WO2005108987A2 - Determination of osteoprotegerin for the prognosis of cardiovascular disorders - Google Patents

Abstract

Thus, the present invention provides a method, an assay and a kit for providing a prognosis of patients suffering from a cardiovascular disorder. It was surprisingly found that the significant increase in Osteoprotegrin concentration in patients (as well as animals) are useful as a prognostic tool to predict the clinical outcome, complications and mortality following a cardiovascular disorder, such as but not limited to acute myocardial infarction.

Description

Determination of Osteoprotegerin for the prognosis of cardiovascular disorders

Field of the invention

The present invention relates to prognosis of a disease, such as a cardiovascular disorder in a mammal e.g. in a human being. In particular the present invention relates to the determination of the concentration of Osteoprotegerin in a human being for predicting the clinical outcome, complications and mortality of a mammal suffering from a cardiovascular disorder.

Background of the invention

Arterial calcification and mineral deposition is a prominent feature of atherosclerosis, and there appears to be a moderate relationship between the extent of coronary artery calcification and occurrence of adverse events [e.g., myocardial infarction (MI)] and poorer 5-year survival. Furthermore, non-collagenous bone-matrix associated proteins such as osteocalcin, osteopontin and osteonectin have been identified at sites of calcification, and endochondral bone formation in the heart has been suggested as a possible mechanism of coronary calcification. Thus, there seem to be similarities between vascular and skeletal calcification suggesting a regulatory role for osteogenic and calcitropic factors in the development of cardiovascular disease.

Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TIMF) receptor superfamily that can function as a soluble decoy receptor by binding receptor activator of nuclear Factor-κB ligand (RANKL) and competitively inhibiting interaction between RANKL and its receptor.

These mediators have been identified as candidate factors for paracrine signaling in bone metabolism, but are also involved in immune responses by modulating T ceil function as well as B cell maturation and antibody response.

Furthermore, mRNA and protein expression of OPG and RANKL have been detected in myocardial tissue and atherosclerotic plaques. Also, high serum OPG levels have been associated with the progression of vascular calcification in patients receiving long-term hemodialysis (Nitta et a\., 2003; Haas et at., 2002), with presence and severity of coronary artery disease(Schoppet et a/., 2003; Jono et al., 2002) and with cardiovascular mortality in elderly women (Browner et a/., 2001).

WO 01/23562 provides the complete transcriptional regulatory region of the human opg gene. The disclosed sequences are also useful in diagnosing patients susceptibility to developing OPG-related bone, cartilage, immune and arterial diseases, however these diagnostic methods are all purely based on determining the nucleotide sequence, not measurements of OPG levels, nor opg mRNA levels.

US2002 0061521 relates to cardiovascular system related polynucleotides and the polypeptides encoded by these polynucleotides including diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to the cardiovascular system. However such methods are all purely based on determining the nucleotide sequence.

Schoppet M et al. showed that OPG serum levels are associated with the severity of coronary artery disease and are increased in elderly men and patients with diabetes mellitus. Schoppet M et al. conclude that increased OPG serum levels may reflect advanced cardiovascular disease in men. However, Schoppet M et al. does not related to an assay for the prognosis of a disease in a human.

WO 01/74896 relates to novel human polypeptides and isolated nucleic acids containing the coding regions of the genes encoding such polypeptides. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human polypeptides. WO 01/74896 does not related to an assay for the prognosis of a disease in a human.

Kiechl S et al. reports a population-based study over a 10-year period documenting cases of atherosclerosis and disorders of the brain and bones. This study recorded vascular mortality of the selected group and concludes that OPG is an independent risk factor for the progression of atherosclerosis and onset of cardiovascular disease.

Patients with e.g. acute myocardial infarction and substantial myocardial injury frequently show evidence of heart failure and have a high risk of morbidity and mortality. Previous studies have shown a considerable overlap when investigating potential predictors of mortality following AMI.

Accordingly, there is a need to provide the means for such a prognosis for patients suffering from a cardiovascular disorder in order to predict the clinical outcome, complications and mortality relative to that patient. This will give the opportunity to determine the severity of a cardiovascular disorder in order to provide the proper management and treatment and follow-up of patients suffering from a cardiovascular disorders, such as of acute myocardial infarction or other similar conditions in a quick and easy manner. In this way it would be possible to direct the correct and necessary management and treatment specifically to each patient.

To further elucidate the potential role of OPG in cardiac disease the present inventors correlates that circulating OPG plasma levels in patients with acute myocardial infarction (AMI) and its prospective relation to adverse outcomes during longitudinal follow-up in patients e.g. randomly assigned to angiotensin converting enzyme (ACE) inhibition or angiotensin II antagonist.

Summary of the invention The present invention provides a method, an assay and a kit for providing a prognosis of patients suffering from a cardiovascular disorder. It was surprisingly found that the increase in plasma OPG concentration in patients was useful as a prognostic tool to predict the clinical outcome, complications and mortality following a cardiovascular disorder, such as acute myocardial infarction.

Patients suffering from a cardiovascular disorder, such as acute myocardial infarction, heart failure and other similar conditions, and who had the highest increase in the concentration of plasma-OPG, was discovered by the inventors of the present invention to subsequently develop clinical complications, typically cardiac problems such as cardiac arrythmia, cardiac hypoxia, cardiac ischemia, cardiac shock, cardiac insufficiency, heart failure, heart attacks or cardiac arrest and significantly increased mortality. Thus, the increase of OPG was reflecting the severity of the cardiovascular disorder.

The inventors of the present invention found that this increase of the level of OPG can be used for predicting or for the prognosis of complications as a consequence of the cardiovascular disorder, in form of e.g. heart diseases, but also in the form of death or survival of the patient. Accordingly, the present inventors found that by determining the concentration of OPG e.g. in plasma it is possible to identify those patients, who will suffer from complications and having an increased rate of mortality.

The present inventors discovered that by determining the concentration of OPG e.g. in plasma it is possible to determine which of the patients will be suffering from further disorders, typically cardiac problems such as cardiac arrythmia, cardiac hypoxia, cardiac ischemia, cardiac shock, cardiac insufficiency, heart failure, heart attacks or cardiac arrest and significantly increased mortality. Thereby, one can distinguish between and classify clinically grade patients with a cardiovascular disorder into groups, relative to the severity of their condition, and this classification can already be done when they arrive first time at the hospital.

In this way it is possible to immediately start the correct monitoring of the critical patients and to clinically follow those critical patients, who will most likely develop complications and sudden death, whereby it will be possible to prevent the complications and increased mortality by means of medical or surgical preventive treatment/prophylaxis.

Accordingly, one can prevent and avoid the complications by treating cardiac problems and thereby limiting the mortality rate among patients suffering from a cardiovascular disorder before it is too late. In the same way is it possible to manage less severe / non- complicated patients quickly and have them quickly discharged from the hospital.

Detailed description The present invention relates in its broadest aspect to an assay for determining the concentration of Osteoprotegrin (OPG) in a sample, said assay comprises means for measuring the Osteoprotegrin (OPG) concentration in said sample.

Further, it is an aspect of the present invention to provide an assay and/or a method for the prognosis of a cardiovascular disease in a mammal and a kit for use in said assay, said assay and/or method comprising the following steps:

(i) providing a sample obtained from said mammal, (ii) measuring the concentration of Osteoprotegrin (OPG) in the sample, and

(iii) evaluating the concentration of Osteoprotegrin (OPG) measured in step (ii) relative to a reference value for the prediction of the clinical outcome, complications and mortality of said mammal.

Cardiovascular disease

In the present context, the term "cardiovascular disease" relates to any kind of abnormal condition which directly, or indirectly, involves the heart. This includes: heart failure irrespective of etiology (cause); acute coronary syndrome; hypertension; heart transplantation or complications related to heart transplantation; conditions associated with reduced pump function. "Heart failure", as used herein, is defined as a clinical syndrome characterised by dyspnea and fatigue, at rest or with exertion due to impaired structure and/or function of the heart of any cause.

In one embodiment of the present invention, the concentration of Osteoprotegrin (OPG) measured in the sample is useful for providing a prognosis of a cardiovascular disease in a mammal, wherein the cardiovascular disease is selected from the group consisting of acute myocardial infarction, heart failure, heart transplantation, unstable coronary syndrome, acute coronary syndrome, hypertension, inflammation and persistent inflammation.

In another presently preferred embodiment, the concentration of Osteoprotegrin (OPG) measured in the sample is useful for providing a prognosis of a cardiovascular disease in a mammal, wherein the cardiovascular disease is based on reduced cardiac muscle function in connection to heart failure independent of ischemia.

The term "independent of ischemia" in the present application refers to that the present invention provides a prognosis of a cardiovascular disease related to the cardiac muscle function independent of whether the reduced function is caused by ischemic or by non- ischemic heart disease.

The dependency of ischemia is surprisingly not of importance for the present invention, thus the failing cardiac muscle function such as but not limited to cardiomyopathy can originate from heart failure due to coronary ischemia, virus infection or any other infection in the cardiac muscle, failing cardiac muscle function secondary to valve failure, failing cardiac muscle function secondary to autoimmune diseases, failing cardiac muscle function secondary to idopatic dilated cardiomyopathy, excessive alcohol consumption and exposure to toxic compounds and pregnancy.

In one embodiment of the present invention, the concentration of Osteoprotegrin (OPG) measured in the sample is useful for providing a prognosis of cardiomyopathy.

In the present context the term cardiomyopathy relates to disease of the heart muscle. Currently four main types of Cardiomyopathy are recognised dilated (DCM), Hypertrophic (HCM or HOCM), Restrictive (RCM) and Arrhythmogenic Right Ventricular (ARVC).

In one embodiment of the present invention, the concentration of Osteoprotegrin (OPG) measured in the sample is useful for providing a prognosis of valve disease due to infection such as but not limited to infective endocarditis and rheumatic fever. In one embodiment of the present invention, the concentration of Osteoprotegrin (OPG) measured in the sample is useful for providing a prognosis of cardiovascular diseases with the proviso that that cardiovascular disease does not originate from atherosclerosis, vascular calcification or other conditions caracterised by characterized by thickening and hardening of artery walls e.g. by depositing fatty material along the walls of arteries. This fatty material thickens, hardens, and may eventually block the arteries.

In one presently preferred embodiment of the present invention, the concentration of Osteoprotegrin (OPG) measured in the sample is useful for providing a prognosis of acute myocardial infarction.

The present inventors further disclosed herein (i) In a rat model for post-infarction HF, they found persistently increased gene expression of OPG, RANK and RANKL in the ischemic and as for OPG, also in the non-ischemic part of left ventricle (LV) involving both non-cardiomyocyte and in particular cardiomyocyte tissue, (ii) Enhanced myocardial gene expression of OPG, RANK and RANKL was also seen in human HF, and by using immunohistochemistry they localized OPG and RANK to cardiomyocytes within LV in both experimental and clinical HF with the most prominent expression in the failing myocardium. (/^'/^'/) In human HF they also found increased systemic expression of RANKL (T-cells and serum) and OPG (serum) with increasing levels according to functional, hemodynamic and neurohormonal disease severity. Imortantly, these findings were unrelated to the etiology of heart failure suggestingthat any potential variations in levels of these proteins (i.e., OPG and RANKL) with regard to etiology may have been overshadowed by the contribution from chronic HF in itself, (iv) RANKL increased the gene expression of MMPs in neonatal rat cardiomyocytes, without affecting their endogenous inhibitors (i.e., TIMPs) suggesting matrix degrading net effects.

These findings suggest a potential role for known mediators of bone homeostasis in the pathogenesis of HF possibly representing new targets for therapeutic intervention in this disorder.

Thus, in another presently preferred embodiment of the present invention, the concentration of Osteoprotegrin (OPG) measured in the sample is useful for providing a prognosis of heart failure independent of aetiology. The present invention also pertains to the use of data obtained from the detection of Osteoprotegrin (OPG) in a sample obtained from a mammal for the prognosis of a cardiovascular disease in said mammal.

Furthermore, in one embodiment of the present invention, the concentration of Osteoprotegrin (OPG) measured in the sample is useful for providing a prognosis of a cardiovascular mortality in a human having a cardiovascular disease.

The kit

When measuring the concentration of Osteoprotegrin (OPG) in a sample, whether a quick determination is required or not, a kit comprising the necessary means is provided.

In the present context, the term "kit" relates to a set of means which are useful for a particular purpose, in this case the purpose of the kit Is to permit the determination of the concentration of Osteoprotegrin (OPG) in a patient sample. The set of means typically comprises means for obtaining the sample being measured, means for storing or holding the sample obtained, means for providing a detectable signal relative to the amount of Osteoprotegrin (OPG) present in the sample and means for evaluating the detectable signal of Osteoprotegrin (OPG) measured, but also other means may be added.

In one embodiment of the present invention, the kit comprises at least one of means selected from the group consisting of means for obtaining the sample being measured, means for storing or holding the sample obtained, means for providing a detectable signal relative to the amount and/or concentration of Osteoprotegrin (OPG) present in the sample and means for evaluating the detectable signal of Osteoprotegrin (OPG) measured, such as two of the means, e.g. three of the means and such as four of the means.

It has been shown that the kit according to the present invention is useful as a prognostic kit for the prediction of the clinical outcome, complications and mortality of the mammal from which the sample measured is being obtained.

When using the kit the means for obtaining the sample being measured comprises e.g. a syringe for obtaining a blood sample or a syringe or a scalpel for obtaining a tissue sample or other conventional means for obtaining a sample perfectly known by the person skilled in the art. Subsequently, the sample is transferred to the means for storing or holding the sample and the sample is analysed using the means for providing a detectable signal relative to the amount of Osteoprotegrin (OPG) present in the sample. The signal obtained is analysed and evaluated in order to determine the clinical severity of the cardiovascular disorder and thereby providing a prognosis for the disease and/or providing the best possible prevention and/or treatment of any complications caused by the cardiovascular disorder.

The means for providing a detectable signal relative to the concentration of Osteoprotegrin (OPG) present in the sample is selected from the group consisting of an assay, a stick, a dry-stick, an electrical device, an electrode, a reader (such as a spectrophotometric reader, an IR-reader, an isotopic reader and similar readers), histochemistry, and similar means.

Other means for providing a detectable signal relative to the concentration of OPG present in the sample may be correlation to the mRNA level of OPG in said sample, such as but not limited to Real-Time PCR.

The assay

In another embodiment of the present invention, the means for providing a detectable signal relative to the concentration of Osteoprotegrin (OPG) present in the sample is an assay. Preferably, the assay is selected from the group consisting of bioassay, immunoassay, microbiological assay, radioassay, and similar assays. Furthermore, the assay also relates to histochemical assay using histochemistry involving the chemistry of tissues as studied with a combination of chemistry and histology.

In one embodiment the bioassay is selected from the group consisting of protein arrays, HPLC and quantitative mass spec assays and/or polnt-of-care instruments such as but not limited to Radiometer ABL™ 77, Roche CoaguCheck, or any point-of-care instrument described at http://www.pointofcare.net/vendors/devices.htm.

In yet another embodiment of the present invention, the immunoassay is selected from the group consisting of radioimmunoassay, flouroimmunoassay, metalloimmuno-assay, spin immunoassay, immunohistochemistry and enzyme immunoassay, enzyme-linked immunosorbent assay (ELISA) and similar immunoassays.

In a presently preferred embodiment the immunoassay is an enzyme-linked immunosorbent assay as described in the examples below. In order to carry out the immunoassay suitable antibodies should be produced. These antibodies may be polyclonal or monoclonal antibodies produced by any conventional procedure.

In another embodiment of the present invention, the means for providing a detectable signal relative to the concentration of Osteoprotegrin (OPG) use the nucleotide level of opg to generate a concentration of OPG present in the sample, in particular mRNA. Thus, use of real time PCR, nucleotide arrays, nucleotide probes including LNA and PNA probes is part of the present invention.

In the context of the present invention, the term "assay" relates to a physical entity used for the measurement of a detectable signal relative to the concentration of Osteoprotegrin (OPG) present in the sample. Furthermore, in the context of the present invention, the term "method" relates to the activity of providing or measuring a detectable signal relative to the concentration of Osteoprotegrin (OPG) present in the sample.

In a further embodiment of the present invention, the method according to the present invention is used in the assay of the present invention and vice versa. Similarly the method and the assay of the present invention may be used in the kit of the present invention and vice versa.

The concentration of Osteoprotegrin (OPG) in a healty human will typically be in the range of 0-1.75 ng/ml in a plasma sample when measured with the methods provided in the present application. However as the skilled addressee would recognise, shifting assay might fluctuate the specific ranges obtainable.

In one embodiment the present invention relates to an assay of the present Invention, wherein the prediction of the clinical outcome, complications and mortality of said human relates to elevated levels of OPG.

In a presently preferred embodiment, the present invention relates to an assay according to the present invention, wherein elevated levels of Osteoprotegrin (OPG) relates to Osteoprotegrin (OPG) levels >1.5 ng/mL.

The concentration of patients suffering badly from a cardiovascular disorder will be at least 3.50 ng/ml, such as at least 3.60 ng/ml, e.g. at least 3.70 ng/ml, such as at least 3.80 ng/ml, e.g. at least 3.90 ng/ml, but less than 10 ng/ml, when measured with the method shown in the examples below. The concentration of OPG in patients suffering from a less severely cardiovascular disorder will be at least 1.75 ng/ml, such as at least 1.80 ng/ml, e.g. at least 1.90 ng/ml, such as at least 1.95 ng/ml, but less than 5 ng/ml.

In an embodiment of the present invention, the concentration of Osteoprotegrin (OPG) has a detectable range of the Osteoprotegrin (OPG) concentration in the range of 0-100 ng/ml, e.g. in the range of 0-50 ng/ml, such as in the range of 0-25 ng/ml, e.g. in the range of 0- 20 ng/ml, such as in the range of 0-19 ng/ml, e.g. in the range of 0-17 ng/ml, such as in the range of 0-15 ng/ml, e.g. in the range of 0-12 ng/ml, such as in the range of 0-10 ng/ml, e.g. in the range of 0-8 ng/ml, such as in the range of 1-25 ng/ml, such as in the range of 5-25 ng/ml, e.g. in the range of 8-25 ng/ml, such as in the range of 10-25 ng/ml.

In an embodiment of the present invention the concentration of Osteoprotegrin (OPG) in a sample from a patient suffering from a disease, such as a cardiovascular disorder, is at least 1.5 times or similar higher than the concentration of Osteoprotegrin (OPG) in a population of normal healthy patients, such as at least 2 times higher, e.g. at least 3 times higher, e.g. as at least 4 times higher , e.g. as at least 5 times higher, e.g. at least 6 times higher, e.g. as at least 7 times higher, e.g. at least 8 times higher, e.g. as at least 9 times higher, e.g. at least 10 times higher, e.g. as at least 15 times higher, e.g. at least 20 times higher, e.g. as at least 25 times higher, e.g. at least 50 times higher.

In a general setting: e.g. in a patient with cardiovascular disease the levels of OPG may temporarily drop to levels overlapping with the normal reference population in response to therapeutic intervention (pharmacological or non-pharmacological). If after some time, the OPG levels start to rise, while still being within the normal range, it is conceivable that the temporal changes in OPG values are diagnostic/prognostic by suggesting a change of therapeutic strategy.

In the context of the present invention, the term "data" relates to the measurement of the concentration of Osteoprotegrin (OPG) and which data is subsequently used for the prognosis of the disease in order to predict the clinical outcome, complications and mortality of the mammal suffering from the disease. In an embodiment the data obtained comprises one detection of the concentration of Osteoprotegrin (OPG) or more than one detection of the concentration of Osteoprotegrin (OPG).

When more than one measurement of the concentration of Osteoprotegrin (OPG) is being provided this may be in order to confirm accuracy of the measurements to provide uniform measurements. Alternatively, more than one measurement is made to provide e.g. a surveillance of the patient in order to prevent or to treat patients before it is too late. Osteoprotegrin (OPG)

In the context of the present invention, the terms " Osteoprotegrin" and "OPG" are used interchangeably and relate to a secreted member of the tumor necrosis factor receptor superfamily. Osteoprotegerin was initially discovered as an Osteoclast-inhibitory factor' (OCIF) released as a heparin-binding protein from human skin fibroblasts and found to inhibit osteoclast formation in vitro (Tsuda et al., 1997). OPG was cloned in a fetal rat intestine cDNA-sequencing project and given the name osteoprotegerin (Simonet et al., 1997). OCIF was then cloned and found to be identical to OPG (Yasuda et al., 1998). OPG is also identical to TR-1, which was identified in a search of an expressed sequence tag database (Kwon et al., 1998), and to follicular dendritic receptor 1 (FDCR-1; Yun et al., 1998). OPG is synthesized in humans, rats, and mice as a 401-amino-acid protein, which, after cleavage of a 21-amino-acid signal peptide, results in a 380-amino-acid mature protein. In humans, the OPG gene is located on chromosome 8q23-24. The amino acid sequence of OPG displays several homologiesto members of the TNF-R superfamily, including RANK. Human, rat, and mouse OPG has large homologies (85-95%). The 60-kDa OPG protein exists as a monomer which forms 120-kDa disulfide-linked homodimers containing several N-glycosylation sites. OPG contains four cysteine-rich domains in the N- terminal end, two homologous 'death domains' (DDH) in the C-terminus, a heparin-binding site, and a cysteine residue required for homodimerization but, in contrast to other members of the TNF-R superfamily, lacks a transmembrane spanning domain and a cytoplasmic tail. These features make OPG a unique protein in the TNF-R superfamily, and, unlike the other members, it is a secreted protein. The biological effect of OPG is dependent on the integrity of the four cysteine-rich domains. The DDH regions of OPG are similar to the 'death domains' of TNF-Rl and FAS, which mediate apoptotic signals. The function of the DDH region of OPG, however, is elusive but is not involved in the inhibitory effect on osteoclast formation and function.

In accordance with the present invention, OPG relates to all forms of OPG, such as but not limited to monomeric and dimeric forms (predominant extracellular form of OPG is a disulfide-linked dimer), membrane bound and secretory forms; postsynthetically modified forms (e.g. glycosylated), and any other molecular forms due to postsynthetic modification (e.g. proteolytic processing) or alternatively spliced variants and isoforms and/or both native and recombinant, with the entire or partial amino acid sequence, in a normal or mutated sequence, as well as the natural, synthetic or semi-synthetic derivatives and mixtures thereof. In the context of the present invention, the term "other molecular forms of Osteoprotegrin" relates to a protein having the same amino acid sequence or having substantially the same amino acid sequence as Osteoprotegerin. The term also relates to proteins having folded differently and/or proteins having similar dominating sites or patches in the protein capable of providing the desired activity.

The sample

In the present context, the term "a sample" relates to any liquid or solid sample collected from a patient to be analysed. Preferably, the sample is liquefied at the time of assaying.

In another embodiment of the present invention, a minimum of handling steps of the sample is necessary before measuring the concentration of Osteoprotegrin (OPG). In the present context, the term "handling steps" relates to any kind of pre-treatment of the liquid sample before or after it has been applied to the assay, kit or method. Pretreatment procedures includes separation, filtration, dilution, distillation, concentration, inactivation of interfering compounds, centrifugation, heating, fixation, addition of reagents, or chemical treatment.

In accordance with the present invention, the sample to be analysed is collected from any kind of mammal, including a human being, a pet animal, a zoo animal and a farm animal.

In yet another embodiment of the present invention, the sample is derived from any source such as body fluids.

Preferably, this source is selected from the group consisting of milk, blood, serum, plasma, saliva, urine, sweat, ocular lens fluid, cerebral spinal fluid, cerebrospinal fluid, ascites fluid, mucous fluid, synovial fluid, peritoneal fluid, amniotic fluid and other secreted fluids, substances and tissue biopsies from organs such as the brain, heart and intestine.

In a presently preferred embodiment, the sample is derived from blood, such as blood, serum, or plasma.

Prognosis

In the context of the present invention, the term "prognosis" relates to an opinion

(professional or non-professional, preferably a professional) on how an illness or a disease will develop and how the illness or disease will influence on other health conditions and death/survival in a mammal. The present invention provides the means for a prognosis of the clinical outcome, complications and mortality of said mammal. In the context of the present invention, the term "clinical outcome" relates to the 'final result' or the 'final situation' or the condition of the patient after the patient have experienced a disease, e.g. a acute myocardial infarction or heart failure. Thus, the clinical outcome may be death or survival, and survival can be everything from poor health condition (moribund) to a perfect health.

In the context of the present invention, the term "complications" relates to symptoms of anything arising after the disease, e.g acute myocardial infarction or heart failure. Thereby the acute myocardial infarction may be the primary problem/attack, while any other symptom or disorder or condition that may be seen afterwards is a new or 'secondary' problem/attack. This secondary problem/attack is defined as a complication. After e.g. a acute myocardial infarction, the complication is typically heart problems and symptoms of heart disorders such as cardiac arrythmia, cardiac hypoxia, cardiac ischemia, cardiac shock, cardiac insufficiency, heart failure, heart attacks, or cardiac arrest and other heart disorders/diseases as well as significantly increased mortality, but also other complications can occur, such as mental changes, mental retardation, personality change, further cerebral apoplexia reactions, epilepsy (epilepsy may occur both as a cerebral apoplexia condition a consequence of a cerebral apoplexia condition), paralysis, physical disorder, visual disorder, speaking disorder, cognition disorder and perception disorder. It can also likely be brain disorders, and almost any brain disease can arise after an acute myocardial infarction as a complication.

In one embodiment the present invention thus also relates to a method for evaluating the response to a medicament comprising monitoring the Osteoprotegrin (OPG) level in a subject.

In a presently preferred embodiment, such method for evaluating a medicament comprising monitoring the Osteoprotegrin (OPG) level relates to a subject, wherein said subject having acute myocardial infarction.

The reference

In order to determine the clinical severity of the disease, e.g. a caridovascular disorder the means for evaluating the detectable signal of Osteoprotegrin (OPG) measured involves a reference or reference means. The reference also makes it possible to count in assay and method variations, kit variations, handling variations and other variations not related directly or indirectly to the concentration of OPG. In the context of the present invention, the term "reference" relates to a standard in relation to quantity, quality or type, against which other values or characteristics can be compared, such as e.g. a standard curve.

In one preferred embodiment of the present invention, the reference means is an internal reference means and/or an external reference means.

In the present context the term "internal reference means" relates to a reference which is not handled by the user directly for each determination but which is incorporated into a device for the determination of the concentration of OPG, whereby only the 'final result' or the 'final measurement' is presented. The terms the "final result" or the "final measurement" relates to the result presented to the user when the reference value has been taken into account.

In a further embodiment of the present invention, the internal reference means is provided in connection to a device used for the determination of the concentration of OPG.

In yet an embodiment of the present invention the device is selected from the group consisting of an assay, a stick, a dry-stick, an electrical device, an electrode, a reader (spectrophotometric readers, IR-readers, isotopic readers and similar readers), histochemistry, and similar means incorporating a reference.

In the present context, the term "external reference means" relates to a reference which is handled directly by the user in order to determine the concentration of OPG, before obtaining the 'final result' or the 'final measurement'.

In yet a further embodiment of the present invention external reference means are selected from the group consisting of a table, a diagram and similar reference means where the user can compare the measured signal to the selected reference means. The external reference means relates to a reference used as a calibration, value reference, information object, etc. for OPG and which has been excluded from the device used.

The device In the present context, the term "device" relates to equipment usable in the present invention both for the measurement of OPG and for the determination or measurement of the reference. In yet an embodiment of the present invention the device is selected from the group consisting of an assay, a stick, a dry-stick, an electrical device, an electrode, a readers (spectrophotometric readers, IR-readers, isotopic readers and similar readers), histochemistry, and similar means where there has been incorporated a reference.

In one embodiment of the present invention relates to a method for the prognosis of a disease > i inn a a m maammmmaall,, s saaiidd m meetthhoodd ro commnprriisspess t thhee s srteeDpSs o off:

(i) providing a sample obtained from a mammal, and

(ii) measuring the concentration of Osteoprotegrin (OPG) in the sample.

(iii) evaluating the concentration of Osteoprotegrin (OPG) measured in step (ii) relative to a reference value for the prediction of the clinical outcome, the complications and the mortality of a mammal.

Another embodiment of the present invention relates to use of data obtained from the detection of Osteoprotegrin (OPG) in a sample obtained from a mammal for the prognosis of a cardiovascular disease in said mammal.

In a presently preferred embodiment of the present invention, the use of the data obtained from the detection of Osteoprotegrin (OPG) in a sample obtained from a mammal relates to the prognosis of a cardiovascular disease in said mammal, wherein the cardiovascular disease is acute myocardial infarction.

Another aspect of the present invention relates to use of the present invention for choosing the optimal therapy and monitoring the treatment both pharmacological and non- pharmacological, thus one embodiment relates to a method for evaluating the effect of pharmacological or non-pharmacological intervention in patients with a cardiovascular disease comprising monitoring the Osteoprotegrin (OPG) level in a subject.

Such prognostic value may be use to in monitoring of treatment e.g. lipid lowering agents (e.g. statins), beta-blockers, TNF etc. in addition to any other treatment/interventions (pharmacological and non-pharmacological), including surgery with the intention to improve prognosis/outcome.

In a presently preferred embodiment method for evaluating the effect of pharmacological or non-pharmacological intervention in patients with a cardiovascular disease comprising monitoring the Osteoprotegrin (OPG) level in a subject, wherein said subject having acute myocardial infarction.

In one embodiment, the present invention relates to an assay according to the present invention, wherein the concentration of Osteoprotegrin measured in the sample is useful for providing a prognosis of a cardiovascular mortality in a mammal having a cardiovascular disease.

In another embodiment the invention relates to a method for the prognosis of a disease in a mammal, said method comprises the steps of:

(i) providing a sample obtained from a mammal, and

(ii) measuring the concentration of Osteoprotegrin (OPG) in the sample.

(iii) evaluating the concentration of Osteoprotegrin (OPG) measured in step (ii) relative to a reference value for the prediction of the clinical outcome, the complications and the mortality of a mammal. wherein the measurement of the concentration of Osteoprotegrin (OPG) in the sample is provided by value selected from the group consisting of an assay, a stick, a dry-stick, an electrical device, an electrode, a reader, histochemistry, and similar value.

One embodiment of the present invention relates to use of data obtained from the detection of Osteoprotegrin (OPG) in a sample obtained from a mammal for the prognosis of a cardiovascular disease in said mammal, particularly wherein the cardiovascular disease is acute myocardial infarction.

In one embodiment, the present invention relates to a kit for use in an assay as defined in the present application.

All the features described herein relating to the methods and/or assays of the present invention are also applicable as embodiments relating to kits and vice versa, thus in one embodiment, the present invention relates to a kit for use in an assay as defined in the present invention.

As will be apparent, preferred features and characteristics of one aspect of the invention may be applicable to other aspects of the invention. All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention will hereinafter be described by way of the following non-limiting Figures and Examples.

Figure legends

Figure 1

Plasma OPG levels during the longitudinal follow-up for 2 years in relation to angiotensin II blockage with captopril or losartan.

Figure 2

Kaplan-Meier curves showing the cumulative incidence of death during the entire study (average follow-up 27 months), according to the quartiles of plasma OPG at enrollment.

Figure 3

Unadjusted risk ratios for levels of OPG (above or below 4 quartile = 4.1 ng/mL) in relation to incidence of angina, non-fatal MI, stroke, cardiac death, total mortality or composite endpoint (all-cause mortality, stroke, and non-fatal MI). øP<0.05, *P<0.001.

Figure 4

Plasma OPG levels during the longitudinal follow-up for 2 years in survivors and non- survivors. *P<0.01, **P<0.001 non-survivors vs. survivors.

Figure 5

Gene expression, quantified by real-time quantitative RT-PCR relative to GAPDH, of OPG, RANKL and RANK in sham-operated rats (n=4), and in non-ischemic and ischemic regions of the LV in rats with heart failure (n=5), 2, 7 and 28 days (d) after induction of myocardial infarction. Values are expressed as mean±SEM. *p<0.05 vs. sham animals, ⁺p 0.01 vs. non-ischemic region. Figure 6

(A) Representative photomicrographs of rat myocardial tissue sections from the LV of a sham-operated rat and from the border area between the infarcted and the noninfarcted myocardium of a rat with heart failure (HF). Immunostaining demonstrated fairly strong RANK and OPG immunoreactivities in cardiomyocytes of both non-failing and failing myocardial tissue. RANK immunoreactivity was also seen in numerous infiltrating cells in the border region between ischemic and non-ischemic rat myocardial tissue, whereas OPG immunoreactivity was only found in a few infiltrating cells. Magnification χ400. (B) Gene expression, quantified by real-time quantitative RT-PCR and relative to GAPDH, of OPG, RANKL and RANK in cardiomyocyte and non-cardiomyocyte tissue from the LV of sham rats and rats with post infarction HF. Values are expressed as mean±SEM. *p<0.05 vs. Sham.

Figure 7

(A) Serum levels of OPG and soluble RANKL in patients with CHF according to NYHA functional class II-IV and healthy controls. (B) Relationships between serum OPG and Cardiac Index (CI) and serum levels of N-BNP in HF patients. (C) Gene expression, quantified by real-time quantitative RT-PCR and relative to β-actin mRNA, of RANKL and RANK in CD3⁺ T-cells and CD14⁺ monocytes from healthy blood donors (n=9) and HF patients (π=13). Values are expressed as mean±SEM. *p<0.05, **p<0.01 vs. controls. ^fp<0.05, p<0.01 vs. NYHA IV.

Figure 8

(A) Myocardial mRNA levels of OPG and RANK relative to GAPDH in LV from CHF patients and donors. (B) Representative photomicrographs of human myocardial tissue sections from a non-failing donor heart (CTR) and a failing explanted heart (HF). Immunostaining of LV sections demonstrated fairly strong RANK and OPG immunoreactivities in cardiomyocytes of non-failing and failing myocardial tissue with the most prominent staining in the failing myocardium. RANK immunoreactivity was also seen in VSMC and endothelial cells (enhanced) from donor and explanted human hearts. OPG immunoreactivity was also found VSMC and endothelial cells (enhanced) but only in explanted failing hearts. Magnification χ400. Figure 9

In vitro effects of RANKL in neonatal rat cardiomyocytes. Effects of 24 and 48 hours (h) stimulation with RANKL (300 ng/mL ) or vehicle (CTR) on mRNA levels of matrix metalloproteinases (MMP), their endogenous tissue inhibitors (TIMPs) and transforming growth factor (TGF)-βl. All gene expression data are normalized against GAPDH. Data are given as mean±SEM. *p<0.05 vs. vehicle.

Examples

Methods

Study population

The design and main results of the OPTIMAAL trial have previously been reported. The present study comprised a subset of 234 patients from the original OPTIMAAL trial with confirmed AMI and heart failure during the acute phase. Patients were randomly assigned and titrated to a target dose of losartan (50 mg qd) or captopril (50 mg tid) as tolerated. There were no significant differences in baseline characteristics between the treatment groups. For comparison, plasma OPG levels were also measured in 20 healthy controls.

Blood Sampling Blood samples were drawn after an overnight fast into pyrogen-free vacuum blood collection tubes with EDTA [OPG and high-sensitivity C-reactive protein (hsCRP)] or EDTA and aprotinin [N-terminal pro-brain natriuretic peptide (N-BNP)]. Tubes were immediately immersed in melting ice, centrifuged (lOOOg and 4°C for 15 minutes) within 15 minutes and plasma was stored at -80°C in multiple aliquots until analyzed. Samples were thawed < 3 times.

Biochemical analysis

Plasma OPG was quantified by enzyme immunoassay (EIA) using commercially available matched antibodies (R&D Systems, Minneapolis, MN) as previously describedl6. The creatinine clearance rate was calculated with the age-corrected Cockroft and Gault equation: (140-age) x (weight, kg)/serum creatinine x 72 [x 0.85 for women]. N-BNP was analyzed as previously described. Plasma levels of hsCRP were measured by an immunonephelometric assay performed on the Behring nephelometer (BN II, Dade Behring, Deerfield, IL). Statistical Methods

Kaplan-Meier estimates were used for evaluation of the occurrence of death. Differences in continuous variables were compared using Mann-Whitney U while the χ2 test was used for proportions. Associations between baseline risk factors, OPG concentrations, and cardiovascular events where analyzed by univariate analysis a priori and if p<0.2, were subsequently included in a forced or stepwise multivariate logistic regression. For investigating treatment effects, univariate repeated-measures ANOVA was performed a priori with time and treatment as fixed factors. OPG was not normally distributed at baseline as evaluated by the Kolmogorov-Smirnov Test, and therefore transformed prior to inclusion in the general linear model. Probability values are two-sided with p<0.05 being considered statistically significant.

Results

Example 1

Plasma OPG during angiotensin II blockage

At baseline the study population had significantly raised plasma OPG levels comparing healthy controls, ranging between 1.2 and 8.0 pg/mL, with a median of 3.0 and interquartiles of 2.1 and 4.1 ng/mL (Figure 1).

When following patients longitudinally, plasma OPG levels demonstrated a marked and early decrease (i.e., after 1 month) remaining at the same level throughout the rest of the sampling period (i.e., 24 months), with no differences between ACE-inhibition and selective angiotensin II antagonism (Figure 1).

Example 2

Association between OPG levels and baseline clinical and biochemical variables

When baseline OPG levels in the study population were divided into two groups according to quartiles (see below), we found that patients with high OPG (forth quartile, 4.1 ng/mL) were significantly older, had a poorer creatinine clearance, higher N-BNP and hsCRP levels, and a higher incidence of diuretic medication than those in the lower 3 quartiles (<4.1 ng/mL) (Table 1). There were no significant differences in other baseline variables between these two "OPG groups" (Table 1). Table 1

Baseline characteristics according to OPG levels. OPG < 4.1 ng/mL OPG > 4.1 ng/mL P-value

Demographics n 169 56

Age (years) 66±10 72±11 <0.001

Male, % 72 66 0.433

BMI (kg/m2) 26±4 26±4 0.564

History

Hypertension, % 33 43 0.188

Previous myocardial infarction, % 12 15 0.477

Diabetes, % 10 18 0.096

Hypercholesterolemia, % 27 16 0.148

Medication

Diuretics, % 7 25 <0.001 β-blocker, % 20 23 0.553

Statins, % 9 9 1.000

ASA, % 23 27 0.573

Warfarin, % 2 4 0.600

Anterior, lateral infarct location, % 63 52 0.159

Kilip class (II-IV), % 80 84 0.504

Creatinine clearance <70 mL/min 30 48 0.002 hsCRP (mg/L) 60±58 98±77 0.002

N-BNP (pmol/L) 1198±643 1801±749 <0.001

ASA, acetylsalicylic acid; BMI, body mass index; statin, hydroxymethylglutaryl coenzyme

A reductase inhibitors; hsCRP, high sensitivity C-reactive protein N-BNP, N-terminal pro-brain natriuretic peptide; OPG, osteoprotegerin. *Data are mean±SD. Example 3

OPG levels at baseline and cardiovascular events

Patients were followed for an average of 27 months and all-cause mortality was 14 % (n=32) during this period. Kaplan-Meier survival curves according to OPG quartiles at baseline are presented in Figure 2 showing increased mortality in those with high OPG levels (i.e. >4.1 ng/mL). Moreover, OPG levels were significantly lower in long-term survivors than in patients dying from all-cause morality (2.8 vs. 4.2 ng/mL, p<0.001) or isolated cardiovascular events (2.8 vs. 4.2 ng/mL, p<0.001). Also, patients experiencing stroke during follow-up had significantly higher plasma OPG concentrations at baseline than those without this complication (4.3 vs. 3.1 ng/mL, p<0.001). Finally, we made a composite end point consisting of all-cause mortality, stroke, and non-fatal MI and found that these patients also had significantly elevated OPG comparing the rest of the study group (3.1 vs. 3.7 ng/mL, p=0.003). The association between OPG levels at baseline and these clinical events during follow-up is also illustrated in Figure 3 showing the unadjusted risk estimate of patients with high OPG levels at baseline in relation to total mortality and cardiovascular.

Example 4

Other baseline predictors of cardiovascular events.

The association between potential confounders and all-cause mortality and cardiovascular events is summarized in Table 2. Several variables were univariate predictors of all-cause mortality and cardiovascular death, including age, diabetes, previous MI, hypercholesterolemia, creatinine clearance and N-BNP. In general, these variables were significant predictors of the composite end point as well. When investigating stroke, no univariate predictors except OPG were identified. Still, for all these end points we included variables with association's p<0.2 in a multivariate analysis (see below).

Example 5

Multivariate analysis In a multivariate model, adjusting for potential confounders (see above), high plasma OPG was still associated with long-term all-cause mortality, cardiovascular death and the composite end point (Table 3). In fact, of all variables entered into the multivariate model, OPG remained the strongest predictor of these end points. Similar results were obtained with both stepwise (Table 3) and forced addition of independent predictors as well as by entering all potential confounders, regardless of association with adverse events (all variables in Table 2). Thus, high plasma OPG levels measured during the acute phase following MI remains an independent and strong determinant of all-cause mortality and cardiovascular death in this study population. Table 2. Univariate Associations between baseline variables and All Cause Mortality, Cardiovascular Death, Stroke or the Composite Endpoint

(All-cause Mortality, Stroke, or Non-fatal MI).

All Cause Mortality Cardiovascular Death Stroke Composite Endpoint OR (95% CI) P value OR (95% P value OR (95% P value OR (95% P-value

Age>65 years 9.9 (2.9- <0.001 8.2 (2.4- <0.001 2.9 (0.6- 0.305 2.6 (1.4- 0.004 Male sex 0.9 (0.4-2.1) 0.837 1.1 (0.4- 1.000 3.5 (0.4- 0.286 1.1 (0.6- 0.744 Hypertension 2.0 (0.9-4.3) 0.074 2.8 (1.2- 0.018 0.5 (0.1- 0.316 1.2 (0.6- 0.595 Previous myocardial 3.7 (1.5-9.3) 0.007 3.6 (1.4- 0.011 0.9 (0.8- 0.605 2.6 (1.1- 0.020 Diabetes 2.6 (1.0-6.7) 0.069 3.1 (1.2- 0.027 2.3 (0.4- 0.279 2.4 (1.0- 0.034

Hypercholesterolemia 0.2 (0.0-0.8) 0.012 0.2 (0.0- 0.031 0.8 (0.7-0 0.119 0.5 (0.2- 0.093 Anterior, lateral infarct 0.7 (0.3-1.5) 0.438 0.7 (0.3- 0.537 0.5 (0.1- 0.489 1.0 (0.5- 0.950 Kilip class (II-IV), % 1.3 (0.5-3.7) 0.808 1.1 (0.4- 1.000 1.9 (0.2- 1.000 1.0 (0.5- 0.967 Treatment (Losartan or 1.1 (0.5-2.4) 0.849 1.0 (0.4- 1.000 1.2 (0.3- 0.517 0.8 (0.5- 0.564 Creatinine clearance <70 7.1 (3.0- <0.001 6.7 (2.7- <0.001 0.2 (0.0- 0.171 3.2 (1.7- <0,001 N-BNP >1988 pM (75%) 3.3 (1.1-7.1) 0.004 4.4 (1.9- 0.001 0.9 (0.2- 1.000 2.0 (1.0- 0.039 hsCRP >100 mg/L (75%) 1.1 (0.5-2.7) 0.822 1.1 (0.4- 1.000 0.9 (0.2- 1.000 1.5 (0.7- 0.273 high sensitivity C-reactive protein N-BNP, N-terminal pro-brain natriuretic peptide; OPG, osteoprotegerin.

Table 3

Multivariate models for all-cause mortality, cardiac death and composite endpoint (all- cause mortality, stroke, or non-fatal MI) during follow-up (average 27 months)

End point Odds Ratio (95% P-value

All cause Mortality Hypercholesterolemia 0.1 (0.0-0.9) 0.038 Creatinine clearance <70 mL/min 4.9 (1.9-12.5) 0.001 OPG > 4.1 ng/mL (> 75 percentile) 5.4 (2.2-13.1) <0.001 Cardiac Death Hypercholesterolemia 0.1 (0.0-0.9) 0.037 Creatinine clearance <70 mL/min 4.6 (1.7-12.5) 0.003 Hypertension 2.7 (1.0-7.0) 0.047

OPG > 4.1 ng/mL (> 75 percentile) 5.3 (2.0-13.7) 0.001 Composite Endpoint (All-cause Mortality, Stroke, or Non-fatal MI)

Creatinine clearance <70 mL/min 2.6 (1.3-5.0) 0.005

OPG > 4.1 ng/mL (> 75 percentile) 2.7 (1.3-5.3) 0.005

Example 6

OPG during longitudinal follow-up in relation to cardiovascular events

As described above, plasma OPG levels demonstrated a marked early decrease (I.e., after 1 month) remaining at this level during the rest of the study period (Figure 1), However, when comparing OPG levels in survivors and non-survivors (all-cause mortality), we found that the latter group had persistently higher OPG levels at all time points during follow-up (Figure 4). Furthermore, the prognostic value of plasma OPG measurements in relation to all-cause mortality and cardiovascular events during follow-up was persistently high, and as for OPG levels at 1 month and 1 year after inclusion, odds-ratios were even higher than for OPG levels at baseline (Table 4).

Table 4 Unadjusted risk ratios for all cause mortality and cardiovascular events after various time- points in relation to high (>4.1 ng/ml) and low (<4.1 ng/mL) OPG levels during follow-up 1 month 1 year 2 years OR (95% CI) p value OR (95% CI) p value OR (95% CI) P-value

Angina 2.5 (0.6-10.9) 0.207 5.6 (1.2-26.2) 0.015 2.3 (0.4-13.0) 0.343 Non-fatal MI 17.9 (3.4- <0.001 8.9 (1.9-42.5) 0.001 7.1 (1.3-37.4 0.009 Stroke 0.9 (0.9-1.0) 0.542 1.0 (0.9-1.0) 0.620 1.0 (0.9-1.0) 0.661

Cardiac death 22.4 (4.8- <0.001 13.8 (2.1- <0.001 1.0 (0.9-1.0) 0.736 Death 22.4 (4.8- <0.001 9.7 (1.6-59.0) 0.003 1.0 (0.9-1.0) 0.696 Composite 12.5 (2.4- <0.001 6.3 (1.3-29.5) 0.009 5.2 (1.0-27.2) 0.032

Discussion

The present study demonstrates that elevated plasma levels of the soluble decoy receptor OPG is associated with a significantly poorer outcome during 24 months of follow-up in patients subsequent to AMI. Furthermore, the predictive value of OPG at baseline, was independent of, and provided better risk prediction than kidney function, N-BNP, hsCRP and other known predictors of mortality and cardiovascular events after AMI. Finally, in non-survivors plasma OPG levels were not only increased in the acute phase at admission following AMI, but also during longitudinal testing, suggesting that OPG may be valuable for monitoring patients at risk both immediately after AMI as well as during follow-up. An increasing number of osteogenic factors have been detected in atherosclerotic plaques and received new attention in relation to vascular biology (Dhore et al., 2001; Gadeau et al., 2001; Tyson et al., 2003). The OPG/RANK system plays an important role in bone metabolism and in the present study we for the first time report a strong association between high plasma levels OPG and increased mortality and occurrence of cardiovascular events after AMI, further supporting a role for mediators in bone metabolism in the pathogenesis of cardiovascular disease. We found significant associations between baseline levels of OPG and other risk factors for cardiovascular events such as age, creatinine clearance, and circulating levels of N-BNP and hsCRP. However, when correcting for these variables during multivariate analysis, OPG levels was still a significant predictor of fatal and non-fatal cardiovascular events. In fact, OPG at admission was a stronger predictor of adverse events than N-BNP and hsCRP, two markers of cardiovascular events that have received much attention during the recent years. The source and mechanism for the enhanced systemic OPG levels found in this study can only be speculated upon since OPG is produced in many tissues including the cardiovascular system, lung, kidney, intestine, bone and circulating immune cells (Schoppet et al., 2002; Simonet et al., 1997). Importantly, OPG expression has been demonstrated in different vascular cell types, and both coronary smooth muscle cells and endothelial cells have been implicated as cellular sources and targets of vascular OPG production (Hofbauer et al., 2001; Malyankar et al., 2000). Furthermore, OPG has been shown to play a role in the regulation of the immune response and is regulated by the ligation of several cytokines involved in the inflammatory response following AMI. Thus both CD40L, TNFα and interleukin lβ have been shown to enhance OPG expression in various cells (Yun et al., 1998; Brandstrom et al., 1998) and these cytokines are persistently activated in the chronic phase during infarction healing (Detaen et al., 2002; Kritharides et al., 2003; Latini etal., 1994; Aukrust et al., 1999). Moreover, in postmenopausal osteoporosis, human immunodeficiency virus infection and cushing's syndrome, high serum OPG is suggested to reflect enhanced RANKL activity (Ueland et al., 2001; Yano et al., 1999). Thus, although OPG may protect against harmful effects of RANKL and TRAIL, another ligand for OPG (Emery et al., 1998), the strong association between high OPG and cardiovascular mortality could reflect that plasma OPG level is a reliable parameter of the overall activity in the OPG/RANK system as well as a stable marker of inflammation.

Cross-sectional associations between circulating OPG and mortality have recently been shown in other populations (Browner et ah, 2001; Terpos et al., 2003). A major finding in the present study was that in addition to being an excellent risk marker at baseline, OPG levels were persistently high in non-survivors. In fact, the association with subsequent cardiovascular events was even higher during follow-up than at baseline suggesting that OPG measurements may be used to monitor patients at risk and help identify a subgroup that may benefit from stronger preventive treatment. It is unknown if plasma OPG levels were modified by angiotensin II blockage/ACE inhibition in the present study, since there was no placebo group and the effect of cardiovascular medications on OPG levels will have to be further investigated. Of interest, anti-TNF therapy, which experimental studies indicate may be a potential strategy for treatment of AMI (Sugano et al., 2002), has been shown to normalize serum OPG (and RANKL)(Ziollowaska et al., 2002) in patients with rheumatoid arthritis whom are at increased risk of cardiovascular disease (Solomon et al., 2003) and other therapeutical approaches targeting the OPG/RANK system could also be of interest in AMI and heart failure.

In summary, plasma OPG levels provide Impressive independent prognostic information in patients with AMI, both in the acute phase as well as during longitudinal follow-up. This identification of OPG as a novel marker for cardiovascular mortality and clinical events support a role for mediators in bone homeostasis the pathogenesis of cardiovascular disease and may suggest a link between bone and vascular calcification Example 7

OPG in relation to HF

Methods

Rat model of experimental HF

The course of myocardial OPG, RANK and RANKL mRNA expression was investigated at various time points after ligation of the left coronary artery during the development of HF in male Wistar rats.

The procedure generally resulted in transmural infarction of the left ventricular (LV) free wall comprising 40-50% of the ventricular circumference (as assessed by perimetry of LV tissue sections). Except for ligation of the coronary artery, sham-operated rats underwent the same procedure. Assessment of hemodynamic function and tissue sampling procedures were performed as previously described (Oie E, et al., 2000).

In a separate set of experiments LV was divided into cardiomyocytes and non- cardiomyocytes. Total RNA was isolated from ischemic and non-ischemic areas in LV by acid-phenol extraction in the presence of chaotropic salts (TRIzol, GIBCO-BRL) and subsequent isopropanol-ethanol precipitation. The animal experiments and housing were in accordance with institutional guidelines and national legislation conforming to the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes of 18 March 1986. HF patients

One hundred and two patients with stable HF for >4 months in New York Heart Association (NYHA) functional class II-IV were consequtively included from Rikshospitalet University Hospital (Table 5). Most of the patients were evaluated by standard right- (n=84) and left- (n=70) sided cardiac catheterization. Patients with acute coronary syndromes during the past 6 months and patients with significant concomitant disease such as infection, malignancy or collagen vascular disease were not included. The underlying cause of HF was classified as ischemic (n=54) or idiopathic dilated cardiomyopathy (n=48) based on disease history and coronary angiography. Control subjects were 20 sex- and age-matched healthy blood donors. Informed consent for participating in the study was obtained from all individuals. The study was approved by the local ethical committee and conducted according to the Declaration of Helsinki.

Biochemical measurements Serum levels of OPG were quantified by ELISA using commercially available matched antibodies (R&D Systems). Serum levels of N-terminai pro-brain natriuretic peptide (N- BNP) and RANKL were quantified by ELISA (Biomedica GmBH). Serum samples were collected and stored as previously described.

Isolation of cells

PBMC were obtained from heparinized blood by Isopaque-Ficoll (Lymphoprep, Nycomed) gradient centrifugation.13 Further separation of monocytes (CD14-labeled magnetic beads; MACS, Miltenyi Biotec) and CD3+ T-cells (negative selection by monodisperse immunomagnetic beads; Dynai) was performed as described elsewhere (Aukrust et al., 1999) The selected T-cells consisted of >90% CD3+ cells and the isolated monocytes of >95% CD14+ cells (flow cytometry). Cell pellets were stored in liquid nitrogen until used. Total RNA was isolated from frozen T-cells and monocytes using RNeasy Minikit (Qiagen).

Human myocardial tissue samples

Tissue aliquots from the failing myocardium were removed from still-beating hearts immediately upon explantation from 16 patients with end-stage HF [NYHA class III-IV, LV ejection fraction <34%] undergoing cardiac transplantation, snap-frozen in liquid nitrogen and stored at -80°C until use. Control (non-failing) human LV tissue was obtained from subjects whose hearts were rejected as cardiac donors for surgical reasons (n=3). The cause of death of donors was cerebrovascular accident or trauma and none had a history of heart disease. Myocardium from these subjects was kept on ice-water for 1-4 hours before tissue sampling as described above. Total RNA was isolated from myocardial tissue by the acid guanidinium thiocyanate phenol-chloroform method.

Real time quantitative RT-PCR

All total RNA samples were subjected to DNase I treatment (RQI DNase; Promega) and stored at -80°C until analysis. Sequence specific PCR primers were designed using the Primer Express software version 1,5 (Applied Biosystems), see Table 6 for details. Quantification of mRNA was performed using the ABI Prism 7000 (Applied Biosystems). Gene expression of the housekeeping gene GAPDH (Applied Biosystems) and β-actin (Table 6) was used for normalization.

Immunohistochemistry Immunohistochemical analysis was performed on LV myocardial tissue from cardiac explants and rat hearts as previously describedlS using purified polyclonal antibodies against human OPG, RANK and RANKL (Santa Cruz Biotechnology). Unmasking of antigens was improved by heating the sections in citrate buffer, pH 6.0, in a microwave oven. Diaminobenzidine was used as the chromogen in a commercial metal enhanced system (Pierce Chemical). The sections were counterstained with hematoxylin. Omission of the primary antibody served as a negative control.

Stimulation of neonatal rat cardiomyocytes

Cardiomyocytes were isolated from 1-3 days old Wistar rats as previously described. (Florholmen et al., 2004) The purity of the cultures were >92% cardiomyocytes. Cells were treated with 300 ng/mL recombinant RANKL (R&D System) or vehicle for 24 or 48 h. Total RNA was isolated from harvested cells on a MagnaPure LC robot (Roche) using RNA isolation kit II including DNase treatment.

Statistical analysis

When comparing >2 groups, the Kruskal-Wallis test was used. If a significant difference was found, the Mann-Whitney U test was used to determine the differences between each pair of groups. Coefficients of correlation were calculated by the Spearman rank test. Data are given as medians and 25th to 75th percentiles if not otherwise stated. Throughout, we report 2-tailed p-values that are considered significant when <0.05.

Results

Expression of OPG/RANK/RANKL in experimental HF

To characterize the OPG/RANK/RANKL axis in HF, we first examined the myocardial gene expression by real-time quantitative RT-PCR in an experimental rat model of post- infarction HF. As shown in Figure 5, rats with myocardial failure had markedly elevated levels of OPG mRNA in both the ischemic and non-ischemic regions of LV compared to sham, with persistently raised levels throughout the observation period. RANKL gene expression was in general low, but significantly elevated mRNA levels were observed in the ischemic region with increasing levels during the study period reaching noticeably high amounts after 28 days (>200-fold increase). Finally, increased RANK gene expression was observed in both ischemic and non-ischemic tissue at 2 days compared to sham, but remained elevated only in the ischemic LV during the entire study. Thus, it seems that post-infarction HF in rats is characterized by persistently elevated expression of both OPG, RANKL and RANK in the ischemic region, and as for OPG, a similar pattern was also seen in non-ischemic parts of the LV.

Localization of OPG/RANK/RANKL within LV in experimental HF

Strong RANK immunoreactivity was seen in cardiomyocytes from both non-failing and failing rat hearts, with the most prominent expression in the failing myocardium (Figure 6a). In HF rats, RANK immunoreactivity was also seen in infiltrating cells in the border region between ischemic and non-ischemic rat myocardial tissue (Figure 6a). OPG immunoreactivity was also strong in cardiomyocytes with no differences between non- failing and failing rat hearts (Figure 6a). As for RANKL, no immunostaining was detected in sham-operated or HF rat myocardium possibly reflecting low sensitivity for the actual Ab. We also analysed the gene expression of these mediators after separating LV tissue into cardiomyocytes and non-card iomyocytes. As shown in Figure 2b, HF rats had enhanced gene expression of OPG and RANKL in both cardiomyocyte and non-cardiomyocyte tissue with particularly strong expression in cardiomyocytes. In contrast, only cardiomyocytes showed increased RANK expression in these animals (Figure 6b).

The OPG/RANK/RANKL system in human HF

Our findings suggest that the OPG/RANK/RA KL axis is activated within the myocardium in an experimental rat model of post-infarction HF. We then examined the possible relevance of these findings in human HF. First, when analysing serum levels of these mediators we found that HF patients (n=102) had significantly raised OPG compared to healthy controls (n=20) with increasing levels according to clinical severity (NYHA functional class), myocardial dysfunction (cardiac index) and neurohormonal activation (N-BNP)(Figure 7a). HF patients also had raised RANKL levels, but this increase seems to be restricted to those with the most severe heart failure (i.e., NYHA group IV; Figure 7a), significantly correlated to decreased cardiac index (r=-0.32, p<0.05). Second, when analysing the expression of these mediators by real-time quantitative RT-PCR in T-cells and monocytes from 13 HF patients and 9 healthy controls we found that T cells from HF patients had markedly enhanced gene expression of RANKL (Figure 7b). In contrast, there were no differences in RANK expression in T-cells or monocytes between CHF patients and controls (Figure 7b). The gene expression of RANKL in monocytes and OPG in T-cells and monocytes was too low to yield quantifiable results. Finally, when analysing OPG/RANK/RANKL expression in LV from end-stage HF patients we found enhanced mRNA levels for OPG, but not for RANK, in the failing compared with the non-failing myocardium (Figure 4a). Furthermore, strong RANK immunoreactivity was seen in cardiomyocytes, VSMC and endothelial cells from donor and explanted human hearts with particularly high protein expression in the failing myocardium (Figure 8b). OPG immunoreactivity was also fairly strong in cardiomyocytes, particularly in the failing LV (Figure 8b). Moreover, while OPG immunostaining was weak in VSMC and not detectable in endothelial cells of non-failing hearts, intense OPG immunoreactivity was seen in both VSMC and endothelial cells of failing human LV (Figure 8b). We were unable to detect RANKL in both failing and non-failing myocardium either by immunostaining or real-time quantitative RT-PCR analyses. For all analysis in human HF there were no significant differences between ischemic and idiopatic dilated cardiomyopathy (data not shown).

The effect of RANKL in neonatal rat cardiomyocytes

RANKL has been shown to induce the expression of matrix metalloproteinases (MMP) in osteoclasts (Wittrant et al., 2003) and an imbalance between MMPs and their endogenous tissue inhibitors (i.e., TIMPs) seems also to be of major importance for LV remodeling. (D'Armiento et a/., 2002; Spinale et a/., 2002) To map any possible pathogenic consequences of the enhanced RANK expression in cardiomyocytes during HF, we therefore examined the effect of RANKL on MMP/TIMP expression in neonatal rat cardiomyocytes by real-time quantitative RT-PCR. While RANKL induced a significant increase in MMP-2 and MMP-9 expression, there was no change in their inhibitors (i.e., TIMP-2 and TIMP-1), suggesting matrix degrading net effects (Figure 9). In contrast to MMPs, transforming growth factor (TGF) β induces the deposition of extracellular matrix proteins and has also been shown to inhibit MMP activity,(Seeland et al., 2002) and notably, RANKL was shown to reduce the gene expression of TGFβ in neonatal rat cardiomyocytes (Figure 9).

The effect of cardiovascular and immunomodulating therapy on serum OPG levels In human HF

Serum levels of OPG is regarded as a stable and reliable marker of the activity in the OPG/RANK/RANKL system. (Hofbauer et al., 2001) We therefore finally examined the ability of cardiovascular (i.e., β-blockers) and immunomodulating [(i.e., intravenous Ig (IVIg)] therapy to modulate serum OPG levels in human HF. Serum samples were obtained from 40 randomly selected HF patients from the MERIT-HF trial (Gullestad et al., 2001a) and from all 40 HF patients in our previously published IVIg trial (Gullestad et al., 2001b) While there was no difference between metoprolol and placebo in the MERIT-HF trial (10 months of therapy), IVIg induced a significant decrease in OPG levels compared with a slight increase in the placebo group resulting in a significant difference in changes (p<0.05, IVIg versus placebo after 6 months of therapy). Table 5. Clinical and hemodynamic characteristics of the heart failure population (n=102)

Age (years) 52±9

Sex (male), % 89

NYHA functional class (II / III / IV) (25/47/3

Cause of heart failure

Coronary artery disease (%) 54

Idiopathic dilated cardiomyopathy (%) 43

Duration of heart failure (years) 4±3

Left ventricular ejection fraction (%) 32±16

Pulmonary capillary wedge pressure 18±9

Cardiac index (l/min/m²) 2.1±0.6

Medication

ACE-inhibitors 79 β-blocker 40

Diuretics 87

Nitrate 24

Aldosterone antagonist 22

Statins 29

Digitalis 54

Data are presented as the mean value±SD or number of patients or subjects. NYHA, New York Heart Association; ACE, angiotensin converting enzyme; statins, hydroxymethylglutaryl coenzyme A reductase inhibitors. Hemodynamic parameters were available in 84 of the patients.

Table 6. Characteristics of real-time PCR assays in the study.

Target Sequence (5'→ 3') Ace. Nr.

Human OPG (+)-AATCAACTCAAAAATGTGGAATAGATGT U94332 (-)-GCGTAAACTTTGTAGGAACAGCAA

RANK (+)-CCCGTTGCAGCTCAACAAG NM_003839 (-)-GCATTTGTCCGTGGAGGAA

RANKL (+)-GTGCAAAAGGAATTACAACATATCGT NM_003701 -)-AACCATGAGCCATCCACCAT β-actin +)-AGGCACCAGGGCGTGAT NM_001101 -)-TCGTCCCAGTTGGTGACGAT

Rat OPG +)-GCCAACACTGATGGAGCAGAT NM_012870 -)-TCTTCATTCCCACCAACTGATG

RANK +)-CGTCCTGCTCCTCTTCATCTCT XM_219402 -)-CCCTGAGGACTCCTTATTTCCA

RANKL +)-GCTCACCTCACCATCAATGCT NM_057149 -)-GGTACCAAGAGGACAGACTGACTTTA

MMP2 +)-TGGGACAAGAACCAGATCACATA X71466 -)-GCCCGAGCAAAAGCATCAT

MMP9 +)-GACCAAGGGTACAGCCTGTTTC U24441 -) -CCGGCACTGAAGAATGATCTAAG

TIMP1 +)-AGGGCTACCAGAGCGATCACT U06179 )-AAGGTATTGCCAGGTGCACAA

TIMP2 +)-GTTTTGCAATGCAGACGTAGTGA NM_021989 -)-CCATAGATGTCATTCCCGGAAT

TGFβl +)-AAGAAGTCACCCGCGTGCTA NM 021578 -)-TGTGTGATGTCTTTGGTπTGTCA

(+), forward primers; (-), reverse primers; Acc.nr., accession number in

GenBank. MMP, matrix metalloproteinases; TIMP, tissue inhibitor of metalloproteinases; TGF, transforming growth factor.

References

WO 01/74896

WO 01/23562

US2002 0061521

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WEB-sites: http://www.pointofcare.net/vendors/devices.htm - point-of-care instruments

Claims

Claims

1. An assay for the prognosis of a cardiovascular disease in a mammal, said assay comprises the following steps: (i) providing a sample obtained from said mammal,

(ii) measuring the concentration of Osteoprotegrin (OPG) in the sample, and

(iii) evaluating the concentration of Osteoprotegrin (OPG) measured in step (ii) relative to a reference value for the prediction of the clinical outcome, complications and mortality of said mammal wherein the cardiovascular disease is based on reduced cardiac muscle function in connection to heart failure independent of ischemia.

2. The assay according to claim 1, wherein the cardiovascular disease is related to acute myocardial infarction.

3. The assay according to claim 1, wherein the cardiovascular disease is cardiomyopathy.

4. The assay according to claim 1, wherein the cardiovascular disease is valve disease due to infection.

5. The assay according to claim 1, wherein the cardiovascular disease is heart failure independent of aetiology

6. The assay according to any of the preceding claims, wherein the sample is selected from the group consisting of urine, serum, blood, plasma, milk, saliva, cerebrospinal fluid (CSF), other secreted fluids, substances and tissue biopsies from organs such as the brain, heart and intestine.

7. The assay according to any of the preceding claims, wherein the measurement of the concentration of Osteoprotegrin (OPG) in the sample is provided by means selected from the group consisting of an assay, a stick, a dry-stick, an electrical device, an electrode, a reader, histochemistry, and similar means.

8. The assay according to any of the preceding claims, wherein the assay is selected from the group consisting of a bioassay, an immunoassay, a microbiological assay, a radioassay, and similar assays.

9. The assay according to any of the preceding claims, wherein the immunoassay is selected from the group consisting of a enzyme immunoassay, a radioimmunoassay, a flouroimmunoassay, a metalloimmunoassay, a spin immunoassay, an enzyme-linked 5 immunosorbent assay, an immunohistochemistry and an enzyme immunoassay.

10. The assay according to any of the preceding claims, wherein the reference value is an internal reference means and/or an external reference means.

10 11. The assay according to any of the preceding claims, wherein the external reference value is selected from the group consisting of a table, a diagram and similar reference value.

12. The assay according to any of the preceding claims, wherein the internal reference

15 value is provided in connection to a device used for the determination of the concentration of Osteoprotegrin (OPG).

13. Osteoprotegrin (OPG), wherein the device is selected from the group consisting of an assay, a stick, a dry-stick, an electrical device, an electrode, a reader, and similar devices.

20 14. The assay according to any of the preceding claims, wherein the concentration of Osteoprotegrin measured in the sample is useful for providing a prognosis of a cardiovascular mortality in a human having a cardiovascular disease.

25 15. The assay according to any of the preceding claims, wherein the prediction of the clinical outcome, complications and mortality of said human relates to elevated levels of OPG.

16. The assay according to any of the preceding claims, wherein elevated levels of OPG 30 relates to OPG levels >1.75 ng/mL.

17. A method for the prognosis of a cardiovascular disease based on reduced cardiac muscle function in connection to heart failure independent of ischemia in a mammal, said method comprises the steps of:

35 (i) providing a sample obtained from a mammal, and

(ii) measuring the concentration of Osteoprotegrin (OPG) in the sample. (iii) evaluating the concentration of Osteoprotegrin (OPG) measured in step (ii) relative to a reference value for the prediction of the clinical outcome, the complications and the mortality of a mammal.

5 18. Use of data obtained from the detection of Osteoprotegrin (OPG) in a sample obtained from a human for the prognosis of a cardiovascular disease based on reduced cardiac muscle function in connection to heart failure independent of ischemia in said human.

10 19. A kit for use in an assay as defined in any one of claims 1-16.

20. A method for evaluating the effect of pharmacological or non-pharmacological intervention in patients with a cardiovascular disease based on reduced cardiac muscle function in connection to heart failure independent of ischemia comprising monitoring the 15 Osteoprotegrin (OPG) level in a subject.