an immunological assay for circulating IGFBP7 was performed. Low levels of this analyte were detected in random human serum, and increased serum levels were associated with insulin resistance (Lopez-Bermejo, A. et al., J. Clinical Endocrinology and Metabolism 88 (2003) 3401-3408, Lopez-Bermejo, A. et al., Diabetes 55 (2006) 2333-2339).

The term "cardiac troponin" also encompasses variants of the above-mentioned specific troponins, i.e., preferably variants of troponin I and more preferably troponin T. Such variants have at least the same basic biological and immunological properties as the specific cardiac troponin. In particular, if they can be detected by the same specific assays mentioned in this specification, such as by ELISA assays using polyclonal or monoclonal antibodies that specifically recognize the cardiac troponin, they share the same basic biological and immunological properties. In addition, it should be understood that variants as mentioned in accordance with the present invention should have an amino acid sequence that differs due to at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of the variant is preferably still 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% identical to the amino acid sequence of the specific troponin. Variants can be allelic variants or any other species-specific homologs, paralogs or orthologs. In addition, the variants mentioned herein include fragments of specific cardiac troponins or variants of the above types, as long as these fragments have the basic immunological and biological properties mentioned above. Preferably, the cardiac troponin variants have immunological properties (i.e., epitope composition) comparable to those of human troponin T or troponin I. Therefore, the variants should be recognizable by the above-mentioned means or ligands for determining cardiac troponin concentrations. Therefore, the variants should be recognizable by the above-mentioned means or ligands for determining cardiac troponin concentrations. Such fragments can be, for example, degradation products of troponin. Further included are variants that differ due to post-translational modifications such as phosphorylation or myristylation. Preferably, the biological properties of troponin I and its variants are the ability to inhibit actomyosin ATPase or inhibit angiogenesis in vivo and in vitro, which can be detected, for example, based on the assay described by Moses et al. 1999 PNAS USA 96 (6): 2645-2650). Preferably, the biological property of troponin T and its variants is the ability to form a complex with troponins C and I, bind calcium ions, or bind tropomyosin, preferably if present as a complex of troponins C, I, and T, or a complex formed from troponin C, troponin I, and a variant of troponin T. It is known that low concentrations of circulating cardiac troponins can be detected in subjects at multiple concentrations, but further research is needed to understand their respective roles and rates (Masson et al., Curr Heart Fail Rep (2010) 7:15–21).

The marker endostatin is well known in the art. Endostatin was originally isolated from mouse hemangioendothelioma as a 20 kDA proteolytic fragment of type XVIII collagen (O'Reilly, M.S. et al., Cell 88 (1997) 277-285). Collagen represents a family of extracellular matrix proteins with a characteristic triple helical conformation, forming supramolecular aggregates that play a leading role in maintaining the integrity of tissue structure. Excessive collagen deposition leads to fibrosis that destroys the normal function of peripheral tissues. Collagen XVIII is a member of the Multiplexin collagen family, with multiple interruptions in the central triple helical domain and a unique non-triple helical domain at the C-terminus, mainly in the basement membrane. The sequence of the short isoform of the human α1 type chain of collagen XVIII (SwissProt: P39060) is disclosed, for example, in WO2010/124821, which is hereby incorporated by reference with respect to its entire disclosure.

Endostatin is released from the α1 chain of collagen XVIII by the action of a variety of proteolytic enzymes (for details see Ortega, N. and Werb, Z., Journal of Cell Science 115 (2002) 4201-4214 - the full disclosure of which is hereby incorporated by reference). As used herein, endostatin is represented by a collagen XVIII fragment spanning 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 metalloproteinases, are known to generate endostatin by cleaving the collagen chains in this region. These proteases are not unique in releasing endostatin, but can release other larger fragments containing the endostatin sequence. As will be apparent to those skilled in the art, such larger fragments are also measured by immunoassays of endostatin.

Osteopontin (also referred to herein as "OPN"), also known as bone sialoprotein 1 (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP1), 2ar, and rickettsial resistance (Ric), is a highly negatively charged polypeptide of the extracellular matrix protein that lacks extensive secondary structure. It consists of approximately 300 amino acids (297 in mice; 314 in humans) and is expressed as a 33-kDa nascent protein; a functionally important cleavage site is also present. OPN can undergo post-translational modifications that increase its apparent molecular weight to approximately 44 kDa. The sequence of osteopontin is well known in the art (human osteopontin: UniProt P10451, GenBank NP_000573.1). Osteopontin is found in normal placenta, 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) 19433-19436; Giachelli, CM. et al., Trends Cardiovasc. Med. 5 (1995) 88-95). Human OPN protein and cDNA have been isolated and sequenced (Kiefer M.C. et al., Nucl. Acids Res. 17 (1989) 3306). OPN plays a role in cell adhesion, chemotaxis, and macrophage-directed interleukin-10. OPN is known to interact with many integrin receptors. Increased OPN expression has been reported in many human cancers, and its cognate receptors (av-b3, av-b5, and av-bl integrins and CD44) have been identified. In vitro studies by Irby, R.B. et al., Clin. Exp. Metastasis 21 (2004) 515-523 indicate that both endogenous OPN expression (via stable transfection) and exogenous OPN (added to the culture medium) enhance the motility and invasiveness of human colon cancer cells in vitro.

Endostatin is a potent inhibitor of angiogenesis and blood vessel growth. The relationship between endostatin and the cytokine network remains to be determined, but it is known that endostatin can alter the expression of a wide range of genes (Abdollahi, A. et al., MoI. Cell 13 (2004) 649-663).

As used herein, endostatin preferably also encompasses variants of the specific endostatin polypeptide.For a description of the term "variant", see above.

Mimecan is a small proteoglycan with a leucine-rich repeat and a precursor comprising 298 amino acids. Other names for mimecan are OGN, osteoglycin, OG, OIF, SLRR3A.

Mimecan is a member of the secreted small leucine-rich proteoglycan (SLRP) family with a structurally related core protein. A common feature shared by all SLRPs is the tandem leucine-rich repeat (LRR) units in the C-terminal half of the core protein. However, in the N-terminal region, each class of SLRP has a unique domain containing a cysteine cluster with conserved intervals called LRRN domains. Class III SLRPs contain six carboxyl LRRs and include mimecan, epican, and opticin.

For class I and II members such as decorin, biglycan, lumecan and fibromodulin, functional studies from mouse knockouts have shown that SLRP-deficient mice exhibit a number of defects attributable to abnormal collagen fibril formation, suggesting that these SLRPs play an important role in establishing and maintaining the collagen matrix (Ameye, L. and Young, M.F., Glycobiology 12 (2002) 107R-116R). Deficiency in class III mimecan also causes collagen fiber abnormalities (Tasheva, E.S. et al., MoI. Vis. 8 (2002) 407-415).

Mimecan is a multifunctional component of the extracellular matrix. It binds to a variety of other proteins (IGF2, IKBKG, IFNB1, INSR, CHUK, IKBKB, NFKBIA, IL15, Cd3, retinoic acid, APP, TNF, lipopolysaccharide, c-abl oncogene 1, receptor tyrosine kinase, v-src sarcoma viral oncogene). These different binding activities may be responsible for the ability of mimecan to perform different functions in various tissues.

Mimecan has been found in cornea, bone, skin and more tissues. Its expression pattern changes under different pathological conditions. Although the amount of data for the biological effect of mimecan increases, its function is still unclear. Mimecan has been shown to be involved in regulating collagen fibril formation, which is a necessary process in development, tissue repair and metastasis (Tasheva et al., MoI.Vis.8 (2002) 407-415). It works in conjunction with TGF-β-1 or TGF-β-2 in bone formation.

The sequence of the human mimecan polypeptide is well known in the art and can be obtained, for example, via GenBank accession number NP_054776.1 GI:7661704. Further, the sequence is disclosed in WO2011/012268. As used herein, mimecan preferably also encompasses variants of specific mimecan polypeptides. For an explanation of the term "variant," see above. In the context of the present invention, mimecan is preferably assayed as described in WO2011/012268.

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

As used herein, the term "sFlt-1" also encompasses variants of the specific sFlt-1 polypeptides described above. Such variants have at least the same basic biological and immunological properties as the specific sFlt-1 polypeptide. In particular, they share the same basic biological and immunological properties if they are detectable by the same specific assays mentioned herein, such as by an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize the sFlt-1 polypeptide. See above for a more detailed description of the term "variant."

As used herein, the term "PlGF" (placental growth factor) refers to a placenta-derived growth factor that is a 149 amino acid polypeptide and is highly homologous (53% identity) to the placenta-derived growth factor-like region of human vascular endothelial growth factor (VEGF). Like VEGF, PlGF has angiogenic activity in vitro and in vivo. For example, biochemical and functional characterization of PlGF derived from transfected COS-1 cells revealed that it is a glycosylated, dimeric, secreted protein capable of stimulating endothelial cell growth in vitro (Maqlione 1993, Oncogene 8(4):925-31). Preferably, PlGF refers to human PlGF, more preferably human PlGF having the amino acid sequence shown in Genebank Accession No. P49763, GI:17380553 (Genebank is available from NCBI, USA at www.ncbi.nlm.nih.gov/entrez).

In the context of the methods of the present invention, determining the amount of a human peptide or polypeptide is particularly envisaged.

Uric acid is the end product of purine metabolism in the subject organism. Its IUPAC name is 7,9-dihydro-3H-purine-2,6,8-trione. This compound is also commonly known as urate, uric acid, 2,6,8 _- trioxopurine, 2,6,8-trihydroxypurine, 2,6,8- _trioxopurine , and 1H-purine- _2,6,8 -triol (compound formula: _{C₅H₄N₄O₃} , PubChem CID 1175, CAS number 69-93-2).

Uric acid measurement is used in the diagnosis and treatment of numerous renal and metabolic disorders, including renal failure, gout, leukemia, psoriasis, starvation, or other wasting conditions, as well as in patients receiving cytotoxic drugs. The oxidation of uric acid provides the basis for two methods for the quantitative determination of this purine metabolite. One method is the reduction of phosphotungstic acid to tungsten blue in an alkaline solution, which is measured photometrically. The second method, described by Praetorius and Poulson, utilizes uricase to oxidize uric acid; this method eliminates the interference inherent in chemical oxidation (Praetorius E, Poulsen H. Enzymatic Determination of Uric Acid with Detailed Directions. Scandinav J Clin Lab Investigation 1953; 3: 273-280). Uricase can be used in methods involving UV measurements of uric acid consumption or in combination with other enzymes to provide colorimetric determinations. Another method is the colorimetric method developed by Town et al. (Town MH, Gehm S, Hammer B, Ziegenhorn J. J Clin Chem Clin Biochem 1985; 23: 591). The sample is initially incubated with a reagent mixture containing ascorbate oxidase and a cleaning system. In this test system, it is important that any ascorbic acid present in the sample is eliminated in the initial reaction; this eliminates any ascorbic acid interference with the subsequent POD indicator reaction. After the initial reagent is added, uric acid oxidation by uricase begins.

In the context of the present invention, uric acid can be measured by any method considered as appropriate. Preferably, biomarker is measured by the above method. More preferably, uric acid is measured by a slight modification of the colorimetry described above. In this reaction, superoxide reacts in the presence of peroxidase (POD), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) and 4-aminopyrine to form benzoquinone-diimine dye. The red intensity formed is proportional to the uric acid concentration and photometric determination.

Galectin-3 (Gal-3) is a structurally unique 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. Galectin-3 has been implicated in multiple biological processes important in heart failure, including myofibroblast proliferation, fibrosis, tissue repair, cardiac remodeling, and inflammation. Galectin-3 is approximately 30 kDa and, like all galectins, contains a carbohydrate recognition binding domain (CRD) of approximately 130 amino acids that allows for specific binding of β-galactosides. Galectin-3 is encoded by a single gene, LGALS3. It comprises an N-terminal domain with tandem repeats of short amino acid stretches (110–130 amino acids total), linked to a single C-terminal CRD of approximately 130 amino acids. It is expressed in the nucleus, cytoplasm, mitochondria, cell surface, and extracellular space. This protein has been shown to be involved in the following biological processes: cell adhesion, cell activation and chemoattraction, cell growth and differentiation, cell cycle and apoptosis. Elevated levels of galectin-3 have been found to be significantly associated with a higher risk of death in both acute decompensated heart failure and chronic heart failure populations (see, e.g., DeFilippi C, Christenson R, Shah R, et al. (2009). Clinical validation of a novel assay for galectin-3 for risk assessment in acutely destabilized heart failure.).

The protein sequence of Galectin-3 is well known in the art, see for example Uniprot Accession No. P17931 (version 5, November 25, 2008), GenBank Accession Nos. 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 conditions of mechanical stress. ST2 is a member of the interleukin-1 receptor family and exists as both a membrane-bound isoform and a soluble isoform (sST2). In the context of the present invention, the amount of soluble ST2 should be measured (see Dieplinger et al. (Clinical Biochemistry, 43, 2010: 1169-1170). ST2 is also known as interleukin-1 receptor-like 1 or IL1RL1 and is encoded by the IL1RL1 gene in humans. The sequence of human ST2 polypeptides is well known in the art and, for example, can be obtained via GenBank, see NP_003847.2 GI: 27894328. Soluble ST2 (sST2) is believed to act as a decoy receptor that binds to IL-33 and cancels the otherwise cardioprotective effects of IL-33 signal transduction via the cell membrane-bound form ST2.

The marker cystatin C is well known in the art. Cystatin C is encoded by the CST3 gene and is produced at a constant rate by all nucleated cells, and its productivity in humans is surprisingly constant throughout life. It is eliminated from the circulation almost completely via glomerular filtration. For this reason, within the age range of 1-50 years, the serum concentration of cystatin C is independent of muscle mass and gender. Therefore, cystatin C in plasma and serum has been proposed as a more sensitive marker for GFR. The sequence of human cystatin C polypeptide can be obtained via Genbank (see, for example, accession number NP_000090.1). Biomarkers can be measured by particle-enhanced immunoturbidimetric assay. Human cystatin C aggregates with latex particles coated with anti-cystatin C antibodies. Turbidimetric assay aggregates.

The marker prealbumin is well known to those skilled in the art. It is a tryptophan-rich protein that is synthesized in hepatocytes and has a molecular weight of 55,000 daltons. At pH 8.6, due to its greater diffusivity to the anode, the electrophoretic band appears before albumin in a relative amount of <2.5%. Its function is to bind and transport low molecular weight retinol binding protein (molecular weight less than 21,000 daltons), preventing its glomerular filtration. 30-50% of circulating prealbumin is complexed with retinol binding protein. In addition, it binds and transports thyroxine (T4), however, its affinity for this hormone is less than that of thyroxine binding globulin. The sequence of the human prealbumin polypeptide can be obtained via Genbank (see, for example, accession number NP_000362.1). Various methods can be used to measure prealbumin, such as radial immunodiffusion (RID), nephelometry, and turbidimetry.

Transferrin (also commonly referred to as serum transferrin or beta-1 metal binding globulin) is a glycoprotein with a molecular weight of approximately 79,570 daltons. It consists of a polypeptide chain with two N-glycosidically bonded oligosaccharide chains. Transferrin is an iron-binding transport protein that can bind two Fe ³⁺ ions with the combination of anions, typically bicarbonate. Various methods can be used to measure transferrin, including radial immunodiffusion, nephelometry, and turbidimetry. Transferrin is a sequence well known in the art, see, for example, Schaeffe et al. Gene 56:109-116 (1987) or Uniprot registration number P02787, particularly version 178).

P1NP (procollagen type 1 N-terminal propeptide) is a marker for bone formation. It is a specific indicator of type 1 collagen deposition. It is released as a trimeric structure but degrades into monomers. Preferably, the total amount of P1NP (total procollagen type 1 N-terminal propeptide) is measured.

References herein to determining the amount of a peptide or polypeptide, particularly GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, Gal-3 (galectin-3), sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, or prealbumin, refer to preferably semi-quantitative or quantitative measurement of the amount or concentration. The measurement can be performed directly or indirectly.

How to determine the amount of uric acid is described above. If the biomarker is a peptide or polypeptide such as GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3) or sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C or prealbumin, the following applies:

Direct measurement refers to measuring the amount or concentration of a peptide or polypeptide based on a signal derived from the peptide or polypeptide itself, and its intensity is directly related to the number of peptide molecules present in the sample. Such a signal - sometimes referred to herein as an intensity signal - can be obtained, for example, by measuring the intensity value of a specific physical or chemical property of the peptide or polypeptide. Indirect measurement includes measuring a signal derived from a second component (i.e., a component other than the peptide or polypeptide itself) or a biological readout system, such as a measurable cellular response, ligand, label, or enzymatic reaction product.

According to the present invention, the amount of the peptide or polypeptide can be determined by all known means of the amount of the peptide in the sample. Said means include immunoassays and methods, which can utilize labeled molecules in the form of multiple sandwich, competition or other assays. Such assays are preferably based on detection reagents such as antibodies that specifically recognize the peptide or polypeptide to be determined. Detection reagents should be able to generate, directly or indirectly, a signal indicating the presence or absence of the peptide or polypeptide. In addition, signal intensity can preferably be directly or indirectly (e.g., inversely proportional) associated with the amount of polypeptide present in the sample. Further suitable methods include measuring physical or chemical properties specific to the peptide or polypeptide, such as its precise molecular weight or NMR spectrum. Said method preferably includes a biosensor, an optical device coupled to an immunoassay, a biochip, an analytical device such as a mass spectrometer, an NMR-analyzer or a chromatographic device. Further methods include microplate ELISA-based methods, fully automated or robotic immunoassays (e.g., available on the Elecsys ^™ analyzer), CBA (enzymatic cobalt binding assay, available on the Roche-Hitachi ^™ analyzer), and latex agglutination assays (e.g., available on the Roche-Hitachi ^™ analyzer).

Preferably, the amount of the peptide or polypeptide is determined by contacting the cell with the peptide or polypeptide for a sufficient period of time, the intensity of the cell response indicating the amount of the peptide or polypeptide, and (b) measuring the cell response. For measuring the cell response, the sample or processed sample is preferably added to the cell culture and the internal or external cell response is measured. The cell response can include measurable reporter gene expression or secretion of substances such as peptides, polypeptides or small molecules. The expression or substance should generate an intensity signal associated with the amount of the peptide or polypeptide.

Also preferably, determining the amount 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 signal can be the signal intensity observed at an m/z variable specific for the peptide or polypeptide observed in a mass spectrum or NMR spectrum specific for the peptide or polypeptide.

Determining the amount of a peptide or polypeptide may preferably comprise the steps of: (a) contacting the peptide with a specific ligand, (b) (optionally) removing unbound ligand, and (c) measuring the amount of bound ligand.

According to a preferred embodiment, the contacting, removing and measuring steps can be performed by an analyzer unit of the system disclosed herein. According to some embodiments, the steps can be performed by a single analyzer unit of the system or by more than one analyzer unit that are operably in communication with each other. For example, according to a specific embodiment, the system disclosed herein may include a first analyzer unit for performing the contacting and removing steps, and a second analyzer unit operably connected to the first analyzer unit by a transport unit (e.g., a robotic arm), the second analyzer unit performing the measuring step.

The binding ligand, particularly the ligand or ligand/peptide complex, generates an intensity signal. Binding according to the present invention includes covalent and non-covalent binding. The ligand according to the present invention can be any compound that binds to the peptide or polypeptide described herein, such as a peptide, polypeptide, nucleic acid, or small molecule. Preferred ligands include antibodies, nucleic acids, peptides, or polypeptides, such as receptors or binding partners for the peptide or polypeptide and fragments thereof comprising the binding domain of the peptide, and aptamers, such as nucleic acid or peptide aptamers. Methods for preparing such ligands are well known in the art. For example, the identification and production of suitable antibodies or aptamers are also provided by commercial suppliers. Those skilled in the art are familiar with methods for developing derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced into nucleic acids, peptides, or polypeptides. These derivatives can then be tested for binding according to screening procedures known in the art, such as phage display. Antibodies, as mentioned herein, include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab, and F(ab)2 fragments capable of binding to an antigen or hapten. The present invention also includes single-chain antibodies and humanized hybrid antibodies, in which the amino acid sequence of a non-human donor antibody exhibiting the desired antigenic specificity is combined with the sequence of a human receptor antibody. The donor sequence typically includes at least the antigen-binding amino acid residues of the donor, but may also include other structural and/or functional related amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. Preferably, the ligand or reagent specifically binds to the peptide or polypeptide. Specific binding according to the present invention means that the ligand or reagent should not be combined in large quantities with another peptide, polypeptide or substance present in the sample to be analyzed ("cross reaction"). Preferably, the specifically bound peptide or polypeptide should be combined with an affinity that is at least 3 times, more preferably at least 10 times and even more preferably at least 50 times higher than any other related peptide or polypeptide. Non-specific binding can be tolerable if it can still be clearly distinguished and measured, for example, according to its size on Western blot, or by its relatively higher abundance in the sample. The binding of the ligand can be measured by any method known in the art. Preferably, the method is semi-quantitative or quantitative. Further suitable techniques for measuring polypeptides or peptides are described below.

First, the combination of the part can be measured directly, for example, by NMR or surface plasmon resonance. According to a preferred embodiment, the measurement of ligand binding is performed by the analyzer unit of the system disclosed herein. Thereafter, the bound amount measured can be calculated by the computing device of the system disclosed herein. Secondly, if the part also serves as the substrate of the enzymatic activity of the target peptide or polypeptide, the enzymatic reaction product can be measured (for example, the amount of protease can be measured by, for example, measuring the amount of the cleaved substrate on a Western blot). Alternatively, the part can itself show enzymatic properties, and "part/peptide or polypeptide" complex or the part bound by the peptide or polypeptide can be contacted with a suitable substrate, allowing detection of the generation of intensity signals. For the measurement of the enzymatic reaction product, preferably, the amount of substrate is saturated. The substrate can also be labeled with a detectable label before the reaction. Preferably, the sample contacts the substrate for a sufficient time period. Sufficient time period refers to the time required for producing a detectable, preferably measurable amount of product. Instead of measuring the amount of product, the time required for the occurrence of a given (for example, detectable) amount of product can be measured. Third, the ligand can be covalently or non-covalently coupled to a label, allowing detection and measurement of the ligand. Labeling can be accomplished by direct or indirect methods. Direct labeling involves direct (covalent or non-covalent) coupling of a label to a ligand. Indirect labeling involves the binding (covalent or non-covalently) of a second ligand to a first ligand. The second ligand should specifically bind to the first ligand. The second ligand can be coupled to a suitable label and/or be the target (receptor) of a third ligand that binds to the second ligand. The use of second, third, or even higher-order ligands is generally used to increase the signal. Suitable second and higher-order ligands can include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The ligand or substrate can also be "tagged" with one or more tags as known in the art. Such tags can then be the target of higher-order ligands. Suitable tags include biotin, digoxygenin, His tags, glutathione S-transferase, FLAG, GFP, myc tags, influenza virus type A hemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and/or the C-terminus. Suitable labels are any labels that can be detected by an appropriate detection method. Typical labels include gold particles, latex beads, acridan esters (acridan ester), luminol, ruthenium, enzymatic activity labels, radiolabels, magnetic labels ("such as magnetic beads", including paramagnetic and superparamagnetic labels) and fluorescent labels. Enzymatic activity labels include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase and derivatives thereof. Suitable detection substrates include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stock solutions from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), ECF™ (Amersham Biosciences). The enzyme-substrate combination can result in a colored reaction product, fluorescence or chemiluminescence, which can be measured according to methods known in the art (e.g., using a photosensitive film or a suitable camera system). About measuring enzymatic reactions, the standard given above is similarly applied. Typical fluorescent markers include fluorescent proteins (e.g., GFP and derivatives thereof), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa 568). Further fluorescent markers are, for example, available from Molecular Probes (Oregon). The use of quantum dots as fluorescent markers is also contemplated. Typical radioactive labels include 35S, 125I, 32P, 33P, etc. Radioactive labels can be detected by any known and appropriate method, such as photosensitive film or phosphorimager. Suitable measuring methods according to the invention also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electrogenerated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immunoassay, electrochemiluminescence sandwich immunoassay (ECLIA), dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetry, nephelometry, latex-enhanced turbidimetry or turbidimetry, or solid phase immunoassay. Further methods known in the art (e.g., gel electrophoresis, 2D gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and mass spectrometry) can be used alone or in combination with labels or other detection methods as described above.

The amount of peptide or polypeptide can also be preferably determined as follows: (a) a solid support comprising a ligand of a peptide or polypeptide as described above is contacted with a sample comprising the peptide or polypeptide, and (b) the amount of the peptide or polypeptide bound to the support is measured. The ligand preferably selected from nucleic acids, peptides, polypeptides, antibodies and aptamers is preferably present on the solid support in an immobilized form. The materials used to make the solid support are well known in the art and particularly include commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and/or silicon chips and surfaces, nitrocellulose test strips, membranes, sheets, duracytes, holes and walls of reaction trays, plastic tubing, etc. The ligand or reagent can be combined with many different supports. Well-known examples of supports include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose and magnetite. For the purposes of the present invention, the nature of the support can be soluble or insoluble. Suitable methods for immobilizing/fixing the ligands are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like. It is also contemplated to use a "suspension assay" as an assay according to the present invention (Nolan 2002, Trends Biotechnol. 20 (1): 9-12). In such a suspension assay, a carrier, such as a microbead or microsphere, is present in suspension. The array consists of different microbeads or microspheres carrying different ligands, possibly labeled. Methods for producing such assays, for example based on solid phase chemistry and photolabile protecting groups, are generally known (US 5,744,305).

Preferably, the amount of individual biomarkers as mentioned herein is determined as described in the Examples section.

As used herein, the term "amount" encompasses the absolute amount of a biomarker, the relative amount or concentration of the biomarker, and any value or parameter associated therewith or that can be derived therefrom. Such values or parameters include intensity signal values derived from all specific physical or chemical properties of the peptide by direct measurement, such as intensity values in a mass spectrum or NMR spectrum. In addition, encompassed are all values or parameters obtained by indirect measurements specified elsewhere in this specification, such as response levels determined by a biological readout system that responds to a peptide or intensity signal derived from a specific binding ligand. It should be understood that the values associated with the above-mentioned amounts or parameters can also be obtained by all standard mathematical operations. According to a preferred embodiment of the present invention, the determination of "amount" is performed by the disclosed system, whereby a computing device determines "amount" based on the contact and measurement steps performed by one or more analyzer units of the system.

As used herein, the term "comparison" encompasses comparing the amount of a biomarker, particularly a peptide or polypeptide, contained in a sample to be analyzed with the amount of a suitable reference source specified elsewhere in this specification. It should be understood that comparison, as used herein, refers to the comparison of corresponding parameters or values, such as the absolute amount compared to the absolute reference amount, the concentration compared to the reference concentration, or the intensity signal obtained from the test sample compared to the intensity signal of the same type from the reference sample. The comparison mentioned in step (b) of the method of the present invention can be performed manually or with the assistance of a computer. Therefore, the comparison mentioned in step (b) of the method of the present invention can be performed by a computing device (such as the system disclosed herein). The values of the amount and the reference can be compared, for example, to each other, and the comparison can be performed automatically by a computer program that executes a comparison algorithm. The computer program that performs the evaluation provides the desired evaluation in a suitable output format. For computer-assisted comparisons, the value of the measured amount can be compared by a computer program with the value corresponding to the suitable reference stored in a database. The computer program can further evaluate the results of the comparison, i.e., automatically provide the desired evaluation in a suitable output format. For computer-assisted comparisons, the value of the measured amount can be compared by a computer program with the value corresponding to the suitable reference stored in a database. The computer program may further evaluate the results of the comparison, ie automatically provide the required evaluation in the form of a suitable output. The results may preferably serve as an aid for identifying subjects suitable for administration of at least one medicament, as described elsewhere herein.

As used herein, the term "reference amount" (or reference ratio) preferably refers to an amount (or ratio) that allows the assignment of a subject to a group of subjects suitable for administration of the at least one pharmaceutical agent, or a group of subjects not suitable for administration of the at least one pharmaceutical agent, as described in the context of the methods of the present invention. Accordingly, the reference amount (or reference ratio) should allow the identification of subjects suitable for administration of the at least one pharmaceutical agent.

Such a reference amount (or ratio) can be a threshold amount that separates these groups from each other. Identification can be provided by a computing device of the system disclosed herein based on the comparison of the calculated "amount" (or ratio) with a reference or threshold. For example, the computing device of the system can provide an indicator in the form of a word, symbol or numerical value, which is an indication of the identification of the object.

Depending on various physiological parameters such as age, sex or subpopulation, and the means used to determine the polypeptide or peptide mentioned herein, the reference amount (or reference ratio) applicable to individual subjects may vary. A suitable reference amount can be determined from a reference sample to be analyzed together with (i.e., simultaneously or subsequently to) the test sample.

In principle, a reference amount (or ratio) can be calculated by applying standard statistical methods based on the mean or average of a given biomarker by a subject cohort as specified above. In particular, the accuracy of a test, such as a method for determining whether an event is present, is best described by its receiver operating characteristic (ROC) (see, inter alia, Zweig 1993, Clin. Chem. 39:561-577). The ROC curve is a curve of all sensitivity versus specificity pairs resulting from continuously changing the decision threshold across the entire data range observed. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign an object to a certain prognosis or diagnosis. The ROC curve plots sensitivity versus specificity for a full threshold range suitable for making a distinction, indicating the overlap between the two distributions. On the y-axis is sensitivity, or the true positive fraction, which is defined as the ratio of the number of true positive test results to the product of the number of true positive and false negative test results. In the presence of a disease or condition, this is also referred to as the positive rate. It is calculated separately by the affected subgroup. On the x-axis is the false positive portion or 1 specificity, which is defined as the ratio of the false positive result number to the true negative number and the product of the false positive test result number. It is a specific index, and is calculated completely by the uninvolved subgroup. Because true positive and false positive portions are calculated separately completely, so by using the test results from two different subgroups, the ROC curve does not rely on the event prevalence rate in the queue. Each point on the ROC curve represents the sensitivity/specificity right corresponding to a specific decision threshold. There is a completely different test (no overlap in the two result distributions) with an ROC curve through the upper left corner, wherein the true positive portion is 1.0, or 100% (complete sensitivity), and the false positive portion is 0 (complete specificity). The theoretical curve of the indifferent test (the identical distribution of the result of two groups) is a 45 ° diagonal line from the lower left corner to the upper right corner. Most of the curves are between these two extremes. If the ROC curve is completely below the 45 ° diagonal line, then this is easily remedied by reversing the "positive rate" standard from "greater than" to "less than" or vice versa. Qualitatively, the closer the curve is to the upper left corner, the higher the overall accuracy of the test. Depending on the required confidence interval, the threshold value can be derived from the ROC curve that allows the identification of the object suitable for administration as mentioned herein, with the appropriate balance of sensitivity and specificity. Accordingly, preferably, by establishing the ROC for the queue as described above and deriving the threshold value therefrom, a reference for the above-mentioned method of the present invention can be generated, i.e., a threshold value that allows the distinction between the object suitable for administration as mentioned herein, or the object that is not suitable for the administration. Depending on the required sensitivity and specificity of the diagnostic method, the ROC curve allows the derivation of a suitable threshold value.

The diagnostic algorithm, i.e. whether the administration of the agent should be initiated, or whether the dosage of the agent should be increased ("up-regulated"), is disclosed in the Examples section. A preferred diagnostic algorithm is summarized below:

If at least one of the agents is a beta-blocker and if at least one of the biomarkers is endostatin, mimecan, GDF-15, cardiac troponin and/or a BNP-type peptide, the following preferably applies:

Preferably, an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is increased compared to a reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the at least one agent, and/or an amount (or amounts) of the biomarker (or multiple biomarkers) that is decreased compared to a reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the at least one agent.

More preferably, the subject to be tested has been treated with a beta-blocker. In this case, an increased amount (or amounts) of the biomarker (or biomarkers) compared to a reference amount (or amounts) indicates that the subject is suitable for administration of the agent at a higher dose, and/or a decreased amount (or amounts) of the biomarker (or biomarkers) compared to a reference amount (or amounts) indicates that the subject is not suitable for administration of the agent at a higher dose.

If the agent is a beta blocker, and if the biomarker is IGFBP7, P1NP, sFlt-1, or osteopontin, the following preferably applies:

Preferably, the amount (or amounts) of one or more biomarkers in the test sample that is reduced compared to one or more reference amounts indicates that the subject is suitable for administration of the beta-blocker, and/or the amount (or amounts) of one or more biomarkers that is increased compared to one or more reference amounts indicates that the subject is not suitable for administration of the beta-blocker. In particular, the amount (or amounts) of one or more biomarkers in the test sample that is reduced compared to one or more reference amounts indicates that the subject is suitable for administration of the beta-blocker.

More preferably, the subject to be tested has been treated with a beta-blocker. In this case, the amount (or amounts) of one or more biomarkers in the test sample that are reduced compared to one or more reference amounts indicate that the subject is suitable for administration of the agent at a higher dose, and/or the amount (or amounts) of one or more biomarkers that are increased compared to one or more reference amounts indicate that the subject is not suitable for administration of the agent at a higher dose. In particular, the amount (or amounts) of one or more biomarkers in the test sample that are reduced compared to one or more reference amounts indicate that the subject is suitable for administration of the beta-blocker at a higher dose.

If at least one agent is an aldosterone antagonist, and if at least one biomarker is IGFBP7, cardiac troponin, Gal-3, cystatin C, PlGF, GDF-15 and/or sST2, the following preferably applies:

Preferably, an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is increased compared to a reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the at least one agent, and/or an amount (or amounts) of the biomarker (or multiple biomarkers) that is decreased compared to a reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the at least one agent.

More preferably, the subject to be tested has been treated with an aldosterone antagonist. In this case, the amount (or amounts) of the biomarker (or biomarkers) that increases compared to the reference amount (or reference amounts) indicates that the subject is suitable for administration of the one or more pharmaceutical agents at a higher dose, and/or the amount (or amounts) of the biomarker (or biomarkers) that decreases compared to the reference amount (or reference amounts) indicates that the subject is not suitable for administration of the one or more pharmaceutical agents at a higher dose.

If the ratio of the amount of PlGF to the amount of sFlt-1 is determined, the following is applied as a diagnostic algorithm.

Preferably, an increased ratio of the amount of PlGF to the amount of sFlt-1 in the test sample compared to the reference ratio indicates that the subject is suitable for administration of the aldosterone antagonist, and/or a decreased ratio compared to the reference ratio indicates that the subject is not suitable for administration of the aldosterone antagonist.

More preferably, the subject to be tested has been treated with an aldosterone antagonist. In this case, an increased ratio of the amount of PlGF to the amount of sFlt-1 in the test sample compared to the reference ratio indicates that the subject is suitable for administration of the aldosterone antagonist at a higher dose, and/or a decreased ratio compared to the reference ratio indicates that the subject is not suitable for administration of the aldosterone antagonist at a higher dose.

If at least one of the agents is an aldosterone antagonist, and if at least one of the biomarkers is endostatin, uric acid and/or sFlt-1, the following preferably applies:

Preferably, an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is reduced compared to a reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the at least one agent, and/or an amount (or amounts) of the biomarker (or multiple biomarkers) that is increased compared to a reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the at least one agent.

More preferably, the subject to be tested has been treated with an aldosterone antagonist. In this case, the amount (or amounts) of the biomarker (or biomarkers) that is reduced compared to the reference amount (or reference amounts) indicates that the subject is suitable for administration of the medicament at a higher dose, and/or the amount (or amounts) of the biomarker (or biomarkers) that is increased compared to the reference amount (or reference amounts) indicates that the subject is not suitable for administration of the medicament at a higher dose.

If at least one of the pharmaceutical agents is an inhibitor of the renin-angiotensin system and if at least one of the biomarkers is GDF-15, cardiac troponin, uric acid, a BNP-type peptide and/or osteopontin, the following preferably applies:

Preferably, an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is increased compared to a reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the at least one agent, and/or an amount (or amounts) of the biomarker (or multiple biomarkers) that is decreased compared to a reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the at least one agent.

More preferably, the subject to be tested has been treated with an inhibitor of the renin-angiotensin system. In this case, an amount (or amounts) of the biomarker (or biomarkers) that is increased compared to a reference amount (or reference amounts) indicates that the subject is suitable for administration of the agent at a higher dose, and/or an amount (or amounts) of the biomarker (or biomarkers) that is decreased compared to a reference amount (or reference amounts) indicates that the subject is not suitable for administration of the agent at a higher dose.

If at least one of the agents is a diuretic and at least one of the biomarkers is selected from the group consisting of IGFBP-7, endostatin, mimecan, GDF-15, prealbumin, transferrin, a BNP-type peptide and uric acid, the following is preferably applied:

Preferably, an amount (or amounts) of a biomarker (or multiple biomarkers) in a test sample that is increased compared to a reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the agent, and/or an amount (or amounts) of a biomarker (or multiple biomarkers) that is decreased compared to a reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the agent.

More preferably, the subject to be tested has been treated with a diuretic. In this case, the amount (or amounts) of the biomarker (or multiple biomarkers) that increases compared to the reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the medicament at a higher dose, and/or the amount (or amounts) of the biomarker (or multiple biomarkers) that decreases compared to the reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the medicament at a higher dose, and/or the amount (or amounts) of the biomarker (or multiple biomarkers) that decreases compared to the reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the medicament at a lower dose.

If at least one agent is an inhibitor of the renin-angiotensin system, and if at least one biomarker is sFlt-1 and/or IGFBP7, the following applies.

Preferably, an increased amount of the biomarker in the test sample compared to the reference amount indicates that the subject is not suitable for administration of the agent, and/or a decreased amount of the biomarker compared to the reference amount indicates that the subject is suitable for administration of the agent.

More preferably, the subject to be tested has been treated with a renin angiotensin system inhibitor. In this case, an increased amount of the biomarker compared to the reference amount indicates that the subject is not suitable for administration of the agent at a higher dose, and/or a decreased amount of the biomarker compared to the reference amount indicates that the subject is suitable for administration of the agent at a higher dose.

It is also preferred that the reference amount is derived from a subject or group of subjects known to be suitable for administration of the at least one medicament and/or from a subject or group of subjects known to be unsuitable for administration of the at least one medicament.

In this case, the preferred diagnostic algorithm is as follows:

If at least one of the agents is a beta-blocker and if at least one of the biomarkers is endostatin, mimecan, GDF-15, cardiac troponin and/or a BNP-type peptide, the following preferably applies:

Preferably, the reference amount is derived from a subject or group of subjects known to be suitable for administration of the at least one agent, wherein an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is substantially the same or increased compared to the reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the at least one agent, and/or the reference amount is derived from a subject or group of subjects known to be unsuitable for administration of the at least one agent, wherein an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is substantially the same or decreased compared to the reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the at least one agent.

If the agent is a beta blocker, and if the biomarker is IGFBP7, P1NP, sFlt-1 and/or osteopontin, the following preferably applies:

Preferably, the reference amount for IGFBP7, P1NP, sFlt-1 and/or osteopontin is derived from a subject or a group of subjects known to be suitable for administration of said beta-blocker, wherein an amount of IGFBP7, P1NP, sFlt-1 and/or osteopontin in the test sample that is substantially the same or decreased compared to the reference amount indicates that the subject is suitable for administration of said beta-blocker, and/or the reference amount is derived from a subject or a group of subjects known to be unsuitable for administration of said beta-blocker, wherein an amount of IGFBP7, P1NP, sFlt-1 and/or osteopontin in the test sample that is substantially the same or increased compared to the reference amount indicates that the subject is unsuitable for administration of said beta-blocker.

If at least one agent is an aldosterone antagonist, and if at least one biomarker is IGFBP7, cystatin C, cardiac troponin, Gal-3, PlGF, GDF-15 and/or sST2, the following preferably applies:

Preferably, the reference amount is derived from a subject or group of subjects known to be suitable for administration of the at least one agent, wherein an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is substantially the same or increased compared to the reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the at least one agent, and/or the reference amount is derived from a subject or group of subjects known to be unsuitable for administration of the at least one agent, wherein an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is substantially the same or decreased compared to the reference amount (or multiple reference amounts) indicates that the subject is not suitable for administration of the at least one agent.

If at least one of the agents is an aldosterone antagonist, and if at least one of the biomarkers is endostatin, uric acid and/or sFlt-1, the following preferably applies:

Preferably, the reference amount is derived from a subject or group of subjects known to be suitable for administration of the at least one agent (i.e., an aldosterone antagonist), wherein an amount (or amounts) of the biomarker (or biomarkers) in the test sample that is substantially the same or decreased compared to the reference amount (or amounts) indicates that the subject is suitable for administration of the at least one agent, and/or the reference amount is derived from a subject or group of subjects known to be unsuitable for administration of the at least one agent, wherein an amount (or amounts) of the biomarker (or biomarkers) in the test sample that is substantially the same or increased compared to the reference amount (or amounts) indicates that the subject is unsuitable for administration of the at least one agent.

More preferably, the subject to be tested has been treated with an aldosterone antagonist. In this case, an amount (or amounts) of the biomarker (or biomarkers) that is increased compared to the reference amount (or reference amounts) indicates that the subject is not suitable for administration of the medicament at a higher dose, and/or an amount (or amounts) of the biomarker (or biomarkers) that is decreased compared to the reference amount (or reference amounts) indicates that the subject is not suitable for administration of the medicament at a higher dose.

If at least one of the pharmaceutical agents is an inhibitor of the renin-angiotensin system and if at least one of the biomarkers is GDF-15, cardiac troponin, uric acid, a BNP-type peptide and/or osteopontin, the following preferably applies:

Preferably, the one or more reference amounts for one or more biomarkers are derived from a subject or a group of subjects known to be suitable for administration of said renin-angiotensin system inhibitor, wherein the amounts of both biomarkers in the test sample that are substantially the same and/or increased compared to the reference amounts indicate that the subject is suitable for administration of said renin-angiotensin system inhibitor, and/or the reference amounts are derived from a subject or a group of subjects known to be unsuitable for administration of said renin-angiotensin system inhibitor, wherein i) the amount of the biomarker cardiac troponin in the test sample is substantially the same or decreased compared to the reference amount of cardiac troponin, or ii) wherein the amounts of both biomarkers in the test sample that are substantially the same and/or decreased compared to the reference amount, indicates that the subject is unsuitable for administration of said renin-angiotensin system inhibitor.

If at least one of the agents is a diuretic and at least one of the biomarkers is selected from endostatin, mimecan, GDF-15, prealbumin, transferrin, a BNP-type peptide and uric acid, the following is preferably applied:

Preferably, the reference amount is derived from a subject or group of subjects known to be suitable for administration of the at least one agent (i.e., a diuretic), wherein an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is substantially the same or decreased compared to the reference amount (or multiple reference amounts) indicates that the subject is suitable for administration of the at least one agent, and/or the reference amount is derived from a subject or group of subjects known to be unsuitable for administration of the at least one agent, wherein an amount (or amounts) of the biomarker (or multiple biomarkers) in the test sample that is substantially the same or increased compared to the reference amount (or multiple reference amounts) indicates that the subject is unsuitable for administration of the at least one agent.

Also preferably, the reference amount is derived from a subject or group of subjects known to be unsuitable for administration of the at least one agent, wherein an amount (or amounts) of the biomarker (or biomarkers) in the test sample that is substantially the same or increased compared to the reference amount (or amounts) indicates that the subject is suitable for administration of the agent at a lower dose.

More preferably, the subject to be tested has been treated with a diuretic. In this case, an amount (or amounts) of the biomarker (or biomarkers) that is increased compared to the reference amount (or reference amounts) indicates that the subject is not suitable for administration of the medicament at a higher dose, and/or an amount (or amounts) of the biomarker (or biomarkers) that is decreased compared to the reference amount (or reference amounts) indicates that the subject is not suitable for administration of the medicament at a higher dose.

If at least one agent is an inhibitor of the renin-angiotensin system, and if at least one biomarker is sFlt-1 and/or IGFBP7, the following applies:

Preferably, the reference amount is derived from a subject or a group of subjects known to be suitable for administration of the renin-angiotensin system inhibitor, wherein an amount of sFlt-1 and/or IGFBP7 in the test sample that is substantially the same or decreased compared to the reference amount indicates that the subject is suitable for administration of the renin-angiotensin system inhibitor, and/or the reference amount is derived from a subject or a group of subjects known to be unsuitable for administration of the renin-angiotensin system inhibitor, wherein an amount of sFlt-1 and/or IGFBP7 in the test sample that is substantially the same or increased compared to the reference amount indicates that the subject is unsuitable for administration of the renin-angiotensin system inhibitor.

Preferred reference amounts to be applied in the context of the present invention are those disclosed in the examples. Further preferred reference amounts are as follows:

• GDF-15: in the range of about 3000 to about 5000 ng/ml, particularly about 4000 ng/ml

• Endostatin: in the range of about 230 to about 270 ng/ml, particularly about 250 ng/ml

• Mimecan: in the range of about 30 to about 70 ng/ml, particularly about 50 ng/ml

• IGFBP7: in the range of about 70 to about 130 ng/ml, particularly about 100 ng/ml

• NT-proBNP: in the range of about 2500 to about 3500 pg/ml, in particular about 3000 pg/ml

• Troponin: in the range of about 22 to about 30 ng/ml, particularly about 26 ng/ml

• Uric acid: in the range of about 5 to about 10 ng/ml, particularly about 7.3 ng/ml

• Gal3 (Galectin-3): in the range of about 26 to about 38 ng/ml, particularly about 31.6 ng/ml

• Osteopontin: in the range of about 80 to about 120 ng/ml, particularly about 100 ng/ml

• sST2 (soluble ST2): in the range of about 30 to about 38 ng/ml, particularly about 34.0 ng/ml

• sFLT-1: in the range of about 85 to about 111 ng/ml, particularly about 98 ng/ml

• PlGF: in the range of about 15 to about 31 ng/ml, particularly about 20.7 ng/ml

• P1NP: in the range of about 29.0 ng/ml to about 43.5 ng/ml, particularly about 36.72 ng/ml

• Cystatin C: in the range of about 1.20 mg/l to about 2.3 mg/l, in particular about 1.76 mg/l

• Prealbumin: in the range of about 0.11 g/l to about 0.27 g/l, in particular about 0.19 g/l

• Transferrin: in the range of about 2.15 g/l to about 2.80 g/l, in particular about 2.48 g/l.

If the ratio of PlGF to sFlt-1 is calculated, the reference ratio is in particular in the range of about 0.17 to about 0.29. In one embodiment, the reference ratio is 0.23.

Preferably, the above-mentioned reference amount (ratio) is a threshold amount (ratio) that separates the groups mentioned herein from each other.

If, in the context of the present invention, more than one biomarker is measured for the purpose of evaluating whether a subject is susceptible to administration of a pharmaceutical agent, it is particularly contemplated that the measured amounts of the individual biomarkers may be compared to reference amounts of the individual biomarkers. For example, the amount of the biomarker IGFBP7 should be compared to a reference amount of IGFBP7, the amount of the biomarker mimecan should be compared to a reference amount of mimecan, the amount of the biomarker endostatin should be compared to a reference amount of endostatin, the amount of the biomarker sFlt-1 should be compared to a reference amount of sFlt-1, and so on. In such cases, the diagnosis is made. Furthermore, if more than one biomarker is measured, it is preferred to combine the diagnostic algorithms described herein for the individual biomarkers.

Furthermore, the present invention relates to a method of treating a subject with at least one agent for the treatment of heart failure, comprising:

a) determining the amount of at least one biomarker in a sample from a subject suffering from heart failure as described above in connection with the method of the present invention, and

b) comparing one or more amounts of at least one biomarker as determined in step a) with one or more reference amounts, thereby identifying a subject suitable for administration of said at least one agent,

c) identifying the subject as suitable for administration of at least one pharmaceutical agent,

d) administering the at least one agent to the subject.

The pharmaceutical agents are disclosed above. The administration may be an initial administration of the at least one pharmaceutical agent, or an administration of the at least one pharmaceutical agent at a higher dose.

Step c) is based on comparing the results of step b).Diagnostic algorithms are disclosed elsewhere herein.

In the context of the studies underlying the present invention it was shown that by using a single biomarker it is possible to make decisions about the administration of several classes of pharmaceutical agents.Thus, the method of the present invention envisages, in particular by using a single marker, the assessment of whether a subject is suitable for the administration of more than one pharmaceutical agent.

Therefore, in a preferred embodiment of the method of the present invention, subjects are identified who are suitable for administration of more than one agent, in particular two or three agents. Preferred combinations are as follows:

If the biomarker is GDF-5, the agent is preferably:

i. beta-blockers and inhibitors of the renin-angiotensin system,

ii. Beta-blockers and diuretics,

iii. Inhibitors of the renin-angiotensin system and diuretics, or

iv. Beta-blockers, inhibitors of the renin-angiotensin system, and diuretics.

If the biomarker is IGFBP7, the pharmaceutical agent is preferably a beta blocker and an aldosterone antagonist. Furthermore, the pharmaceutical agent is preferably a beta blocker, an aldosterone antagonist, and a diuretic. Furthermore, it is contemplated that the pharmaceutical agent is a beta blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system.

If the biomarker is mimecan, then the preferred agents are: beta blockers and diuretics.

If the biomarker is endostatin, the agent is preferably:

i. Beta-blockers and aldosterone antagonists,

ii. Beta-blockers and diuretics,

iii. Aldosterone antagonists and diuretics, or

iv. Beta-blockers, aldosterone antagonists, and diuretics.

If the biomarker is cardiac troponin, the agent is preferably:

i. Beta-blockers and aldosterone antagonists,

ii. beta-blockers and inhibitors of the renin-angiotensin system,

iii. Aldosterone antagonists and inhibitors of the renin-angiotensin system, or

iv. Beta-blockers, aldosterone antagonists, and inhibitors of the renin-angiotensin system.

If the biomarker is uric acid, the agent is preferably:

i. Diuretics and aldosterone antagonists,

ii. diuretics and inhibitors of the renin-angiotensin system,

iii. Aldosterone antagonists and inhibitors of the renin-angiotensin system, or

iv. Diuretics, aldosterone antagonists, and inhibitors of the renin-angiotensin system.

Accordingly, the present invention relates to a method for identifying a subject suitable for administration of two or three agents, comprising:

a) determining the amount of GDF-15 (Growth Differentiation Factor 15) in a sample from a subject suffering from heart failure, and

b) comparing the amount determined in step a) with a reference amount, thereby identifying a subject suitable for administration of the medicament,

wherein the two or three agents are:

i. beta-blockers and inhibitors of the renin-angiotensin system,

ii. Beta-blockers and diuretics,

iii. Inhibitors of the renin-angiotensin system and diuretics, or

iv. Beta-blockers, inhibitors of the renin-angiotensin system, and diuretics.

Further, the present invention relates to a method for identifying a subject suitable for administration of two agents, comprising:

a) determining the amount of IGFBP7 in a sample from a subject suffering from heart failure, and

b) comparing the amount determined in step a) with a reference amount (or multiple reference amounts), thereby identifying a subject suitable for administration of the medicament,

The two agents are a beta blocker and an aldosterone antagonist.

Additionally, the present invention relates to a method for identifying a subject suitable for administration of two agents, comprising:

a) determining the amount of mimecan in a sample from a subject suffering from heart failure, and

b) comparing the amount determined in step a) with a reference amount (or multiple reference amounts), thereby identifying a subject suitable for administration of the medicament,

The two agents are a beta blocker and a diuretic.

Accordingly, the present invention relates to a method for identifying a subject suitable for administration of two or three agents, comprising:

a) determining the amount of cardiac troponin in a sample from a subject suffering from heart failure, and

b) comparing the amount determined in step a) with a reference amount (or multiple reference amounts), thereby identifying a subject suitable for administration of the medicament,

wherein the two or three agents are:

i. Beta-blockers and aldosterone antagonists,

ii. beta-blockers and inhibitors of the renin-angiotensin system,

iii. Aldosterone antagonists and inhibitors of the renin-angiotensin system, or

iv. Beta-blockers, aldosterone antagonists, and inhibitors of the renin-angiotensin system.

Accordingly, the present invention relates to a method for identifying a subject suitable for administration of two or three agents, comprising:

a) determining the amount of endostatin in a sample from a subject suffering from heart failure, and

b) comparing the amount determined in step a) with a reference amount (or multiple reference amounts), thereby identifying a subject suitable for administration of the medicament,

wherein the two or three agents are:

i. Beta-blockers and aldosterone antagonists,

ii. Beta-blockers and diuretics,

iii. Aldosterone antagonists and diuretics, or

iv. Beta-blockers, aldosterone antagonists, and diuretics.

If a subject susceptible to the administration of two or three pharmaceutical agents should be identified based on the determination of the amount of a single biomarker, the following should apply with regard to the reference amount:

Preferably, the reference amount for multiple agents described in step b) can be the same for each marker. Accordingly, the amount of the biomarker as measured in step a) of the method of the present invention is preferably compared with a single reference amount, and therefore the reference amount for an individual marker allows the evaluation object to be suitable for the administration of the two or three agents (i.e., the reference amount is applied to all agents. Also preferably, the reference amount for an individual biomarker with respect to multiple agents can be different, i.e., it can be agent-specific. Accordingly, the amount of the biomarker as measured in step a) of the method of the present invention is preferably compared with two or three reference amounts. Therefore, if an object suitable for the administration of two agents should be identified, the amount of the biomarker should be compared with two reference amounts. In addition, if an object suitable for the administration of three agents is identified, the amount of the biomarker should be compared with three reference amounts. An individual reference amount should be agent-specific. Accordingly, an individual reference amount for multiple agents (with respect to a single marker) can be applied. For example, if a subject is to be identified as sensitive to administration of a beta-blocker and an aldosterone antagonist, the amount of the biomarker determined in step a) should be compared to i) a reference amount of the biomarker for identifying subjects suitable for administration of a beta-blocker, and ii) a reference amount of the biomarker for identifying subjects suitable for administration of an aldosterone antagonist (this applies if the biomarker is IGFBP7). For example, if a subject is to be identified as sensitive to administration of a beta-blocker and a diuretic, the amount of the biomarker determined in step a) should be compared to i) a reference amount of the biomarker for identifying subjects suitable for administration of a beta-blocker, and ii) a reference amount of the biomarker for identifying subjects suitable for administration of a diuretic (this applies if the biomarker is mimecan).

The diagnostic algorithms for individual biomarkers in combination with individual agents have been described elsewhere herein. In addition, preferred reference amounts are described above. If a subject sensitive to the administration of two or more agents should be identified based on the determination of the amount of a single biomarker, then the diagnostic algorithm and preferred reference amounts are preferably also applied. If a subject suitable for the administration of two or more agents should be identified, then the diagnostic algorithms described above for the respective biomarkers in combination with the agents are combined.

For example, with respect to the biomarker IGFBP7, the following applies:

Preferably, a decreased amount of the biomarker in the test sample compared to a reference amount (particularly a reference amount for identifying subjects suitable for administration of a beta-blocker) indicates that the subject is suitable for administration of the beta-blocker, and/or an increased amount of the biomarker compared to the reference amount indicates that the subject is not suitable for administration of the beta-blocker, whereas an increased amount of the biomarker in the test sample compared to a reference amount (particularly a reference amount for identifying subjects suitable for administration of an aldosterone antagonist) indicates that the subject is suitable for administration of the aldosterone antagonist, and/or a decreased amount of the biomarker compared to the reference amount indicates that the subject is not suitable for administration of the aldosterone antagonist.

Alternatively, the reference amount for identifying a subject suitable for administration of a beta-blocker with respect to the biomarker IGPBP7 is derived from a subject or a group of subjects known to be suitable for administration of said beta-blocker, wherein an amount of IGFBP7 in a test sample that is substantially the same as or decreased compared to the reference amount indicates that the subject is suitable for administration of said beta-blocker, and/or the reference amount for identifying a subject suitable for administration of a beta-blocker is derived from a subject or a group of subjects known to be unsuitable for administration of said beta-blocker, wherein an amount of IGFBP7 in a test sample that is substantially the same as or increased compared to the reference amount indicates that the subject is unsuitable for administration of said beta-blocker, and The reference amount of IGFBP7 for identifying a subject suitable for administration of an aldosterone antagonist is derived from a subject or a group of subjects known to be suitable for administration of the aldosterone antagonist, wherein an amount of IGFBP7 in a test sample that is substantially the same or increased compared to the reference amount indicates that the subject is suitable for administration of the aldosterone antagonist, and/or the reference amount of IGFBP7 for identifying a subject suitable for administration of an aldosterone antagonist is derived from a subject or a group of subjects known to be unsuitable for administration of the aldosterone antagonist, wherein an amount of IGFBP7 in a test sample that is substantially the same or decreased compared to the reference amount indicates that the subject is not suitable for administration of the aldosterone antagonist.

In one aspect of the invention, a method for establishing an aid for identifying a subject suitable for administration of at least one agent selected from the group consisting of a beta blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system is contemplated, the method comprising:

a) determining the amount of at least one marker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, a BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin, and transferrin by (i) contacting the sample with a detection reagent(s) that specifically binds to the at least one marker for a period of time sufficient to allow formation of a complex between the detection reagent and the at least one marker from the sample, (ii) measuring the amount of the complex formed, wherein the amount of the complex formed is proportional to the amount of the at least one marker present in the sample, and (iii) converting the amount of the complex formed into an amount of at least one marker that reflects the amount of the at least one marker present in the sample;

b) comparing the amount to a reference; and

c) establishing an aid for identifying a subject suitable for administration of said at least one medicament based on the results of the comparison made in step b).

In another aspect of the invention, a system for establishing an aid for identifying a subject suitable for administration of at least one agent selected from the group consisting of a beta blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system is contemplated, the system comprising:

a) an analyzer unit configured to contact a sample with a detection reagent(s) that specifically binds to said at least one marker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, a BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin, and transferrin for a time sufficient to allow formation of a complex of said detection reagent and the at least one marker from the sample,

b) an analyzer unit configured to measure the amount of the formed complex, wherein said amount of the formed complex is proportional to the amount of at least one marker present in the sample,

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

d) a non-transitory computer-readable medium comprising a plurality of instructions executable by a processor, which, when executed, convert the amount of the formed complex into an amount of at least one marker that reflects the amount of at least one marker present in the sample, compare the amount to a reference amount, and based on the result of the comparison to the reference, establish an aid for identifying a subject suitable for administration of the at least one agent.

In one aspect, a suitable detection reagent can be an antibody that specifically binds to at least one marker in a sample of a subject to be studied by the methods of the present invention, i.e., a detection reagent that binds GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin, and transferrin. In one aspect, another detection reagent that can be used can be an aptamer that specifically binds to at least one marker in the sample. In another aspect, the sample is removed from the complex formed between the detection reagent and the at least one marker before measuring the amount of the formed complex. Accordingly, in one aspect, the detection reagent can be immobilized on a solid support. In another aspect, the sample can be removed from the complex formed on the solid support by applying a washing solution. The complex formed should be proportional to the amount of the at least one marker present in the sample. It will be appreciated that the specificity and/or sensitivity of the detection reagent to be used defines the proportion of at least one marker capable of specific binding contained in the sample. Further details on how the assay can be performed are also found elsewhere herein. The amount of the complex formed should be converted into an amount of at least one marker that reflects the amount actually present in the sample. In one aspect, such an amount can be substantially the amount present in the sample, or in another aspect, it can be an amount that is a proportion of the amount present due to the relationship between the complex formed and the amount present in the original sample.

In yet another aspect of the above method, step a) may be performed by an analyzer unit, in one aspect, the analyzer unit is as defined elsewhere herein.

In one aspect of the method of the present invention, the one or more amounts determined in step a) are compared to a reference amount. In one aspect, the reference is a reference as defined elsewhere herein. In another aspect, the reference takes into account the proportional relationship between the amount of the complex measured and the amount present in the original sample. Therefore, in one aspect of the method of the present invention, the reference used is an artificial reference that reflects the limitations of the detection reagents used. In another aspect, before actually comparing the values of the measured amount and the reference, the relationship can also be taken into account when comparing, for example by including a standardization and/or correction calculation step for the measured amount. Again, the standardization and/or correction calculation step for the measured amount adopts a comparison step so that the limitations of the detection reagents used are appropriately reflected. In one aspect, the comparison is automatically performed, for example with the help of a computer system or the like.

As described elsewhere herein, an aid for identifying subjects suitable for administration of the at least one pharmaceutical agent is established based on the comparison performed in step b) by assigning subjects to groups of subjects that are suitable for administration or not suitable for said administration. As discussed elsewhere herein, the assignment of study subjects need not be correct in 100% of the study cases. Furthermore, the groups of subjects to which the study subjects are assigned are artificial groups because they are established based on statistical considerations, i.e., a certain pre-selected degree of probability that the method of the present invention should be based on its operation. In one aspect of the invention, the aid for identifying subjects suitable for administration of the at least one pharmaceutical agent is automatically established with the aid of a computing device or the like, as described and disclosed herein.

In one aspect of the method of the invention, the method further comprises the step of recommending and/or managing the subject, and/or adapting the intensity of disease monitoring, based on the results established in step c) as elaborated elsewhere herein.

In one aspect of the above method, steps b) and/or c) are performed by one or more analyzer units as described elsewhere herein.

The present invention also relates to the detection of at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin and transferrin in a sample of a subject suffering from heart failure, or ii) and/or specifically binding to at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin and transferrin. Use of a detection reagent for biomarkers of β-ST2 (soluble ST2), sFlt-1, PlGF, P1NP, cystatin C, prealbumin and transferrin for identifying subjects suitable for administration of at least one agent selected from the group consisting of a beta blocker, an aldosterone antagonist, a diuretic and an inhibitor of the renin-angiotensin system.

The present invention also relates to i) at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, P1NP, cystatin C, prealbumin and transferrin, or ii) and/or specifically binds to at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, P1NP, cystatin C, prealbumin and transferrin. Use of a detection reagent for a biomarker of sFlt-1, PlGF, PlNP, cystatin C, prealbumin, and transferrin for the manufacture of a medicament or diagnostic composition for identifying a subject suitable for administration of at least one agent selected from the group consisting of a beta-blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system.

Further, the present invention relates to at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin and transferrin, or ii) and/or specifically binds to at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin and transferrin. A detection reagent for a biomarker selected from the group consisting of soluble ST2, sFlt-1, PlGF, PlNP, cystatin C, prealbumin, and transferrin is used to identify subjects suitable for administration of at least one agent selected from the group consisting of a beta-blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system. The at least one biomarker or the at least one detection reagent may be included in a kit.

As used herein, the term "detection reagent" refers to a reagent that is capable of specifically recognizing and binding one or more biomarker polypeptides present in a sample. In addition, the reagent should allow for direct or indirect detection of a complex formed by the reagent and the biomarker. Direct detection can be achieved by including a detectable label in the reagent. Indirect labeling can be achieved by a further reagent that specifically binds to a complex comprising the biomarker and the detection reagent, wherein the further reagent is subsequently capable of generating a detectable signal. Suitable compounds that can be used as detection reagents are well known in the art. Preferably, the detection reagent is an antibody or aptamer that specifically binds to the biomarker. The term "antibody" has been described elsewhere herein.

According to a preferred embodiment of the present invention, there is provided an apparatus suitable for carrying out the method of the present invention, comprising:

a) an analyzer unit comprising a detection reagent (or reagents) that specifically binds to a detection reagent selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, a BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin, and transferrin, said unit being suitable for determining one or more amounts of one or more markers in a sample from a subject suffering from heart failure; and

b) an analyzer unit for comparing one or more measured amounts to one or more reference amounts, thereby identifying a subject suitable for administration of at least one agent selected from the group consisting of a beta blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system, said unit comprising a database with reference amount(s) and a computer-implemented algorithm for performing the comparison.

Preferred reference amounts and algorithms are disclosed elsewhere herein.

Preferred embodiments of the present disclosure include systems for identifying subjects suitable for administration of at least one agent selected from the group consisting of beta blockers, aldosterone antagonists, diuretics, and inhibitors of the renin-angiotensin system. Examples of systems include clinical chemistry analyzers, coagulation chemistry analyzers, immunochemistry analyzers, urine analyzers, and nucleic acid analyzers for detecting chemical or biological reaction results or monitoring chemical or biological reaction progress. More specifically, exemplary systems of the present disclosure may include Roche Elecsys ^™ systems and ^Cobas® e immunoassay analyzers, Abbott Architect ^™ and Axsym ^™ analyzers, Siemens Centaur ^™ and Immulite ^™ analyzers, and Beckman Coulter UniCel ^™ and Access ^™ analyzers, among others.

The embodiment of the system may include one or more analyzer units for practicing the present disclosure. The analyzer unit of the system disclosed herein is operatively communicated with the computing device disclosed herein by any one of known wired connections, Bluetooth, LANS or wireless signals. In addition, according to the present disclosure, the analyzer unit may include an independent instrument, or a module in a larger instrument, which performs detection, such as one or both of the sample qualitative and/or quantitative evaluations for diagnostic purposes. For example, the analyzer unit may perform or help the absorption, administration, mixing of sample and/or reagent. The analyzer unit may include a reagent holding unit for accommodating the reagent for performing the assay. Reagent may, for example, be arranged in the form of a container or a cartridge containing an individual reagent or reagent group, placed in a position in a suitable container or storage compartment or a conveyor belt. Detection reagent may also be in a fixed form on a solid carrier, which contacts the sample. Further, the analyzer unit may include a process and/or detection component optimized for specific analysis.

According to some embodiments, the analyzer unit can be configured for the optical detection of analytes such as markers in the sample. The exemplary analyzer unit configured for optical detection includes a device configured for converting electromagnetic energy into electrical signals, and the device includes both single and multi-element or array photodetectors. According to the present disclosure, the photodetector can monitor optical electromagnetic signals and provide a power outlet signal or a response signal relative to a baseline signal indicating the presence and/or concentration of the analyte in the sample in the light path. Such devices can also include, for example, photodiodes, including avalanche photodiodes, phototransistors, photoconductivity detectors, linear sensor arrays, CCD detectors, detector CMOS, including CMOS array detectors, photomultiplier tubes, and photomultiplier arrays. According to certain embodiments, photodetectors such as photodiodes or photomultiplier tubes can contain other signal conditioning or processing electronics. For example, the photodetector can include at least one preamplifier, electronic filter, or integrated circuit. Suitable preamplifiers include, for example, integrated, transimpedance, and current gain (current mirror) preamplifiers.

In addition, 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 be composed of at least one light-emitting element (e.g., a light-emitting diode, an electrically powered radiation source such as an incandescent lamp, an electroluminescent lamp, a gas discharge lamp, a high-intensity discharge lamp, a laser) for measuring the analyte concentration of the sample to be tested or allowing energy transfer (e.g., by fluorescence resonance energy transfer or catalytic enzymes).

Further, the analyzer unit of the system can include one or more incubation units (e.g., for maintaining samples or reagents at a specified temperature or temperature range). In some embodiments, the analyzer unit can include a thermal cycler, including a real-time thermal cycler, for performing repeated temperature cycles on the sample and monitoring changes in the amount of amplification product within the sample.

In addition, the analyzer unit of the system disclosed herein can include or be operably connected to a reaction vessel or a small cup feed unit. Exemplary feed units include liquid processing units, such as suction units, to deliver samples and/or reagents to the reaction vessel. The suction unit can include a reusable washable needle such as a steel needle, or a disposable pipette tip. The analyzer unit can further include one or more mixing units, such as an oscillator to oscillate the small cup containing the liquid, or a mixing slurry to mix the liquid in the small cup or reagent container.

Following the above, according to some embodiments of the present disclosure, parts of some steps of the method disclosed and described herein can be performed by a computing device. The computing device can be, for example, a general-purpose computer or a portable computing device. It should also be understood that multiple computing devices can be used together, for example, over a network or other method of transferring data, for performing one or more steps of the method disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants ("PDAs") such as BLACKBERRY brand devices, cellular devices, tablet computers, servers, and the like. In general, a computing device includes a processor capable of executing multiple instructions (e.g., software programs).

The computing device has access to the memory. The memory is a computer-readable medium and may include, for example, a single storage device or multiple storage devices that are locally located within the computing device or accessible to the computing device across a network. The computer-readable medium can be any available medium that can be accessed by the computing device and includes both volatile and non-volatile media. Further, the computer-readable medium can be one or both of removable and non-removable media. For example and without limitation, the computer-readable medium may include computer storage media. Exemplary computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory or any other storage technology, CD-ROM, digital versatile disk (DVD) or other optical disk storage, cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store multiple instructions that can be accessed by the computing device and executed by the 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 thus may be operable via these control signals to transform material removed from the computer itself. These descriptions and representations are provided as a means for use by those skilled in the art of data processing, for example, to most effectively convey their working material to those skilled in the art.

A plurality of instructions may also comprise an algorithm generally conceived as a self-consistent sequence of steps leading to a desired result. These steps are those requiring physical manipulation of physical quantities. Typically, although not necessarily, these quantities take the form of electrical or magnetic pulses or signals capable of storage, transfer, conversion, combination, comparison, and other manipulations. In principle, for reasons of common usage, it is sometimes convenient to refer to these signals as values, characters, display data, numbers, etc. as references to the physical items or manifestations that such signals embody or express. However, it should be remembered that all of these and similar terms are associated with appropriate physical quantities and are used herein only as convenient labels for application to these quantities. According to some embodiments of the present disclosure, an algorithm for comparing the measured amount of one or more markers disclosed herein with a suitable reference is embodied and executed by executing instructions. The result can be given as an output of parameter diagnostic raw data or as an absolute or relative amount. According to a plurality of embodiments of the system disclosed herein, a "diagnosis" can be provided by a computing device of the system disclosed herein based on the comparison of the calculated "amount" with a reference or threshold value. For example, the computing device of the system can provide an indicator in the form of a word, symbol, or numerical value, which is an indication of a specific diagnosis.

The computing device may also access an output device. Exemplary output devices include, for example, a fax machine, a display, a printer, and a file. According to some embodiments of the present disclosure, the computing device may perform one or more steps of the methods disclosed herein and thereafter provide output regarding the results, indications, ratios, or other factors of the methods via the output device.

Finally, the present invention relates to a kit suitable for performing the method of the present invention, comprising at least one detection reagent that specifically binds to a marker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sST2 (soluble ST2), sFlt-1, PlGF, P1NP, cystatin C, prealbumin and transferrin, reference standards and instructions for performing the method.

As used herein, the term "kit" refers to a collection of the above-mentioned components that are preferably provided separately or in a single container. The container also includes instructions for carrying out the method of the present invention. These instructions can take the form of a manual or can be provided by a computer program code, which, when implemented on a computer or data processing device, can perform the comparisons mentioned in the method of the present invention and establish a diagnosis accordingly. The computer program code can be on a data storage medium or device such as an optical storage medium (e.g., a compact disc), or directly on a computer or data processing device. Further, the kit should include at least one standard for reference as defined above, i.e., a solution having a predetermined amount of a biomarker as mentioned herein that represents a reference amount.

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

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

In some embodiments, the kit may include one or more non-fixed probes and one or more solid supports with or without fixed probes. Some such embodiments may include some or all of the reagents and supplies required to fix the one or more probes to the solid support, or some or all of the reagents and supplies required to bind the fixed probes to a specific protein in a sample.

In certain embodiments, single probe (comprising multiple copies of identical probe) can be fixed on single solid carrier, and is provided in single container.In other embodiments, two or more probes specific for different forms (such as specific epitopes) of different target proteins or single target proteins are provided in single container.In some such embodiments, fixed probe can provide (such as in the form of single use) in multiple different containers, or multiple fixed probes can provide in multiple different containers.In further embodiments, probe can be fixed on multiple different types of solid carriers.For kit disclosed herein, any combination of one or more fixed probes and one or more containers is considered, and any combination thereof can be selected to realize the suitable kit for desired purposes.

The container of the test 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 can completely surround one or more fixed probes, or can simply cover the probes to minimize contamination by dust, oil, etc. and exposure to light. In some further embodiments, the test kit can include a single container or multiple containers, and when multiple containers are present, each container can be the same as all other containers, different from each other, or different from some but not all other containers.

Preferred embodiments of the present invention

In the following the preferred embodiments of the present invention are disclosed.The definitions and descriptions given elsewhere herein apply mutatis mutandis.

1. A method for identifying a subject suitable for administration of at least one pharmaceutical agent, comprising:

a) determining the amount of at least one biomarker in a sample from a subject suffering from heart failure, and

b) comparing the amount determined in step a) with a reference amount, thereby identifying a subject suitable for administration of the at least one pharmaceutical agent,

wherein the agent is a beta blocker, and wherein the biomarker is IGFBP7 (IGF binding protein 7) or mimecan.

2. A method for identifying a subject suitable for administration of at least one agent selected from the group consisting of a beta blocker, an aldosterone antagonist, a diuretic, and a renin-angiotensin system inhibitor, comprising:

a) determining the amount of at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, PlGF, sFlt-1, sST2 (soluble ST2), PlNP, cystatin C, prealbumin, and transferrin in a sample from a subject suffering from heart failure, and

b) comparing the amount determined in step a) with a reference amount (a plurality of reference amounts), thereby identifying a subject suitable for administration of the at least one pharmaceutical agent,

In particular,

i) the biomarker is osteopontin and the agent is an inhibitor of the renin-angiotensin system,

ii) the biomarker is endostatin and the agent is an aldosterone antagonist,

iii) the biomarker is s-Flt-1 and the agent is an aldosterone antagonist and/or an inhibitor of the renin-angiotensin system,

iv) the biomarker is PlGF and the agent is an aldosterone antagonist,

v) the biomarker is cardiac troponin and the agent is an inhibitor of the renin-angiotensin system,

vi) the biomarker is a BNP-type peptide and the pharmaceutical agent is an inhibitor of the renin-angiotensin system and/or a beta-blocker,

vii) the biomarker is uric acid and the pharmaceutical agent is a diuretic and/or an inhibitor of the renin-angiotensin system,

viii) the biomarker is GDF-15 and the agent is a diuretic and/or an inhibitor of the renin-angiotensin system,

ix) the biomarker is sST2 and the agent is an aldosterone antagonist and/or a beta blocker,

x) the biomarker is IGFBP7 and the agent is an inhibitor of the renin-angiotensin system,

xi) the biomarker is PINP and the agent is a beta blocker,

xii) the biomarker is cystatin C and the agent is an aldosterone antagonist,

xiii) the biomarker is prealbumin and the agent is a diuretic,

xiv) the biomarker is transferrin and the agent is a diuretic, and/or

xv) The biomarkers are PlGF and sFlt-1, and the agent is an aldosterone antagonist, wherein the ratio of the amount of PlGF to the amount of sFlt-1 (or vice versa) is calculated, and wherein the ratio is calculated using a reference ratio.

3. The method of embodiment 1 or 2, wherein the administration is the initial administration of the at least one agent, or the administration of the at least one agent at a higher dose.

4. The method according to any one of embodiments 1-3, wherein the subject is a human.

5. The method according to any one of embodiments 1 to 4, wherein the sample is blood, serum or plasma.

6. The method according to any one of embodiments 1-5, wherein

• the biomarker is IGFBP7, P1NP, sFlt-1 and/or osteopontin, and the agent is a beta blocker,

• the biomarker is endostatin and/or sFlt-1 and the agent is an aldosterone antagonist,

• the biomarker is uric acid and/or GDF-15, and the agent is a diuretic,

• wherein the biomarker is sFlt-1 and/or IGFBP7 and the agent is an inhibitor of the renin-angiotensin system,

wherein the amount of at least one biomarker that is decreased compared to the reference amount indicates that the subject is suitable for administration of the at least one agent, and/or wherein the amount of at least one biomarker that is increased compared to the reference amount indicates that the subject is not suitable for administration of the at least one agent.

7. The method according to any one of embodiments 1-6, wherein

• The biomarker is mimecan, a BNP-type peptide and/or sST2, and the agent is a beta-blocker,

• the biomarker is osteopontin, cardiac troponin, a BNP-type peptide, uric acid and/or GDF-15, and the agent is an inhibitor of the renin-angiotensin system, and/or

• the biomarker is sST2, cystatin C and/or PlGF, and the agent is an aldosterone antagonist,

wherein the amount of at least one biomarker that is increased compared to the reference amount indicates that the subject is suitable for administration of the at least one agent, and/or wherein the amount of at least one biomarker that is decreased compared to the reference amount indicates that the subject is not suitable for administration of the at least one agent.

8. The method according to any one of embodiments 1 to 7, wherein the biomarker is IGFBP-7 and the method is used to identify subjects suitable for administration of a beta blocker and an aldosterone antagonist.

9. The method of embodiment 8, wherein the amount of IGFBP7 determined in step a) is compared in step a) to i) a single reference amount, or ii) a reference amount of IGFBP7 for identifying subjects suitable for administration of a beta-blocker, and to a reference amount of IGFBP7 for identifying subjects suitable for administration of an aldosterone antagonist.

10. The method according to embodiments 8 and 9, wherein

an amount of the biomarker in the test sample that is decreased compared to the reference amount indicates that the subject is suitable for administration of the beta blocker, and/or an amount of the biomarker that is increased compared to the reference amount indicates that the subject is not suitable for administration of the beta blocker,

and

An increased amount of the biomarker in the test sample compared to the reference amount indicates that the subject is suitable for administration of the aldosterone antagonist, and/or a decreased amount of the biomarker compared to the reference amount indicates that the subject is not suitable for administration of the aldosterone antagonist.

11. The method according to any one of embodiments 1-10, wherein the subject suffers from heart failure stage B, C or D according to the ACC/AHA classification.

12. The method of embodiment 2, wherein (i) the at least one agent is an aldosterone antagonist, and wherein the at least one biomarker is selected from the group consisting of GDF-15, IGFBP7, cardiac troponin, uric acid, PlGF, sST2, and Gal-3, or (ii) the at least one agent is a beta blocker, and wherein the at least one biomarker is at least one biomarker selected from the group consisting of endostatin, mimecan, cardiac troponin, a BNP-type peptide, and sST2, or (iii) the at least one agent is an inhibitor of the renin-angiotensin system, and wherein the at least one biomarker is selected from the group consisting of cardiac troponin, a BNP-type peptide, osteopontin, and/or uric acid.

13. The method of embodiment 12, wherein an amount of at least one biomarker that is increased compared to the reference amount indicates a subject that is suitable for administration of the at least one agent, and/or wherein an amount of at least one biomarker that is decreased compared to the reference amount indicates a subject that is not suitable for administration of the at least one agent.

14. The method of embodiment 2, wherein the at least one agent is a diuretic, and wherein the biomarker is endostatin, mimecan, GDF-15, uric acid and/or a BNP-type peptide.

15. The method of embodiment 14, wherein a decreased amount of at least one biomarker compared to the reference amount indicates a subject suitable for administration of the at least one agent, and/or wherein an increased amount of at least one biomarker compared to the reference amount indicates a subject unsuitable for administration of the at least one agent.

16. In a sample from a subject with heart failure, i) at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin and sST2 (soluble ST2), sFlt-1, PlGF, PlNP, cystatin C, prealbumin and transferrin, or ii) at least one detection agent that specifically binds to a natriuretic peptide, and/or at least one detection agent that specifically binds to a natriuretic peptide selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sFlt-1, PlGF, sST2 Use of a detection reagent for biomarkers of β-blockers, soluble ST2, P1NP, cystatin C, prealbumin, and transferrin for identifying subjects suitable for administration of at least one agent selected from the group consisting of beta blockers, aldosterone antagonists, diuretics, and inhibitors of the renin-angiotensin system.

17. An apparatus suitable for carrying out the method of any one of embodiments 1 to 12, comprising:

a) an analyzer unit comprising a detection reagent (or reagents) that specifically binds to a marker selected from the group consisting of GDF-15 (growth differentiation factor 15), endostatin, mimecan, IGFBP7 (IGF binding protein 7), cardiac troponin, a BNP-type peptide, uric acid, Gal-3 (galectin-3), osteopontin, sFlt-1, PlGF, sST2 (soluble ST2), PlNP, cystatin C, prealbumin, and transferrin, said unit being suitable for determining one or more amounts of one or more markers in a sample from a subject suffering from heart failure; and

b) an analyzer unit for comparing one or more measured amounts to one or more reference amounts, thereby identifying a subject suitable for administration of at least one agent selected from the group consisting of a beta blocker, an aldosterone antagonist, a diuretic, and an inhibitor of the renin-angiotensin system, said unit comprising a database with reference amount(s) and a computer-implemented algorithm for performing the comparison.

The attached figure shows:

Figure 1: Additive/upregulatory effects of aldosterone antagonists based on marker levels.

Figure 2: Effects of adding/up-regulating beta-blockers based on marker levels.

Figure 3: Effects of adding/up-regulating diuretics based on marker levels.

Figure 4: Effects of adding/upregulating inhibitors of the renin-angiotensin system (RAS) based on marker levels.

All references mentioned above are hereby incorporated by reference with respect to their entire disclosure content as well as to the specific disclosure content to which they are explicitly mentioned in the above description.

Example

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

Example 1: Determination

The following markers were measured in plasma.

Troponin T was measured using the electrochemiluminescent ELISA sandwich test Elecsys Troponin T hs (High Sensitivity) STAT (Short Turn Around Time) from Roche. The test employs two monoclonal antibodies specific for human cardiac troponin T. The antibodies recognize two epitopes (amino acid positions 125-131 and 136-147) located in the central portion of the cardiac troponin T protein, which consists of 288 amino acids. The hs-TnT assay allows measurement of troponin T levels in the range of 3-10,000 pg/mL.

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

To determine GDF-15 concentrations 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&D Systems (AF957). In each experiment, a standard curve was generated using recombinant human GDF-15 from R&D Systems (957-GD/CF). Results for new batches or recombinant GDF-15 protein were tested in standard plasma samples, and any deviation exceeding 10% was corrected by introducing an adjustment factor for the assay. After correcting for the final dilution factor, GDF-15 measurements in serum and plasma samples from the same patient obtained virtually identical results. The detection limit of the assay was 200 pg/ml.

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

A streptavidin-coated 96-well microtiter plate was incubated with 1 μg/ml of 100 μl of biotinylated anti-IGFBP7 polyclonal antibody in 1x PBS for 60 minutes. Following incubation, the plate was washed three times with 1x PBS + 0.02% Tween-20, blocked with PBS + 1% BSA (bovine serum albumin), and then washed again three times with 1x 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 diluted serum or plasma samples (1:50) from patients or controls, respectively. Following IGFBP7 binding, the plate was washed three times with 1x PBS + 0.02% Tween-20. For specific detection of bound IGFBP7, wells were incubated with 100 μl of digoxigenin-labeled anti-IGFBP7 polyclonal antibody at 1 μg/ml in 1x PBS + 1% BSA for 60 minutes. The plates were then washed three times to remove unbound antibody. In the next step, wells were incubated with 75 mU/ml anti-digoxigenin-POD conjugate (Roche Diagnostics GmbH, Mannheim, Germany, catalog number 1633716) in 1x PBS + 1% BSA for 60 minutes. The plates were then washed six times with the same buffer. For detection of antigen-antibody complexes, wells were incubated with 100 μl of ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, catalog number 11685767), and after 15 minutes, the optical density (OD) was measured at 405 and 492 nm using an ELISA reader.

Gal-3 was measured using the BGM Galectin-3 assay (BG Medicine, Waltham, MA, USA). It quantitatively measures Galectin-3 in serum or EDTA-plasma using an enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies against Galectin-3 in a microtiter plate platform. A rat monoclonal anti-mouse Galectin-3 antibody was coated onto the well surface of the microtiter plate and served as a capture antibody to bind Galectin-3 molecules in the sample, while a mouse monoclonal anti-human Galectin-3 antibody was provided in solution and served as a tracer antibody to detect Galectin-3 molecules bound to the capture antibody.

In order to detect the mimecan in human serum or plasma, sandwich ELISA is used. For antigen capture and detection, aliquots of the anti-mimecan polyclonal antibody (catalog number: AF 2660) from R&D Systems are conjugated to biotin and digoxin respectively. 96-well microtiter plates coated with streptavidin were incubated in 1x PBS solution for 60 minutes with 100 μl of biotinylated anti-mimecan polyclonal antibody of 0.2 [mu] g/ml. After incubation, plates were washed three times with 1x PBS+0.02%Tween-20, blocked 45 minutes with PBS+2%BSA (bovine serum albumin), and subsequently washed three times with 1x PBS+0.02%Tween-20 again. The wells were then incubated for 1 hour with a serial dilution of 100 μl recombinant mimecan as a standard antigen or diluted serum or plasma samples (1:5 in 1x PBS + 1% BSA) from patients or control individuals. After mimecan binding, the plate was washed three times with 1x PBS + 0.02% Tween-20. For specific detection of bound mimecan, the wells were incubated for 45 minutes in 1x PBS + 1% BSA with 100 μl digoxigenin-labeled anti-mimecan polyclonal antibodies at 0.2 μg/ml. Thereafter, the plate was washed three times to remove unbound antibodies. In the next step, the wells were incubated for 30 minutes in 1x PBS + 1% BSA with 100 μl 75 mU/ml anti-digoxigenin-POD conjugate (Roche Diagnostics GmbH, Mannheim, Germany, catalog number 1633716). The plate was then washed six times with the same buffer as above. For detection of antigen-antibody complexes, the wells were incubated with 100 μl ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 11685767) and the optical density (OD) was measured after 15 minutes at 405 and 492 nm using an ELISA reader.

To measure endostatin in human serum or plasma, a commercially available sandwich ELISA (Quantikine Human Endostatin Immunoassay, catalog number DNSTO, R&D Systems) was used. The measurement was performed according to the manufacturer's instructions.

sST2 is measured by using the Presage from Critical Diagnostics (San Diego, CA, USA). The quantitative sandwich monoclonal ELISA in 96 well plate formats is used to measure the ST2 in serum or the blood plasma. The plasma of dilution is loaded in the appropriate wells in the plate coated with anti-ST2 antibodies, and the time of incubation appointment. Reagent is washed away from plate, and after the series of steps of adding other reagent and washing out subsequently, the final detection analyte of the colorimetric reagent is added, and the resulting signal of 450 nm place spectrophotometry is measured.

The biomarker mimecan was determined as described in WO2011/012268.

PlGF and sFlt1 were tested using the ELECSYS immunoassay, which utilizes two antibodies specific for PlGF and sFlt1, respectively. The assay can be automated using various Roche analyzers, including the ELECSYS 2010, as well as the cobra e411 and cobra e601. For PlGF, the assay has a sensitivity of 3 pg/ml. sFlt-1 levels range from 10 to 85,000 pg/ml.

Prealbumin was tested using an in vitro assay for the quantitative determination of prealbumin in human serum on Roche/Hitachi cobas c systems (ACN 710 for c 311/501 analyzers; ACN 8710 for c 520 analyzers; catalog number 20764655322). The assay is an immunoturbidimetric assay. Human prealbumin forms a precipitate with specific antiserum, which is measured turbidimetrically.

Cystatin C was used by immunoturbidimetric assay for the quantitative in vitro determination of cystatin C in human serum and plasma on a Roche automated clinical chemistry analyzer (for Roche/Hitachi 917, MODULAR P analyzer: ACN 431, catalog number 04975774 190). Human cystatin C was agglutinated with latex particles coated with anti-cystatin C antibodies. Aggregates were determined turbidimetrically at 546 nm.

Example 2: Patient Cohort/Outcomes

Example from the TIME-CHF study: GDF-15, TnT-hs, uric acid, endostatin, IGFBP-7, Mimecan, sST2, galectin-3, and osteopontin levels were measured in samples from n = 450 patients in the TIME-CHF randomized trial (aged 60 years or older, with systolic HF (ejection fraction <= 45%), NYHA class II or greater within 1 year before HF hospitalization, and NTproBNP levels 2 or more times the upper limit of normal). The TIME-CHF study is described in BNP-Guided vs Symptom-Guided Heart Failure Therapy JAMA, 2009; 301(4):383-392.

At baseline, most patients were receiving recommended HF therapy: ACE inhibitors or angiotensin II receptor blockers, beta-blockers, and diuretics.

Biomarkers are measured when baseline and after 6 months.The number provided in the following table is the patient % with " poor prognosis " (death, repeated hospitalization). Whether the biomarkers measured by analysis will allow identification of patients, and the patients will benefit from increasing the following dosage or reducing the following dosing interval: the inhibitor of beta blockers, aldosterone antagonists, diuretics and/or renin-angiotensin system, and/or add some medicament to therapy.For this analysis, patients are divided into i) the medicament that accepts increased dosage and/or the subject group that accepts other medicament, and ii) the subject group that treatment scheme does not revise.In a further step, based on respective biomarker levels (lower than median value/higher than threshold value), two groups are divided into groups separately.The results are shown in Table 1:

Functional principal component analysis (fPCA) is used to further analyze the data. fPCA reduces the dimensionality of the complex administration data of the drug to allow for interaction analysis of marker levels and results at multiple time points (beyond baseline). In this case, three principal components are used, representing 1) the overall dose level of the drug during the study (the most important component), 2) the trend of drug dosage increase or decrease during the study, and 3) the trend of drug dosage first increasing and then decreasing or first decreasing and then increasing. The advantage and supplementary information of fPCA over the stratified analysis (previous analysis) at baseline is that it reveals the interaction of markers and drugs when exceeding the time point of baseline. Therefore, other interactions and therapy response predictions can be evaluated. Subsequently, the components of fPCA are tested with respect to interaction and therapy response predictions, wherein using median marker levels and results, using the same endpoints as described above. The results and intensity of interaction are represented in the table below. These results confirm the interactions of many previous findings and propose other marker drug combinations.

The results are shown in the table below.

Results for IGFBP7 : Data analysis showed that patients whose baseline IGFBP-7 levels (independent of other biomarkers) were below the reference value (< 100 ng/mL in the study) did not benefit from the addition or titration of spironolactone. If IGFBP-7 levels were above the reference value (> 100 ng/mL in the study, with a cutoff to be defined; this is age-dependent, with lower levels generally found in younger populations), an aldosterone antagonist (e.g., spironolactone) and/or beta-blocker should be added or titrated. Survival at 18 months was improved in the second group, whereas survival was unchanged in the first group.

Results for GDF-15 : Data evaluation showed that patients whose GDF-15 levels at baseline (independent of other biomarkers) were below the reference value (< 4000 pg/mL in the study) did not benefit from the additional administration or up-titration of beta-blockers or aldosterone antagonists (e.g., spironolactone), whereas patients whose GDF-15 levels were above the reference value (> 4000 pg/mL in the study) were likely to benefit from the addition or up-titration of beta-blockers or aldosterone antagonists. Survival at 18 months was improved in the second group, while survival was unchanged in the first group.

Endostatin results : Data evaluation showed that patients whose endostatin levels at baseline (independent of other biomarkers) were below the reference value (< 250 ng/mL in the study) did not benefit from additional administration or up-regulation of beta-blockers. Patients already taking beta-blocker compounds should maintain, but the dose does not need to be increased. Patients whose endostatin levels were above the reference value (> 250 ng/mL in the study) may benefit from additional administration of beta-blockers. In addition, up-regulation of diuretics should be avoided. 18-month survival was improved in the second group, while survival remained unchanged in the first group.

Mimecan results : Data evaluation showed that patients whose mimecan levels at baseline (independent of other biomarkers) were below the reference value (< 50 ng/mL in the study) did not benefit from the addition or increase of beta-blockers. If mimecan levels were above the reference value (> 50 ng/mL in the study), a beta-blocker should be added or the beta-blocker dose should be increased. In addition, increase of diuretics should be avoided.

NTproBNP results : Data evaluation showed that patients with NTproBNP levels above the reference value (>3000 pg/mL in the study) did not benefit from the addition or upward adjustment of diuretics.

sST2 Outcomes : Data evaluation showed that patients with sST2 levels above the reference value (> 34.0 g/mL in the study) benefited from the addition or up-titration of beta-blockers.

Gal-3 results : Data evaluation showed that patients with Gal-3 levels below the reference value (<31.6 g/mL in the study) benefited from the addition or up-titration of renin-angiotensin system inhibitors.

sFlt-1 results : Data evaluation showed that patients with sFlt-1 levels below the reference value (<98 μg/mL in the study) benefited from the addition or up-titration of RAS inhibitors and/or aldosterone antagonists.

Results for PlGF : Data evaluation showed that patients with sFlt-1 levels above the reference value (>20.7 g/mL in the study) benefited from the addition or up-titration of an aldosterone antagonist.

Results for osteopontin : Data evaluation showed that patients with osteopontin levels above the reference value (>100 g/mL in the study) did not benefit from the addition or upward adjustment of diuretic therapy.

The following conclusions can be drawn:

• If endostatin is above the reference value, BB should be added and/or its dose should be adjusted upward. In addition, up-regulation of aldosterone antagonists and/or diuretics should be avoided.

• If Mimecan is above the reference value, BB should be added and/or the BB dose should be adjusted upward. In addition, upward adjustment of diuretics should be avoided.

• If IGFBP7 is below the reference value, BB should be added or increased. If IGFBP7 is above the reference value, aldosterone antagonist should be added or increased.

• If IGFBP7 is below the reference value, aldosterone antagonists and RAS inhibitors should not be added or up-titrated.

If GDF-15 is above the reference value, BB, RAS inhibitors, and aldosterone antagonists should be added or adjusted upward. In contrast, diuretics should not be added or the diuretic dose should not be increased.

• RAS inhibitors, BB compounds, or aldosterone antagonists can be up-titrated if cTnT-hs is above the median, whereas this should not be done when cTnT-hs is below the median.

• If uric acid is above the median, RAS inhibitor compounds may be up-titrated. Additionally, up-titration of diuretics and/or aldosterone antagonists should be avoided.

• Patients with NTproBNP levels above the reference level (> 3000 pg/mL in the study) do not benefit from up-titration of diuretics, but RAS inhibitors and BB should be up-titrated. Patients with NTproBNP levels below the reference level (< 3000 pg/mL in the study) do not benefit from up-titration of RAS inhibitors.

•If Gal-3 is above the median, an aldosterone antagonist should be added and/or titrated upward.

• If osteopontin is above the median, a RAS inhibitor should be added and/or up-titrated. If osteopontin is below the median, a BB compound should be added and/or up-titrated.

• If sFlt-1 is below the reference amount (median values were used in this study), RAS inhibitors, BB compounds, and/or aldosterone antagonists should be added and/or titrated upwards.

• If PLGF is above a reference amount (eg, median), then an aldosterone antagonist should be added and/or titrated upward.

•If sST2 is above a reference value (e.g., median), aldosterone antagonists and/or beta-blockers should be added and/or titrated upward.

• If P1NP is below a reference amount (e.g., median), beta-blockers should be added and/or titrated upward.

• If prealbumin is above a reference amount (eg, median), diuretics should not be added, reduced, or upregulated.

• If transferrin is above a reference value (eg, median), diuretics should not be added, reduced, or upregulated.

•If cystatin C is below the reference amount (e.g., median value), the aldosterone antagonist should not be added or titrated upward, and high doses should be reduced.

• If the ratio PlGF/sFlt-1 C is higher than the reference ratio (eg, median), an aldosterone antagonist should be added or the dose adjusted upward.

Thus, by applying the diagnostic algorithm outlined above, subjects suitable for administration of the above-mentioned agents (ie, initial administration or administration at a higher dose, "up-regulation") can be identified. Suitable reference amounts can be determined as described in the specification.

Example 3: Individual case study

An 89-year-old male patient with Class C heart failure received low-dose chlorthalidone (25 mg/d), enalapril (5 mg/d), and metoprolol (25 mg/d). The patient showed signs of progressive heart failure, with elevated NT-proBNP levels. The patient also had asthma, and the treating physician was unsure whether BB should be increased. Mimecan was measured in a plasma sample obtained from the patient. The mimecan value was above 80 pg/mL. Therapy was intensified by sequential uptitration of enalapril (to 20 mg/d) and metoprolol (100 mg/d). In contrast, the chlorthalidone dose was not increased. The patient remained stable with a good outcome until the end of the study (no deaths or hospitalizations).

A 90-year-old female patient with Class C heart failure was receiving a fixed-dose combination of hydrochlorothiazide (12.5 mg/day) and valsartan (80 mg/day), along with atenolol (100 mg/day). The patient had a history of decompensation episodes and hospitalizations. At her last visit, her exercise intolerance worsened, and she achieved a decreased walking distance on the 6-minute walk test compared to her most recent visit 3 months prior. NT-proBNP and IGFBP-7 were measured in a plasma sample obtained from the patient. NT-proBNP values were above 1000 pg/mL, and IGFBP-7 values were above 100 pg/mL. The treating physician was unsure whether to safely add spironolactone because the patient occasionally presented with elevated potassium levels. Therapy was intensified, and spironolactone was initially added at 25 mg/day and subsequently titrated upward to 100 mg/day, while closely monitoring serum potassium levels. Consequently, the NT-proBNP value decreased to below 1000 pg/mL. The patient remained stable with a good outcome until the end of the study (no deaths, no hospitalizations).

A 93-year-old male patient with Class D heart failure was receiving hydrochlorothiazide (25 mg/day) and valsartan (160 mg/day), as well as bisoprolol (2.5 mg/day). The patient had a history of decompensated episodes and prolonged hospitalizations. During previous visits, plasma samples obtained from the patient were measured for NT-proBNP and IGFBP-7. The NT-proBNP value was above 1000 pg/mL, and the IGFBP-7 value was below 100 pg/mL. The treating physician did not add spironolactone. Instead, therapy was intensified by increasing bisoprolol to 10 mg/day. The patient remained stable with a favorable outcome until the end of the study (no deaths, no hospitalizations).

A 70-year-old female patient with class B heart failure was receiving captopril (2 x 6.25 mg/d). The patient had been stable for 10 months but was hospitalized for acute heart failure after watching television and consuming a bag of potato chips. GDF-15 and cTnThs were measured in a plasma sample obtained from the patient at a pre-scheduled visit. The GDF-15 value was above 4000 pg/mL, and the cTnT-hs was above the median. The addition of the beta-blocker metoprolol and the aldosterone antagonist spironolactone was chosen to enhance therapy. The patient had just experienced another hospitalization due to dizziness and hypotension.

A 77-year-old male vegetarian patient with Class C heart failure received valsartan (160 mg/d) and carvedilol (12.5 mg/d). The patient had been stable over the past 5 months, but arrived at the emergency room in a decompensated state with acute dyspnea, rales, and tachycardia. Uric acid and Gal-3 were measured in plasma samples obtained from the patient. Uric acid and Gal-3 values were lower than the median. The treating physician increased the dose of carvedilol to 50 mg/d to strengthen the therapy. In addition, spironolactone was initially added at 25 mg/d and later increased to 50 mg/d. The patient remained stable for 1 month, but subsequently developed edema, atrial fibrillation, dyspnea, and cough. On the way to the hospital, the patient died due to sudden death.

in conclusion:

The present invention provides a method for selecting a drug therapy that will be beneficial in patients suffering from heart failure. The method predicts the response to therapy and/or helps select appropriate therapy or therapy reinforcement. Compared with the prior art and previous biomarker-guided heart failure methods, the present invention provides specific target levels of multiple markers and specific instructions for therapy selection and administration. The present invention also improves the identification and utilization of drug therapies that are beneficial to patients, and on the contrary avoids potentially harmful therapies to patients. Therefore, the present invention aims to reduce the mortality and morbidity of patients with heart failure.

Claims (10)

1. The use of reagents that specifically bind to the biomarkers PlGF and sFlt-1, or reagents that specifically bind to the biomarker PlGF, in the preparation of kits for methods of identifying subjects suitable for administration of aldosterone antagonists, including: a) Determining the amount of biomarkers PlGF or PlGF and sFlt-1 in samples from subjects with heart failure, wherein the samples are blood, serum, or plasma, and b1) Calculate the ratio of PlGF to sFlt-1, or vice versa, and compare the calculated ratio with a reference ratio to identify suitable candidates for aldosterone antagonist administration. b2) Compare one or more quantities measured in step a) with one or more reference quantities to identify suitable subjects for the application of the aldosterone antagonist. 2. The use of claim 1, wherein the application is the initiation of the application of the aldosterone antagonist or the application of the aldosterone antagonist at a higher dose. 3. The use of any one of claims 1-2, wherein the object is a person. 4. Use according to any one of claims 1-2, wherein an indicator of the ratio of PlGF to sFlt-1 in a test sample that is increased compared to a reference ratio is suitable for the administration of the aldosterone antagonist, and/or an indicator of the ratio that is decreased compared to a reference ratio is not suitable for the administration of the aldosterone antagonist. 5. Use according to any one of claims 1-2, wherein the subject to be tested has been treated with an aldosterone antagonist, wherein an increased ratio of PlGF to sFlt-1 in the test sample compared to a reference ratio indicates that the subject is suitable for administration of the aldosterone antagonist at a higher dose, and/or a decreased ratio compared to a reference ratio indicates that the subject is not suitable for administration of the aldosterone antagonist at a higher dose. 6. Use according to any one of claims 1-2, wherein the biomarker is PlGF, and wherein an increase in the amount of the biomarker compared to the reference amount indicates a subject suitable for administration of the aldosterone antagonist, and/or a decrease in the amount of the biomarker compared to the reference amount indicates a subject unsuitable for administration of the aldosterone antagonist. 7. Use according to any one of claims 1-2, wherein the subject suffers from heart failure stage B, C or D according to the ACC/AHA classification. 8. The use of reagents that specifically bind to sFlt-1 and/or PlGF in samples from subjects with heart failure in the preparation of kits for identifying subjects suitable for administration of aldosterone antagonists, wherein the samples are blood, serum, or plasma. 9. An apparatus suitable for carrying out the method described in any one of claims 1-7, comprising: a) An analyzer unit comprising one or more detection reagents that specifically bind to sFlt-1 and/or PlGF in a sample from a subject suffering from heart failure, wherein the sample is blood, serum, or plasma; and b) An analyzer unit for comparing a measured quantity with one or more reference quantities, thereby identifying subjects suitable for administration of an aldosterone antagonist, the analyzer unit for comparing a measured quantity with one or more reference quantities including a database of one or more reference quantities and a computer-implemented algorithm for performing the comparison. 10. The use of any one of claims 1-2, or the apparatus of claim 9, wherein the aldosterone antagonist is spironolactone.