HK1124114A - Natriuretic peptides and placental-growth factor/soluble vegf-receptor discriminate cardiac dysfunction related to heart disease from a placenta-associated cardiac dysfunction in pregnant women - Google Patents

Description

Natriuretic peptides and placental growth factor/soluble VEGF receptor regions associated with heart disease or placenta-associated cardiac dysfunction in pregnant women

Technical Field

The present invention relates to the use of biomarkers for diagnosing heart dysfunction in pregnant women, in particular, to use thereof for distinguishing placenta-related heart dysfunction from heart disease-related heart dysfunction.

Background

Preeclampsia is one of the most common pregnancy disorders, affecting about 5% of pregnancies. It is the leading cause of maternal death and morbidity, perinatal death, premature labor and intrauterine growth restriction. Preeclampsia is a syndrome of hypertension, edema, and proteinuria, with symptoms appearing after the 20 th week of gestation and generally detectable by routine monitoring of the woman's blood pressure and urine. When the pregnant woman develops hypertension (140/90 or higher for two independent readings taken at least 6 hours apart) and 300mg protein is present in the 24 hour urine sample: (Proteinuria) Then, preeclampsia is diagnosed. Preeclampsia is also more common in the pre-existence ofHypertension (hypertension)、Diabetes mellitusOrRenal diseaseAmong women with a family history of preeclampsia, and among women with multiple pregnancies (twins, triplets or more).

The findings of relevance support a causal or pathogenic model of global placenta driven by immune maladaptation and subsequent decrease in angiogenic growth factor concentration and an increase in placental debris in the maternal circulation that elicits a maternal inflammatory response. Currently, women at risk for developing preeclampsia are identified based on epidemiological and clinical risk factors, but the diagnostic criteria for preeclampsia are still unclear.

Pregnancy involves the synchronous formation of new blood vessels, a process known as angiogenesis. Several growth factors and specific receptors such as Vascular Endothelial Growth Factor (VEGF), placental growth factor (PlGF), and soluble Flts-1(sFlts-1) play an important role in this process. Preeclampsia is associated with the abnormal expression of these molecules, which can be measured in the blood samples of pregnant women.

VEGF is an endothelial cell-specific mitogen, an inducer of angiogenesis, and a regulator of vascular permeability. VEGF has been shown to be important for glomerular capillary repair. VEGF binds to transmembrane tyrosine kinase receptors, a homodimer of fms-like tyrosine kinase (Flt-1) that is differentially expressed in endothelial cells obtained from a number of different tissues. High Flt-1 expression by trophoblast cells contributes to placental development. PlGF is expressed by cytotrophoblasts and syncytiotrophoblasts and induces endothelial cell proliferation, migration and activation. PlGF binds the Flt-1 receptor as a homodimer. Both pigf and VEGF contribute to mitogenic activity and angiogenesis, which are critical for the developing placenta. sFlt-1, a splice variant of the Flt-1 receptor, lacks the transmembrane and cytoplasmic domains of the receptor, binds VEGF with high affinity, but does not stimulate mitogenesis in endothelial cells. It is expressed in placental tissue and human umbilical vein endothelial cells. sFlt-1 is believed to down-regulate the VEGF signaling pathway.

Some studies have shown that women who develop preeclampsia are also at risk for cardiovascular complications during their lifetime. Many of the risk factors and pathophysiological abnormalities of preeclampsia resemble coronary artery disease. There are indications that insulin resistance is a common factor. Microvascular dysfunction associated with insulin resistance can contribute to coronary heart disease and preeclampsia.

In addition to placenta-induced cardiac dysfunction, primary cardiac dysfunction is also considered for the etiology of maternal cardiac dysfunction. The main causes of primary heart dysfunction are congenital and acquired heart diseases and cardiomyopathies (myocardic dialases).

A method for monitoring pre-eclampsia in pregnant women by determining the level of sFlt-1, VEGF or PLGF polypeptides in a sample is disclosed in US 2004/0126828A 1. The authors stated that the levels of sFlt-1 were elevated in blood samples taken from women with pre-eclampsia. sFlt-1 binds VEGF and PlGF with high affinity and blocks the mitogenic and angiogenic activity of these growth factors. The authors concluded that circulating sFlt-1 in preeclamptic patients may oppose vasodilation and thus contribute to hypertension.

Furthermore, US2005/0025762A1 discloses methods of treating pre-eclampsia and eclampsia by using compounds that reduce the levels of sFlt-1 and compounds that inhibit the binding of VEGF or PLGF to sFlt-1. However, cardiac dysfunction in pregnant women still seems to be undetectable by assaying these angiogenic growth factors alone.

DE102004051847 discloses a method for diagnosing atherosclerosis by determining the level of PlGF and sFlt-1, which comprises a method for determining the correlation between PlGF and sFlt-1. Decreased PlGF levels in myocardial infarction patients are associated with increased risk of further vascular events. However, the authors predict that the claimed method relates only to vascular diseases with atherosclerotic etiology, and that preeclampsia or eclampsia is excluded from the claimed method.

In addition, attempts have been made to determine whether Brain Natriuretic Peptide (BNP) can be used as a biochemical marker in pregnant women with preeclampsia. Resnik et al (American Journal of Obstetrics and Gynology (2005)193, 450-4) found elevated BNP levels in severe preeclampsia compared to normal pregnancy. The authors speculate that ventricular stress and/or subclinical cardiac dysfunction is associated with preeclampsia. However, restik does not describe whether the cardiac dysfunction of pregnant women is caused by preeclampsia, nor whether the symptoms are due to other pre-existing cardiac events.

However, since other causes such as placental insufficiency were not detected, the mere determination of natriuretic peptides could not prove the cause of cardiac dysfunction in pregnant women. Thus, as far as the state of the art is concerned, there is no diagnostic method available to distinguish whether a pregnant woman's heart dysfunction is caused by placenta-related heart dysfunction or by primary heart disease.

It is therefore an object of the present invention to provide improved methods and means (means) for diagnosing heart dysfunction in pregnant women, in particular for differentiating placenta-related heart dysfunction from primary heart disease.

Summary of The Invention

The present invention relates to a method for diagnosing whether a pregnant woman suffers from cardiac dysfunction, comprising the steps of: a) determining the level of natriuretic peptide in the sample, b) determining the level of placental growth factor and/or sFlt-1 or a variant thereof in the sample, wherein an increased level of natriuretic peptide and a decreased level of placental growth factor and/or an increased level of sFlt-1 or a variant thereof is indicative for the presence of placenta-associated heart dysfunction, or wherein an increased level of natriuretic peptide and a non-decreased level of placental growth factor and/or a non-increased level of sFlt-1 or a variant thereof is indicative for the presence of heart disease-associated heart dysfunction.

Furthermore, the invention comprises an array for in vitro diagnosis of a cardiac disease, in particular for distinguishing between cardiac disease-related and placenta-related cardiac dysfunction by determining natriuretic peptides and placental growth factor and/or sFlt-1 or variants thereof, said array comprising a ligand that specifically binds to a natriuretic peptide, in particular NT-pro BNP (NT-proBNP) or variants thereof, and a ligand for PlGF and/or sFlt-1 or variants thereof, said ligands (a) being for determining the level of a natriuretic peptide in a sample of a pregnant woman, and (b) being for determining the level of PlGF and/or sFlt-1 or variants thereof in a sample of a pregnant woman.

In addition, the present invention relates to a method for determining a viable treatment for supporting a pregnant woman suffering from heart disease, wherein said pregnant woman presents with symptoms of heart disease related heart disease, the method comprising the steps of: a) determining the level of natriuretic peptide in the sample, b) determining the level of placental growth factor and/or sFlt-1 or a variant thereof in the sample, c) wherein an increased level of natriuretic peptide and a decreased level of placental growth factor and/or an increased level of sFlt-1 or a variant thereof is indicative for the presence of placenta-associated heart dysfunction, or wherein an increased level of natriuretic peptide and a non-decreased level of placental growth factor and/or a non-increased level of sFlt-1 or a variant thereof is indicative for the presence of heart disease-associated heart dysfunction, d) optionally initiating a maternal check by a cardiologist, e) recommending the initiation of the treatment if the presence of heart disease-associated heart dysfunction is indicated according to the assessment of step c).

Brief Description of Drawings

FIG. 1 shows a block diagram (box plot) of the reference values of the NT-pre BNP concentrations. N represents the number of patients. The first column shows NT-pre BNP concentrations in 508 apparently healthy 18-44.9 year old female blood donors. The reference value was compared with the NT-probnp concentrations of 55 pregnant women, which were divided into 9 pregnant women in the middle of gestation (2.trimester) and 46 pregnant women in the late of gestation (3. trimester). There were no significant differences in NT-pre BNP levels between these groups. Furthermore, the median sum 75, 95 th and 5 th and 25 th percentiles (percentiles) are indicated.

FIG. 2 shows a block diagram of reference values for sFlt-1 concentration and PlGF concentration measured in 46 pregnant women. These 46 pregnant women had a NT-proBNP concentration of less than 125pg/ml, a fraction of 14 pregnant women in the middle of gestation and 32 pregnant women in the late gestation. PlGF and sFlt-1 concentrations decreased only slightly from mid to late gestation. In addition, the block diagram shows the sFlt-1/PlGF ratio. The concentration ratio of sFlt-1/PlGF increases from mid to late gestation.

Furthermore, the median and 75 th, 95 th and 5 th and 25 th percentiles are indicated.

Detailed Description

In a first embodiment, the object of the invention is achieved by a method for diagnosing a pregnant woman suffering from cardiac dysfunction, comprising the steps of:

a) determination of natriuretic peptide levels in a sample

b) Determining the level of placental growth factor and/or sFlt-1 or a variant thereof in a sample

Wherein an increased level of natriuretic peptide and a decreased level of placental growth factor and/or an increased level of sFlt-1 or a variant thereof is indicative for the presence of placenta-associated cardiac dysfunction,

or wherein an increased level of natriuretic peptide and a non-decreased level of placental growth factor and/or a non-increased level of sFlt-1 or a variant thereof is indicative for the presence of a heart disorder associated with heart disease.

The method of the invention also distinguishes between placenta-related and heart disease-related heart dysfunction in pregnant women suffering from heart dysfunction.

Similarly, the invention also relates to the use of the combined information of the determined natriuretic peptide and placenta growth factor and/or sFlt-1 levels for the diagnosis of heart dysfunction, in particular heart dysfunction associated with heart disease and/or placenta-associated heart dysfunction in pregnant women presenting with symptoms of heart dysfunction. Such applications are similarly applicable to all other features and preferred embodiments disclosed in the specification and examples of the present application.

The method may further comprise the step of taking a sample from the pregnant woman, such as a body fluid or tissue sample. In the present invention, it may be preferred to take a sample of body fluid or tissue by non-medical personnel (i.e. without receiving education required by a doctor's profession). This applies in particular to the case where the sample is blood.

According to the present invention, a tissue sample refers to any kind of tissue obtained from a dead or living human or animal body. The tissue sample may be obtained using any method known to those skilled in the art, for example using biopsy or curettage.

According to the present invention, body fluids may include blood, serum, plasma, lymph, cerebral plasma (cerebralliquor), saliva, vitreous humor and urine. In particular, body fluids include blood, serum, plasma and urine. The body fluid sample may be obtained using any method known in the art.

The invention also provides improved safety in diagnosing pregnant women suffering from cardiac dysfunction. As noted above, it has been found in the present invention that increased levels of natriuretic peptide and decreased levels of placental growth factor and/or increased levels of sFlt-1 or a variant thereof are indicative for the presence of placenta-associated cardiac dysfunction, wherein increased levels of natriuretic peptide and non-decreased levels of placental growth factor and/or non-increased levels of sFlt-1 or a variant thereof are indicative for the presence of cardiac dysfunction associated with heart disease.

In the present invention it has been found that the determination of natriuretic peptides, in particular NT-pre BNP, alone, does not allow to distinguish whether a heart dysfunction is associated with a cardiac disease or with a placenta-related heart dysfunction. The combined determination of natriuretic peptides and placental growth factor and/or sFlt-1 or variants thereof may help to avoid misdiagnosis, particularly at the time of rescue.

The present invention makes use of certain "biomarkers" (or simply "markers"), particularly biochemical or molecular markers. The terms "biomarker", "biochemical marker" and "molecular marker" are known to those skilled in the art. In particular, biochemical or molecular markers are gene expression products that are differentially expressed (i.e., up-regulated or down-regulated) in the presence or absence of certain conditions, diseases, or complications. Generally, a molecular marker is defined as a nucleic acid (e.g., mRNA), while a biochemical marker is a protein or peptide. The level of a suitable biomarker can be indicative of the presence or absence of a condition or disease and can therefore be used for diagnosis.

The invention makes use in particular of placental growth factor (PlGF), sFlt-1 and variants thereof and natriuretic peptides, in particular NT-pre-ANP and NT-pre-BNP, as biomarkers, in particular as biochemical markers.

NT-Pre-ANP and NT-Pre-BNP belong to the natriuretic peptide group (see, e.g., Bonow, R.O. (1996). New impedances into the cardiac natural peptides, circulation 93: 1946-1950). As already mentioned, NT-pre-ANP and NT-pre-BNP are generated by proteolytic cleavage of a precursor molecule, prepropeptide, which is proteolytically cleaved to generate the active hormone (ANP or BNP) and the corresponding N-terminal fragments (NT-pre-ANP and NT-pre BNP, respectively).

The prepropeptide (134 amino acids in the case of pre-BNP pro) comprises a short signal peptide which is cleaved enzymatically to release the prepropeptide (108 amino acids in the case of pre-BNP). This propeptide is further cleaved into an N-terminal propeptide (NT-propeptide, 76 amino acids in the case of NT-pre BNP) and an active hormone (32 amino acids in the case of BNP, 28 amino acids in the case of ANP).

The different cleavage products show different respective properties. BNP is produced primarily in the ventricles (but not exclusively) and is released as the wall tension increases. In contrast, ANP is produced and released only in the atria. ANP and BNP are active hormones and have shorter half-lives than their respective inactive counterparts, NT-pre-ANP and NT-pre-BNP. BNP is metabolized in the blood, whereas NT-pre-BNP circulates in the blood as a complete molecule and is excreted intact through the kidneys. NT-proBNP has an in vivo half-life of 120 minutes, longer than BNP (which is 20 minutes) (Smith MW, Espiner EA, Yandle TG, Charles CJ, Richards AM. delayed metabolism of human brain branched biological peptide reflection residue to neutral end oppression. J Endocrinol. 2000; 167: 239-46.).

According to the invention. The term "natriuretic peptide" encompasses ANP and/or BNP or fragments thereof, and/or NT-pre-BNP and/or NT-pre-ANP or variants thereof. Thus, the term "natriuretic peptide" encompasses the group consisting of ANP, BNP or fragments thereof, NT-pre-BNP, NT-pre-ANP or variants thereof. Furthermore, "natriuretic peptide" includes NT-pre-BNP or variants thereof.

Thus, a preferred embodiment of the present invention is the determination of a natriuretic peptide, preferably ANP and/or BNP or fragments thereof, more preferably NT-pre-BNP and/or NT-pre-ANP or variants thereof, most preferably NT-pre-BNP or variants thereof.

Placental growth factor (PlGF, also known as PGF) is well known to those skilled in the art. It is a protein associated with vascular permeability factor (VPF or VEGF). This protein is 149 amino acids long and shares 53% identity with the platelet-derived growth factor-like region of VPF. PlGF appears to be involved in angiogenesis during development, certain stages in adults, and during tumorigenesis.

In the early stages of pregnancy, natural killer cells accumulate around the cytotrophoblasts as a dense infiltrate. Since the human placenta is completely formed at about 20 weeks of gestation, these killer cells gradually disappear from the middle of gestation, which is consistent with the invasion of the cytotrophoblasts. Uterine natural killer cells produce several cytokines involved in angiogenesis and vascular stability, including PlGF, VEGF (vascular endothelial growth factor) and angiopoietin2 (angiopoetin 2). In healthy pregnancy, proper interaction between the vascular trophoblast and decidua leukocytes, especially natural killer cells, results in the release of large amounts of PlGF and VEGF. During pre-eclampsia, placenta-derived sFlt-1 (a soluble fms-like tyrosine kinase, also known as a soluble VEGF receptor), a PlGF and VEGF antagonist, is upregulated, resulting in increased systemic amounts of sFlt-1 that are reduced post-partum. Increased circulating sFlt-1 in pre-eclampsia is associated with decreased circulating concentrations of free PlGF and VEGF, leading to endothelial dysfunction. The magnitude of sFlt-1 increase correlates with disease severity.

Median NT-probnp levels did not increase during normal pregnancy and remained stable throughout pregnancy. The normal NT-pre BNP value in pregnant women corresponds to a plasma level of NT-pre BNP of less than 125pg/ml, in particular of less than 76pg/ml, more in particular of less than 50 pg/ml.

Elevated levels of NT-proBNP in pregnant women with preeclampsia correlate with the severity of the disease.

According to the invention, an increased level of NT-pre BNP corresponds to a plasma level of NT-pre BNP from 125pg/ml to 300pg/ml, a high increased level of NT-pre BNP indicating a heart dysfunction associated with a primary heart disease or placenta-related heart dysfunction corresponds to a plasma level of NT-pre BNP from 300pg/ml to more than 500 pg/ml.

According to the invention, the term "non-reduced PlGf levels and/or non-increased sFlt-1 levels and/or sFLt-1/PlGF ratio" refers to the level of a control sample of a healthy control group. The control group comprises samples of healthy pregnant women who do not suffer from preeclampsia or primary heart disease.

According to the invention, "reduced PlGf levels and/or increased sFlt-1 levels or altered sFlt-1/PlGf ratio levels" means that the values, if they differ collectively from healthy controls, preferably deviate from the 90 th percentile, more preferably deviate from the 95 th percentile, and most preferably deviate from the 99 th percentile.

As seen in the present invention, the sole determination of natriuretic peptides, in particular NT-pre BNP, does not distinguish between heart disease-related cardiac dysfunction and placenta-related cardiac dysfunction. In accordance with the present invention, a combined assay of natriuretic peptides, placental growth factor, and/or sFlt-1, or variants thereof, can distinguish between heart disease-related cardiac dysfunction and placenta-related cardiac dysfunction.

During normal pregnancy, PlGF levels and sFlt-1 levels in the middle and late gestation were not reduced or only slightly reduced. These data are shown in figure 2 of the present invention.

Elevated natriuretic peptide levels and decreased levels of PlGF and/or increased levels of sFlt-1 determined in the middle and late gestation are indicative of the presence of placenta-associated cardiac dysfunction with pre-eclampsia. These data are confirmed by the present invention table 1 showing that 8 of 9 pregnant women have PlGF levels less than 100 pg/ml. Furthermore, these 8 pregnant women had increased levels of NT-pre-BNP of 125 to 1000pg/ml, corresponding to the plasma levels of NT-pre-BNP.

Elevated natriuretic peptide levels and unreduced levels of PlGF and/or non-increased levels of sFlt-1 or variants thereof measured in the middle and late gestation are indicative of the presence of heart disease-associated cardiac dysfunction with primary heart disease. These data are confirmed by the present invention in that patient 92316544 of Table 1 showed increased levels of NT-pre-BNP but normal levels of PlGF and sFlt-1.

The term "variant" relates to peptides substantially similar to natriuretic peptides, in particular to NT-pre-ANP, NT-pre-BNP, PlGF, and sFlt-1. The term "substantially similar" is well known to those skilled in the art. In particular, the variant may be an isoform or allelic variant that shows amino acid exchanges compared to the amino acid sequence of the most prevalent peptide isoform in the human population. More preferably, such substantially similar peptides have at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence similarity to the most prevalent isoform of the peptide. Substantially similar are also degradation products, such as proteolytic degradation products, which can still be recognized by diagnostic methods or by ligands directed against the corresponding full-length peptide.

The term "variant" also relates to post-translationally modified peptides, such as glycosylated peptides. A "variant" is also a peptide that has been modified after collection of a sample, for example by covalently or non-covalently attaching a label, particularly a radioactive or fluorescent label, to the peptide. Determining the level of modified peptide after collection of the sample is understood to be determining the level of peptide which was not initially modified.

Examples of specific NT-pre ANP and NT-pre BNP variants and methods for their determination are known (Ala-Kopsala, M., Magga, J., Peuhkurinen, K. et al (2004): Molecular biology has a major impact on the measurement of differentiation N-terminal fragments of A-type and B-type native peptide. clinical Chemistry, vol.50(9), 1576-1588).

The term "diagnosis" is known to the person skilled in the art. Diagnosis is understood as the beginning of awareness of any medical condition, in particular heart disease. Diagnosis also relates to "differential diagnosis", i.e. to distinguish between different disorders with the same or similar symptoms. Specifically, differential diagnosis includes distinguishing heart disease-related heart dysfunction from placenta-related heart dysfunction.

More preferably, the diagnostic information obtained by the tool and method according to the invention is interpreted by a trained physician. More preferably, any decision regarding further treatment of the individual subject is also made by a trained physician. The physician will also decide, if deemed appropriate, which further diagnostic method is relevant.

According to the invention, the term "pregnant woman" preferably relates to a pregnant individual. The individual may have an unknown history of cardiovascular disease. More preferably, according to the present invention, the term "pregnant woman" relates to a pregnant individual showing symptoms of a heart dysfunction which may be caused by a heart disease-related heart dysfunction or which may involve a placenta-related heart dysfunction.

The present invention relates generally to the diagnosis of cardiac dysfunction in pregnant women. The term "cardiac dysfunction" is known to the person skilled in the art. It relates to any type of cardiac dysfunction, more particularly cardiac dysfunction affecting the pumping capacity, more particularly it relates to acute and chronic cardiac events.

Patients with heart disease may be individuals with Stable Angina Pectoris (SAP) and individuals with Acute Coronary Syndrome (ACS). ACS patients may show Unstable Angina Pectoris (UAP) or these individuals may already have Myocardial Infarction (MI). MI may be ST-elevated MI or non-ST-elevated MI. MI occurs followed by Left Ventricular Dysfunction (LVD). Eventually, LVD patients experience Congestive Heart Failure (CHF), with a mortality rate of approximately 15%. According to the invention, heart diseases also include coronary heart disease, heart valve defects (such as mitral valve defects), dilated cardiomyopathy, hypertrophic cardiomyopathy, and cardiac rhythm defects (arrhythmias).

Pregnant women suffering from heart disease-related cardiac dysfunction show increased levels of natriuretic peptides and non-reduced levels of placental growth factor and/or non-increased levels of sFlt-1 or variants thereof.

The term "heart disease-related cardiac dysfunction" may relate to the ability of the heart to supply sufficient amounts of oxygenated blood to peripheral tissues without adaptation. Cardiac dysfunction may be symptomatic or asymptomatic, and may be associated with diastolic or systolic dysfunction or both.

Pregnant women suffering from placenta-associated heart dysfunction show increased levels of natriuretic peptides and decreased levels of placenta growth factor and/or increased levels of sFlt-1 or variants thereof. The term "placenta-associated cardiac dysfunction" relates to a cardiac dysfunction which originally originates from placenta dysfunction and associated abnormalities and is not primary to the heart.

Symptomatic heart disease can cause "cardiac insufficiency". The term "cardiac insufficiency" is familiar to the person skilled in the art. More preferably, cardiac insufficiency relates to the inability of the heart to circulate blood adequately, in particular in the case of increased oxygenation requirements, for example during physical exercise. Cardiac insufficiency includes unstable adequate ejection of blood (inability to advance) and inadequate initiation of venous return of blood to the heart (backward failure).

Cardiac insufficiency may be classified according to the cardiovascular disease functional classification system established according to the New York Heart Association (NYHA). Patients of class I do not have obvious symptoms of cardiovascular disease. Physical activity is not limited and normal physical activity does not cause undue fatigue, palpitation, or dyspnea (breathlessness). Patients of class II have slightly limited physical activity. They are comfortable at rest, but normal physical activity causes fatigue, palpitation, or dyspnea. Class III patients exhibit markedly restricted physical activity. They are comfortable at rest, but very few general activities cause fatigue, palpitation, or dyspnea. Patients of class IV are unable to perform any physical activity without discomfort. They show symptoms of cardiac insufficiency at rest. If any physical activity is performed, discomfort increases.

Another indicator of cardiac insufficiency is the "left ventricular ejection fraction" (LVEF), which is also known as the "ejection fraction". People with a healthy heart usually have no reduction in LVEF, which is usually higher than 50%. Most people with symptomatic systolic cardiac dysfunction have 40% or less LVEF.

The term "cardiac decompensation" is familiar to the person skilled in the art. "cardiac decompensation" generally refers to the most severe level of cardiac insufficiency. During cardiac decompensation, the heart is unable to circulate blood sufficiently, to the extent that body stress cannot compensate for insufficient pumping capacity. Symptoms of cardiac decompensation are known to those skilled in the art. In particular, patients showing "cardiac decompensation" symptoms show symptoms according to NYHA class II, III, IV or worse. More particularly, the patient shows symptoms according to NYHA class III, IV or worse. Also particularly, the patient shows symptoms according to NYHA class IV or worse. Most particularly, patients require clinical support to stabilize or maintain circulation.

The terms "non-increased" and "decreased" levels refer to biomarker levels determined in a pregnant woman compared to known levels indicative of the absence of cardiac dysfunction, in particular compared to known levels indicative of the absence of cardiac dysfunction associated with heart disease or associated with placenta-related cardiac dysfunction.

One skilled in the art can determine a known level (or such as a ratio). For example, a known level can be determined as the median or average of the levels determined in a population of individuals who do not have a heart disease. Qualifying levels in more individuals or patients, such as in a cohort study, can help improve known levels or ratios. Similarly, it is also possible to define and/or improve a reference level indicating the presence of a heart disorder associated with a heart disease or a heart dysfunction associated with the placenta.

The known level may also be a "reference value". The person skilled in the art is familiar with the concept of biomarker reference values (or "normal values"). In particular, the term reference value may relate to the actual value of the level in one or more control samples, or it may relate to a value from the actual level in one or more control samples. More preferably, samples of at least 2, more preferably at least 5, more preferably at least 50, more preferably at least 100, most preferably at least 500 subjects are analyzed to determine a reference value.

In most simple cases, the reference value is the same as the level determined in the control sample, or the same as the average level determined in a plurality of control samples. However, it is also possible to calculate the reference value from more than one control sample. For example, the reference value can be the arithmetic mean of the levels in a control sample representing a control state (e.g., healthy, a particular disorder or a particular disease state). More preferably, the reference value relates to a range of values, such as mean ± one or more standard deviations, that can be found in most comparable control samples (representing the same or similar disease state). Similarly, the reference value may also be calculated by other statistical parameters or methods, such as a specified percentile of the levels found in most control samples, such as a 90%, 95%, 97.5% or 99% percentile. The choice of a particular reference value can be determined by the desired sensitivity, specificity or statistical significance (in general, the higher the sensitivity, the lower the specificity and vice versa). The calculation can be performed according to statistical methods known and deemed appropriate by the person skilled in the art.

The terms "control" and "control sample" are known to those skilled in the art. More preferably, a "control" relates to a test or test that is performed to provide a standard against which the test results (as measured in a patient) can be colonized. In the present invention, the criteria preferably relate to the level of the biomarker of interest associated with a particular health or disease state. Thus, a "control" is preferably a sample used to provide the above-mentioned criteria. For example, the control sample may be from one or more healthy subjects, or from one or more patients representative of a particular disease state.

In the present invention, patients representing a specific disease state specifically include pregnant women suffering from heart disease-related cardiac dysfunction or placenta-related cardiac dysfunction. All the patients in this group were tested in the middle and/or late gestation period. The determination comprising a control sample of a healthy pregnant woman not suffering from preeclampsia is also performed in the middle and/or late gestation. Thus, one embodiment of the invention is that all assays are performed in the middle and/or late gestation.

Examples of known levels or ratios are also given below. It is possible to further improve such levels or ratios. The specific known levels or ratios given in the specification can be used as a diagnostic guide. As is known and well accepted in the art, it is preferred that the physician actually diagnoses the individual subject by individual analysis, e.g., depending on the individual subject's weight, age, general health, and past medical history.

As already mentioned, the underlying cause of cardiac dysfunction in pregnant women may involve the presence of placenta-related cardiac dysfunction, such as pregnant women suffering from preeclampsia. In addition, pregnant women may suffer from heart-related cardiac dysfunction, such as pregnant women whose cardiac function has been impaired prior to the onset of pregnancy.

Thus, the method according to the invention can preferably treat two groups of patients showing symptoms of cardiac dysfunction during pregnancy:

pregnant women suffer from:

(1) the primary heart disease is the heart disease,

(2) placental-associated cardiac dysfunction caused by preeclampsia.

In the present invention, it was found that pregnant women (patient group 1 above) presenting with symptoms of primary heart disease show increased levels of natriuretic peptides, in particular increased levels of NT-pre-BNP, but not decreased levels of PlGF and not increased levels of sFlt-1.

Furthermore, in the present invention, it was found that the patients (patient group 2 described above) in which there was a symptom of cardiac dysfunction associated with placenta-associated cardiac dysfunction exhibited increased NT-pre BNP levels, but decreased PlGF levels and increased sFlt-1 levels.

According to the invention, the term "non-increased level of NT-pre BNP" preferably corresponds to a plasma level of NT-pre BNP of less than 125pg/ml, in particular less than 76pg/ml, more in particular less than 50 pg/ml.

Elevated levels of NT-proBNP in pregnant women with preeclampsia correlate with the severity of the disease.

According to the invention, an increased level of NT-pre BNP corresponds to a plasma level of NT-pre BNP between 125pg/ml and 300pg/ml, a high increased level of NT-pre BNP corresponds to a plasma level of NT-pre BNP between 300pg/ml and more than 500 pg/ml.

It is clear that the combined information from natriuretic peptides and PlGF and/or sFlt-1 can also be expressed differently.

In general, the higher the ratio of NT-pre BNP to PlGF in a sample of a pregnant woman determined to exhibit symptoms of a cardiac disorder, the more likely the patient will suffer from placenta-related cardiac dysfunction caused by preeclampsia.

Furthermore, the skilled person is able to determine the corresponding levels of the sample other than plasma.

As can be seen from the examples, determining the levels of PlGF, sFlt-1, and natriuretic peptides, particularly NT-pre ANP and NT-pre BNP, at least one additional time point provides additional diagnostic information. For example, determination of NT-pre BNP may help to avoid underestimating the extent of heart disease. Thus, in a preferred embodiment, the level of PLGF, sFlt-1 and natriuretic peptides, in particular NT-pre ANP and NT-pre BNP, is determined in at least one additional sample, preferably taken within a short time interval after the first determination. Suitable times may be, for example, within 2 to 12 hours, preferably within 4 to 12 hours, after the first sampling.

In another preferred embodiment, additional diagnostic parameters of a cardiac disease are determined, in particular selected from: (a) left Ventricular Ejection Fraction (LVEF), (b) echocardiogram (c) anamnesis (medical history), in particular with respect to angina pectoris, (d) electrocardiogram, (e) parameters of thyroid or renal function, (f) blood pressure, in particular arterial hypertension, (g) thallium scintigram, (h) angiography, (i) additional diagnostic parameters of catheterization.

These additional diagnostic parameters can be determined before, after, or parallel to the determination of PlGF, sFlt-1, and natriuretic peptides. Additional diagnostic parameters may establish a suspicion of the presence of a cardiac malfunction, or they may be used to further assess the diagnostic relevance of a particular level or ratio determined.

Assays for PlGF, sFlt-1, and natriuretic peptides can be performed in parallel or sequentially. More preferably, the assays are performed in a parallel fashion. The term "parallel" in this context relates to the use of samples taken simultaneously, preferably within less than 2 hour intervals, more preferably within less than 1 hour intervals. Most preferably, "parallel" in this context relates to the use of the same sample. More preferably, the determination of the amount or concentration of the peptide in the sample is also performed simultaneously.

In another preferred embodiment, at least one preeclampsia biomarker is additionally determined. Biomarkers for preeclampsia are known to those skilled in the art. Such markers indicate the presence of preeclampsia in the pregnant woman. In preeclampsia, many placental factors seen in the maternal circulation during healthy pregnancy are increased. These include several inflammatory cytokines, corticotropin releasing hormone, free radical species and activin a, including factors that stimulate the maternal inflammatory response.

Examples of preeclampsia biomarkers include factors such as alpha 2-macroglobulin, CD40 ligand, urotensin II (Urotensin II), and the like.

The level of biochemical or molecular markers can be determined by measuring the concentration of the protein (peptide or polypeptide) or corresponding transcript. In this context, the term "determining" preferably relates to quantitatively or semi-quantitatively determining the level.

The level may be determined by measuring the amount or concentration of the peptide or polypeptide. More preferably, the level in a given sample is determined as the concentration. For the present invention, it may not be necessary to determine absolute levels. It may be sufficient to determine the relative level compared to an appropriate control level. The determination may also be made by determining derivatives or specific fragments of the peptide or polypeptide of interest, such as specific fragments contained in a nucleic acid or protein digest.

The determination of nucleic acids, in particular mRNA, can be carried out according to any known method and with due consideration by the person skilled in the art.

Examples for RNA assays include northern blot hybridization, rnase protection assays, in situ hybridization, and aptamers, such as Sephadex (Sephadex) bound RNA ligands (Srisawat, c., Goldstein i.j., and Engelke, D.R. (2001). Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA hybrids nucleic acids Research, vol.29, No. 2e4).

In addition, RNA can be reverse transcribed into cDNA. Thus, methods for assaying DNA may also be used for assaying RNA, such as DNA hybridization, Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR) (see, e.g., Cao, W. (2004) percent of recovery strategies in ligand-mediated amplification and detection. trends in Biotechnology, vol.22(1), p.38-44), RT-PCR, real-time RT-PCR, quantitative RT-PCR, and microarray hybridization (see, e.g., free, B., Brehm, U., and Kubler, G., et al. (2002). Gene expression arrays: high throughput detection of expression patterns with improved protocols for target expression. Biochemical, vol.2, p.27-29).

DNA and RNA assays can also be performed in solution, e.g., using molecular beacons, Peptide Nucleic Acids (PNA), or Locked Nucleic Acids (LNA) (see, e.g., Demidov, V.V. (2003).

Protein or protein fragment assays may be performed according to any known method for the assay of peptides or polypeptides of interest. The person skilled in the art will be able to select an appropriate method.

Those skilled in the art are familiar with different methods for determining the level of a peptide or polypeptide. The term "level" relates to the amount or concentration of a peptide or polypeptide in a sample.

The determination may be performed directly or indirectly. Indirect assays include measuring cellular responses, bound ligands, labels, or enzymatic reaction products.

The assay may be performed according to any method known in the art, such as a cellular assay, an enzymatic assay, or a ligand binding-based assay. Representative methods are described below.

In one embodiment, the method for determining the level of a peptide or polypeptide of interest comprises the steps of: (a) contacting the peptide or polypeptide with a suitable substrate for a sufficient period of time, (b) determining the amount of product.

In another embodiment, the method for determining the level of a peptide or polypeptide of interest comprises the steps of: (a) contacting the peptide or polypeptide with a specific binding partner, (b) (optionally) removing unbound partner, (c) determining the amount of bound partner.

In another embodiment, the method for determining the level of a peptide or polypeptide of interest comprises the steps of: (a) (optionally) disrupting the peptides or polypeptides of the sample, (b) (optionally) separating the peptides or polypeptides or fragments thereof according to one or more biochemical or biophysical properties (e.g. according to binding to a solid surface or running time in a chromatography step), (c) determining the amount of one or more peptides, polypeptides or fragments, (d) determining the identity of one or more peptides, polypeptides or fragments of step (c) by mass spectrometry. A review of mass spectrometry is given, for example, by Richard D.Smith (2002) Trends in mass spectrometry analysis for proteins Biotechnology, Vol.20, No.12(Suppl.), pp.S. 3-S7.

Other representative methods for assaying include assaying the amount of ligand that specifically binds to the peptide or polypeptide of interest. According to the invention, binding includes both covalent and non-covalent binding.

According to the present invention, a ligand may be any peptide, polypeptide, nucleic acid or other substance that binds to a peptide or polypeptide of interest. It is well known that modifications such as glycosylation can be obtained if the peptide or polypeptide is obtained or purified from the human or animal body. According to the invention, suitable ligands may bind to the peptide or also to the polypeptide via the same site.

More preferably, the ligand should specifically bind to the peptide or polypeptide to be detected. According to the present invention, "specifically binds" means that the ligand should not substantially bind to another peptide, polypeptide or substance present in the sample under investigation ("cross-react"). More preferably, the binding of the specifically binding protein or isoform should have an affinity which is at least 3-fold higher, more preferably at least 10-fold higher and still preferably at least 50-fold higher than any other relevant peptide or polypeptide.

Non-specific binding can be tolerated, particularly if the peptide or polypeptide of interest can still be clearly distinguished and determined, e.g., by separation by size (e.g., by electrophoresis), or by its correspondingly high abundance in the sample.

Binding of the ligand can be determined by any method known in the art. More preferably, the method is semi-quantitative or quantitative. Suitable methods are described below.

First, the binding of the ligand can be determined directly, e.g., by NMR or surface plasmon resonance.

Secondly, if the ligand also serves as a substrate for the enzymatic activity of the peptide or polypeptide of interest, the product of the enzymatic reaction can be determined (e.g.the amount of protease can be determined by measuring the amount of cleaved substrate, e.g.by Western blot). For the determination of the enzyme reaction product, it is preferred that the amount of substrate is saturated. The substrate may also be labeled with a detectable label prior to reaction. More preferably, the sample is contacted with the substrate for a sufficient period of time. A sufficient period of time is the time necessary for a detectable, preferably determinable, amount of product to be produced. In addition to measuring the amount of product, the time necessary for a given (e.g., detectable) amount of product to occur can be determined.

Again, the ligand may be covalently or non-covalently coupled to a label for ligand detection and determination. Labeling may be performed by direct or indirect methods. Direct labeling includes coupling the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding (covalently or non-covalently) of a second ligand to a first ligand. The second ligand should specifically bind to the first ligand. The secondary ligand may be coupled to a target (receptor) for a suitable label and/or a tertiary ligand that binds to the secondary ligand. The use of secondary, tertiary or even higher order ligands is often used to increase the signal. Suitable secondary or higher ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vectorlaboratories, Inc.)

The ligand or substrate may also be "tagged" with one or more labels known in the art. Such labels may then target higher order ligands. Suitable labels include biotin, digoxigenin, a histidine tag, glutathione-S-transferase, FLAG, GFP, a myc tag, influenza A virus Hemagglutinin (HA), maltose binding protein, and the like. In the case of peptides or polypeptides, the label is preferably located at the N-terminus and/or C-terminus.

Suitable labels are any labels detectable by a suitable detection method. Representative labels include gold particles, latex particles, acridinium ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g., magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels.

Enzymatically active labels include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, and derivatives thereof. Suitable substrates for detection include Diaminobenzidine (DAB), 3 '-5, 5' -tetramethylbenzidine, NBT-BCIP (4-nitrotetrazolium blue chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as off-the-shelf stock solutions from Roche Diagnostics), CDP-Star^TM(Amersham Biosciences)、ECF^TM(Amersham Biosciences). Suitable enzyme substrate combinations can produce chromogenic reaction products, fluorescence or chemiluminescence that can be measured according to methods known in the art (e.g., using a photographic film or suitable camera system). For the enzyme reaction assay, the criteria given above are similarly applied.

Representative fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, texas red, fluorescein, and Alexa dyes (e.g., Alexa 568). More fluorescent labels are available, for example, from Molcular Probes (Oregon). The use of quantum flashes (quantum dots) as fluorescent labels is also contemplated.

Representative radiolabels include 35S, 125I, 32P, 33P and the like. The radioactive label can be detected by any known and suitable method, such as photographic film or a fluorescence imager.

Suitable assays according to the invention also include precipitation reactions, in particular immunoprecipitation reactions, electrochemiluminescence (electrochemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immunoassays, electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-amplified lanthanide fluorescence immunoassays (DELFIA), Scintillation Proximity Assays (SPA), turbidimetry, nephelometry, latex-amplified turbidimetry or nephelometry, solid phase immunoassays and mass spectrometry assays such as SELDI-TOF, MALDI-TOF or capillary electrophoresis-mass spectrometry (CE-MS). Further methods known in the art (e.g., gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting) may be used alone or in combination with the above-described labeling or other detection methods.

In addition, suitable methods include ELISA-based microplate methods, fully automated or robotic immunoassays (available as, for example, Elecsys)^TMOr Cobas^TMAnalyzer), CBA (enzyme Cobalt binding assay, available for example from Roche-Hitachi^TMAnalyzer) and latex agglutination assays (available as, for example, Roche-Hitachi)^TMAn analyzer).

Preferred ligands include antibodies, nucleic acids, peptides or polypeptides, and aptamers, such as nucleic acid or peptide aptamers. Methods for such ligands are well known in the art. Identification and production of suitable antibodies or aptamers is also provided, for example, by commercial suppliers. Those skilled in the art are familiar with methods for developing derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced into a nucleic acid, peptide, or polypeptide. These derivatives can then be tested for binding according to screening methods known in the art, such as phage display.

As used herein, the term "antibody" includes polyclonal and monoclonal antibodies and fragments thereof, such as Fv, Fab, and f (ab)2 fragments that are capable of binding antigen or hapten.

In another preferred embodiment, a ligand, preferably a ligand selected from the group consisting of nucleic acids, peptides, polypeptides, more preferably a ligand selected from the group consisting of nucleic acids, antibodies or aptamers, is present on the array.

The array contains at least one additional ligand, which may be directed against a peptide, polypeptide or nucleic acid of interest. The additional ligands may also be directed against peptides, polypeptides or nucleic acids not specifically contemplated in the present invention. More preferably, the array comprises ligands for at least 3, preferably at least 5, more preferably at least 8 peptides or polypeptides of interest of the invention.

Binding of the ligands on the array can be detected by any known readout or detection method, such as methods involving optical (e.g. fluorescent), electrochemical or magnetic signals or surface plasmon resonance.

In another preferred embodiment, the invention relates to the use of a ligand which specifically binds to a natriuretic peptide, in particular NT-pre-BNP or a variant thereof, and a ligand of PlGF and/or sFlt-1 or a variant thereof for the manufacture of a diagnostic kit (kit) for the diagnosis of a cardiac disease, in particular for distinguishing cardiac-related cardiac dysfunction from placental-related cardiac dysfunction in pregnant women suffering from cardiac dysfunction. In addition, ligands that specifically bind to a preeclampsia biomarker may be used to make such kits.

According to the present invention, the term "array" refers to a solid phase or gel-like support to which at least two compounds are attached or bound in a one-, two-or three-dimensional arrangement. Such arrays (including "gene chips", "protein chips", antibody arrays, etc.) are generally known to those skilled in the art and are typically formed on glass slides, particularly coated slides, such as polycation-, nitrocellulose-or biotin-coated slides, coverslips, and membranes, e.g., nitrocellulose-or nylon-based membranes. The array may comprise bound ligands or at least two cells each expressing at least one ligand.

According to the invention, the use of "Suspension arrays" is also contemplated as arrays (Nolan JP, Sklar LA. (2002); Suspension array technology: evolution of the flat-array partner. trends Biotechnology.20 (1): 9-12). In such suspension arrays, carriers such as microbeads or microspheres are present in suspension. The array consists of different microbeads or microspheres, possibly labeled, carrying different ligands.

In another preferred embodiment, the invention relates to an array for in vitro diagnosis of a cardiac disease, in particular for distinguishing between cardiac and placental-related cardiac dysfunction by determining natriuretic peptides and placental growth factor and/or sFlt-1 or variants thereof, said array comprising a ligand that specifically binds to a natriuretic peptide, in particular NT-probnp or variants thereof, and PlGF and/or sFlt-1 or variants thereof, said ligand (a) being for determining the level of a natriuretic peptide in a sample of a pregnant woman, and (b) being for determining the level of PlGF and/or sFlt-1 or variants thereof in a sample of a pregnant woman.

The invention further relates to a method for producing the above-described array, wherein at least one ligand, in addition to other ligands, is bound to a support.

Methods for producing such arrays, e.g. based on solid phase chemistry and photolabile protecting groups, are generally known (US5,744,305). Such arrays may also be contacted with libraries of materials and tested for interactions, e.g., for binding or conformational changes. Thus, arrays comprising the above peptides or polypeptides can be used to identify ligands that specifically bind to the peptides or polypeptides.

Peptides and polypeptides (proteins) can be determined in tissue, cell and body fluid samples, i.e. preferably in vitro. More preferably, the peptide or polypeptide of interest is determined in a sample of body fluid.

Some samples, such as urine samples, contain only degradation products, in particular fragments of the peptide or polypeptide of interest. However, as described above, determination of the level is still possible as long as the fragment is specific for the peptide or polypeptide of interest.

If necessary, the sample may be subjected to further processing prior to the assay. For example, nucleic acids, peptides or polypeptides may be purified from a sample according to methods known in the art, including filtration, centrifugation or extraction methods such as chloroform/phenol extraction.

Furthermore, it is contemplated to obtain a sample and measure a peptide or polypeptide of interest using a so-called point-of-care or lab-on-a-chip device. Such devices may be designed similarly to devices used in blood glucose determinations. Thus, the patient can obtain a sample and determine the peptide or polypeptide of interest without the immediate assistance of a trained physician or nurse.

In another preferred embodiment, the invention relates to a kit comprising (a) an agent (means) or device for determining the level of natriuretic peptides in a sample of a pregnant woman, and (b) an agent or device for determining the level of PlGF and/or sFlt-1 or variants thereof in a sample of a pregnant woman, for the in vitro diagnosis of a heart disease, in particular for distinguishing heart dysfunction associated with a heart disease from heart dysfunction associated with a placenta.

More preferably, the agent according to (a) is a ligand that specifically binds to a natriuretic peptide, and/or the agent according to (b) is a ligand that specifically binds to PlGF and/or sFlt-1 or variants thereof. In addition, the kit may comprise reagents or devices for determining the level of a biomarker for preeclampsia in a patient sample, in particular a specific binding partner.

In another preferred embodiment, the invention relates to the use of such a kit for the in vitro diagnosis of heart diseases, in particular for differentiating between heart disease-related heart dysfunction and placenta-related heart dysfunction in pregnant women presenting with symptoms of heart dysfunction.

In another preferred embodiment, kit package instructions for interpreting data on the determined levels of natriuretic peptide, PlGF and/or sFlt-1 or variants thereof for a pregnant woman suffering from heart disease are included for distinguishing heart disease related heart dysfunction from placenta related heart dysfunction.

A further preferred embodiment of the invention is an immunological rapid assay for the in vitro diagnosis of heart disease, in particular by assaying natriuretic peptides and placental growth factor and/or sFlt-1 or variants thereof for distinguishing heart disease-related heart dysfunction from placenta-related heart dysfunction, characterized in that an antibody specific for a natriuretic peptide, and/or sFlt-1 or variants thereof is used (a) for determining the level of a natriuretic peptide or a variant thereof in a sample of a pregnant woman, and (b) for determining the level of PlGF and/or sFlt-1 or variants thereof in a sample of a pregnant woman.

According to the invention, an "immunological immunoassay" is an immunoassay for immunologically detectable substances which have been known for a long time for a number of different parameters, for example from WO97/06439, EP 0291194, U.S. Pat. No. 5,591,645, U.S. Pat. No. 4,861,711, U.S. Pat. No. 5,141,850, U.S. Pat. No. 6,506,612, U.S. Pat. No. 5,458,852, U.S. Pat. No. 5,073,484. In these cases, the immunological detection reagents (substantially labeled and unlabeled antibodies or antigens) are usually provided in a dried form on a carrier, so that the sample liquid (in particular body fluids such as blood, serum, plasma, urine, saliva, etc.) on or in the carrier is allowed to flow. As support, preference is given to capillary-active, for example films or plastic supports equipped with capillary channels. They are commonly referred to as immunochromatographic test strips or test devices in the specialist world.

Since the ELECSYS ® NT-pre-BNP test can only be performed in the central laboratory, it is difficult to rapidly determine NT-pre-BNP outside of the daily working hours. It would be particularly advantageous for an emergency room if a rapid measurement could be obtained that could be performed directly in the emergency room outside of the daily working hours. However, the rapid test should ensure the same reference range and demarcation as the central laboratory reference (ELECSYS ® NT-pre BNP) in order to be able to achieve good result comparability, without being limited by the type of test actually carried out.

Quantitative determinations of human placental growth factor (PlGF) concentrations in cell culture supernatants, serum, plasma, and urine can be made by using the human PlGF immunoassay "Quantikine" (catalog No. DPG00) from R & D Systems. Quantitative determination of the concentration of human soluble vascular endothelial growth factor receptor 1(sVEGF R1) can be performed by using the human soluble VEGF R1/Flt-1 immunoassay "Quantikine" (product catalog number DVR100B) from R & D Systems. The rapid test method was applied to PlGF and VEGF R1/Flt-1, however, the same reference ranges and boundaries as the above-cited reference methods were to be ensured in order to be able to achieve good result comparability, regardless of the type of test actually performed.

The invention also relates to a method for determining a viable treatment for supporting a pregnant woman suffering from cardiac dysfunction. Once the patient is diagnosed, there may be a result for further treatment. If the method according to the invention indicates that the patient has a heart disease, the treatment can be initiated or adjusted. The level and/or ratio of natriuretic peptides, in particular NT-pre-BNP and NT-pre-ANP, PlGF and/or sFlt-1 or variants thereof, in the pregnant woman may be monitored at regular intervals. In addition, the subject may be studied in depth by further diagnosis according to methods known to the skilled cardiologist, for example as described earlier in the specification, such as electrocardiography or echocardiography. Treatment may include any means commonly associated with improving or restoring cardiac function.

The treatment of heart disease-related cardiac dysfunction in pregnant women may be different from the treatment of placenta-related cardiac dysfunction. If the method according to the invention indicates the presence of cardiac dysfunction associated with heart disease, the treatment may focus on the administration of ACE-inhibitors, diuretics, beta-blockers, digoxin and the like.

If the method according to the invention indicates the presence of placenta-related cardiac dysfunction in the pregnant woman, the treatment may instead focus on the delivery of aspirin, a steroid, at an early stage with or without cardiac treatment.

More specifically, in yet another embodiment, the present invention relates to a method for determining a viable treatment for supporting a pregnant woman with heart failure, wherein the pregnant woman presents with heart failure symptoms associated with heart disease, comprising the steps of: a) determining the level of natriuretic peptide in the sample, b) determining the level of placental growth factor and/or sFlt-1 or a variant thereof in the sample, c) wherein an increased level of natriuretic peptide and a decreased level of placental growth factor and/or an increased level of sFlt-1 or a variant thereof is indicative for the presence of placenta-associated heart dysfunction, or wherein an increased level of natriuretic peptide and a non-decreased level of placental growth factor and/or a non-increased level of sFlt-1 or a variant thereof is indicative for the presence of heart disease-associated heart dysfunction, d) optionally initiating a maternal check by a cardiologist, e) recommending the initiation of the treatment if the presence of heart disease-associated heart dysfunction is indicated according to the evaluation of step c).

More preferably, if the method indicates that the pregnant woman is suffering from cardiac-related cardiac dysfunction, it is recommended that the examination and/or treatment be initiated by a cardiologist. The method relates to all diseases and conditions mentioned earlier in this specification.

The invention further relates to the use of a natriuretic peptide and placental growth factor and/or sFlt-1 or a variant thereof for the preparation of a medicament for the diagnosis of a pregnant woman for heart dysfunction,

wherein said diagnosis comprises the steps of:

a) determination of natriuretic peptide levels in a sample

b) Determining the level of placental growth factor and/or sFlt-1 or a variant thereof in a sample

Wherein an increased level of natriuretic peptide and a decreased level of placental growth factor and/or an increased level of sFlt-1 or a variant thereof is indicative for the presence of placenta-associated cardiac dysfunction,

or wherein an increased level of natriuretic peptide and a non-decreased level of placental growth factor and/or a non-increased level of sFlt-1 or a variant thereof is indicative for the presence of a heart disorder associated with heart disease.

The invention also relates to the use of antibodies specific for natriuretic peptides and placental growth factor and/or sFlt-1 or variants thereof for the preparation of reagents for use in an immunological rapid assay characterized in that antibodies specific for natriuretic peptides, and/or sFlt-1 or variants thereof are used in said immunological rapid assay for the in vitro diagnosis of heart diseases, in particular for distinguishing heart disorders related to heart diseases from heart disorders related to placenta by determining the levels of natriuretic peptides and placental growth factor and/or sFlt-1 or variants thereof in samples of pregnant women.

The invention also relates to the use of a natriuretic peptide and placental growth factor and/or sFlt-1 or a variant thereof for the manufacture of a medicament for use in a method for determining a viable treatment for a pregnant woman suffering from a cardiac dysfunction,

wherein the pregnant woman presents with heart disease related cardiac dysfunction symptoms comprising the steps of:

a) determining the level of the natriuretic peptide in the sample,

b) determining the level of placental growth factor and/or sFlt-1 or a variant thereof in the sample,

c) wherein an increased level of natriuretic peptide and a decreased level of placental growth factor and/or an increased level of sFlt-1 or a variant thereof is indicative for the presence of placenta-associated cardiac dysfunction,

or wherein an increased level of natriuretic peptide and a non-decreased level of placental growth factor and/or a non-increased level of sFlt-1 or a variant thereof is indicative for the presence of a heart disorder associated with heart disease,

d) optionally initiating a maternal check by a cardiologist,

e) if the evaluation according to step c) indicates the presence of cardiac dysfunction associated with a cardiac disease, it is recommended to initiate the treatment.

Examples

Clinical studies have been conducted on 55 pregnant women of the age group (cohort) for the presence of placenta-related cardiac dysfunction or for the presence of cardiac-related cardiac dysfunction. Reference values for sFlt-1, PlGF and NT-pre-BNP were determined for pregnant women (N ═ 55) classified in the middle (N ═ 9) and late (N ═ 46) gestation. The sFlt-1 and PlGF values of pregnant women with elevated NT-pre BNP values (> 125pg/ml) are shown in Table 1.

Has passed Elecsys NT-pre BNP^TMThe NT-proBNP concentration of the blood samples of pregnant women was analyzed by assay (Roche Diagnostics). Has been obtained by using a compound from R&Human soluble VEGF R1/Flt-1 immunoassay "Quantikine" by D Systems (catalog number DVR100B) analyzed the concentration of sFlt-1. By using a compound from R&Human PlGF immunoassays by D Systems "Quantikine" (Cat. DPG00) quantitative determination of human placental growth factor (PlGF) concentration was analyzed.

Table 1: sFlt-1 and PlGF values in pregnant women with increased NT-pre BNP values (> 125pg/ml)

Pregnant woman serial number	Number of weeks of gestation	March period	sFlt-1[pg/ml]	PlGF[pg/ml]	NT-proBNP [ pg/ml]
						92316544	24	2	2645.7	324.31	181.85
6	26-33	3	1062.1	96.80	336.94
						17	26-33	3	7551.6	93.01	125.12
1	34-36	3	8653.8	73.97	655.69
						16	34-36	3	3285.1	21.61	184.57
2	26-33	3	341.6	15.31	415.13
						2	26-33	3	165.9	11.99	940.15
5	26-33	3	164.7	11.27	324.75
						16	26-33	3	127.3	10.69	940.52

Table 1 summarizes sFlt-1 and PlGF levels in pregnant women with elevated NT-pre BNP values. 9 of the 55 pregnant women shown in Table 1 had elevated NT-pre BNP levels greater than 125 pg/ml. In addition, 8 of these 9 pregnant women (patients 6, 17, 1, 16, 2, 2, 5, 16) had reduced levels of PlGF, indicating the presence of placenta-associated cardiac dysfunction with preeclampsia. Elevated levels of NT-proBNP in pregnant women with preeclampsia correlate with the severity of the disease.

In 1 of 9 pregnant women, patient 92316544 showed increased levels of NT-probBNP, but normal levels of PlGF, indicating the presence of cardiac-related cardiac dysfunction with primary cardiac dysfunction.

Table 2: reference values for NT-pre BNP in apparently healthy female blood donors (18-44.9 years old)

	18-44.9 years old
				Median age		33
	Total of	Male sex	Female with a view to preventing the formation of wrinkles
				N	1323	815	508
Percentile
				0	20.00	20.00	20.00
2.5	20.00	20.00	20.00
				5	20.00	20.00	20.00
10	20.00	20.00	20.00
				25	20.00	20.00	21.67
50	20.43	20.00	37.06
				75	39.35	25.67	61.97
90	70.20	41.69	98.80
				95	97.32	62.89	116.40
97.5	115.00	85.75	129.70
				100	534.40	534.40	196.30

Table 2 contains reference values for NT-pre BNP for the 1323 apparently healthy blood donor cohorts aged 18-44.9 years. The median age of the blood donors was 33. Blood samples from 508 women were analyzed for NT-pre BNP concentration. Lux 0, 2.5, 5, 10, 25, 50, 75, 90, 95, 97.5 and 100 percentiles are indicated.

FIG. 1 shows a block diagram of reference values for the concentration of NT-pre BNP. N represents the number of patients. The first column shows NT-pre BNP concentrations in 508 apparently healthy 18-44.9 year old female blood donors. The reference value was compared with the NT-probnp concentrations of 55 pregnant women, which were divided into 9 pregnant women in the middle of gestation and 46 pregnant women in the late gestation. There were no apparent differences in the concentration of NT-pre BNP between these groups. Furthermore, the median and 75 th, 95 th and 5 th and 25 th percentiles are indicated.

FIG. 2 shows a block diagram of reference values for sFlt-1 concentration and PlGF concentration measured in 46 pregnant women. These 46 pregnant women had a NT-proBNP concentration of less than 125pg/ml, a fraction of 14 pregnant women in the middle of gestation and 32 pregnant women in the late gestation. PlGF and sFlt-1 concentrations decreased only slightly from mid to late gestation. In addition, the block diagram shows the sFlt-1/PlGF ratio. The concentration ratio of sFlt-1/PlGF increases from mid to late gestation.

Furthermore, the median and 75 th, 95 th and 5 th and 25 th percentiles are indicated.

Claims (17)

1. Use of a natriuretic peptide and placental growth factor and/or sFlt-1 or a variant thereof for the manufacture of a medicament for the diagnosis of a pregnant woman suffering from a cardiac dysfunction,

wherein said diagnosis comprises the steps of:

a) determination of natriuretic peptide levels in a sample

b) Determining the level of placental growth factor and/or sFlt-1 or a variant thereof in a sample

Wherein an increased level of natriuretic peptide and a decreased level of placental growth factor and/or an increased level of sFlt-1 or a variant thereof is indicative for the presence of placenta-associated cardiac dysfunction,

or wherein an increased level of natriuretic peptide and a non-decreased level of placental growth factor and/or a non-increased level of sFlt-1 or a variant thereof is indicative for the presence of a heart disorder associated with heart disease.

2. Use according to claim 1, wherein the assays for natriuretic peptides and placental growth factor and/or sFlt-1 or variants thereof are performed in a parallel manner.

3. Use according to any one of claims 1 and 2, wherein the natriuretic peptide is a BNP-type peptide or a variant thereof.

4. Use according to any one of claims 1 to 3, wherein the natriuretic peptide is NT-pre-BNP or a variant thereof.

5. Use according to any one of claims 1 to 4, wherein the natriuretic peptide is an ANP-type peptide or a variant thereof.

6. Use according to any one of claims 1 to 5, wherein the sample is a blood sample, preferably a plasma or serum and/or urine sample.

7. Use according to any one of claims 1 to 6, wherein the determination is made in the middle or late gestation.

8. The use according to any of claims 1-7, wherein an increased level of NT-pre BNP corresponds to a plasma level of NT-pre BNP between 125pg/ml and 300pg/ml, and a high increased level of NT-pre BNP corresponds to a plasma level of NT-pre BNP between 300pg/ml and more than 500 pg/ml.

9. The use according to any of claims 1-8, wherein the non-increased level of NT-pre BNP corresponds to a plasma level of less than 125 pg/ml.

10. Use according to any of claims 1-9, wherein the level of natriuretic peptide and placental growth factor and/or sFlt-1 or a variant thereof is determined for at least one additional sample.

11. Use according to any of claims 1 to 10, wherein additionally at least one preeclampsia biomarker is determined, said preeclampsia biomarker preferably being selected from the group consisting of alpha 2-macroglobulin, CD40 ligand, urotensin ii (urotensin ii) and the like.

12. A kit comprising (a) a reagent or device for determining the level of natriuretic peptide in a sample of a pregnant woman, and (b) a reagent or device for determining the level of PlGF and/or sFlt-1 or variants thereof in a sample of a pregnant woman, wherein said kit is for use in the in vitro diagnosis of heart disease, in particular for distinguishing between heart disease related cardiac dysfunction and placenta related cardiac dysfunction according to any one of claims 1 to 11.

13. Kit according to claim 12, characterized in that the kit comprises a package insert for interpreting the data determined for the level of natriuretic peptide, PlGF and/or sFlt-1 or variants thereof of a pregnant woman suffering from a heart disease, for distinguishing heart dysfunction related to heart disease from placenta related heart dysfunction.

14. Use of a ligand that specifically binds to a natriuretic peptide, in particular NT-proBNP or a variant thereof, and a ligand of PlGF and/or sFlt-1 or a variant thereof for the manufacture of a diagnostic kit for the diagnosis of a cardiac disease, in particular for distinguishing between a cardiac-related cardiac dysfunction and a placenta-related cardiac dysfunction in a pregnant woman suffering from a cardiac dysfunction.

15. An array for in vitro diagnosis of a cardiac disease, in particular for distinguishing between cardiac disease-related and placenta-related cardiac dysfunction by determining natriuretic peptides and placenta growth factor and/or sFlt-1 or variants thereof, said array comprising a ligand that specifically binds to a natriuretic peptide, in particular NT-pre-BNP or a variant thereof, and a ligand for PlGF and/or sFlt-1 or a variant thereof, said ligand (a) being for determining the level of the natriuretic peptides in a sample of a pregnant woman, and (b) being for determining the level of PlGF and/or sFlt-1 or variants thereof in a sample of a pregnant woman.

16. Use of a natriuretic peptide and an antibody specific for placental growth factor and/or sFlt-1 or a variant thereof for the preparation of a reagent for use in an immunological rapid assay characterized in that an antibody specific for a natriuretic peptide, and/or sFlt-1 or a variant thereof is used in said immunological rapid assay, (a) for determining the level of a natriuretic peptide or a variant thereof in a sample of a pregnant woman, and (b) for determining the level of PlGF and/or sFlt-1 or a variant thereof in a sample of a pregnant woman,

said immunological rapid assay is used for the in vitro diagnosis of heart diseases, in particular for distinguishing heart disorders related to heart diseases from heart disorders related to placenta by assaying natriuretic peptides and placenta growth factor and/or sFlt-1 or variants thereof.

17. Use of a natriuretic peptide and placental growth factor and/or sFlt-1 or a variant thereof for the manufacture of a medicament for use in a method for determining a viable treatment for a pregnant woman suffering from a cardiac dysfunction,

wherein the pregnant woman presents with heart disease related cardiac dysfunction symptoms comprising the steps of:

a) determining the level of the natriuretic peptide in the sample,

b) determining the level of placental growth factor and/or sFlt-1 or a variant thereof in the sample,

c) wherein an increased level of natriuretic peptide and a decreased level of placental growth factor and/or an increased level of sFlt-1 or a variant thereof is indicative for the presence of placenta-associated cardiac dysfunction,

or wherein an increased level of natriuretic peptide and a non-decreased level of placental growth factor and/or a non-increased level of sFlt-1 or a variant thereof is indicative for the presence of a heart disorder associated with heart disease,

d) optionally initiating a maternal check by a cardiologist,

e) if the evaluation according to step c) indicates the presence of cardiac dysfunction associated with a cardiac disease, it is recommended to initiate the treatment.