WO2011024107A1

WO2011024107A1 - Method for prognosing heart failure

Info

Publication number: WO2011024107A1
Application number: PCT/IB2010/053750
Authority: WO
Inventors: Zoltán PROHÁSZKA; Tímea GOMBOS; Lívia JÁNOSKÚTI; Zsolt FÖRHÉCZ; Zoltán POZSONYI
Original assignee: Semmelweis University
Priority date: 2009-08-29
Filing date: 2010-08-19
Publication date: 2011-03-03
Also published as: HU0900534D0

Abstract

The invention provides a method, reagent and kit for diagnosing or prognosing heart failure in a patient.

Description

METHOD FOR PROGNOSING HEART FAILURE

The invention relates to the field of medicine, more particularly to the prognosis of heart failure. In particular, the invention provides a method, reagent and kit for prognosing heart failure in a patient who had been diagnosed to have heart failure.

Heart failure is initiated by the loss of myocardium resulting from a variety of insults. Following a myocardial infarction, there is a compensatory response of the 'surviving' muscle to preserve overall function. These compensatory responses continue over time and eventually lead into progressive myocardial dysfunction. There are continuous efforts to identify the condition as early as possible in order to treat it more effectively. Biomarkers are believed to have potential utility in further improving the diagnostic and prognostic capabilities that clinicians apply in routine practice. A number of markers for heart failure diagnosis and prognosis have been identified which are from diverse biochemical groups and include brain natriuretic peptide (BNP), norepinephrine, troponins, heart-type fatty acid binding proteins, myosin light chain- 1, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), C-reactive protein, tumour necrosis factor [alpha] (TNF[alpha]), soluble ST2 receptor, soluble IL-2 receptor, CD40-CD154, ICAM-I, P-selectin, tissue factor, von Willebrand factor and urocortin.

Chronic heart failure is a progressive disease characterized by declining systolic and diastolic heart function, remodeling of heart muscles finally resulting in chronic neurohormonal activation, inflammation, malnutrition and renal failure. There are at least three well characterized pathways for immune activation in heart failure: immune activation by direct antigenic stimulation, as is the case of a virus infecting the myocardium; secondary to cardiac injury whereby the myocardium exposes altered or damaged self antigens capable of triggering inflammatory reaction and immune response against the heart; and finally, cytokine release by cardiac cells, both myocytes and nonmyocytes, in response to hemodynamic stress (Celis R et al., 2008). Furthermore, endotoxin levels may increase in adult patients with acute oedematous decompensated heart failure (Anker et al., 1997; Rauchhaus et al., 2000) and indeed, increased levels of proinflammatory cytokines were observed in patients with heart failure who had peripheral oedema (Niebauer et al., 1999).

The complement system, comprising a protein cascade of more than 30 proteins, represents an important part of the innate immune system playing central role in the initiation of inflammation (Markiewski et al., 2007) and the host defense against microbes (Harboe et al., 2008). The central component of the complement system is C3 that can be activated by either the classical, the alternative or the lectin pathways. Once C3 is activated and the activation continues through C5 until the terminal pathway, activating surfaces are opsonized, proinflammatory anaphylatoxins including C3a and C5a are generated, and the terminal complex SC5b9 is formed. The system is regulated on multiple levels involving soluble and membrane bound regulators (Muller-Eberhard et al., 1988; Matsushita et al., 1996). The anaphylatoxins are powerful proinflammatory mediators inducing hyperaemia, chemotaxis and increasing vascular permeability. Increased levels of C5a were reported in sepsis and blockade of C5a represents a promising therapeutic strategy in sepsis (Guo et al., 2006).

Although activation of the innate immune system has been implicated in the pathogenesis of both ischaemic and nonischaemic chronic heart failure (CHF) (Torre -Amione et al., 2005) large studies aimed to investigate the role of complement system in CHF are scarce. Enhanced systemic complement activation involving classical, alternative, lectin and terminal pathways was shown in patients with congestive HF (Aukrust et al., 2001) and high SC5b9 levels were linked with adverse clinical events in patients with symptomatic HF (Clark DJ. et al., 2001). Increased amounts of the terminal complement complex SC5b9 have been shown myocardial biopsies (Zwaka et al., 2002), but the extent of staining did not correlate with clinical outcomes (Zimmermann et al., 2007).

Since heart failure is a very serious, life threatening condition with adverse and increasing morbidity and mortality, there is always a clinical need for further diagnostic and prognostic markers of heart failure to improve the current state of the art.

The evaluation of complement activation in a well characterized heart failure cohort allowing the comparison of the predictive value of complement activation to the gold standard marker NT-proBNP surprisingly led the present inventors to the identification of C3a as a novel marker for prognosing heart failure.

Results of our study showed that complement activation is strongly linked to unfavourable outcomes in heart failure. High levels of anaphylatoxin C3a predicted rehospitalization and mortality in adjusted survival model. Increased C3a levels were associated with biomarkers of acute -phase reaction, inflammation, cellular stress response, endothelial-cell activation and oedematous complications independently from disease severity.

According to our knowledge this is the first prospective cohort study with sufficiently high number of patients allowing the analysis of serum complement activation in HF in adjusted survival models. Clark DJ. et al. (2001) described complement activation in 36 patients (13 events) with congestive heart failure and showed an association of increased SC5b9 levels with near-term (6 months) adverse events. However, adjusted models were not presented and anaphylatoxins were not analysed in this study. At the same time but independently from this study, Aukrust and colleagues (2001) also reported enhanced systemic complement activation in CHF but anaphylatoxins were not studied. The 39 patients included in that study were followed for 6 months but clinical events were not reported. In a more recent study (Tanner et al., 2007) SC5b9 complex was measured in a cohort of 118 CHF patients followed for 1 year. Complement activation significantly correlated to CRP levels, however, the authors did not analyze SC5b9 as a potential predictor of end-points. There are further studies describing systemic complement activation in a couple of series of advanced HF patients receiving left-ventricular assist devices, convincingly supporting the presence of systemic complement activation in HF (Clark AL. et al., 2001; Loebe et al., 1998; Loebe et al., 2001).

In contrast to the above studies, as presented herein, our cohort included 182 patients (75 events during 12 months of follow up) with CHF allowing the analysis of complement activation in adjusted survival models for CHF prognosis. As presented in Table 2, increased levels of anaphylatoxin C3a, a complement activation product with powerful biological effects, are significantly related to increased risk of combined end-points (first hospitalization related to HF and/or death after study inclusion). According to the results of a multivariable Cox model, this relationship is independent of age, body mass index, diastolic blood pressure, haemoglobin, creatinine and NT -pro BNP levels. Addition of C3a as a co-variate to the model may cause further improvement in the prediction of clinical events.

Schmid et al. (1998) described that the level of C3a is associated with oedematous complications. More specifically, decreased levels of C3a are associated with decreased oedema. However, the present results show that the C3a levels have well-defined correlation with the prognosis of heart failure, which was not disclosed or even suggested in the prior art.

In our study activation of innate immunity and inflammation has been shown to be related to the progression of HF. Our results on the disease severity-independent association of C3a with oedematous complications in HF (Fig. 2) proved C3a-desArg as a powerful marker for the prognosis of HF. Taken together, our data on the predictive power for clinical events and the relationship to oedematous complications support the important role of complement activation in the progression of HF.

Accordingly, the present invention provides a method for diagnosing or prognosing heart failure in a patient, said method comprising

a) providing a sample from said patient,

b) determining the level of C3a-desArg in said sample; and

c) evaluating if the C3a-desArg level is indicative with a particular state or outcome for said patient.

In a specific embodiment, the invention provides a method wherein said evaluation comprises the determination of the lower tertile boundary of the group of heart failure patients as a cut off value, and patients having a C3a- desArg level over this cut off value are assessed as having bad prognosis.

In a further specific embodiment, the invention provides a method wherein said cut off level of C3a-desArg is within the range of 150 to 250 ng/ml, preferably 175 to 200 ng/ml, more preferably about 185 ng/ml.

In another specific embodiment, the invention provides a method further comprising the determination of the level of at least one other marker for heart failure in said sample of said patient, wherein the levels of all markers are considered when evaluating if said levels are associated with a particular outcome for said patient.

In another embodiment, the invention provides the use of a reagent that specifically identifies C3a-desArg for diagnosing or prognosing heart failure in a patient.

In another embodiment, the invention provides the use of a reagent that specifically identifies C3a-desArg in combination with at least one other reagent that specifically identifies at least one other marker for heart failure, for diagnosing or prognosing heart failure in a patient.

In another embodiment, the invention provides a reagent kit for diagnosing or prognosing heart failure in a patient, comprising a reagent capable of specifically identifying C3a-desArg and at least one other reagent capable of specifically identifying at least one other marker for heart failure.

In a specific embodiment, the invention provides a reagent kit in the form of a rapid test.

In another embodiment, the invention provides a method for identifying a compound useful in treating heart failure in a patient who had been diagnosed to have heart failure, comprising

a) providing a sample from said patient,

b) determining the level of C3a-desArg in said sample;

c) administering a test compound to said patient, wherein said test compound was assessed as safe to be administered to said patient by previous in vitro and in vivo toxicology experiments;

d) providing a sample from said patient,

e) determining the level of C3a-desArg in said sample; and

f) identifying said test compound as useful in improving the prognosis of said heart failure in a patient, if said level of C3a-desArg in step e) is decreased compared to said level of C3a-desArg in step b). In a specific embodiment, the invention provides a method further comprising the determination of the level of at least one other marker for heart failure in said sample of said patient in steps b) and e), wherein the levels of all markers are considered when evaluating if said test compound is useful in improving the prognosis of said heart failure in a patient.

In further specific embodiments, the invention provides a method, reagent or kit according to the invention, wherein the patient had been diagnosed to have heart failure.

In further specific embodiments, the invention provides a method, reagent or kit according to the invention, wherein said patient is human.

In further specific embodiments, the invention provides a method, reagent or kit according to the invention, wherein said patient having heart failure is selected from patients having cardiac disease, myocardial disorder or cardiomyopathy.

In further specific embodiments, the invention provides a method, reagent or kit according to the invention, wherein said sample is a body fluid, preferably selected from the group consisting of blood, serum, plasma, saliva, tear and urine.

In further specific embodiments, the invention provides a method, reagent or kit according to the invention, wherein said level of C3a-desArg is measured by immunoassay, antibody, mass-spectrometry, or surface plasmon resonance.

In further specific embodiments, the invention provides a method, reagent or kit according to the invention, wherein said at least one other marker is selected from the group consisting of Brain natriuretic peptide or fragments thereof, preferably NT -pro BNP, acute -phase proteins, preferably C-reactive protein, alphal-acid- glycoprotein, prealbumin, cytokines, preferably tumor necrosis factor alpha, interleukin 1 -alpha, interleukin 6, cytokine receptors, preferably soluble TNF receptor I, soluble TNF receptor II, Heat shock protein 70,

ADAMTS 13, Von Willebrand factor, Endothelin- 1 , cardiac markers, preferably troponins, atrial natriuretic peptide, arginine -vasopressin, adrenomedullin, markers of anaemia, preferably hemoglobin, markers of impaired renal function, preferably creatinine , markers of undernutrition, preferably body-mass index, markers of cardiac pump insufficiency, preferably diastolic blood pressure, and age.

In another embodiment, the invention provides a method, reagent or kit for diagnosing or prognosing heart failure in a patient as disclosed herein. Detailed description

Due to its central position in the complement system and important biological activities, complement C3, C4, anaphylatoxin C3a and terminal pathway complex SC5b9 were selected as analytes for the study carried out by the inventors.

C3a-desArg is one of three activation fragments formed during the activation of the complement cascade. C3a- desArg is formed from C3a via carboxypeptidase cleavage of the C-terminal arginine group. The structure of C3a-desArg is similar in human, primates, cow, rat and mouse.

In the context of the present invention, the term C3a-desArg includes and is interchangeable with complement C3a, a splice variant, a fragment, a derivative or a precursor thereof, or any combination of the above. In a first aspect, the present invention provides a method for prognosmg heart failure m a patient, said method comprising

a) providing a sample from said patient,

b) determining the level of C3a-desArg m said sample, and

c) evaluating if the C3a-desArg level is indicative with a particular outcome for said patient

In a specific embodiment, the patient already had been diagnosed to have heart failure, and the method facilitates the follow-up treatment of the patient

In the context of the present description, the term "patient who had been diagnosed to have heart failure" (HF) includes patients having any cardiac disease, myocardial disorder or cardiomyopathy The patient may be a human or mammalian patient such as a primate, horse, dog, cow, sheep or a pet Preferably, the patient is a human patient In a specific embodiment, where a patient is tested for the first time for heart failure according to the invention, the present method is capable to provide a prognosis for the patient, even if he had not been diagnosed earlier to have heart failure, although the condition was already present In this way, the present method can also be performed as a diagnostic method to help diagnose heart failure m a patient m itself or m addition to other available diagnostic means

Common causes of heart failure include myocardial infarction and other forms of ischemic heart disease, hypertension, valvular heart disease and cardiomyopathy Heart failure can cause a large variety of symptoms such as shortness of breath (typically worse when lying flat, which is called orthopnea), coughing, ankle swelling and reduced exercise capacity The patient diagnosed to have heart failure may have acute heart failure characterized by sudden onset of typical symptoms or may have chronic heart failure with sustained typical signs and complaints

Chronic heart failure is a progressive disease characterized by declining systolic and diastolic heart function, remodeling of heart muscles finally resulting m chronic neurohormonal activation, inflammation, malnutrition and renal failure The progression of HF may cause several clinical adverse events including arrhythmias, sudden worsening of systolic and diastolic heart function, acute episodes of dyspnea and oedematous attacks The risk of sudden death and cardiac death is increased m HF patients In the context of the present invention, prognosmg heart failure involves the prediction of the occurrence of at least one of these clinical events or any other events that are generally accepted m the art as being associated with heart failure

According to the present invention, heart failure already has been diagnosed m the patient according to state of the art procedures, for example by determining whether there is a reduction m ejection fraction of cardiac output, usually taken as less than 40% Typically, this would be measured by echocardiography but other cardiac imaging methods can also be used It may be possible to have heart failure symptoms with diastolic dysfunctions Preferred method for prognosmg CHF m a patient is Seattle Heart Failure Model (SHFM, 1 ittp. dcpt^ Vi ash nigton cd'i slifJu )

It will be appreciated by the person skilled m the art that if the prognosis is of a poor outcome, the clinician will be able to select a treatment regimen for the patient accordingly Conversely, when the prognosis is of a good outcome, the treatment may be tailored accordingly For example, if the prognosis is poor, it may be beneficial to treat the patient by increasing the dose of diuretic treatment, by adding an angiotensin converting enzyme (ACE) inhibitor to the treatment regime, or spironolactone or starting [beta] -blocker treatment, or any other treatment beneficial to the health of the patient with heart failure, either separately or in any appropriate combination. The sample according to the present method may be in any form suitable for carrying out the clinical analysis as necessary. In particular, the sample is a body fluid, preferably selected from the group consisting of blood, serum, plasma, saliva, tear and urine. Sample collection is carried out according to standard clinical laboratory practices. The preferred sample is a plasma sample, prepared from a blood sample in a standard way (for example by collection in EDTA or heparin tubes followed by centrifugation). It is particularly preferred if the sample is contacted with an agent that prevents or inhibits complement activation, for example an inhibitor of a nonspecific protease, after it has been obtained from the patient and that it is kept in the presence of the agent prior to the level of C3a-desArg being measured.

The determination of the level of C3-desArg according to the present invention may be carried out by using any suitable state of the art protocol. In particular, the level of C3-desArg may be measured by binding assays where a reagent is provided that can identify C3-desArg (such as in immunoassays, by antibodies, etc.) or alternatively, by physically detecting C3-desArg, for example by HPLC or capillary electrophoresis, followed by UV or fluorescent detection, or by mass-spectrometry or surface plasmon resonance.

In the context of the present invention, the "antibody" for use in the determination of the level of C3-desArg (or any other biochemical markers that may be assayed according to the invention) includes any types of antibodies generally applied for this purpose in the art. For example, it may be a whole antibody or a fragment thereof, genetically altered, humanized, chimeric, single -stranded, monoclonal, polyclonal; originating from any animal species, belonging to any useful class or subclass of antibodies, and so on. The person skilled in the art will be readily capable to select the best reagent for the specialized determination which he intends to perform.

In addition, although C3a-desArg does not have any enzyme activity, it has anaphylatoxin bioactivity and this activity can be used to quantify its levels in the sample. Protocols for this determination are available in the art. After performing said determination according to the invention, it is evaluated whether the C3a-desArg level measured is indicative of a particular outcome for said patient. Said evaluation may preferably be a simple comparison with a pre-determined cut off value. A cut-off value for a bad prognosis of CHF can be determined as the lower tertile boundary of the group of heart failure patients. In this evaluation, patients having a C3a- desArg level over this cut off value are evaluated as having bad prognosis. The person skilled in the art will be readily able to determine any suitable cut off level as it deemed reasonable.

In specific embodiments of the present invention, said pre-determined cut off level of C3a-desArg is within the range of 100 to 300 ng/ml, 150 to 250 ng/ml, 175 to 200 ng/ml, 180 to 190 ng/ml, and preferably around 185 ng/ml.

In a further embodiment of the invention, the level of at least one other marker for heart failure is measured in the sample of the patient, and the levels of all markers are considered when evaluating if said levels are associated with a particular outcome for the patient. Preferably, the levels of C3a-desArg and of the further heart failure marker are measured in the same sample taken from the patient. Alternatively, the levels may be measured in separate samples taken from the patient. In the latter case, the samples are taken from the patient at substantially the same time, or within several hours of each other. In specific embodiments of the invention said at least one other marker is selected from the group consisting Brain natriuretic peptide or fragments thereof, preferably NT -pro BNP, acute-phase proteins, preferably C- reactive protein, alphal-acid-glycoprotein, prealbumin, cytokines, preferably tumor necrosis factor alpha, interleukin 1 -alpha, interleukin 6, cytokine receptors, preferably soluble TNF receptor I, soluble TNF receptor II, Heat shock protein 70, ADAMTS13, Von Willebrand factor, Endothelin- 1 , cardiac markers, preferably troponins, atrial natriuretic peptide, arginine -vasopressin, adrenomedullin, markers of anaemia, preferably hemoglobin, markers of impaired renal function, preferably creatinine , markers of undernutrition, preferably body-mass index, markers of cardiac pump insufficiency, preferably diastolic blood pressure, and age. The person skilled in the art will be able to assemble together a suitable panel for the assessment of heart failure.

Depending on the type of the heart failure marker, different protocols are available for the person skilled in the art for the determination according to the invention. For example, standard clinical markers, such as blood pressure or body-mass index can be determined by performing the appropriate measurements. Chemical or biochemical markers may be determined by specific measurement protocols developed for that purpose. In the case of these biochemical markers, specialized assay methods are widely available for the person skilled in the art from commercial sources, or may be developed specifically for carrying out the determination necessary for the method of the invention. Preferably, the level of the further heart failure marker is determined using a reagent that is capable of selectively identifying the further marker in the sample taken from the patient. The determination of the levels of these additional markers may be carried out by using any suitable state of the art protocol.

In a preferred embodiment, the levels of C3a-desArg and of the one or more further heart failure markers are all considered together or in some combination when evaluating whether the levels of said markers are indicative of a particular outcome for the patient. Thus, it will be appreciated by the person skilled in the art that the evaluation (i.e. prognosis) may be carried out on the basis of the level of C3a-desArg and the level of said further heart failure marker in said sample from the patient. It is believed that using a combination of markers may improve the accuracy of the evaluation (prognosis), but nevertheless determining the level of C3a-desArg alone in a sample from the patient should be sufficient according to the present invention.

A further aspect of the invention provides the use of a reagent that specifically identifies C3a-desArg for prognosing heart failure in a patient who had been diagnosed to have heart failure. Suitable reagents are those that are routinely used in the field to determine the level of C3a-desArg. It is particularly preferred to use an antibody to C3a-desArg for the prognosis of heart failure in a patient. Thus, the invention provides the use of a reagent which specifically identifies C3a-desArg, such as an antibody, for use in evaluating patients with respect to heart failure as discussed above, for example for use in prognosing heart failure in a patient who had already been diagnosed with heart failure. Typically, the reagent is used in the methods described above. The other specifics of this embodiment are the same as enumerated above with respect to the method according to the invention.

A further aspect of the invention provides the use of a reagent that specifically identifies C3a-desArg in combination with at least one other reagent that specifically identifies at least one other marker for heart failure, for prognosing heart failure in a patient who had been diagnosed to have heart failure. Suitable markers are those that are described above. Suitable reagents are those that are routinely used in the field to determine the level of the marker selected. It is preferred if the further heart failure marker is human BNP. Thus, in a specific embodiment, the invention provides an antibody to C3a-desArg and an antibody to BNP both for use in evaluating heart failure in a patient for example for prognosing heart failure in said patient. It is particularly preferred if the antibody to C3a-desArg and the antibody to the further heart failure marker (such as BNP) are used in an immunoassay. The immunoassay may be for each marker individually, or it may be for two or more markers combined for example in a single immunoassay which is able to detect C3a-desArg and the other heart failure marker(s). Protocols are available in the art for simultaneous detection of different markers within the same assay. For example, the secondary antibodies are labeled with different enzyme or flourophors, and the primary antibodies are of different isotypes so that they can be distinguished by the different secondary antibodies. In this way, it is possible for a single immunoassay to determine the level of C3a-desArg and one or more further heart failure markers (such as BNP) in the same sample from the patient.

The other specifics of this embodiment are the same as enumerated above with respect to the method according to the invention.

It is further provided a reagent kit for prognosing heart failure in a patient who had been diagnosed to have heart failure, comprising a reagent capable of specifically identifying C3a-desArg and at least one other reagent capable of specifically identifying at least one other marker for heart failure. The reagent kit is preferably an immunoassay. In addition to the antibody to C3a-desArg and the antibody to the one or more further heart failure markers (such as BNP), the kit may also contain secondary antibodies to these primary antibodies.

In a particularly preferred embodiment the reagent kit is in the form of a rapid test. In the context of the present invention, the term "rapid test" includes any type of assay that may be performed without any specialized clinical laboratory instrumentation, for example at the bedside of the patient, at home, in the ER room, etc. Preferably, the operation of such a rapid test would not require expert knowledge of the heart failure condition, and in its simplest form it would only require the application of the sample from the patient, and simple evaluation of the results of the test, for example by assessing the development of a color reaction by naked eyes, or by using a simple reader device.

Biological effects of anaphylatoxins include the induction of hyperaemia, vascular permeability, proinflammatory cytokine release. Experimental observation of our study support that increased C3a levels may contribute to such consequences in HF: we observed NT-proBNP-independent associations between C3a levels and oedematous complications, low diastolic blood pressure and markers of acute phase reaction, proinflammatory cytokines, endothelial cell activation and stress markers. Taken together these data indicate that C3a is not only a marker but, at least partly, a mediator of complications in HF, therefore targeting of the action of anaphylatoxins may open up as yet untapped modalities in the therapy of patients with heart failure.

Accordingly, the invention provides a method for identifying a compound useful in treating heart failure in a patient who had been diagnosed to have heart failure, comprising

a) providing a sample from said patient,

b) determining the level of C3a-desArg in said sample;

d) providing a sample from said patient,

e) determining the level of C3a-desArg in said sample; and f) identifying said test compound as useful in improving the prognosis of said heart failure in a patient, if said level of C3a-desArg in step e) is decreased compared to said level of C3a-desArg in step b).

In preferred embodiments, the patient may be a laboratory animal such as a rat, mouse, guinea pig, dog or primate.

As a starting point, a test compound is selected that is proven to be effective in inhibiting the complement system, and is tested thoroughly in pharmacological and toxicological experiments. The choice of test compound is within the ability of the person skilled in the art, especially because substances known for modulating the complement system are available, for example C 1 -inhibitor, compstatin, sCRl, anti-C5 monoclonal antibody, etc. New compounds may be tested for their activity to inhibit or block C3-convertase. Specific C3a inhibitors may also be tested, when available, such as anti-C3a antibodies, C3aR antagonists, etc.

In a next step, an experimental animal model of heart failure may be used for assessing the effectiveness of the test compound. See for example the pressure -overload induced by thoracic aortic constriction (TAC) to model heart failure in mice (Sheridan et al., 2000). Briefly, female C57BL/6 mice are housed under standard conditions. Animals are anesthetized with ketamine (100 mg/kg body weight [BW] IP) and xylazine (10 mg/kg BW IP) for transverse aortic constriction (TAC). As a result of pressure overload progressive increases in lung and heart weights, left ventricular (LV) hyperthrophy, increased LV end-diastolic pressure (LVEDP) and pleural effusion, indicative for heart failure, can be observed.

In the model, markers of heart failure and/or complement components may be measured (such as C3a, C5a, C3bBbP complex, sC5b9 complex, etc.) before and after the induction of heart failure. Next, the test compound is administered to the treatment group, and several outputs are followed, such as complement activation, changes in the level of cardiac markers and/or heart failure markers, and/or other parameters (e.g. mass of heart and lungs, LV hypertrophy, etc.). Based on the data collected, assessment of the test compound may be carried out whether it is able to modulate the progression of heart failure.

With respect to the screening method according to the invention, human patients should not be excluded, as long as the standard safeguards are followed as customary in clinical trials. After identifying effective candidates in animal model systems, those should be tested in human patients. In this context, the clinical outputs measured are those that are customary in the assessment of human patients in standard clinical protocols. Accordingly, body-mass, lean body mass, echocardiographic parameters indicative for HF [LV ejection fraction, end-diastolic chamber diameters, parameters of LV hypertrophy], peripheral edema, pleural effusion, NYHA functional class together with mortality are usually measured or registered in addition to the analytical assays for the markers of heart failure.

Brief description of the figures

Fig. 1. Univariate Cox proportional-hazards regression analysis for the effect of complement C3a anaphylatoxin on event-free survival. C3a levels are categorized according to tertile boundaries (<185.4; 185.4-317.2 and >317.2 ng/ml), pO.0001.

Fig. 2. Relationship between complement C3a anaphylatoxin levels and peripheral oedema (top left) and pulmonary congestion (top right) in chronic heart failure. Medians with IQ-ranges (box) and non-outlier ranges (whiskers) are indicated, p values were calculated with Mann- Whitney test. NT-proBNP adjusted odds ratios (with 95% CI) of complement C3a levels (low versus medium tertile O; and low versus high tertile A) are displayed for the presence of peripheral oedema or pulmonary congestion (bottom panel). The present invention is further illustrated by the experimental examples described below, however, the scope of the invention will by no means be limited to the specific embodiments described in the examples.

Example 1: Methods

Study population

The study was carried out in accordance of the Helsinki Declaration at the IIIrd Department of Internal Medicine, Semmelweis University, based on a study protocol approved by the highest Ethical Committee of Hungary. Consecutive patients with clinical signs of CHF referred to trans-thoracic echocardiography were considered for inclusion. All patients with <45% left ventricular ejection fraction who provided written informed consent were included independently of the etiology of the disease from the out- or inpatient cardiology departments. Patients with co-existing malignant or acute infectious conditions were not included. A total of 182 patients (133 men, 49 women) were enrolled between February 2005, and March 2007. The full clinical record of the patients was registered at inclusion with the detailed physical status and routine laboratory tests. All patients were contacted after 1-year from study entrance. For patients alive at year 1 all major clinical events (re -hospitalization due to worsening of HF, or lack of it) were registered. Information on mortality (with specific cause of death) was collected from hospital database, medical staff or family members.

Blood samples were taken after 6 hours of fasting between 8 and 10 AM by antecubital venipuncture into native, EDTA- and sodium citrate anticoagulated tubes. The samples were processed to obtain aliquots of serum and plasma, later stored at -70 ⁰C until further analysis. Determination of the complement proteins

C3 and C4 levels were measured by Roche Cobras Integra 800 (Tina-quant® C3c 2. ver. and Tina-quant® C4 2. ver., Cat. No.: 3001938 and 3001962). Levels of C3a (C3a des-arg) and SC5b-9 were determined with Quidel ELISA kits (San Diego, California, USA) according to the manufacturer's instructions (Cat.No.:A015, A029, respectively).

Determination of other laboratory parameters

Levels of NT-proBNP (Biomedica ELISA kit (Cat No. BI-20852)), serum interleukin-6 (IL-6) (R&D Systems High Sensitivity ELISA kit serum (Cat No. HS600B)), tumor-necrosis factor-alpha (TNF-α) (R&D System high sensitivity ELISA kit (Cat No. HSTAOOC)), serum soluble TNF-receptor-I (TNFR-I) and TNFR-II (R&D Quantikine ELISA kit (Cat No. DRTlOO and DRT200) were measured according to the manufacturer's instructions. C-terminal-proET-1 (CT-proET-1) was measured by a novel sandwich immunoluminometric assay using 2 polyclonal antibodies to amino acids 168-212 of pre-proET-1 (BRAHMS AG, Hennigsdorf, Germany), as previously described (Papassotiriou et al., 2006). Von Willebrand factor (VWF) antigen levels were measured by ELISA according to Cejka et al. (1984). Serum soluble heat shock protein 70 (sHSP70) levels were determined by ELISA as described previously (Gombos et al., 2008). Standard laboratory parameters were measured by Roche Integra 800 (clinical chemistry, CRP, prealbumin), or by Cell-Dyn 3500 hematology analyzer (complete blood count).

Statistical analysis

For descriptive purposes the values of each measurement are given as median and 25^th-75^th percentile, or as numbers (percent), since most of the variables were not normally distributed. Non-parametric tests were used for group comparisons; continuous variables between two groups were compared with Mann- Whitney U test, for three groups with the Kruskal-Wallis ANOVA by ranks test, whereas categorical variables were compared with Pearson's χ² test. Multiple regression method with log-C3a as dependent variable was used on log-transformed variables to analyze associations between continuous variables.

Univariate Cox regressions were used to find predictors of clinical events from the complement components. The best predictor (C3a) was fitted to multivariate Cox proportional hazard model to assess the effect on CHF event free survival. Survival times were measured from inclusion in the study until both of the endpoints studied, all-cause mortality or rehospitalization due to worsening of CHF. The studied patient characteristics and laboratory markers together with the logarithm transformed variants of the continuous variables were pre- evaluated using a multitude of univariate Cox regressions. The best predictors by their chi-square values of likelihood ratio tests were noted and the plain or logarithmic variants of the predictors were selected for later inclusion in the multivariate models. One predictor was included in the multivariate models from each group of variables of clinically justified pathological pathways to adjust for the known effects and the studied variables. The results of the Cox regression models are presented as hazard ratios standardized on 1-SD increase of the predictors, the corresponding 95% confidence intervals (CI) and the WaId chi-square and p values of likelihood ratio tests.

Statistical analyses were carried out using the software STATISTICA 7.0 (StatSoft Inc., Tulsa, OK, USA), SPSS 13.01 (Apache Software Foundation, USA) and GraphPad Prism 4.03 (GraphPad Softwares Inc, CA, USA) softwares. Two tailed p values were calculated and the significance level was put at a value of p<0.05, if not otherwise stated.

Example 2: Baseline characteristics of the patient population

The basic clinical characteristics of the whole patient population and of subgroups stratified according to the presence or absence of clinical events during one year follow up are presented in Table 1. A clinical event was defined as death of any cause, or rehospitalisation due to the progression of heart failure. Unfavourable outcome

(n=75 until month 12 of follow-up) was associated with higher NYHA-classes, the presence of peripheral oedema or pulmonary congestion, increased NT-proBNP, TNF-alpha and decreased serum sodium and LVEF levels. The patients received complex medication according to their requirements and evidence-based guidelines, 75% received loop-diuretics, 69% ACE -inhibitors or angiotensin receptor blockers, 68% beta-blockers and 39% low-dose aspirin. Ischemic etiology was present in 62% of the patients, 20% had implanted devices. The patients were followed for a median of 14.5 months and the event rate for the whole group was 40.5/100 person-year. Table 1. Basic characteristics of the patient population.

All patients n= 182 Event* free n=107 With event* n=75

media interquartile median interquartile interquartile p-value** median or n

n or n range or % or n range or % range or %

Sex (males) 133 73 77 72 56 75 0.686

Age (years) 69.4 59.1-77.2 69.5 58.2-77.4 69.3 59.8-76.1 0.897

BMI (kg/m2) 26.82 24.4-31.1 26.57 24.4-30.8 27.31 24.1-32.6 0.721

Heart rate (1/min) 80 70-90 80 67-90 80 70-96 0.520

Diastolic blood

pressure (mmHg) 80 70-80 80 70-80 80 65-80 0.061

NYHA class (I-

II/III-IV) 94/88 51/49 69/38 64/36 25/50 33/67 O.0001

Left ventricular

ejection fraction

(%) 34 27-40 35 29-41 30 23-39 0.003

NT-proBNP

(fmol/ml) 722 398-1567 578 359-1321 1102 570-2357 O.0001

Peripheral

oedema (present) 77 42 36 34 41 55 0.006

Pulmonary

congestion

(present) 77 42 33 31 44 59 0.0002

Sodium (mmol/1) 140 137-142 141 139-143 139 135-141 O.0001

Creatinine

(umol/1) 98 78-139 92 75-112 109 89-166 O.0001

Hemoglobin (g/1) 141 128-154 142 132-154 138 124-152 0.183

Complement C4

(g/i) 0.29 0.24-0.35 0.30 0.25-0.35 0.27 0.24-0.34 0.115

Complement C3

(g/i) 1.20 1.04-1.41 1.24 1.09-1.36 1.17 1.01-1.42 0.321

Complement C3a

(ng/ml) 245.1 153.8-367.4 208.8 129.1-315.9 301.5 195.8-432.0 O.0001 sC5b-9 (ng/ml) 297.2 244.6-408.9 288.3 226.5-373.7 331.7 250.5-424.7 0.094

*Rehospitalization due to the progression of heart failure or death

**P-values were determined by Mann- Whitney U test of Pearson Chi square test.

Example 3: The predictive value of complement C3a levels in CHF

Levels of complement components C4, C3 and activation products C3a and SC5b9 were measured in the cohort (Table 1). In the univariate analysis high level of anaphylatoxin C3a was significantly associated with clinical events (pO.0001) whereas SC5b9 showed a tendency of association (p=0.094). Next, C3a and SC5b9 levels were analyzed in Cox proportional-hazards regression models as potential predictors of clinical events. Significant predictive value of C3a was obtained in univariate Cox regression analysis with a HR of 1.234 (95% CI 1.044-1.459) for 1-SD increase. Patients with low (<185.4 ng/ml, lowest tertile) C3a levels suffered 24.6 events/100 person-year whereas in case of the medium (185.4-317.2 ng/ml) or highest (>317.2 ng/ml) tertile groups event rates of 43 and 54/100 person-year were observed during follow-up, respectively. Fig. 1 shows event- free survival for these groups as obtained in the univariate Cox analysis. For SC5b9 the HR was 1.111 (0.914-1.352) in the univariate Cox model.

Table 2 represents results of the multivariable Cox analysis, where prediction of clinical events by C3a levels was adjusted for baseline clinical and laboratory variables related to the events including age, NT-proBNP, diastolic blood pressure, body mass index (BMI), hemoglobin and creatinine levels. A 1-SD increase of C3a levels was found to be related with a significant -20% increase of risk of clinical events in heart failure in this adjusted model. According to the results of the likelihood-ratio test, addition of C3a to the model presented in Table 2, significantly (WaId chi-square=4,195, p=0.041) improves its predictive power. Table 2. Results of multivariable Cox proportional -hazards regression analyzing effects of complement anaphylatoxin C3a for clinical events*

HR** 95% CI i P

NT-proBNP 1.275 1.161-1.670 10.95 0.001

Age (per y) 1.002 0.999-1.006 2.18 0.337

Body-mass index 1.339 1.067-1.681 6.154 0.013

Diastolic blood 0.916 0.747-1.123 0.721 0.396

pressure

Hemoglobin 0.778 0.591-1.025 3.090 0.079

Creatinine 1.267 1.036-1.550 4.883 0.027

C3a 1.189 1.023-1.383 4.195 0.041

*Rehospitalization due to the progression of heart failure and/or death

**Hazad ratios for variables shown as standardized hazard ratios (HR per 1 SD); age was considered as time- dependent covariate, WaId χ² and p values of likelihood-ratio tests are presented.

Example 4: Biological associations of C3a in CHF

The associations of complement anaphylatoxin C3a with clinical and laboratory variables are presented in Table 3. Setting a more conservative limit for p values according to Bonferroni's rule (0.05/20=0.0025), significant associations with linear trend through tertile groups of C3a were observed for acute phase reactants (CRP and prealbumin) and interleukin-6; soluble TNFR-I; endothelial cell activation markers endothelin-1 and VWF and complement terminal pathway complex SC5b9. Since C3a levels were marginally associated with disease severity (p value for association with NT-proBNP 0.023), NT-proBNP adjusted linear regression models were calculated for C3a as dependent variable. For all of the above mentioned markers plus soluble Hsp70 and soluble TNFR-II significant associations were observed in these adjusted models (Table 4.). Table 3. The basic characteristics and selected biomarker levels of patient groups, as stratified according to the C3 a levels

C3a <l 85.4 ng/ml C3a l85 .4-317.2 ng/ml C3a >317.2 ng/ml

p-value* Median IQ range Median IQ range Median IQ range

Age (years) 66.7 (59.1-78.5) 71.2 (60.6-77.7) 70.0 (59.6-76.1) 0.725

Body-mass index

27.18 (24.37-31.24) 26.31 (24.60-31.08) 27.53 (23.91-31.0) 0.865 (kg/m2)

Heart rate (1/min) 72 (64-88) 81 (72-96) 78 (67-90) 0.012

Diastolic blood

80 (70-80) 80 (70-80) 70 (62.5-80) 0.038 pressure (mmHg)

Le ft-ventricular

35 (29.5-41) 34 (24-40) 32 (25-40) 0.211 ejection fraction (%)

NT-proBNP

489 (322-1353) 897 (571-1729) 952 (414-2153) 0.023

(fmol/ml)

Event free survival

14.3 (10.6-16.8) 12.3 (6.1-16.7) 8.6 (2.5-15.3) 0.007 (months)

Creatinine (umol/1) 91 (72-108) 104 (87-145) 103 (81-159) 0.015

Hemoglobin (g/1) 140 (132-150) 146 (130-158) 138 (123-151) 0.077

TNF -alpha (pg/ml) 2.02 (0.95-4.24) 2.34 (1.69-3.10) 3.05 (2.09-4.38) 0.023

C-reactive protein

4.1 (1.2-10.0 6.4 (3.4-14.3) 8.7 (4.4-21.4) 0.0002 (mg/1)

Prealbumin (g/1) 0.26 (0.22-0.31) 0.22 (0.18-0.27) 0.18 (0.13-0.24) O.0001 sTNFR-I (ng/ml) 4.3 (2.8-7.4) 6.0 (3.9-7.9) 7.2 (4.5-10.4) 0.0018 sTNFR-II (ng/ml) i.i (2.5-5.0) 4.3 (3.0-5.3) 4.9 (3.5-6.1) 0.0041

Interleukin-6

5.9 (3.8-10.1) 11.2 (7.8-17.1) 13.3 (8.5-25.3) O.0001 (pg/ml)

Soluble Hsp70

0.31 (0.23-0.45) 0.33 (0.26-0.50) 0.41 (0.28-0.69) 0.025 (ng/ml)

von-Willebrand

156 (115-222) 191 (144-280) 240 (167-333) 0.0002 factor (%)

sC5b-9 (ng/ml) 274.2 (215.2-331.7) 309.3 (245.7-412.2) 356.9 (269.5-467.8) 0.0007

* p-values were obtained by Kruskal-Wallis test Table 4. NT-proBNP-adjusted linear regression analysis of complement anaphylatoxin C3a (dependent variable) with selected biomarkers in patients with heart failure (n=182)

Variable Unstandardized Coefficient (B) 95% Confidence Interval for B p-value

C-reactive protein 0.180 0.101-0.259 O.0001 Prealbumin -0.771 -1.037- -0.506 O.0001 Interleukin-6 0.337 0.195-0.478 O.0001 TNF-alpha 0.125 0.019-0.231 0.023 sTNFR-I 0.222 0.088-0.356 0.0013 sTNFR-II 0.347 0.133-0.561 0.0016 von Willebrand factor 0.466 0.222-0.712 0.0002

Soluble Hsp70 0.244 0.088-0.401 O.0001

Example 5: Anaphylatoxin C3a and oedematous complications in CHF

The relationships between baseline C3a levels and presence of oedematous complications in patients with CHF were next analyzed. Peripheral oedema was significantly associated with higher C3a levels, the difference in median C3a concentrations between the two groups was ~1.4 fold. Even larger difference was observed in the C3a concentrations between the groups with or without the presence of pulmonary congestion. Since disease severity is strongly related to the presence of oedematous complications we analyzed the relationship of their presence with C3a levels in adjusted logistic regression models. As presented on Fig. 2, patients with medium (middle tortile) or high (highest tortile) levels of C3a have 3.81 (95% CI 1.66-8.69) or 4.76 (2.07-10.94) times higher risk to have pulmonary congestion as compared to patients in the lowest tertile, respectively. This association is independent of disease severity since the analysis was adjusted for NT-proBNP. The same NT- pro BNP -independent relationship was observed for patients in the highest C3a tertile (as compared to the lowest) and presence of peripheral oedema (OR 3.06, 95% CI 1.43-6.57). References

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Claims

1. Method for diagnosing or prognosing heart failure in a patient, said method comprising

a) providing a sample from said patient,

b) determining the level of C3a-desArg in said sample; and

2. The method according to claim 1, wherein said evaluation comprises the determination of the lower tertile boundary of the group of heart failure patients as a cut off value, and patients having a C3a-desArg level over this cut off value are assessed as having bad prognosis.

3. The method according to claim 1 or 2, wherein said cut off level of C3a-desArg is within the range of 150 to 250 ng/ml, preferably 175 to 200 ng/ml, more preferably about 185 ng/ml.

4. The method according to any of claims 1 to 3, further comprising the determination of the level of at least one other marker for heart failure in said sample of said patient, wherein the levels of all markers are considered when evaluating if said levels are associated with a particular outcome for said patient.

5. Use of a reagent that specifically identifies C3a-desArg for diagnosing or prognosing heart failure in a patient.

6. Use of a reagent that specifically identifies C3a-desArg in combination with at least one other reagent that specifically identifies at least one other marker for heart failure, for diagnosing or prognosing heart failure in a patient.

7. Reagent kit for diagnosing or prognosing heart failure in a patient, comprising a reagent capable of specifically identifying C3a-desArg and at least one other reagent capable of specifically identifying at least one other marker for heart failure.

8. The reagent kit according to claim 7, in the form of a rapid test.

9. Method for identifying a compound useful in treating heart failure in a patient who had been diagnosed to have heart failure, comprising

a) providing a sample from said patient,

b) determining the level of C3a-desArg in said sample;

d) providing a sample from said patient,

e) determining the level of C3a-desArg in said sample; and

f) identifying said test compound as useful in improving the prognosis of said heart failure in a patient, if said level of C3a-desArg in step e) is decreased compared to said level of C3a-desArg in step b).

10. The method according to claim 9, further comprising the determination of the level of at least one other marker for heart failure in said sample of said patient in steps b) and e), wherein the levels of all markers are considered when evaluating if said test compound is useful in improving the prognosis of said heart failure in a patient.

11. The method, reagent or kit according to any of claims 1 to 10, wherein the patient had been diagnosed to have heart failure.

12. The method, reagent or kit according to any of claims 1 to 11, wherein said patient is human.

13. The method, reagent or kit according to any of claims 1 to 12, wherein said patient having heart failure is selected from patients having cardiac disease, myocardial disorder or cardiomyopathy.

14. The method, reagent or kit according to any of claims 1 to 13, wherein said sample is a body fluid, preferably selected from the group consisting of blood, serum, plasma, saliva, tear and urine.

15. The method, reagent or kit according to any of claims 1 to 14, wherein said level of C3a-desArg is measured by immunoassay, antibody, mass-spectrometry, or surface plasmon resonance.

16. The method according to any of claims 1 to 15, wherein said at least one other marker is selected from the group consisting of Brain natriuretic peptide or fragments thereof, preferably NT -pro BNP, acute -phase proteins, preferably C-reactive protein, alphal-acid-glycoprotein, prealbumin, cytokines, preferably tumor necrosis factor alpha, interleukin 1 -alpha, interleukin 6, cytokine receptors, preferably soluble TNF receptor I, soluble TNF receptor II, Heat shock protein 70, ADAMTS13, Von Willebrand factor, Endothelin-1, cardiac markers, preferably troponins, atrial natriuretic peptide, arginine-vasopressin, adrenomedullin, markers of anaemia, preferably hemoglobin, markers of impaired renal function, preferably creatinine , markers of undernutrition, preferably body-mass index, markers of cardiac pump insufficiency, preferably diastolic blood pressure, and age.

17. The method, reagent or kit for prognosing heart failure in a patient as disclosed herein.