HK1224375B - Use of mimecan in the assessment of heart failure - Google Patents

Description

Use of mimecan in the assessment of heart failure

The present application is a divisional application of PCT application having an international application date of 23/7/2010, an international application number of PCT/EP2010/004521, an application number of 201080029136.5 at the stage of entering the country, and an invention name of "use of mimecan in evaluating heart failure".

Technical Field

The present invention relates to a method of assessing heart failure in an individual, the method comprising the steps of: a) measuring the concentration of the marker mimecan in a sample obtained from the individual, b) optionally measuring the concentration of one or more other marker of heart failure in the sample, and assessing heart failure by comparing the concentration determined in step (a) and optionally the concentration determined in step (b) with the concentration of said one or more marker established for a control sample. Also disclosed is the use of mimecan as a marker protein for assessing heart failure, a marker combination comprising mimecan, and a kit for measuring mimecan.

Background

Heart Failure (HF) is an important problem and is becoming more and more serious in the health risks of the general public. For example, in the United states, approximately 5 million patients suffer from HF, and over 550000 patients are first diagnosed with HF each year (as described in American Heart Disease, Heart Disease and Stroke statics: 2008 Update, Dallas, Texas, American Heart Association (2008)). Also, U.S. statistics indicate that HF is a major cause for 1200 to 1500 million clinic visits and 650 million hospitalizations per year. From 1990 to 1999, the number of hospitalized patients with HF as the primary diagnostic decision increases from about 81 to over 100 million and the number of people with HF as the primary or secondary diagnostic decision increases from 240 to 360 million each year. In 2001, nearly 53000 patients died from disease with HF as the main cause. Heart failure is primarily a condition of the elderly, and thus, it is widely believed that "population aging" also contributes to increased HF incidence. In people older than 65 years, the incidence of HF approaches 10% o. In the united states alone, the total direct and indirect loss due to HF is estimated to be about $279 billion in 2005, and the annual cost of drugs for treating HF is about $29 billion (see AHA-statistics cited above).

Heart failure

Heart failure is characterized by a loss of the ability of the heart to pump the amount of blood needed by the body. Failure does not mean that the heart stops pumping, but rather that it is unable to pump blood effectively.

Both NYHA [ New York Heart Association ] and ACC/AHA [ American Heart Association/American Heart Association ] establish a functional classification of HF to assess the progression of the disease. The NYHA classification scheme has four levels of disease status: grade 1 is asymptomatic at any degree of behaviour, grade 2 is symptomatic under heavy physical behaviour, and grade III and IV are symptomatic under mild behaviour and no behaviour respectively.

In the four-phase ACC/AHA regimen, phase a is asymptomatic but at risk of developing HF. Stage B is asymptomatic with evidence of cardiac dysfunction. In phase C, there is evidence of cardiac dysfunction with accompanying symptoms. In stage D, the patient still has symptoms despite the maximum degree of treatment.

Etiology of HF

In medicine, Heart Failure (HF) must be understood as a complex disease. It may be caused by a triggering event, such as a myocardial infarction (heart attack), or be a consequence of other causes, such as hypertension, diabetes, or cardiac malformations (e.g., valvular disease). Myocardial infarction or other causes of HF can result in an initial decline in the pumping capacity of the heart, for example, due to damage to the heart muscle. Such a decrease in pumping capacity may not be perceptible due to activation of one or more compensatory mechanisms. However, the progression of HF has been found to be independent of the patient's hemodynamic status. Thus, the damaging changes caused by the disease have already developed and the patient is even asymptomatic. Indeed, the compensatory mechanisms that maintain normal cardiovascular function during the early stages of HF may ultimately contribute to disease progression, for example by exerting deleterious effects on the heart and its function to maintain adequate levels of blood circulation.

Some of the more important pathophysiological changes in the presence of HF are (i) activation of the hypothalamic-pituitary-adrenal axis, (ii) systemic endothelial dysfunction and (iii) myocardial remodeling.

(i) Therapies directed specifically at antagonizing hypothalamic-pituitary-adrenal axis activation include β -adrenergic blockers (B-blockers), Angiotensin Converting Enzyme (ACE) inhibitors, certain calcium channel blockers, nitrates, and endothelin-1 blockers. Calcium channel blockers and nitrates, while producing clinical improvement, have not been clearly shown to prolong survival, while B-blockers and ACE inhibitors have been shown to significantly prolong life, as have aldosterone antagonists. Experimental studies using endothelin-1 blocking agents have shown beneficial effects.

(ii) Systemic endothelial dysfunction is a well-known feature of HF and manifests itself in the presence of signs of left ventricular dysfunction. Endothelial dysfunction is important for the close relationship between myocardial microcirculation and cardiac myocytes. This evidence suggests that microvascular dysfunction contributes significantly to myocyte dysfunction and leads to morphological changes in progressive myocardial failure.

Based on the underlying pathophysiology, there is evidence that endothelial dysfunction may be caused by a relative lack of NO, which can be attributed to vascular O caused by NADH-dependent oxidase₂Increased production and subsequent over-scavenging of NO. Potential increase of O₂The contributing factors generated include elevated sympathetic tone, norepinephrine, angiotensin II, endothelin-1, and TNF- α furthermore, the level of IL-10, a key anti-inflammatory cytokine, is too low relative to the level of TNF- α it is now believed that TNF- α and related pro-inflammatory cytokines (including IL-6 and soluble TNF- α receptors) have an elevated level that plays an important role in the development of HF by causing decreased myocardial contractility, biventricular enlargement, and hypotension, and may be involved in endothelial cell activation and dysfunction.

(iii) Myocardial remodeling is a complex process that accompanies the transition from asymptomatic to symptomatic heart failure and can be described as a series of adaptive changes in the myocardium, such as changes in ventricular shape, mass, and volume (pinano, m.r. et al, j. cardiovasc. nurs. 14 (2000) 1-23; Molkentin, j.d., ann. rev. physiol.63 (2001) 391-426). The main components of myocardial remodeling are alterations in myocyte biology, such as myocyte hypertrophy, loss of myocytes due to necrosis or apoptosis, alterations in extracellular matrix and geometric alterations in the left ventricular cavity. It is not clear whether myocardial remodeling is simply an end organ effect that occurs years after toxic effects from long-term neurohormonal stimulation, or whether myocardial remodeling independently contributes to the progression of heart failure. Evidence to date suggests that appropriate treatment may slow or stop the progression of myocardial remodeling.

Markers and disease states

As mentioned above, myocyte hypertrophy may represent one of the initial steps to progression to HF. Myocyte hypertrophy is characterized by elevated expression of certain genes encoding contractile proteins (e.g., p-myosin heavy chain and troponin T (TnT)) and some non-contractile proteins (e.g., type A and type B natriuretic peptides), increased cell volume and cytoskeletal changes (Piano, M.R. et al, J.Cardiovasc. Nurs. 14 (2000) 1-23; Molkentin, J.D., Ann. Rev. physiol.63 (2001) 391-426).

Studies in human and animal models of heart failure have shown that muscle cell function is reduced in the late stages of heart failure. It has been shown that the mechanisms leading to muscle cell dysfunction involve alterations in the calcium processing network, myofilaments and cytoskeleton (de Tombe, P.P., Cardiovasc. Res. 37 (1998) 367-. For example, in human and animal models of heart failure, the enzymatic activity of sarcoplasmic reticulum calcium-ATPase is reduced, while the sarcolemma Na⁺/Ca²⁺Both the mRNA and protein levels of the exchanger were increased. Moreover, there is isotype switching of TnT, decreased troponin i (tni) phosphorylation, decreased myofibrillar actomyosin atpase activity, and increased microtubule formation in both human and animal models of heart failure.

Initially, the cardiac changes that lead to myocardial remodeling are to compensate for the myocardial lesions to maintain the body's need for oxygen and nutrients. However, the compensatory phase of heart failure is limited, and eventually, the failing heart fails to maintain a cardiac output sufficient to meet the physical demands. Thus, there will be a transition from a period of compensation to a period of decompensation. Within the decompensation phase, the cardiac cascade changes persist but are no longer favorable, which leads to the development of a chronic state of heart failure and ultimately death in the patient.

According to "updated guidelines for diagnosis and management of adult chronic heart failure" for ACC/AHA 2005 "(S. Hunt et al)The person or persons can be provided with the following functions,www.acc.orgACC/AHA guidelines), today, in the field of heart failure, the disease process is divided into four stages as described above. In phases a and B, the individual is found at risk of developing heart failure, while phases C and D indicate that these patient groups show signs and symptoms of heart failure. A detailed description of the various phase definitions a to D is given in the above references and is incorporated herein by reference.

Method for diagnosing heart failure

The only most effective diagnostic test to evaluate HF patients is to use a comprehensive 2-dimensional echocardiogram in conjunction with a doppler flow study to determine whether abnormalities of the myocardium, heart valves or pericardium occur, and which chambers are involved. Three basic issues must be considered: 1) whether LVEF is maintained or reduced, 2) whether LV structures are normal or abnormal, and 3) whether there are other structural abnormalities that may explain clinical manifestations, such as abnormalities of the valve, pericardium, or right ventricle? This information should be quantified by numerical expectations of EF, measurements of ventricular dimensions and/or volume, measurements of wall thickness, and assessment of chamber geometry and local wall activity. The size of the right ventricle and contractile performance should be evaluated. In addition, the size of the atrium should be semi-quantitatively determined and the dimensions and/or volume of the left atrium measured.

Non-invasive hemodynamic data obtained while performing echocardiography is an important additional correlation indicator for patients with maintained or reduced EF. The combined quantification of the combined mitral valve infusion model, pulmonary vein infusion model, and mitral annulus flow rate provides data on characteristics of LV filling and left atrial pressure. Assessment of the tricuspid valve regurgitation gradient, as well as measurement of the inferior vena cava dimension and its response during respiration, allows assessment of systolic pulmonary artery pressure and central venous pressure.

Stroke volume can be determined by combining dimensional measurements and pulsed doppler of the LV outflow tract. However, in the absence of HF, anomalies in any of these parameters may occur. None of these parameters necessarily correlates specifically with HF; however, a generally normal filling model may rule out clinical HF.

From a clinical point of view, the disease is clinically asymptomatic in the compensatory phase and in the early phase of decompensation (stage a is completely asymptomatic, stage B has structural heart disease but no signs and symptoms of HF, refer to ACC/AHA guidelines for practice). Until the decompensation phase is completely entered (i.e., according to ACC/AHA guidelines, stages C and D), external signs of the disease (e.g., breathlessness) do not appear. The current diagnostic modality is based on the external symptoms of the patients of stages C and D.

Typically, heart failure patients receive standard treatment with drugs that meet heart failure-specific mechanisms. No diagnostic test reliably reflects those specific mechanisms and helps the physician select the appropriate drug (and dose) for the appropriate patient (e.g., ACE inhibitors, AT II, beta-blockers, etc.).

Early diagnosis of HF with markers

It seems possible to perform an early assessment of patients at risk of heart failure only by biochemical markers, since individuals at risk of developing heart failure are still free of clinical symptoms of HF at that stage. There are no established biochemical markers that can be reliably assessed before the symptoms of the disease appear. By the time HF diagnosis is established, the disease has advanced profoundly.

In recent years, the natriuretic peptide family, particularly the atrial natriuretic peptide family and the brain natriuretic peptide family, has proven to be of significant value for the assessment of HF.

Prognosis and need of HF

At least in part because of the delayed diagnosis, 50% of HF patients die within two years after diagnosis. The 5-year survival rate is less than 30 percent. There is an urgent need for new biochemical markers that aid in the early diagnosis of heart failure.

Improved protocols for the early assessment of individuals at risk of heart failure (i.e., individuals without clinical symptoms of heart failure) have been approved.

In recent years, type B natriuretic peptide markers have been identified as a good means of monitoring the disease progression in HF patients and used to assess the risk of cardiovascular complications, such as heart attack, in patients.

However, for many other diagnostic aspects, a single marker is not sufficient.

Although low values of NT-proBNP are very good negative predictive index values for the exclusion of HF or LV, in the above and other studies (cf. Triepels R.H. et al, Clin. chem. 49, suppl. A (2003) A37-A38) positive predictive values for heart failure were found to be in the range of 50-60%. Thus, markers that can be used to assess the risk of heart failure in an individual are of high clinical/practical importance, such markers having either a high HF positive prediction rate on their own or a better HF positive prediction rate in combination with NT-proBNP compared to NT-proBNP alone.

In this clinically very important and urgently needed diagnostic field, markers that aid in the assessment of patients with heart failure are also of great significance for achieving further technological advances.

Disclosure of Invention

It has now been found and established that the marker mimecan aids in the assessment of heart failure. In one embodiment, it may help assess whether an individual is at risk for developing heart failure. In a further aspect, it can assist in assessing disease progression. In another embodiment, it may assist in predicting the occurrence of heart failure. In another embodiment, it can aid in the assessment and selection of an appropriate treatment regimen to prevent or treat heart failure.

Disclosed herein is a method of assessing heart failure in an individual comprising the steps of: measuring the concentration of the marker mimecan in a sample obtained from the individual; optionally measuring the concentration of one or more other markers of heart failure in the sample; and assessing heart failure by comparing the concentration of mimecan and optionally the concentration of said one or more other markers with the concentration of said one or more markers determined for a control sample.

The invention also relates to the use of the protein mimecan as a marker molecule in the assessment of heart failure.

Further disclosed herein is the use of a marker combination comprising mimecan and one or more other markers of heart failure in the assessment of heart failure.

Also provided herein is a kit for performing a method for assessing heart failure in vitro, the method comprising the steps of: measuring the concentration of the marker mimecan in the sample; optionally measuring the concentration of one or more other markers of heart failure in the sample; and assessing heart failure by comparing the concentration of mimecan and optionally of one or more other markers with the concentration of said one or more markers determined in a reference population, the kit comprising reagents required for the specific determination of mimecan and optionally reagents required for the specific determination of one or more other markers of heart failure.

Other aspects and advantages of the invention will become apparent from the ensuing description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Detailed Description

In a first embodiment, the present invention relates to a method of assessing heart failure in an individual, comprising the steps of: a) measuring the concentration of the marker mimecan in a sample obtained from the individual, b) optionally measuring the concentration of one or more other marker of heart failure in the sample, and c) assessing heart failure by comparing the concentration measured in step (a) and optionally the concentration measured in step (b) with the concentration of said one or more marker determined in a control sample.

In this document, each of the following terms has its meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an antibody" means one antibody or more than one antibody.

The expression "one or more", "one or more" means 1 to 50, preferably 1 to 20, also preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 15.

The term "label" or "biochemical label" as used herein refers to a molecule that is used as a target for analyzing a test sample from a patient. In one embodiment, examples of these molecular targets are proteins or polypeptides. Proteins or polypeptides used as labels in the present invention are intended to include natural fragments of the protein, particularly immunologically detectable fragments. The immunologically detectable fragment preferably comprises at least 6, 7, 8, 10, 12, 15 or 20 consecutive amino acids of the marker polypeptide. One skilled in the art will recognize that proteins released by cells or present in the extracellular matrix may be damaged, such as during the inflammatory phase, and may be degraded or cleaved into such fragments. Some labels are synthesized in an inactive form and may subsequently be activated by proteolysis. It will be appreciated by those skilled in the art that the protein or fragment thereof may also be present as part of a complex. These complexes may also be used as labels in the sense of the present invention. In addition, or alternatively, the marker polypeptide may carry post-translational modifications. Examples of post-translational modifications are glycosylation, acylation and/or phosphorylation, and the like.

The term "assessing/evaluating heart failure" is used to indicate that the method of the invention will assist a physician in assessing whether an individual is at risk of developing heart failure, or in assessing an HF patient in one or several other diagnostic-related aspects of HF. In assessing HF individuals, preferred diagnostic aspects are staging of heart failure, differential diagnosis of acute and chronic heart failure, determining risk of disease progression, guiding selection of appropriate drugs, monitoring treatment response, and follow-up of HF patients.

For the purposes of the present invention, a "marker for heart failure" is a marker which, if combined with the marker mimecan, will add relevant information for assessing HF for an ongoing diagnosis. For the assessment of HF, the markers are incorporated into the marker combination already comprising the marker mimecan, which information is considered relevant or of added value if the sensitivity at a given specificity or if the specificity at a given sensitivity, respectively, is increased. Preferably, the increase in sensitivity or specificity is of a statistically significant level, respectively: p is 0.05, 0.02, 0.01 or lower. Preferably, the one or more other markers of heart failure are selected from the group consisting of a natriuretic peptide marker, a cardiac troponin marker and an inflammation marker.

The term "sample" as used herein refers to a biological sample obtained for in vitro evaluation. In the method of the invention, the sample or patient sample may preferably comprise any body fluid. Preferred test samples include blood, serum, plasma, urine, saliva, and synovial fluid. Preferably the sample is whole blood, serum, plasma or synovial fluid, plasma or serum being the most convenient sample species. The skilled person will appreciate that any such assessment is performed in vitro. The patient sample is then discarded. Patient samples are used only for the in vitro method of the invention and the substances in the patient samples are not returned to the patient's body. Typically, the sample is a liquid sample, e.g., whole blood, serum or plasma.

The expression "comparing a concentration with a concentration determined for a control sample" is only used to further illustrate what is obvious to the skilled person. The control sample may be an internal control sample or an external control sample. In one embodiment, the internal control sample is used, i.e., the test sample is evaluated for the level of the marker, and one or more other samples taken from the same subject are evaluated for the level of the marker to determine if there is any change in the level of the marker. In another embodiment, an external control sample is used. Comparing the presence or amount of the marker in a sample from one individual with the presence or amount of the marker in another individual for an external control sample, the latter individual being known to have or known to be at risk of having a given condition; or one individual is known to be free of a given disorder, i.e., "normal individuals". For example, the level of the marker in a patient sample can be compared to levels known to be associated with a particular course of HF. Typically, the sample marker level is directly or indirectly associated with diagnosis, and e.g., the marker level is used, e.g., to determine whether an individual is at risk for HF. Alternatively, the marker level of the sample may be compared, for example, to marker levels known to be associated with: response to treatment in HF patients, differential diagnosis of acute and chronic heart failure, guidance in selecting the appropriate drug to treat HF, identification of risk of disease progression, or follow-up of HF patients. Depending on the intended diagnostic use, an appropriate control sample is selected and the control value or reference value for the marker therein is determined. The skilled artisan will recognize that in one embodiment these control samples are obtained from a reference population that is age matched and free of confounding disease. It will also be apparent to the skilled person that the absolute value of the label determined in the control sample will depend on the assay used. Control (reference) values were determined using preferred samples from 100 well-characterized individuals from the appropriate reference population. Also preferably, the reference population may be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals are the preferred reference population for determining control values.

Mimecan

Mimecan is a small proteoglycan with leucine rich repeats and a precursor consisting of 298 amino acids (see SEQ ID NO: 1). Other names of Mimecan are OGN, osteoglyccin, DKFZp586P2421, OG, OIF, SLRR 3A.

Mimecan is a member of the secretory small leucine-rich proteoglycan (SLRP) family, with a structurally related core protein. A common feature shared by all SLRPs is a tandem leucine-rich repeat (LRR) unit at the C-terminal half of the core protein. However, in the N-terminal region, each class of SLRPs has a unique domain called LRR N-domain, which contains cysteine clusters with conserved spacing. Class III SLRPs contain six carboxy LRRs and include mimecan, epiphycan and opticin.

Functional studies of knockout from mice on class I and class II members, such as decorin, biglycan, lumecan and fibromodulin, show that SLRP-deficient mice exhibit a number of defects due to abnormal collagen fibril formation, suggesting that these SLRPs play an important role in the establishment and maintenance of the collagen matrix (Ameye, l. and Young, m.f., Glycobiology 12 (2002) 107R-116R). The absence of class III mimecan also leads to abnormalities in collagen fibers (Tasheva, E.S. et al, mol. Vis. 8(2002) 407- "415).

Mimecan is a multifunctional component of the extracellular matrix. It binds to a variety of other proteins (IGF2, IKB KG, IFNB1, INSR, CHUK, IKB, NFKBIA, IL15, Cd3, retinoic acid, APP, TNF, lipopolysaccharide, c-abl oncogene 1, receptor tyrosine kinase, v-crk sarcoma virus CT10 oncogene, v-src sarcoma virus oncogene). These diverse binding capacities may allow the ability of mimecan to perform diverse functions in a variety of diseases.

Mimecan has been found in the cornea, bone, skin and other tissues. Its expression pattern varies under different pathological conditions. Although there is an increasing amount of data on the biological effects of mimecan, its function is still unclear.

Mimecan has been shown to be involved in the regulation of collagen fibril formation, a process essential in development, tissue repair and metastasis (Tasheva et al, mol. Vis. 8(2002) 407-. It acts in conjunction with TGF-beta-1 or TGF-beta-2 in bone formation.

Mimecan was found to be constitutively expressed in mouse water crystals (Tasheva et al, Molecular Vision 10(2004) 403-416).

Mimecan was shown to be upregulated after vascular injury and after low intensity laser irradiation of osteoblasts, suggesting that the corresponding protein may play a role in wound healing in vascular smooth muscle cells and osteoblasts (Shanahan, C.M. et al, Arter. Thromb. Vasc. biol.17 (1997) 2437-2447; Hamajima, S. et al, Lasers Med. Sci.18 (2003) 78-82).

Other mimecans were found to be upregulated in activated endothelium and to play a role in atherosclerosis (Fernandez, b. et al, mol. cell. biochem. 246 (2003) 3-11).

Transcriptome analysis in rat and human heart tissue showed that mimecan is highly correlated with left ventricular mass (leftventricular mass) and with extracellular remodeling in dilated cardiomyopathy (Petretto, E. et al, Nature Genetics 40 (2008) 546-161552; Barth, A.S. et al, J. American College of Cardiology 48 (2006) 1617).

WO 2006/099336 provides a complex gene expression profile for ischemic and non-ischemic heart diseases. One of the large number of differentially expressed m-RNAs is the mimecan's m-RNA.

WO 2009/061382 claims the use of secreted proteins from cardiac stem cells or muscle cells in regenerative therapy of the heart and methods of screening cardiac patients for hepatocyte therapy. Mimecan is a protein other than the several proteins described as useful in the treatment of cardiac regeneration.

In the area of proliferative/malignant diseases, mimecan is found to be expressed at low levels or not at all in most cancer cell lines. Proteomic studies and western blot validation showed that the down-regulation of mimecan in colorectal cancer (CRC) is consistent with normal mucosa (Wang, y. et al, exp. biol. med. 232 (2007) 1152-1159).

Other applications relate to the therapeutic use of mimecan. WO 2006/043031 relates to the use of therapeutic agents that promote class III SLRP activity, such as for example mimecan, for the prevention and/or treatment of cancer. WO 2004/105784 claims the use of therapeutic agents that promote class III SLRP activity, such as mimecan, for inhibiting angiogenesis or inhibiting conditions characterized by overactivity and/or migration of monocytes and/or macrophages.

It should be noted that in the prior art, the presence or level of the protein mimecan in body fluids is neither known nor suggested to have diagnostic use for assessing heart failure.

The inventors of the present invention have now found and determined that an elevated mimecan measurement in a body fluid sample from an individual is indicative for heart failure.

The mimecan values measured in the control group or control population are used, for example, to determine cut-off values or reference ranges. A value above the cutoff value or outside the reference range at its high end indicates an increase in the value.

In one embodiment, a fixed cutoff value is determined. A cutoff value is selected based on the diagnostic objective.

In one embodiment, the mimecan values measured for a control group or control population are used to determine a reference range. In a preferred embodiment, the mimecan concentration is considered to be increased if the measured value is above 90% of the reference range. In a further preferred embodiment, the mimecan concentration is considered to be increased if the measured value is higher than 95%, 96%, 97% or 97.5% of the reference range.

In one embodiment, the control sample is an internal control sample. In this embodiment, serial samples are obtained from the individual under study and the marker levels are compared. This may be useful, for example, in evaluating efficacy.

The method of the invention is based on liquid samples obtained from individuals and on mimecan measurements in these samples. As used herein, "individual" refers to a single human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Preferably the individual is a human.

The labeled mimecan is preferably specifically determined from the liquid sample by using a specific binding reagent.

Specific binding agents are for example mimecan receptors, lectins binding mimecan or antibodies binding mimecan. The specific binding reagent has a binding affinity for its corresponding target molecule of at least 10⁷Affinity of l/mol. The specific binding reagent preferably has a value of 10 for the target molecule⁸l/mol, even more preferably 10⁹Affinity of l/mol. It will be understood by those skilled in the art that the term specific is used to denote that it is present in a sampleIts biomolecule does not bind significantly to the mimecan specific binding reagent. Preferably, the binding affinity for binding to a biomolecule that is not a target molecule is only 10% or less, more preferably only 5% or less of the affinity for the target molecule. Preferred specific binding reagents should have the minimum criteria for affinity and specificity described above.

The specific binding reagent is preferably an antibody reactive with mimecan. The term "antibody" refers to polyclonal antibodies, monoclonal antibodies, antigen-binding fragments of these antibodies, single chain antibodies, and genetic constructs comprising antibody binding domains.

Any antibody fragment that retains the above criteria for a specific binding reagent may be used. Antibodies can be produced by state-of-the-art procedures, such as those described by Tijssen (Tijssen, P., Practice and the biology of enzymeimmunoassays, Elsevier Science Publishers B.V., Amsterdam (1990), complete books, especially pages 43-78). Furthermore, the skilled person is well aware of methods based on immunoadsorbents that can be used for antibody specific separation. By these methods, the quality of the polyclonal antibodies and thus their performance in immunoassays will be improved (Tijssen, p., above, page 108-.

For the results disclosed in the present invention, polyclonal antibodies produced in goats may be used. It is clear that polyclonal antibodies as well as monoclonal antibodies from different species (e.g. rat, rabbit or guinea pig) can also be used. Because monoclonal antibodies can be produced in any amount as required for constant performance, they are ideal tools for developing clinical routine assays.

The production and use of anti-mimecan monoclonal antibodies, respectively, in the method of the invention represent other preferred embodiments.

Purification of mimecan from natural sources is not easy. Recombinant production of mimecan is an alternative method to obtain higher quantities of mimecan. In a preferred embodiment, the mimecan is produced by recombinant expression using a eukaryotic expression system. Examples of eukaryotic expression systems are baculovirus expression, expression in yeast and expression in mammalian expression systems. In a preferred embodiment, mimecan expression is carried out in a mammalian expression system. Examples of mammalian expression systems are CHO cells, HEK cells, myeloma cells, and the like. In a further preferred embodiment, recombinantly produced mimecan is used as an antigen to produce polyclonal or monoclonal antibodies against mimecan. It is also preferred that the polyclonal antibodies are purified by immunoadsorption on a mimecan immunoadsorbent, using mimecan recombinantly produced by the methods described herein above.

The skilled person will now understand that mimecan has been identified as a marker for efficient assessment of HF and that alternative methods can be used to obtain results comparable to the results of the present invention. For example, alternative strategies for producing antibodies may be used. Such strategies include the use of synthetic or recombinant peptides that represent clinically relevant immune epitopes of mimecan. Alternatively, DNA immunization (also known as DNA vaccination) may be used.

For measurement, a liquid sample obtained from an individual and a binding agent specific for mimecan are incubated under conditions suitable for the formation of a binding agent mimecan complex. Such conditions need not be specified, since the skilled person can easily identify these suitable incubation conditions even without any inventive effort. The amount of binding agent mimecan complex was determined and used for HF evaluation. The skilled person will appreciate that there are many ways to determine the amount of specific binding agent mimecan complex which are described in detail in relevant textbooks (see e.g. Tijssen p., supra, or diamandadis, e.p. and Christopoulos, t.k. (eds.), immunological, Academic Press, Boston (1996)).

Preferably, the mimecan is detected in a sandwich type assay format. In this assay, the mimecan is captured from one side with a first specific binding reagent and a second specific binding reagent with a label that can be detected directly or indirectly is used on the other side. Preferably, the anti-mimecan antibody is used in a qualitative (with or without mimecan) or quantitative (determining the amount of mimecan) immunoassay.

As described in detail in the examples section, two mouse models have been used to identify mRNA and polypeptides present in the heart tissue of experimental animals by advanced microarray and protein formulation methods. However, these models yield at least partially conflicting data, and of course, the tissue data for mRNA or corresponding polypeptides does not represent the presence or absence of these polypeptides in the circulation. Markers found to be differentially expressed in one model may not be differentially expressed in a second model, or even show conflicting data in yet another model. Differentially expressed mrnas can be found to be unrelated to the enhanced levels of the corresponding polypeptides in the circulation. Even though a protein may be differentially expressed in tissues, if measured from body fluids, in most cases this protein is not of any diagnostic relevance, as it may not be released into the circulation, may become fragments or be modified, e.g. when released from cells or tissues, may be unstable in the circulation, may not be detectable in the circulation, may not be specific for a particular disease, etc.

Surprisingly, the inventors of the present invention were able to detect the protein mimecan in a body fluid sample. Even more surprising, they could demonstrate that the presence of mimecan in such a liquid sample obtained from an individual can be correlated with HF. Evaluation of HF using a labeled mimecan without tissue and biopsy samples. Measuring the level of the protein mimecan is considered to be very beneficial in the HF field.

In a preferred embodiment, the present invention relates to a method of assessing heart failure in an individual, comprising the steps of: a) measuring the concentration of the marker mimecan in a sample obtained from the individual, wherein said sample is a sample of a bodily fluid, b) optionally measuring the concentration in said sample of one or more other markers of heart failure selected from the group consisting of a natriuretic peptide marker, a cardiac troponin marker and a marker of inflammation, and c) comparing the concentration measured in step (a) and optionally the concentration measured in step (b) with the concentration of this marker or these markers determined in a control sample, thereby assessing heart failure, an increased concentration of mimecan being indicative for heart failure.

In a preferred embodiment, the method of the invention is carried out with serum as the liquid sample material. In a more preferred embodiment, the method of the invention is carried out with plasma as the liquid sample material. In another preferred embodiment, the method of the invention is carried out with whole blood as the liquid sample material.

In a further preferred embodiment, the present invention relates to the use of the protein mimecan as a marker molecule for the assessment of heart failure from a liquid sample obtained from an individual.

An ideal situation for diagnosis would be one in which a single event or process would lead to a corresponding disease, such as an infectious disease. In all other cases, correct diagnosis can be difficult, especially if the etiology of the disease is not well understood, as is the case with HF. The skilled person will understand that in the field of HF, no biochemical marker has 100% specificity in diagnosis and at the same time 100% sensitivity for a certain diagnostic problem. Instead, biochemical markers are used to assess potential diagnostic problems with some likelihood or predictive value. The skilled person is well familiar with mathematical/statistical methods commonly used to calculate the relative risk or likelihood of a diagnostic indicator being evaluated. In routine clinical practice, physicians often examine various clinical symptoms and biomarkers simultaneously in diagnosing, treating, and treating underlying diseases.

Preferably, in a further preferred embodiment of the invention, the method for assessing HF is performed as follows: the concentrations of mimecan and one or more other markers are measured and used to estimate HF.

In HF assessment, the marker mimecan will assist physicians in one or more of the following: assessing the risk of an individual to suffer from heart failure or assessing a patient suffering from heart failure, e.g. wanting to identify a stage of heart failure; distinguishing between acute and chronic heart failure; determining the risk of disease progression; providing guidance for selecting an appropriate treatment; monitoring the patient's response to the treatment; and monitoring the course of the disease, i.e., follow-up on HF patients.

Screening (assessing whether an individual is at risk for developing heart failure):

in a preferred embodiment, the present invention relates to an in vitro method of assessing whether an individual is at risk of developing heart failure, comprising the steps of: measuring the concentration of the marker mimecan in the sample; optionally measuring the concentration of one or more other markers of heart failure in the sample; and assessing the risk of the individual to develop heart failure by comparing the mimecan concentration and optionally the measured concentration of said one or more other markers with reference values for the concentration of said one or more markers.

In the context of the present invention, screening is directed to an unbiased assessment of the risk of developing or developing heart failure in an associated individual. While such a screen may in theory be performed on any sample, in clinical practice such a screen will usually be given to individuals at risk of developing heart failure in some way. As noted above, such individuals may be asymptomatic, i.e., they do not have signs or symptoms of HF. In a preferred embodiment, individuals at risk of developing heart failure will be screened for HF, for example, as belonging to stage a or B as defined by the ACC/AHA guidelines.

As mentioned above, heart failure is one of the most prominent, costly, life-threatening diseases in developed countries. Because of its high morbidity and long asymptomatic phase, identification of an individual's risk of developing HF will become extremely important in order to intervene and block the course of the disease, if possible. Only early risk assessment may prevent the progression of HF from the asymptomatic phase to the symptomatic phase.

Assessment of heart failure risk is performed by mathematical/statistical methods, which are well understood and appreciated by the skilled artisan. Preferably, the risk of an individual to suffer from heart failure is expressed in relative values and is given in terms of the so-called relative risk (═ RR). To calculate the RR of such heart failure, the individual values of mimecan are compared with mimecan values determined in a reference population, preferably healthy individuals not developing heart failure. It is also preferred that the assessment of such heart failure RR is based on a population of individuals who developed heart failure within the study period (preferably within one year or also preferably within two years) and a population of individuals who did not develop heart failure within the same study period.

In another preferred embodiment, the present invention relates to the use of the marker mimecan screening for heart failure. As known to the skilled person, the term "used as a marker" means that the presence or absence of a disease or the presence or absence of a clinical condition is indicated (i.e.indicated) by the value of the measured marker after the concentration of the marker molecule has been quantified by suitable means. Suitable means for quantification are, for example, specific binding reagents, such as antibodies.

Preferably, HF screening is performed on individuals suspected of being at risk for heart failure. In this sense, patients at risk of future heart failure are patients who have been diagnosed with hypertension, atherosclerotic disease, diabetes, obesity and metabolic syndrome. Preferably, an individual suffering from hypertension, atherosclerotic disease, diabetes and/or metabolic syndrome is assessed for the risk of future heart failure.

It is also preferred to use the marker mimecan to assess the risk of future heart failure in subjects of stage B as defined in the ACC/AHA practice guidelines, i.e. subjects presenting with altered cardiac structure but showing no symptoms of heart failure.

In a more preferred embodiment, the invention relates to the use of mimecan as one of the markers in a HF marker combination for screening for HF.

In the screening, elevated levels of mimecan are a positive indicator of an increased risk of developing heart failure in an individual.

Patient stratification

In a preferred embodiment, the present invention relates to an in vitro method for assisting in the stratification of a heart failure patient, comprising the steps of: a) measuring the concentration of the marker mimecan in the sample, b) optionally measuring the concentration of one or more other markers of heart failure in the sample, and grading heart failure by comparing the concentration determined in step (a) and optionally the concentration determined in step (b) with reference values for the concentration of said one or more markers. Preferably, the level of the marker mimecan is used to assist in the grouping of the subjects into groups of individuals that are clinically "normal" (i.e. individuals classified according to ACA/ACC at stage a), patients with structural heart disease but no symptoms (stage B classified according to ACA/ACC) and heart failure patients (i.e. patients classified according to ACA/ACC at stage C or stage D).

Discriminating between acute cardiac events and chronic cardiac disease

In a preferred embodiment, the present invention relates to an in vitro method for aiding in the differential diagnosis of an acute cardiac event and a chronic cardiac disease, comprising the steps of: measuring the concentration of the marker mimecan in the sample; optionally measuring the concentration of one or more other markers of heart failure in the sample; and establishing a differential diagnosis of acute cardiac events and chronic cardiac disease by comparing the concentrations determined in step (a) and optionally in step (b) with a reference value for the concentration of the marker or markers.

The person skilled in the art is familiar with the meaning of "acute cardiac event" and "chronic cardiac disease".

Preferably, an "acute cardiac event" relates to an acute condition, disease or disorder of the heart, in particular acute heart failure, such as Myocardial Infarction (MI) or arrhythmia. Depending on the extent of MI, it may follow LVD and CHF.

Preferably, a "chronic heart disease" is a weakening of the heart function, for example, due to cardiac ischemia, coronary artery disease or a previous, particularly small myocardial infarction (perhaps followed by a progressive LVD). It may also be debilitating caused by inflammatory diseases, heart valve defects (e.g., mitral valve defects), dilated cardiomyopathy, hypertrophic cardiomyopathy, cardiac rhythm defects (arrhythmias), and chronic obstructive pulmonary disease. Thus, chronic heart disease may obviously also include patients with acute coronary syndromes, such as MI, but who are currently free of acute cardiac events.

It is important to distinguish between acute cardiac events and chronic cardiac diseases, as acute cardiac events and chronic cardiac diseases may require quite different treatment regimes. For example, for patients with acute myocardial infarction, early reperfusion therapy may be of utmost importance. Reperfusion therapy in chronic heart failure patients is at most harmless or only hardly harmful to such patients.

In a more preferred embodiment of the invention, the marker mimecan is used for the differential diagnosis of acute and chronic heart failure.

Assessing risk of disease progression

In a preferred embodiment, the present invention relates to an in vitro method for assessing the risk of disease progression in a HF patient, comprising the steps of: measuring the concentration of the marker mimecan in the sample; optionally measuring the concentration of one or more other markers of heart failure in the sample; and determining the risk of disease progression in said individual by comparing the mimecan concentration and optionally the measured concentration of said one or more other markers with a reference value for the concentration of said one or more markers.

At present, it is difficult to assess with reasonable probability or even predict whether a patient diagnosed with HF has a more or less stable state, or whether the disease will progress, and whether the health status of the patient may deteriorate. Clinically, the severity and progression of heart failure is often determined by assessing clinical symptoms or, alternatively, identifying detrimental changes using imaging techniques such as echocardiography. In one embodiment, worsening heart failure is determined by monitoring Left Ventricular Ejection Fraction (LVEF). A 5% or greater decrease in LVEF is considered disease progression or worsening.

Thus, in a further preferred embodiment, the invention relates to the use of the marker mimecan for assessing the risk of disease progression in patients with HF. In the assessment of disease progression in patients with HF, an increase in mimecan levels is indicative of an increased risk of disease progression.

Guide selection of appropriate HF therapy

In a preferred embodiment, the present invention relates to an in vitro method for aiding in the selection of an appropriate HF therapy, comprising the steps of: measuring the concentration of the marker mimecan in the sample; optionally measuring the concentration of one or more other markers of heart failure in the sample and selecting an appropriate therapy by comparing the mimecan concentration and the optionally determined concentration of said one or more other markers with reference values for the concentration of said one or more markers.

The marker mimecan is expected to assist the physician in selecting the most appropriate treatment regimen from the various treatment regimens currently available in the field of heart failure. Thus, a more preferred embodiment involves the use of a marker mimecan to select a treatment regimen for HF patients.

Monitoring patient response to therapy

In a preferred embodiment, the present invention relates to an in vitro method of monitoring a patient's response to HF therapy, comprising the steps of: a) measuring the concentration of the marker mimecan in the sample, b) optionally measuring the concentration of one or more other markers of heart failure in the sample, and monitoring the patient's response to HF therapy by comparing the concentrations determined in step (a) and optionally step (b) with reference values for the concentrations of said one or more markers.

Alternatively, the above method for monitoring a patient's response to therapy may be carried out by: marker levels of mimecan and optionally one or more other markers before and after treatment are determined and compared.

The diagnosis of heart failure is clinically established. According to the present invention, HF is considered clinically established if a patient meets the criteria for stage C or D as defined by the ACC/AHA practice guidelines. According to these guidelines, stage C refers to patients with structural heart disease and having earlier or current symptoms of heart failure. Stage D patients are patients with refractory heart failure who require specialized intervention.

As further shown above, the measured values of NT-proBNP are closely related to the severity of heart failure. However, both BNP and NT-proBNP appear to be less than ideal in monitoring patient response to therapy, see, e.g., Beck-da-Silva, L. et al, Conget. Heart fail. 11 (2005) 248-.

The marker mimecan appears to be suitable for monitoring the patient's response to therapy. The invention thus also relates to the use of mimecan to monitor patient response to therapy. In the diagnostic field, the marker mimecan can also be used to determine a baseline value before treatment and to determine the mimecan at one or several time points after treatment. In the follow-up of HF patients, elevated levels of mimecan are a positive indication of effective HF treatment.

Mark combination

The biochemical markers may be measured individually or, in a preferred embodiment of the invention, they may be measured simultaneously using chip-based or bead-based array technology. The concentration of the biomarkers is then interpreted independently with the cut-off value for each marker, or they are interpreted in combination, i.e. they form a marker combination.

The person skilled in the art knows that the step of associating the marker level with a certain likelihood or risk can be performed and carried out in different ways. Preferably, the measured values of the marker mimecan and one or more other markers are mathematically combined and the combined value is then correlated with a potential diagnostic problem. The signature value may be combined with the mimecan measurement data using any presently suitable mathematical method.

The mathematical algorithm applied to the combination of the signatures is preferably a logical function. The result of applying such a mathematical algorithm or such a logistic function is preferably a single value. Depending on the underlying diagnostic question, this value can easily be correlated with the risk of, for example, an individual suffering from heart failure, or with other predetermined diagnostic applications, which are, for example, useful for assessing HF patients. In a preferred manner, the logic function is obtained as follows: a) grouping individuals into groups, e.g., a group of normal individuals, a group of individuals at risk of heart failure, a group of patients with acute or chronic heart failure, etc., b) identifying markers that are significantly different between these groups using univariate analysis, c) logistic regression analysis to evaluate independent discrimination values for markers useful in evaluating these different groups, and d) constructing logistic functions to combine the independent discrimination values. In such assays, the markers are no longer independent, but represent a combination of markers.

In a preferred embodiment, the logic function for combining the mimecan value and the further at least one marker value is obtained as follows: a) the individuals are divided into a normal group and a heart failure risk group, respectively, b) the value of mimecan and the value of the further at least one marker are established, c) a logistic regression analysis is performed, and d) a logistic function is constructed to combine the value of mimecan and the value of the further at least one marker.

The logistic function used to associate the marker combination with the disease preferably uses algorithms developed by applying statistical methods. Suitable statistical methods are, for example, Discriminant Analysis (DA) (i.e. linear DA, quadratic DA, regularized DA), nuclear methods (i.e. SVM), nonparametric methods (i.e. k-nearest-neighbor classification), PLS (partial least squares), tree-based methods (i.e. logistic regression, CART, random Forest, Boosting/Bagging), generalized linear models (i.e. logistic regression), principal component-based methods (i.e. SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. Those skilled in the art should be able to select appropriate statistical methods to evaluate the marker combinations of the present invention and thereby obtain appropriate mathematical algorithms. The mathematical algorithm for assessing heart failure is preferably obtained by a statistical method selected from DA (i.e. linear, quadratic, regularized discriminant analysis), a kernel method (i.e. SVM), a non-parametric method (i.e. k-nearest-neighbor classification), PLS (partial least squares), tree-based methods (i.e. logistic regression, CART, random Forest, Boosting methods) or generalized linear models (i.e. logistic regression). Detailed descriptions of these statistical methods can be found in the following references: ruczinski, I.et al, J.of Computational and Graphical statics 12 (2003) 475-; friedman, J.H., J.of the American Statistical Association 84(1989) 165-175; hastie, T, et al, The Elements of Statistical Learning, Springer Verlag (2001); breiman, L.et al, Classification and regression trees, Wadsworth International Group, California (1984); breiman, L., Machine Learning 45(2001) 5-32; pepe, M.S., The Statistical Evaluation of Medical Tests for Classification and Prediction, Oxford Statistical Science Series, 28, Oxford university Press (2003); and duca, r.o. et al, Pattern Classification, John Wiley & Sons, inc., 2 nd edition (2001).

A preferred embodiment of the invention is to use optimized multivariate cut-off values for the potential combination of biomarkers and to distinguish between state a and state B, e.g. between normal and heart failure risk individuals, HF patients responding to treatment and HF patients failing treatment, acute and chronic HF patients with heart failure, HF patients showing disease progression and patients not showing disease progression, respectively.

The area under the subject's working curve (═ AUC) indicates the effectiveness or correctness of the diagnostic procedure. The correctness of the diagnostic method is best described by its Receiver Operating Characteristics (ROC) (see in particular Zweig, M.H. and Campbell, G., Clin. chem. 39 (1993) 561-. ROC plots are plots of all sensitivity/specificity pairings resulting from continuously varying decision thresholds across the range of data observed.

The clinical efficacy of a laboratory test depends on the accuracy of its diagnosis, or on the ability to correctly classify a subject into clinically relevant subpopulations. Diagnostic accuracy measures the ability of the test to correctly distinguish two different conditions of a subject. These two different conditions are for example health and disease or with and without disease progression.

In each case, the ROC plot describes the overlap between the two distributions by plotting sensitivity versus 1-specificity over the full range of decision thresholds. The y-axis is sensitivity, or true positive score [ determined as (number of true positive determinations)/(number of true positive determinations + number of false negative determinations) ]. This is also referred to as a positive for the presence of the disease or condition. It was calculated only from the affected subpopulations. The X-axis is the false positive score, or 1-specificity [ determined as (number of false positive results)/(number of true negative results + number of false positive results) ]. It is an indicator of specificity and is calculated entirely from unaffected subgroups. Because true and false positive scores are calculated completely separately using test results from two different subpopulations, the ROC plot is independent of the prevalence of disease in the sample. Each point on the graph represents a sensitivity/1-specific pair, which corresponds to a particular decision threshold. A well-discriminative assay (no overlap of the two result distributions) has an ROC plot across the top left corner with a true positive score of 1.0 or 100% (very good sensitivity) and a false positive score of 0 (very good specificity). For a non-discriminative assay (results of both sets are equally distributed), the theoretical plot is a 45 ° diagonal from the bottom left to the top right. Most curves are between these two extremes. (if the ROC plot falls well below the 45 ° diagonal, this situation is easily remedied by reversing the "positive" criteria from "greater" to "less" or vice versa). Qualitatively, the closer the graph is to the upper left corner, the higher the overall accuracy of the assay.

One convenient goal of quantifying the diagnostic accuracy of a laboratory test is to express its effect by a single number. The most commonly used overall measure is the area under the ROC curve (AUC). Conventionally, the area is always ≧ 0.5 (if not, one can reverse the decision rule to make it so). The values were between 1.0 (very well separating the two sets of test values) and 0.5 (no significant difference in the distribution of the two sets of test values). The area does not depend only on a particular part of the plot, such as the point closest to the diagonal or the sensitivity at 90% specificity, but the entire plot. This is a quantitative, descriptive expression indicating how close the ROC plot is to perfect (area 1.0).

The overall assay sensitivity depends on the specificity that is required to practice the methods disclosed herein. In certain preferred settings, a specificity of 75% may be sufficient, and statistical methods and result algorithms may be based on this specificity requirement. In a preferred embodiment, the method applied for assessing the risk of heart failure in an individual is based on a specificity of 80%, 85%, or, still preferably, 90% or 95%.

As mentioned above, the marker mimecan is useful for assessing the risk of an individual to develop heart failure and also for the in vitro diagnostic assessment of patients suffering from heart failure. Thus, a preferred embodiment is the use of mimecan as a marker molecule for the assessment of heart failure.

The use of a marker combination comprising mimecan and one or more other HF markers for assessing an HF patient or for assessing the risk of an individual to develop HF represents a further preferred embodiment of the present invention. In such a marker combination, the one or more additional markers are preferably selected from the group consisting of a natriuretic peptide marker, a cardiac troponin marker and an inflammatory marker.

The one or more preferred other HF markers which can be combined with the mimecan measurement are preferably or are selected from the group consisting of a natriuretic peptide marker, a cardiac troponin marker and an inflammation marker. These preferred measurements of other markers are preferably combined with the measurements of the mimecan or form part of the HF marker combination including the mimecan, as discussed in more detail below, respectively.

Natriuretic peptide tags

In the sense of the present invention, a natriuretic peptide marker is a marker selected from the Atrial Natriuretic Peptide (ANP) family or a marker selected from the Brain Natriuretic Peptide (BNP) family.

The polypeptide tags in the atrial natriuretic peptide family or the brain natriuretic peptide family are derived from the prepro form of the corresponding active hormone.

Preferred natriuretic peptide markers according to the invention are NT-proANP, ANP, NT-proBNP, BNP and immunologically detectable physiological fragments thereof. The skilled person will readily understand that an immunologically detectable fragment must comprise at least one epitope to allow specific detection of such a physiological fragment. Physiological fragments are fragments that occur naturally in the circulation of an individual.

The markers in both natriuretic peptide families represent fragments of the corresponding prohormones (proANP and proBNP, respectively). Since the considerations for the two families are similar, only the BNP marker family is described in slight detail. Prohormone of the BNP family, proBNP, consists of 108 amino acids. proBNP is cleaved into 32C-terminal amino acids (77-108), which represent the biologically active hormone BNP, and the N-terminal amino acids 1-76, called N-terminal proBNP (or NT-proBNP). BNP, N-terminal proBNP (1-76) and further breakdown products (Hunt, P.J. et al, biochem. Biophys. Res. Com.214 (1995) 1175-1183) circulate in the blood. Whether the complete precursor molecule (proBNP 1-108) is also present in plasma is not yet fully understood. However, it has been stated (Hunt, P.J. et al, Peptides 18 (1997) 1475-1481) that low release of proBNP (1-108) in plasma is detectable, but some amino acids are deleted due to very rapid local degradation at its N-terminus. It is now generally accepted that, for example for NT-proBNP, the central part of the molecule between amino acids 10 and 50 represents a physiologically more stable moiety. NT-proBNP molecules comprising the central portion can be reliably determined from body fluids. A method for the immunological detection of the central part of the NT-proBNP molecule is disclosed in detail in WO 00/45176, to which the reader is referred for further details. It may be advantageous to determine only a certain sub-component of NT-proBNP, for which the term native NT-proBNP has been proposed. A detailed disclosure of this NT-proBNP subcomponent is found in WO 2004/099253. The skilled person will find all necessary guidance there. Preferably, the determined NT-proBNP is (or corresponds to) NT-proBNP detected using the ELECSYSS NT-proBNP assay from Roche Diagnostics, Germany.

Pre-analysis using NT-proBNP is useful, which facilitates sample transfer to a central laboratory (Mueller, T. et al, Clin. chem. Lab. Med. 42 (2004) 942-944). The blood sample can be stored at room temperature for several days, or can be mailed or transported without loss of recovery. In contrast, storage of BNP at room temperature or 4 ℃ for 48 hours may result in a loss of concentration of at least 20% (Mueller, T. et al, supra; Wu, A.H. et al, Clin. chem. 50(2004) 867-.

The brain-derived natriuretic peptide family (in particular BNP and NT-proBNP) has been thoroughly studied in screening HF for certain populations. The discovery of these markers, in particular NT-proBNP, is quite encouraging. The values of NT-proBNP are increased even in asymptomatic "patients", clearly indicating a "heart problem" (Gremmler, B. et al, Exp. Clin. Cardiol. 8 (2003) 91-94). These authors show that elevated NT-proBNP shows the presence of 'cardio-renal discomfort' and that further examination should be scheduled quickly. In line with several other panelists, Gremmler et al also found that abnormal concentrations of NT-proBNP were an accurate diagnostic test for the exclusion of HF in the population and for the exclusion of left ventricular dysfunction (= LVD) in asthmatic patients. The effect of negative BNP or NT-proBNP values excluding HF or LVD was confirmed by other groups of researchers, see e.g., McDonagh, T.A. et al, Eur. J. Heart fail. 6 (2004) 269-273; and Gustafsson, f. et al, j. card. fail. 11, supplement 5 (2005) S15-20.

BNP is produced primarily (although not exclusively) in the ventricles and is released when wall pressure rises. Thus, an increase in BNP release is primarily reflected in ventricular dysfunction or in dysfunction occurring in the atria but affecting the ventricles, e.g. by a decrease in inflow or an overload in blood volume. In contrast to BNP, ANP is produced and released primarily from the atria. The level of ANP may therefore reflect primarily atrial function.

ANP and BNP are active hormones having a shorter half-life than their corresponding inactive counterparts, NT-proANP and NT-proBNP. BNP is metabolized in the blood, whereas NT-proBNP circulates in the blood as an intact molecule and is removed by the kidneys. NT-proBNP has a 120 min in vivo half-life longer than BNP, and BNP has a 20 min in vivo half-life (Smith, M.W. et al, J. Endocrinol. 167 (2000) 239-246).

Thus, depending on the time course or the properties of interest, it may be beneficial to measure the natriuretic peptide in active or inactive form.

In the assessment of the risk of heart failure in an individual, the determined mimecan values are preferably combined with the values of NT-proANP and/or NT-proBNP. The value of NT-proBNP is preferably combined with the value of mimecan. Similar considerations apply to the selection of appropriate therapies, identification of risk of disease progression and monitoring of the course of disease.

mimecan is used to assess the response of a patient to therapy, preferably combining its detection with the measurement of ANP or BNP.

Where mimecan is used to distinguish between acute and chronic heart failure, preferred marker combinations include mimecan, ANP or proANP and BNP or proBNP.

Cardiac troponin markers

The term cardiac troponin relates to cardiac isoforms of troponin I and troponin T. As already indicated above, the term marker also relates to physiological variants of the marker molecule, such as physiological fragments or complexes. For cardiac troponin markers, their physiologically present complexes are known to be of diagnostic value and are therefore expressly included in the present invention.

Troponin T has a molecular weight of about 37.000 Da. The troponin T isoform (cTnT) found in cardiac tissue is so different from skeletal muscle TnT that antibodies can be produced to discriminate between the two TnT isoforms. TnT is considered a marker of acute myocardial injury; see Katus, H.A. et al, J.mol. cell. Cardiol. 21(1989) 1349-1353; hamm, C.W. et al, N.Engl. J. Med. 327 (1992) 146- "150; ohman, E.M. et al, N, Engl. J. Med. 335 (1996) 1333-1341; christenson, R.H. et al, Clin.chem.44 (1998) 494-501; and EP 0394819.

Troponin i (tni) is the inhibitory element of the troponin complex, 25 kDa, present in muscle tissue. TnI in the absence of Ca²⁺Binds actin and inhibits atpase activity of actomyosin. The TnI isoform (cTnI) present in heart tissue differs from skeletal muscle TnI by 40%, allowing immunological discrimination of the two isoforms. Normal plasma concentrations of cTnI were less than 0.1 ng/ml (4 pM). cTnI in the heartReleased into the bloodstream upon cell death; thus, plasma cTnI concentrations are elevated in patients with acute myocardial infarction (Benamer, H. et al, Am. J. Cardiol. 82 (1998) 845- "850).

The unique cardiac isoforms of troponin I and T allow them to be distinguished from other skeletal muscle troponins by immunization. Thus, troponins I and T released into the blood from the damaged myocardium may be specifically associated with cardiac tissue damage. It is also known by the skilled person that cardiac troponin can be detected from the circulation, either in free form or as a constituent of a complex (cf. e.g.US 6,333,397, US 6,376,206 and US 6,174,686).

In the assessment of the risk of an individual to suffer from heart failure as well as in the assessment of a patient already suffering from heart failure, the determined mimecan value is preferably combined with a value for troponin T and/or a cardiac isoform of troponin I. A preferred cardiac troponin for use in combination with the marker mimecan is cardiac troponin T.

Inflammation marker

The skilled person is familiar with the term inflammation marker. Preferred inflammatory markers are interleukin-6, C-reactive protein, serum amyloid A and S100 protein.

Interleukin-6 (IL-6) is a 21kDa secreted protein with a variety of biological activities, which can be divided into activities involved in erythropoiesis and activities involved in activating the innate immune response. IL-6 is an acute phase reactant and stimulates the synthesis of a variety of proteins, including adhesion molecules. Its main functions are to mediate the production of acute liver protein and to induce its synthesis by cytokines IL-1 and TNF-alpha. IL-6 is usually produced by macrophages and T lymphocytes. Normal serum concentrations of IL-6 were less than 5 pg/ml.

C-reactive protein (CRP) is a homo-pentamer Ca²⁺Binds to acute phase proteins, with a 21kDa subunit involved in host defense. CRP synthesis is induced by IL-6 and indirectly by IL-1, since IL-1 can trigger the synthesis of IL-6 by Kupffer cells from the hepatic sinusoid. In 90% of healthy populationThe normal plasma concentration of CRP in less than 3 μ g/ml (30nM), and less than 10 μ g/ml (100 nM) in 99% of healthy individuals, for example, CRP concentration in plasma can be determined by ELISA it is synthesized primarily by the liver in response to IL-1, IL-6, or TNF- α stimulation, and is involved in regulating T cell-dependent immune responses in acute events, the concentration of SAA increases 1000-fold to 1 mg/ml.

Formation of constantly expanding Ca by S100 protein²⁺A family of binding proteins, which now includes more than 20 members. The physiologically relevant structure of the S100 protein is a homodimer, but some may also form heterodimers with each other, e.g., S100A8 and S100a 9. Its intracellular function encompasses regulation of protein phosphorylation, enzyme activity, or cytoskeletal dynamics involved in cell proliferation and differentiation. Since some S100 proteins can also be released from cells, their extracellular functions have now been described, such as neuronal survival, astrocyte proliferation, induction of apoptosis and modulation of inflammatory processes. S100A8, S100A9, heterodimers S100A8/A9, and S100A12 have been found in inflammation, with S100A8 responding to chronic inflammation, while S100A9, S100A8/A9, and S100A12 are increased in acute inflammation. S100A8, S100A9, S100A8/A9 and S100A12 have been found to be associated with different diseases with inflammatory components, including certain cancers, renal allograft rejection, colitis, and most importantly RA (Burmeister, G. and Gallacchi, G., Inflammophandoggy 3 (1995) 221-1389; Foell, D. et al, Rheumatography 42 (2003) 1383-1389). For example, for the marker combinations used in the present invention, the most preferred S100 markers when used to assess individual HF risk or HF patients are S100A8, S100a9, S100A8/a9 heterodimer, and S100a 12.

sE-selectin (soluble endothelial leukocyte adhesion molecule-1, ELAM-1) is a type I transmembrane glycoprotein of 115 kDa, expressed only on endothelial cells and only after activation by inflammatory cytokines (IL-1. beta., TNF-. alpha.) or endotoxins. E-selectin on the cell surface is a mediator of the rotational attachment of leukocytes to the endothelium, a process essential for the extravasation of leukocytes at sites of inflammation, and thus has an important role in local inflammatory responses. Soluble E-selectin is present in the blood of healthy individuals, probably due to the proteolytic cleavage of surface-expressed molecules. Increased levels of sE-selectin have been reported in serum under a number of pathological conditions (Gearing, A.J. and Heminway, I., Ann. N.Y.Acad. Sci. 667.1992) 324-331).

In a preferred embodiment, the invention relates to the use of mimecan as marker molecule for HF in combination with one or more HF marker molecules for the HF evaluation of a liquid sample obtained from an individual.

As indicated above, in a preferred method of the invention, the measurement of mimecan is combined with at least another value selected from the following markers: natriuretic peptide markers, cardiac troponin markers, and inflammatory markers.

In a preferred embodiment, the present invention relates to the use of a marker combination comprising mimecan and one or more other markers of heart failure selected from the group consisting of a natriuretic peptide marker, a cardiac troponin marker and an inflammation marker for assessing heart failure.

In a preferred embodiment, the present invention relates to the use of a marker combination of mimecan and NT-proBNP for the assessment of heart failure.

In a preferred embodiment, the invention relates to the use of a marker combination of mimecan and troponin T for the assessment of heart failure.

In a preferred embodiment, the present invention relates to the use of a marker combination of mimecan and CRP for assessing heart failure.

In a more preferred embodiment, the invention relates to a marker combination comprising the markers mimecan, troponin T, NT-proBNP and CRP.

In yet another more preferred embodiment, the invention relates to a marker panel for use in a method for assessing HF in vitro by biochemical markers, said method comprising measuring the concentration of mimecan and one or more other HF markers in a sample and assessing HF using the determined concentrations.

The marker panel of the present invention is preferably determined using protein array technology. An array is an addressable collection of individual labels. Such labels may be spatially addressed, such as contained in a microtiter plate or printed on an array of planar surfaces, where each label exists at a different X and Y axis coordinate. Alternatively, the labels may also be addressed by labels, beads, nanoparticles, or physical properties. Microarrays can be prepared according to methods known to those of ordinary skill (see, e.g., U.S. Pat. No. 5,807,522; Robinson, W.H. et al, nat. Med. 8(2002) 295-893; Robinson, W.H. et al, Arthritis Rheum. 46 (2002) 885-893). An array as used herein refers to any immunological assay having a plurality of addressable labels. In one embodiment, the addressable marker is an antigen. In another embodiment, the addressable element is an autoantibody. Microarrays are miniaturized forms of arrays. Antigen as used herein refers to any molecule that can specifically bind to an antibody. The term autoantibody is well defined in the art.

In a preferred embodiment, the invention relates to a protein array comprising further labels labeling mimecan and optionally one or more HF.

In a preferred embodiment, the invention relates to a protein array comprising the markers mimecan and NT-proBNP.

In a preferred embodiment, the invention relates to a protein array comprising the markers mimecan and troponin T.

In a preferred embodiment, the invention relates to a protein array comprising the markers mimecan and CRP.

In a further preferred embodiment, the invention relates to a protein array comprising the markers mimecan, troponin T, NT-proBNP and CRP.

In yet a more preferred embodiment, the invention relates to a kit comprising the reagents required for the specific determination of mimecan. Another preferred kit comprises reagents required for the specific determination of mimecan and reagents required for the determination of one or more other marker of heart failure, which markers are used together in a combination of HF markers.

In a preferred embodiment, the invention relates to a kit comprising the reagents required for the specific determination of mimecan and optionally one or more other markers of heart failure selected from the group consisting of a natriuretic peptide marker, a cardiac troponin marker and a marker of inflammation.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the claims. It is to be understood that the method may be modified without departing from the spirit of the invention.

Drawings

FIG. 1 phenotypic analysis of wild type mice and R9C mice. (A) Survival curves generated after 24 weeks for wild-type mice (n-79) and R9C mice (n-44). (B) Assessment of cardiac shortening by echocardiography (shortening score). R9C transgenic animals began to develop significant functional lesions as early as 8 weeks of age.

Figure 2 echocardiogram and hemodynamic parameters of wild type mice and AB mice. (A) Change in maximal pressure (mmHg) 2, 4 and 8 weeks post-surgery. (B) Left Ventricular Ejection Fraction (LVEF) percent change at 2, 4 and 8 weeks post-surgery. (filled circles indicate data from sham operated mice, open circles indicate data from aortic ligated (AB) mice).

FIG. 3 mimecan values detected from HF samples of the clinical routine group and the extended control group, respectively. The concentrations were calculated from mimecan measured from 241 heart failure patient samples (labeled HF) and healthy control samples (146 samples) (labeled normal human serum markers ═ NHS), respectively. The box whisker plot shows the lower and upper quartile values (boxes) and the highest and lowest values (whiskers).

Example 1

Mouse model of heart failure

1.1R 9C mouse model

A genetic human dilated cardiomyopathy has been reported to result from the conversion of Arg9 to Cys (PLN-R9C) in the human Phospholamban (PLN) gene (Schmitt, J.P. et al, Science 299 (2003) 1410-. The onset of dilated cardiomyopathy patients generally begins at puberty, followed by progressive deterioration of cardiac function, leading to risk and death. This mutant transgenic mouse model showed a similar cardiac phenotype to the diseased patient, with dilated cardiomyopathy, reduced contractility of the heart, and premature death (Schmitt et al, 2003, supra).

We established survival curves for transgenic mice. PLN-R9C mice survived only about 20 weeks on average, with less than 15% surviving for more than 24 weeks (fig. 1A). In the PLN-R9C line, the earliest record of death observed was 12 weeks of age, whereas only one wild-type control mouse died at 24 weeks. Eight weeks were selected as representative time points 'early' in the disease before the earliest death record, while 16 weeks were selected because it is the midpoint between 8 and 24 weeks (classical DCM). Detailed pathological analysis of ex vivo hearts showed evidence of ventricular and atrial enlargement in PLN-R9C mice even at 8 weeks of age. The myocardium was cross-sectioned ex vivo (from wild-type and PLN-R9C mice) and then stained with hematoxylin and eosin, evidence also showing that transgenic animals began left ventricular dilatation or ventricular wall thinning at 8 weeks, with expansion continuing to worsen with age.

Cardiac function was determined by echocardiography in 8, 16 and 24 week old male mice (summarized in table 1). Echocardiographic measurements of anterior and posterior wall thicknesses showed that R9C mice had significant expansion at 8 weeks and continued to deteriorate over the lifetime of the mice. Contractility was assessed by cardiac shortening (fig. 1B), which was also a slight but significant decrease at 8 weeks, with a more significant decrease becoming evident at 16 weeks. The analyzed female mice showed the same results as the males (data not shown).

TABLE 1：

Echocardiography and hemodynamic parameters at 8, 16, and 24 weeks in wild type and R9C male mice

The values in table 1 are mean ± sem, the symbols used in table 1, HR, heart rate, AW, PW, thickness of anterior and posterior walls (left ventricle), LVEDD, LVESD, respectively, left ventricular diastolic and end systolic dimensions, FS, fraction shortened (LVEDD-LVEDD)/LVEDD × 100%, ETC, for HR corrected ejection time, VCFC, speed of peripheral shortening corrected for HR, FS/ETC, PAVC, aortic velocity apex corrected for HR, E-wave, early-filling transvalvular diastolic wave, LVESP, LVEDP, left ventricular systolic and end diastolic pressure, + dP/dtmax, maximum positive derivative of left ventricle, dP/dtmax, maximum negative derivative of left ventricle, AVA, aortic velocity acceleration time (PAVC/WT), and comparison of aortic acceleration time with WTP<0.05。

1.2 aortic ligation (AB) mouse model

In this mouse model, pressure overload by aortic ligation (AB) induces cardiac hypertrophy and heart failure.

C57BL mice were surgically pressure overloaded. Coarctation of the ascending aorta (known as aortic ligation) induces cardiac hypertrophy and myocardial growth, particularly in the left ventricle, as a primary response to coarctation of the aorta. In the later stages of this mouse model, the heart becomes hypertrophied and eventually dilated. The model is well characterized and has proven to be highly reproducible, with empirically low mortality rates of 10-15% or less. Following constriction, this animal model can be used to assess the progression of left ventricular hypertrophy and heart failure in response to hemodynamic stress.

Briefly, C57BL mice were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) and the aorta was ligated using a 25 gauge needle. Sham operated mice underwent the same surgical procedure except that the ligation sites were not tightened with a needle.

Time point of experiment

To examine the hypertrophic response, ligated animals and sham-operated controls were sacrificed at 1, 2, 4 and 8 weeks post-intervention. Cardiac function and hypertrophy progression were assessed by echocardiographic analysis and confirmed by histological examination after sacrifice. Table 2 shows an overview of the cardiac function assessed by echocardiography at different time points. The skilled person is aware of the details given in table 2 regarding echocardiographic parameters and can be found, for example, in Asahi, m. et al, proc. natl. acad. sci. usa 101 (2004) 9199-.

TABLE 2

In addition to functional parameters, histological analysis of hematoxylin/eosin (HE) staining was performed on AB mice and control mouse heart tissue taken at 2, 4 and 8 weeks. The necrosis and remodeling processes expected in AB mice were confirmed histologically, while the heart tissue of sham operated mice did not show any significant changes. Two weeks after surgery, the ventricles of the ligated mice showed significant left ventricular hypertrophy, developed further after four weeks, and became very similar to end-stage dilated cardiomyopathy eight weeks after surgery.

Example 2

Microarray analysis

Microarray analysis was performed using crude tissue preparations without further isolation of organelles. Microarray data analysis methods are described in the literature (see, e.g., U.S. Pat. No. 5,807,522; Robinson, W.H. et al, nat. Med. 8(2002) 295-.

Sample preparation and Mass Spectrometry

Cardiac homogenization and organelle separation

The heart was isolated, the atria removed, the ventricles carefully minced with a razor blade and rinsed thoroughly with ice cold PBS (phosphate buffered saline) to remove residual blood. Using a loose-fitting hand-held glass homogenizer in 10 ml of lysis buffer (250 mM sucrose, 50mM Tris-HCl, pH 7.6, 1mM MgCl)₂1mM DDT (dithiothreitol) and 1mM PMSF (phenylmethylsulfonyl fluoride)) for 30 seconds all subsequent steps were performed at 4 deg.C the lysate was centrifuged at 800 × g for 15 minutes in a bench top centrifuge and the supernatant used as a source of cytosolic, mitochondrial and microsomal fractions the pellet containing the nuclei was diluted with 8 ml lysis buffer and overlaid to 4 ml 0.9M sucrose buffer (0.9M sucrose, 50mM Tris-HCl pH 7.6, 1mM MgCl)₂1mM DDT and 1mM PMSF) at 1000 × g for 20 minutes at 4 deg.C the resulting pellet was resuspended in 8 ml of 2M sucrose buffer (2M sucrose, 50mM Tris-HCl pH 7.4, 5 mM MgCl)₂1mM DTT and 1mM pmsf), overlaid onto 4 ml of 2M sucrose buffer and pelleted at 150,000 × g ultracentrifugation for 1 hour (Beckman SW40.1 rotor), nuclear pellets were recovered, centrifuged at 7500 × g at 4 ℃ for a further 20 minutes to separate the mitochondria from the supernatant, the pellet obtained was washed twice with lysis buffer, the mitochondria from which were removed were pelleted with 100,000 × g ultracentrifugation for 1 hour using Beckman SW41 rotor and the supernatant was used as the cytosol fraction (═ cyto).

Organelle extraction

Soluble mitochondrial proteins were extracted by incubating mitochondria on ice for 30 min with hypotonic lysis buffer (10 mM HEPES, pH 7.9, 1mM DTT, 1mM PMSF). The suspension was briefly sonicated and centrifuged at 13,000 Xg for 30 minutes to remove debris. The supernatant was used as the "mito 1" fraction. The insoluble pellet obtained was resuspended in membrane detergent extraction buffer (20 mM Tris-HCl, pH 7.8, 0.4M NaCl, 15% glycerol, 1mM DTT, 1mM PMSF, 1.5% Triton-X-100) and shaken gently for 30 minutes, followed by centrifugation at 13,000 Xg for 30 minutes; the supernatant was used as the "mito 2" fraction.

Membrane-associated proteins were extracted by resuspending the microsomes in membrane detergent extraction buffer. The suspension was incubated for 1 hour under gentle shaking and centrifuged at 13,000 Xg for 30 minutes to remove insoluble debris. The supernatant was used as the "micro" component.

Digestion and MudPIT analysis of organelle extracts

Aliquots of about 100 μ g total protein from each fraction (as determined by the Bradford assay) were pelleted with 5 volumes of ice cold acetone at about 20 ℃ and then centrifuged at 13,000 × g for 15 minutes, protein pellet was solubilized in a solution of small volumes of 8M Urea, 50mM Tris-HCl, pH 8.5, 1mM DTT at 37 ℃ for 1 hour, followed by carboxyamide methylation with 5 mM iodoacetamide in the dark at 37 ℃ for 1 hour, then the sample was diluted with an equal volume of 100 mM, pH 8.5 ammonium bicarbonate to 4M Urea and with 1:150 times the ratio of endoprotease Lys-C (Roche Diagnostics, Laval, Quebec, Canada) overnight at 37 ℃. the second day, the sample was diluted with an equal volume of 50mM, pH 8.5 ammonium bicarbonate to 2M Urea, supplemented with CaCl₂To a final concentration of 1mM, and incubated overnight with Poroszyme trypsin beads (applied biosystems, Streetsville, Ontario, Canad) at 30 ℃ with rotation. The resulting peptide mixture was subjected to solid phase extraction using a SPEC-Plus PT C18 column (Ansys Diagnostics, Lake Forest, Calif.) according to the manufacturer's instructions and stored at-80 ℃ for later use. As described (Kislinger, t. et al, mol. Cell protein. 2(2003) 96-106), a fully automated, 20-hour long, 12-step, multi-cycle MudPIT program was set. Briefly, the HPLC quaternary pump was connected to an LCQ DECA XP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). A100 μ M inner diameter capillary micro-column of fused silica (Polymicro Technologies, Phoenix, AZ) was drawn very finely with a P-2000 laser stretcher (Sun Instruments, Novato, Calif.)The tips were packed with 8 cm of 5. mu.M Zorbax Eclipse XDB-C18 resin (Agilent Technologies, Mississauga, Ontario, Canada) and 6 cm of 5. mu.M Partisphere Strong cation exchange resin (Whatman, Clifton, NJ). The single sample was manually applied to the separation column using a high pressure vessel. The conditions for the chromatography solvent were exactly as described in Kislinger, T. et al, mol. Cell protein. 2(2003) 96-106.

Identification and validation of proteins

The peptide tandem mass spectra were matched with peptide sequences in a database (consisting of mouse and human protein sequences, obtained from the Swiss-Prot/TrEMBL and IPI databases) that locally maintained the minimal redundancy FASTA format using a sequence database search algorithm. To statistically evaluate the experimental false discovery rate to control and thus minimize false positive identification, all spectra were searched for protein sequences in both the normal (forward) and inverted (flip) amino acid orientations (Kislinger, t. et al, mol. Cell protein. 2(2003) 96-106). The STATQUEST filtering algorithm is then applied to all the assumed search results to obtain a statistical reliability measure (confidence score) (cutoff) for each candidate qualification resultpA value ≦ 15, corresponding to a probability of 85% or more being a correct match). High confidence matches are resolved into using a Perl script based internal SQL type database. The database is designed to accommodate database search results and spectral information (scan heads) for a variety of peptides that match a given protein, as well as information about sample name, experimental number, MudPIT procedure, organelle source, amino acid sequence, molecular weight, isoelectric point, charge, and confidence level. Only those with an expected confidence of 95% or higherpThe value and at least two spectra together detected protein can be retained for further analysis.

Example 3

Statistical evaluation of data obtained in model systems

3.1 statistical methods for generating differentially expressed p-values for R9C mouse model

For each of 137 different experimental procedures, the raw data obtained using the method described in example 2 consisted of 6190 proteins, each protein had a mass spectra number, and the total number of all spectra was related to the protein. The raw data, 6190 protein subgroups, were internationally normalized by first dividing the data in each process into the same number of groups based on their mass spectral fraction, setting to 100 for our analysis. LOESS (Cleveland, W.S. and Devlin, S.J., Journal of the American Statistical Association 83 (1988) 596-.

Based on our raw data, we constructed two linear models, the first using control/disease, time (8 weeks, 16 weeks, end) and location (cyto, micro, mitoI, mitoII) as factors and described using the following formula:

process count β 0 + β 1 time + β 2 time²+ β 3 position + β 4 control (1)

The second model uses only time (8 weeks, 16 weeks, end) and location (cyto, micro, mitoI, mitoII) as factors and is described by the following formula:

process count β 0 + β 1 time + β 2 time²+ β 3 position (2)

Where β 0 is the intercept and β 1, β 2, β 3 and β 4 are the slope estimates of the variable time, square of time, location and control/disease.

Anova was used to compare the two models, and invalidity assumed no difference between the two models. While low p-values show insufficient evidence that the two models are identical. Additional information indicates that the status (i.e. control/disease) appears to be an important part of the model. To extract proteins with significant changes in relative protein abundance between our control and disease models, our 6190 protein list was ranked according to their calculated p-values. This produced a panel of 593 proteins with p values < 0.05.

To explain the multiple hypothesis test from the above model, the p-value was corrected using a False Discovery Rate (FDR) correction method, specifically a Benjamini-Hochberg FDR correction method (Benjamini, Y. and Hochberg, Y., Journal of Royal Statistical Society B.57 (1995) 289-300). This produced a set of 40 proteins, which corrected p-values < 0.05 for the R9C mouse model.

3.2 statistical methods for generating differentially expressed p-values in aortic ligated mouse models

From the course of 68 experiments in the aorta-ligated mouse model, 3152 proteins with mass spectra numbers were identified. The same data analysis as described above applied to the R9C mouse model was used for the data set of the aortic ligation mouse model.

Example 4

4.1. Detection of mimecan in human serum and plasma samples by ELISA

For the detection of mimecan in human serum or plasma, a sandwich ELISA was developed. For capture and detection of the antigen, aliquots of anti-mimecan polyclonal antibody from R & D Systems (catalog # AF2660) were conjugated with biotin and digoxigenin, respectively.

Streptavidin-coated 96-well microtiter plates were incubated with 100. mu.l of a 1xPBS solution of 2. mu.g/ml biotinylated anti-mimecan polyclonal antibody for 60 minutes. After incubation, the plates were washed three times with 1xPBS +0.02% Tween-20, blocked with PBS +2% BSA (bovine serum albumin) for 45 minutes, and then washed three times with 1xPBS +0.02% Tween-20. Wells were incubated for 1 hour with 100 μ l serial dilutions of recombinant mimecan as standard antigen or diluted serum or plasma samples from patients or control individuals (1: 5 in 1xPBS +1% BSA), respectively. After binding to mimecan, the plates were washed three times with 1XPBS +0.02% Tween-20. To specifically detect bound mimecan, wells were incubated with 100 μ l of a 1xPBS +1% BSA solution of 0.2 μ g/ml of a homooctanoated anti-mimecan polyclonal antibody for 45 minutes. Thereafter, the plates were washed three times to remove unbound antibody. Next, each well was incubated with 75 mU/ml of anti-digoxigenin-POD conjugate (Roche diagnostics GmbH, Mannheim, Germany, cat No. 1633716) in 1xPBS +1% BSA for 30 minutes. The plate was then washed six times with the same buffer as above. To detect antigen-antibody complexes, wells were incubated with 100. mu.l of ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, cat # 11685767) and the Optical Densities (OD) were determined after 15 minutes at 405 nm and 492 nm with an ELISA reader.

4.2. Mimecan ELISA with serum from HF patients and clinically routine and apparently healthy donors, respectively

To further evaluate the utility of the mimecan assay under routine clinical conditions, serogroups of HF patients (n 241) and 146 apparently healthy control patients were studied. Serum was diluted 1:5 with 1xPBS +1% BSA as previously described. Table 3 shows the results for these extended groups:

TABLE 3: results of mimecan ELISA (from the clinically routine set of HF samples)

The data summarized in Table 3 has also been used to calculate box-blocks (box-blocks) shown in FIG. 3. FIG. 3 shows that the mean value of mimecan measured from the sera of heart failure patients is very different compared to the mimecan value measured from the sera of apparently healthy control individuals. An increased value of Mimecan is indicative of heart failure.

Example 5

Marker combination comprising the marker mimecan for the assessment of heart failure

Example 5.1 marker combination NT-proBNP and mimecan

NT-proBNP and mimecan marker combinations were evaluated for distinguishing patients in phase B and phases C and D, respectively. The accuracy of the diagnosis was assessed by analyzing individual fluid samples obtained from a well-identified group of individuals, namely 50 individuals at stage B according to the HF classification standard for ACA/ACC and 50 patients with HF and at stage C according to the HF classification standard for ACA/ACC. With serum samples obtained from each of the above-mentioned individuals, according to a commercial assay (Roche diagnostics, NT-proBNP-assay (cat # 03121640160, for Elecsys)^®Systemic immunoassay analyzer) and mimecan determined as described above. ROC-analysis was performed according to Zweight, M.H. and Campbell, G. (supra). By regularized discriminant Analysis (Friedman, J. H., Regularized diagnostic Analysis, Journal of the American Statistical Association 84(1989) 165-175) To calculate the ability to discriminate between stage C patients and stage B individuals for the combination of mimecan and the established marker NT-proBNP.

Example 5.2 labelling of the combination troponin T and mimecan

The evaluation of the troponin T and mimecan marker combinations was used to differentiate patients suffering from acute cardiac events from patients suffering from chronic cardiac diseases, respectively. The accuracy of the diagnosis is assessed by analyzing individual fluid samples obtained from a well-identified group of individuals, namely 50 individuals diagnosed with an acute cardiac event and 50 patients diagnosed with a chronic cardiac disease. Using serum samples obtained from each of the above individuals, a commercial assay (Roche Diagnostics, troponin T-assay (cat # 2017644 for Elecsys) was used^®Systemic immunoassay analyzer) and mimecan determined as described above. ROC-analysis was performed according to Zweight, M.H. and Campbell, G. (see above). The discriminatory power for the combination of mimecan and established marker troponin T to discriminate stage C patients from stage B individuals was calculated by a regularized discriminant analysis (Friedman, J.H., J.of the American Statistical Association 84(1989) 165-175).

Example 5.3 labeling of combinations mimecan and CRP

The combination of C-reactive protein and mimecan markers was evaluated for distinguishing patients diagnosed with cardiomyopathy from controls without any suspected heart disease, respectively. The accuracy of the diagnosis was assessed by analyzing liquid samples of individuals from a well-identified group of individuals, namely 50 individuals with cardiomyopathy and 50 healthy control individuals. Using sera from each individual, CRP determined using a commercial assay (Roche Diagnostics, CRP-assay (Tina-quant C-reactive protein (latex) high sensitivity assay-Roche Cat # 11972855216) and mimecan determined as described above were both quantified ROC-analysis was performed as per the methods of Zweig, M.H., and Campbell, G. (supra). The discriminatory power to distinguish stage C patients from stage B individuals by a combination of mimecan and established labeled CRP was calculated by the regularized discriminant analysis (Friedman, J.H., J.of the American Statistical Association 84(1989) 165-175).

Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention as set forth in the appended claims.

All publications, patents, and applications are herein incorporated by reference in their entirety to the same extent as if each such reference was specifically and individually indicated to be incorporated by reference in its entirety.

Sequence listing

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F. Hoffmann-La Roche AG

University of Toronto

<120> use of mimecan in the assessment of heart failure

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<150>EP09009666.0

<151>2009-07-27

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<170>PatentIn version 3.5

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Claims (5)

1. Use of a reagent required for the specific measurement of mimecan in the manufacture of a kit for the assessment of heart failure in an individual, wherein said kit further comprises optionally reagents required for the specific measurement of one or more other markers of heart failure selected from the group consisting of a natriuretic peptide marker, a cardiac troponin marker and a marker of inflammation, and wherein an increased concentration of mimecan measured in a sample of a bodily fluid selected from the group consisting of serum, plasma and whole blood is indicative for heart failure.

2. The use according to claim 1, further characterized in that said one or more other marker is NT-proBNP.

3. Use according to claim 1 or 2, further characterized in that said one or more other marker is troponin T.

4. The use of claim 1, wherein mimecan is measured in a sample obtained from an individual at risk of heart failure.

5. A kit for carrying out the use of claim 1, said kit comprising the reagents required for the specific measurement of mimecan and optionally the reagents required for the specific measurement of one or more other markers of heart failure.