WO2007116199A1

WO2007116199A1 - Methods and reagents for diagnosing heart failure

Info

Publication number: WO2007116199A1
Application number: PCT/GB2007/000581
Authority: WO
Inventors: Robert John Edwards; Vahitha Banu Abdul Salam; Martin R. Wilkins
Original assignee: Imperial Innovations Limited
Priority date: 2006-04-07
Filing date: 2007-02-19
Publication date: 2007-10-18

Abstract

A method of diagnosing heart failure in a patient, the method comprising providing a sample from the human patient, determining the level of C4a des Arg in the sample and assessing whether the level is indicative of heart failure in the patient. A method of prognosing heart failure in a patient who has heart failure, the method comprising providing a sample from the human patient, determining the level of C4a des Arg in the sample and assessing whether the level is indicative of a particular outcome for the patient. Preferably, the level of C4a des Arg in the sample is measured using an immunoassay. Typically, the level of brain natriuretic peptide (BNP) is also measured in the sample and used in the diagnosis or prognosis. Kits of parts, typically immunoassay kits, are also provided which contain reagents which selectively identify C4a des Arg and BNP. Preferably, the patient is a human.

Description

METHODS AND REAGENTS FOR DIAGNOSING HEART FAILURE

The present invention relates to methods and reagents for use in relation to the diagnosis and prognosis of heart failure in a patient, preferably a human patient.

Heart failure is a very serious, life threatening condition with adverse morbidity and mortality. Its dismal prognosis has led to efforts to identify the condition early in order to try to treat it more effectively. Biomarkers may 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 (Lee & Vasan (2005) Current Opinion in Cardiology 20, 201-210) which are from diverse biochemical groups and include brain natriuretic peptide (BNP)₅ 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 α (TNFα), soluble ST2 receptor, _.soluble IL-2 receptor, CD40-CD154, ICAM-I, P-selectin, tissue factor, von Willebrand factor and urocortin. However, there remains a clinical need for further diagnostic and prognostic markers of heart failure. BNP is of limited value for the diagnosis of heart failure; while a negative value excludes the diagnosis, a positive value needs further investigation. BNP has greater value in following the course of the condition once the diagnosis has been established. Also, clinical factors such as age, gender and thyroid function impact on BNP levels in humans and there are no clear conclusions available at this time as to what represents a normal level. Recommended guidelines from NHS Quality Improvement Scotland (NHS QIS) on BNP testing for patients with suspected heart failure should not. be used to replace an echo cardiogram in diagnosing heart failure.

Heart failure is a clinical syndrome resulting from a cardiac disease which compromises ventricular systolic or diastolic function or both. Heart failure results when the heart is unable to generate a cardiac output sufficient to meet the demands of the body without unduly increasing diastolic pressure. Heart failure may be manifested by symptoms of poor tissue perfusion alone (eg fatigue, poor exercise tolerance, confusion) or by both symptoms of poor tissue perfusion and congestion of vascular beds (eg dyspnea, chest rales, pleural effusion, pulmonary edema, distended neck veins, congested liver, peripheral edema).

Congestive Heart Failure (CHF) is a clinical syndrome caused by heart disease, characterized by breathlessness and abnormal sodium and water retention, and. resulting in edema. This term is used when there is congestion of pulmonary or systemic vascular beds.

Myocardial infarction (MI) refers specifically to myocardial (heart muscle) damage from lack of blood supply. It can be a cause of heart failure if the damage is extensive enough to impair the pump action of the heart. Or it can occur as a separate entity without heart failure. It usually affects the left side of the heart.

Idiopathic pulmonary arterial hypertension (IPAH) is high blood pressure in the pulmonary vessels and when severe causes right heart failure secondary to the pressure load on that part of the heart.

Abdul-Salam et al (2006) Proteomics 6, 2286-2294 describes the identification of plasma protein biomarkers associated with idiopathic pulmonary arterial hypertension (IPAH). Muscari et al (1995) Cardiology 8, 232-237 and Agostini et al (1994) Circulation 90, 2666-2670 relate to complement components, fibrinogens and the kinrn system in myocardial infarction. In none of these papers is there any mention of markers of heart failure.

The inventors have now found that C4a des Arg is a marker for heart failure diagnosis and prognosis. C4a des Arg is a truncated form of the complement factor C4a in which the arginine residue has been removed, probably arising from the action of carboxypeptidase N on C4a. The structure of human C4a des Arg is NVNFQKAINEKLGQYASPTAKRCCQDGVTRLPMMRSCEQRAARVQQPD CPvEPFLSCCQFAESLRKKSRDKGQAGLQ. The structure of C4a was first reported by Moon et al (1981) J Biol. Chem. 256, 8685-8692. C4a des Arg is known in other mammalian species.

Human C4a des Arg is one of three activation fragments formed from the activation of the complement cascade. C4a des Arg is formed from C4a via carboxypeptidase cleavage of the C-terminal arginine group. The structure of C4a des Arg is similar in human, cow, rat and mouse.

A first aspect of the invention provides a method of diagnosing heart failure in a patient, the method comprising providing a sample from the patient, determining the level of C4a des Arg in the sample and assessing whether the level is indicative of heart failure in the patient.

It will be appreciated that the invention includes a method of assessing whether a patient is suffering from heart failure, the method comprising providing a sample from the patient, determining the level of C4a des Arg in the sample and assessing whether the level is indicative of heart failure in the patient. It will be appreciated that this assessment may aid diagnosis, and may be used in association with other tests, or observations by the physician, in reaching a diagnosis.

The patient may be a human or mammalian patient, such as a horse, dog, cow or sheep. Preferably, the patient is a human patient.

The level of C4a des Arg which is indicative of heart failure in the patient may vary depending on the type of patient (eg human, horse, dog and so on). The level may be determined by comparing the levels in known heart failure patients with those in normal patients (ie those with no sign or symptoms of heart failure). Typically, the level which is indicative of heart failure is a level which is greater than 2 standard deviations above the mean for the normal range (ie in patients with no signs or symptoms of heart failure). For those samples collected in tubes not containing futhan (or a serine protease inhibitor), a typical threshold level is around 1.0 μg/ml of plasma C4a des Arg, wherein levels above this threshold typically are indicative of a high likelihood of heart failure in the individual (see Figure 2).

For samples collected in tubes containing fiithan (see below), plasma levels of C4a des Arg of >1 μg/ml typically are indicative of a high likelihood of heart failure and plasma levels of C4a des Arg of between 0.5 μg/ml and 1.0 μg/ml typically are indicative of an intermediate likelihood of heart failure. The level of C4a des

Arg is also correlated with NYHA functional classifications (see Figure 5), and the plasma levels of C4a des Arg may be used in classification.

A second aspect of the invention provides a method of prognosing heart failure in a patient who has heart failure, the method comprising providing a sample from the patient, determining the level of C4a des Arg in the sample and assessing whether the level is indicative of a particular outcome for the patient.

The patient may be a human or mammalian patient such as a horse, dog, cow or sheep. Preferably, the patient is a human patient.

It will be appreciated that the invention includes a method of assessing the likely outcome of a patient who has heart failure, the method comprising providing a sample from the patient, determining the level of C4a des Arg in the sample and assessing whether the level is indicative of a particular outcome for the patient. It will be appreciated that this assessment may aid prognosis, and may be used in association with other tests, or observations by the physician, in reaching a prognosis.

Heart failure may have already been diagnosed in the patient for example by determining whether there is a reduction in 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. The level of C4a des Arg which is indicative of a poor prognosis may vary depending on the type of patient. The level may be determined by comparing levels in heart failure patients who fare well and those also have a poor outcome. Typically, a C4a des Arg level which is greater than 2 standard deviations (SD) above the mean level of C4a des Arg in a population of heart failure patients who fare well is indicative of a poor outcome. .

It will be appreciated that if the prognosis is of a poor outcome, the physician will be able to tailor treatment of the patient accordingly. Similarly, if the prognosis is of a good outcome, the physician will be able to tailor treatment of the patient 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 β-blocker treatment.

The sample from the patient may be any. suitable sample. In particular embodiments of the invention, a suitable sample is obtained from the patient who is to be assessed (eg diagnosed or prognosed), and this sample is provided for analysis of the level of C4a des Arg. Conveniently, the sample is a fluid sample and it may be blood, serum or plasma. It is particularly convenient if the sample is a plasma sample which may be prepared from a blood sample in a standard way (for example by collection in EDTA 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 non-specific 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 C4a des Arg being measured. It is particularly preferred if the agent is a serine protease inhibitor which can prevent or inhibit the activity of a protease that acts on C4 to form C4a des Arg. It is particularly preferred if the agent is futhan. Futhan is the serine protease inhibitor nafamosat mesilate and may be obtained from Banyu Pharmaceutical Co Ltd, 2-2- 3 Nihonbashi Honcho Chuo-ku, 103 Tokyo, Japan. The sample may also be contacted with EDTA after it has been obtained from the patient and that it is kept in the presence of EDTA prior to the level of C4a des Arg being measured. Typically, a combination of futhan and EDTA is used. Typically futhan is added to a concentration of 0.1 g/1. Typically, EDTA is added to a concentration of 1.0 g/1

The level of C4a des Arg may be measured in the sample by any suitable means. One convenient way of measuring the level of C4a des Arg in the sample is to make use of a reagent which can identify C4a des Arg. Conveniently, the reagent is one which binds to C4a des Arg, but it may be any other type of suitable reagent.

Although C4a des Arg does not have any enzyme activity it has anaphylatoxin bioactivity and this activity can be used to quantify levels of C4a des Arg. ^{■ •}

HPLC and capillary electrophoresis, followed by UV or fluorescent detection, may be used to detect and quantify C4a des Arg.

Reagents which bind to C4a des Arg include antibodies and peptides selected from a combinatorial or phage display library. By the term "antibodies" we include whole antibodies which bind to C4a des Arg but also fragments of antibodies which bind C4a des Arg such as Fv, Fab and F(ab)₂ fragments as well synthetic antibodies or antibody fragments such as single chain Fv (scFv) molecules and domain antibodies (dAbs). The antibody fragments and synthetic antibodies retain antigen binding activity (and usually contain some or all of the complementarity determining regions (CDRs) of a parent antibody molecule). Antibodies that bind C4a des Arg selectively are known in the art and are commercially available, for example Metachem Diagnostics (Northamptonshire, UK) supplies an enzyme- linked immunosorbent assay (ELISA) kit which contains antibodies to human complement 4a (C4a) des Arg, as well as human C4a des Arg itself as a standard. It will be appreciated that the antibody is typically one which has been raised to or selected using human c4a des Arg. Typically, the antibody is selective for C4a des Arg. Typically, the antibody exhibits <5% cross-reactivity with C4.

In any event, antibodies for C4a des Arg may be made using well known technology such as the hybridoma method for making monoclonal antibodies, and phage display techniques for making synthetic antibody fragments. Suitable methods for the production and use of antibodies are described and referred to in "Using antibodies: A laboratory manual", Ed Harlow and David Lane, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1999.

Preferably, antibodies which bind preferentially to C4a des Arg (or C4a) compared to other molecules in patient plasma are used. Preferably, the antibody has at least a 10 fold-higher affinity for C4a des Arg (or C4a) than for any other component in patient plasma. (Antibodies are typically reactive with both C4a des Arg and C4a but in practice this is of no consequence since C4a is rapidly converted to C4a des Arg in the blood.)

Conveniently, the level of C4a des Arg in the sample is measured using an immunoassay. The antibody selective for C4a des Arg may itself be labelled, for example with a radioactive label or a fluorescence label or with an enzyme. Alternatively, and preferably, it is detected with a secondary antibody, which binds the antibody selective for C4a des Arg which is labelled. It is particularly convenient if the immunoassay is an ELISA. Immunoassays are well known in the art (see, for example, Immunoassays: A practical approach. James P. Gosling (ed), Oxford University Press, 2000, ISB4 0-19-963710-5), and as described above an ELISA for human C4a des Arg is commercially available.

A suitable assay for C4a des Arg is sold by Assay Designs, Inc, 800 Technology Drive, Ann Arbor MI 48108, USA. The assay kit is highly selective for C4a des Arg (C4a) showing very little cross-reactivity with C4 and practically no cross- reactivity with other complement factors. Other methods of measuring C4a des Arg in a sample are available. For example, as discussed in more detail in the Examples, mass spectrometry may be used since human C4a des Arg has a characteristic molecular ion signature (m/z = 8600 when analysed by SELDI-TOF mass spectrometry). Methods of measuring the levels of compounds in a sample by mass spectrometry are well known in the art (see, for example, Mass Spectrometry Principles and Applications, E. De Hoffmann, J. Charette, V. Stroobant, Wiley & Sons, New York, NY, 1996).

In a' further embodiment of the invention, the level of one or more further heart failure markers is measured in a sample from the patient and it is assessed whether the level of the one or more further markers is indicative of heart failure in the patient (according to the first aspect of the invention) or whether the level of the one or more further markers is indicative of a particular outcome for the patient (according to the second aspect of the invention). Conveniently, the level of C4a des Arg 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. Conveniently, the samples are taken from the patient at substantially the same time, or within several hours of each other.

Preferably, the level of the further heart failure marker is determined using a reagent that selectively identifies the further marker in a sample from the patient. Conveniently, the reagent binds to the further marker, and more conveniently the reagent is an antibody to the further marker. It will be appreciated that the antibody is typically one which has been raised to or selected using the further marker. Preferably, antibodies which bind preferentially to the further marker compared to other molecules in patient plasma are used. Preferably, the antibody has at least a 10-fold higher affinity for C4a des Arg or C4a than for any other^' component in patient plasma.

Preferably, the further heart failure marker is brain natriuretic peptide (BNP) or N- terminal pro BNP (NT-pro BNP). The sample may be any suitable sample, but typically is blood, serum or plasma. It is preferred if the sample is a plasma sample. The other heart failure marker may be any of those mentioned in Lee & Vasan (2005) discussed above, for example brain natriuretic peptide (BNP) or NT- pro BNP or norepinephrine or troponin or heart-type fatty acid binding protein or myosin light chain-1 or matrix metalloproteinase or tissue inhibitor of matrix metalloproteinase or C-reactive protein or TNFα or soluble T2 receptor or soluble 1L-2 receptor or CD40-CD154 or CCAM-I or P-selectin or tissue factor or von Willebrand factor or urocortin.

The level of BNP in a sample from the patient can be measured in an analogous way to the level of C4a des Arg in a sample from the patient. For example, antibodies which bind to BNP are known and may be used in an immunoassay.

Typically, the antibodies are ones which have been raised to or selected using

BNP. Typically, the antibody has at least a 10-fold higher affinity for BNP than for any other component in patient plasma.

BNP may be measured using an assay from Bayer. N-terminal pro-BNP may be measured using an assay from Roche. The BNP assays require the use of plasma.

Typically, thresholds for plasma BNP may be defined as follows: levels below

100 pg/ml indicates that heart failure was unlikely; results in the range 100-500 pg/ml suggest an intermediate probability of heart failure; and values exceeding

500 pg/ml are consistent with a high likelihood of heart failure.

In a preferred embodiment, the levels of C4a des Arg and of the one or more further heart failure markers are all taken into account when assessing whether the levels are indicative of heart failure in the patient or are indicative of a particular outcome for the patient. Thus, it will be appreciated that the assessment (eg diagnosis or prognosis) may be made, on the basis of the level of C4a des Arg and the level of a further heart failure marker (such as BNP) in a sample from the patient. It is believed that using a combination of markers may improve the accuracy of the assessment (eg diagnosis or prognosis), but nevertheless determining the level of C4a des Arg alone in a sample from the patient is useful. A further aspect of the invention provides the use of a reagent which identifies C4a des Arg in the diagnosis or prognosis of heart failure in a patient. The invention includes the use of a reagent which identifies C4a des Arg in the assessment of whether a patient is suffering from heart failure. The invention also includes the use of a reagent which selectively identifies C4a des Arg in the assessment of the outcome for a patient who has heart failure. Suitable reagents are disclosed, above, and it is particularly preferred to use an antibody to C4a des Arg in the diagnosis or prognosis of heart failure in a patient. Thus, the invention includes a reagent which selectively identifies C4a des Arg, such as an antibody, for use in assessing patients with respect to heart failure as discussed above, eg for use in diagnosing or prognosing heart failure. Typically, the reagent is used in the methods described above. Preferably, the reagent selectively identifies C4a des Arg. Preferably, the patient is a human patient.

As discussed in more detail below, the reagent which identifies C4a des Arg may be used to assess whether a test compound has an effect on heart failure in a patient (typically a human patient) by altering the level of C4a des Arg in the patient.

In a preferred embodiment, the previous aspect of the invention also includes the • use of a reagent that selectively identifies a further heart failure marker in the assessment of heart failure in a patient (eg diagnosis or prognosis). Suitable reagents that selectively identify the further heart failure marker are described above, but conveniently the reagent is an antibody. It is preferred if the further heart failure marker is human BNP. Thus, conveniently, the invention includes an antibody to C4a des Arg and an antibody to BNP both for use in assessing heart failure in a patient eg diagnosing or prognosing heart failure in a patient. It is particularly preferred if the antibody to C4a des Arg and the antibody to the further heart failure marker (such as BNP) are used in an immunoassay for assessing, eg diagnosing or prognosing, heart failure. The immunoassay may be for each marker individually, or it may be for two or more markers combined for example a single immunoassay which is able to detect C4a des Arg and another heart failure marker such as BNP. Preferably, the patient is a human.

The invention also includes the use of a reagent which selectively identifies C4a des Arg in the manufacture of a composition for assessing heart failure in a patient. The composition is a reagent which is used to assess heart failure in a patient. The composition manufactured may be used in the diagnosis of heart failure in a patient. The composition manufactured may be used in the prognosis of heart failure in a patient. Typically, the reagent which selectively identifies C4a des Arg is a reagent as described above. Preferably, the patient is a human.

A still further aspect of the invention provides a kit of parts comprising a reagent that selectively identifies C4a des Arg and a reagent which identifies a further marker of heart failure. Conveniently, the reagents bind the C4a des Arg and the further heart failure marker; more conveniently, the reagent is an antibody as described above. It is particularly preferred if the further marker of heart failure is BNP.

In a particularly preferred embodiment, the kit is an immunoassay kit. In addition to the (primary) antibody to C4a des Arg and the (primary) antibody to the one or more further heart failure markers (such as BNP), the kit may also contain secondary antibodies to the primary antibodies. Conveniently, the primary antibodies are of different isotypes so that they can be distinguished by different secondary antibodies. Conveniently, the secondary antibodies are labelled differently so that they can be distinguished. In this way, it is possible for a single immunoassay to determine the level of C4a des Arg and one or more further heart failure markers (such as BNP) in the same sample from the patient.

Another aspect of the invention provides use of a means for selectively identifying ^• C4a des Arg in a sample from a patient in the assessment of heart failure in the human patient. The means for selectively identifying C4a des Arg in the sample may be any suitable means. For example, in one embodiment the means is a mass spectrometer arranged to detect C4a des Arg in the sample. As noted above, C4a des Arg has a molecular ion of m/z = 8600 when analysed by SELDI-TOF mass - spectrometry.

The means may also be capable of selectively identifying a further heart failure marker in the sample, such as BNP.

The measurement of C4a des Arg in a patient (particularly a human patient) is also useful in the development of drugs for treating heart failure. Thus, a further aspect of the invention provides a method of assessing whether a compound has an effect on heart failure in a patient, the method comprising administering to the patient the said compound and determining the effect of the compound on the level of C4a des Arg in the patient. In this embodiment, the patient may be a laboratory animal such as a rat, mouse, guinea pig, dog or primate.

Methods for determining the level of C4a des Arg are described above. Typically, the level of C4a des Arg in the patient's blood is determined. This may be done by determining the level in a plasma sample from the patient.

In a further embodiment the level of a further heart failure marker, such as BNP is determined.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

All documents cited in the patent specification are hereby incorporated herein by reference. The invention will now be described in more detail by reference to the following non-limiting Examples and Figures wherein:

Figure 1 - Individual scatterplots showing the levels of C4a des Arg and N- BNPa from Control, HF, Respiratory and IPAH patients. The levels of plasma

C4a des Arg (a) and BNP (b) were measured in controls (n=21), HF (setl) (n=20),

Respiratory disease (n=15) and IPAH patients (n=27) by ELISA. An independent measurement of plasma C4a des Arg samples was made in a second set of HF samples (n=19). Each point is the mean of duplicate determinations and the lines indicate mean values. Statistical differences between controls and patients were determined using Student's t-test.

Figure 2 - ID scatterplot demonstrating the classification power of C4a des Arg and N-BNP in controls and HF. The levels of plasma C4a des Arg and N- BNP from controls (n=21)and HF patients (n=20) were determined as described in the method section. Plasma C4a des Arg levels at a cut off point of 1.0 μg/mL gave 100% specificity and 100% sensitivity while plasma N-BNP levels at a cut off levels of 270 pg/mL gave 75% specificity; 95% sensitivity). Each point is the mean of duplicates.

Figure 3 - Individual scatterplots showing the levels of C4a des Arg in EDTA and Futhan tubes at different time intervals, (a). The levels of plasma C4a des Arg from blood collected in EDTA tubes in controls (n=5) at different time intervals, (b) The levels of plasma C4a des Arg from blood collected in EDTA tubes and 50 meg Futhan containing EDTA tubes in HF samples (n=4) at different time intervals. Each point is the mean of duplicate determinations and the lines indicate mean values.

Figure 4 - Individual scatterplots showing the levels of C4a des Arg collected in Futhan containing EDTA tubes from Control, HF, Respiratory, EPAH and

CAD patients. The levels of plasma C4a des Arg were measured in controls

(TE=U), HF (11=13), Respiratory disease (n=8), IPAH (n=16) and CAD (n=27) patients. Each point is the mean of duplicate determinations and the lines indicate mean values. Statistical differences between controls and patients were determined using Student's t-test.

Figure 5 - Box & whiskers plot showing the levels of C4a des Arg collected in Futhan containing EDTA tubes from HF patients with varying NYHLA. classes. The levels of plasma C4a des Arg were measured in HF (n=13) as ^■ described in the method section. The levels were correlated with their respective NYHA classes.

Example: Plasma C4a des Arg levels are associated with heart failure.

MATERIALS & METHOD

Materials

A kit to measure C4a des Arg was purchased from Metachem Diagnostics (Piddington, UK). Futhan (nafamostat mesilate ) (5mg) was purchased from Merck Biosciences Ltd (Beeston, UK) and Trasylol (approtinin) (10,000 kaUikrein inhibitory units/ml) was purchased from Bayer UK Ltd (Newbury, UK). All other reagents were purchased from Sigma- Aldrich Company Ltd. (Poole, Dorset, UK).

Plasma samples

The samples for this study were obtained using protocols approved by the Hammersmith and Queen Charlotte's & Chelsea Hospitals Research Ethics

Committee (2001/6157). ^' Heart failure (HF) patients (n=52) were attending hospital with a diagnosis of heart failure for specialist management. The diagnosis of idiopathic pulmonary arterial hypertension (IPAH) (n=46) was made after confirmation of raised pulmonary artery pressure by exclusion of known causes of the condition according to current practice (ESC Guidelines for PAH,

British Cardiac Society Recommendations). Coronary artery disease (CAD) patients (n=26) were selected from a group of patients who underwent angiograms and who show a positive narrowing of at least 1 coronary artery, without any left ventricular dysfunction. Respiratory group patients (n=23) were selected from patients attending a specialist hospital unit. All volunteers were asked to fill in a questionnaire form and their blood pressure was measured. Healthy volunteers who had no indication of any cardiopulmonary risk factors and who were not on any active therapy were recruited in this study.

For the measurement of C4a des Arg, venous blood was collected in EDTA tubes and EDTA tubes containing Futhan (50μg) while for the measurement of N- BNP₅ venous blood was collected in EDTA tubes containing Trasylol (125 kallikrein inhibitory units). All tubes were centrifuged (950xg for 10 min) within half an hour of collection. The separated plasma samples were immediately frozen at -7O⁰C. Once in the laboratory, samples were thawed and aliquoted into 200 μL volumes, and refrozen at -7O⁰C until analysis.

C4a des Arg ELISA

The levels of C4a des Arg were measured by competitive ELISA assay according to manufacturer's protocol (human C4a des Arg Enzyme Immunoassay kit, Catalog No. 900-059, Assay Designs, Inc, 800 Technology Drive, Ann Arobor, MI 48108, USA.

N-BNP levels

The N-BNP levels were measured by Prof Leong Ng from Leicester University using radioimmunoassay.

RESULT

Comparison of plasma levels of C4a des Arg and N-BNP

Initially, the levels of C4a des Arg were determined by ELISA in samples collected in EDTA tubes. The control donors had a mean plasma level of 0.40 ± 0.05 μg/rαL (n=21) and this rose to 2.03 ± 0.06 μg/mL in HF patients (n=20) (pO.0001; Student's t-test) (Fig. Ia). The levels were also elevated in samples from HF patients compared to those from IPAH and Respiratory disease patient groups (pO.0001; Student's t-test) (Fig. Ia). This degree of elevation (pO.0001; Student's t-test) of plasma C4a des Arg levels in HF patients compared to controls, IPAH and Respiratory groups was maintained in an independent second set of HF samples (HF set 2; n=19; mean plasma levels =1.62± 0.05 μg/mL) (Fig. Ia).

The measurement of plasma BNP showed elevation in samples from HF patients (2.45 ± 0.74 ng/mL) compared to controls (0.15 ± 0.11 ng/mL) (pO.0001; Student's t-test) (Fig. Ib). When plasma BNP levels in HF patients were compared to other disease groups, a significant difference was only seen with the Respiratory group (p<0.05; Student's t-test).

Plasma C4a des Arg levels at a cut off levels of 1.0 μg/mL produced higher values for specificity (100%) and sensitivity values (100%) compared to plasma N-BNP levels at a cut off level of 270 pg/mL (75% specificity; 95% sensitivity) (Fig. 2). This indicates that plasma C4a des Arg levels are better than plasma N-BNP levels at differentiating between the HF and control groups.

Plasma levels of C4a des Arg in EDTA and Futhan tubes

The effect of processing time prior to plasma storage on C4a des Arg levels was studied in 5 samples. This showed an increase of plasma C4a des Arg levels with an increase in processing time (Fig. 3a) which was due to in-vitro activation of the complement system within the collection tube. .The addition of an inhibitor of complement activation pathway, Futhan, provided protection from in vitro activation and therefore ensured more accurate measurements that better reflect the circulating levels of C4a des Arg (Fig. 3b).

Subsequently, levels of plasma C4a des Arg collected in EDTA tubes containing Futhan were measured in different disease groups. An increase in the levels was observed in HF samples (n=13; 1.34+0.23 μg/mL) compared to controls (n=14; 0.32+0.04 μg/mL; pO.001, Student's t-test) and CAD samples (n=27, 0.72±0.10 μg/mL; p<0.01, Student's t-test) (Fig. 4).

5 Plasma levels of C4a des Arg in varying NYHA classes

Plasma C4a des ARg levels of 13 HF patients shows segregation based on NYHA classes (Fig. 5).

10 A functional and therapeutic classification for prescription of physical activity fqr cardiac patients is provided by the New York Heart Association (NYHA).

• Class I: patients with no limitation of activities; they suffer no symptoms from ordinary activities.

15

• Class II: patients with slight, mild limitation of activity; they are comfortable with rest or with mild exertion.

• Class III: patients with marked limitation of activity; they are 20 comfortable only at rest.

• Class IV: patients who should be at complete rest, confined to bed or chair; any physical activity brings on discomfort and symptoms occur at rest.

•25

Claims

1. A method of diagnosing heart failure in a patient, the method comprising providing a sample from the human patient, determining the level of C4a des Arg in the sample and assessing whether the level is indicative of heart failure in the patient.

2. A method of prognosing heart failure in a patient who has heart failure, the method comprising providing a sample from the human patient, determining the level of C4a des Arg in the sample and assessing whether the level is indicative of a particular outcome for the patient.

3. A method according to Claim 1 wherein a level of C4a des Arg in the sample of greater than 2 standard deviations (SD) above the mean level of C4a des Arg in a healthy population is indicative of heart failure.

4. A method according to Claim 2 wherein a level of C4a des Arg in the sample which of greater than 2 standard deviations (SD) above the mean level of C4a des Arg in a population of heart failure patients who fare well is indicative of a poor outcome.

5. A method according to any of the preceding claims wherein the sample is a blood, serum or plasma sample.

6. A method according to any of the preceding claims wherein the level of C4a des Arg in the sample is measured using an immuno assay.

7. A method according to any of Claims 1 to 5 wherein the level of C4a des Arg in the sample is measured using mass spectrometry.

8. A method according to Claim 1 further comprising determining the level of one or more further heart failure markers in a sample from the patient and assessing whether the level of said further marker or markers is indicative of heart failure in the patient.

9. A method according to Claim 2 further comprising determining the levels of one or more further heart failure markers in a sample from the patient and assessing whether the levels of said further marker or markers is indicative of a particular outcome for the patient.

10. A method according to Claims 8 or 9 wherein the level of C4a des Arg and the level of the one or more further heart failure markers are all taken into account when assessing whether the levels are indicative of heart failure in the patient or are indicative of a particular outcome for the patient.

11. A method according to any of Claims 8 to 10 wherein the further marker is brain natriuretic peptide (BNP) or NT-pro BNP or norepinephrine or troponin or heart-type fatty acid binding protein or myosin light chain- 1 or matrix metalloproteinase or tissue inhibitor of matrix metalloproteinase or C-reactive protein or TNFα or soluble T2 receptor or soluble 1L-2 receptor or CD40-CD154 or CCAM-I or P-selectin or tissue factor or von Willebrand factor or urocortin.

12. A method according to any one of the preceding claims wherein the patient is a human.

13. Use of a reagent which selectively identifies C4a des Arg in the assessment of heart failure in a patient.

14. Use of a reagent which selectively identifies C4a des Arg and use of a reagent that selectively identifies a further heart failure marker in the assessment of heart failure in a patient.

15. Use according to Claim 14 wherein the further heart failure marker is BNP NT-pro BNP or norepinephrine or troponin or heart-type fatty acid binding protein or myosin light chain- 1 or matrix metalloproteinase or tissue inhibitor of matrix metalloproteinase or C-reactive protein or TNFα or soluble T2 receptor or soluble 1L-2 receptor or CD40-CD154 or CCAM-I or P-selectin or tissue factor or von Willebrand factor or urocortin.

16. Use according to Claims 13 to 15 wherein the reagent is an antibody.

17. Use according to Claim 16 in an immunoassay.

18. Use of a reagent which selectively identifies C4a des Arg in the manufacture of a composition for assessing heart failure in a patient.

19. Use according to any one of Claims 13 to 18 wherein the patient is human.

20. A kit of parts comprising a reagent that selectively identifies C4a des Arg and a reagent which selectively identifies a further marker of heart failure.

21. A kit of parts wherein the further marker of heart failure is BNP .

22. A kit of parts according to Claim 20 or 21 wherein the reagent is an antibody.

23. A kit of parts according to Claims 20 to 21 wherein the kit is an immunoassay kit.

24. Use of a means for selectively identifying C4a des Arg in a sample from a patient in the assessment of heart failure in the patient.

25. Use according to Claim 24 wherein the means is a mass spectrometer arranged to detect C4a des Arg in a sample.

26. Use according to Claim 24 or 25 wherein the means are also for selectively identifying a further marker of heart failure.

27. A method of assessing whether a compound has an effect on heart failure in a patient, the method comprising administering to the patient the said compound and determining the effect of the compound on the level of C4a des Arg in the patient.

28. A method according to Claim 27 wherein the level of C4a des Arg in the patient's blood is determined.

29. A method according to Claim 28 wherein the level of C4a des Arg in a plasma sample from the patient is determined.

30. A method according to any one of Claims 27 to 29 wherein the level of a further heart failure marker is determined.

31. A method according to Claim 30 wherein the further heart failure marker is BNP.

32. Use according to any one of Claims 24 to 26 or a method according to any one of Claims 27 to 31 wherein the patient is human.

33. Any novel method of diagnosing or prognosing heart failure in a human patient as herein disclosed.

34. Any novel kit of parts as herein disclosed.