Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders
  • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

• This invention relates to the field of myocardial disorders. It discloses that antibodies to a cardiac troponin found in a sample obtained from an individual can be used as a diagnostic marker, especially in the assessment of an individual's risk of developing a myocardial disorder.
• a method aiding in the assessment of an individual's risk of developing a myocardial disorder comprising measuring in vitro antibodies to a cardiac troponin and optionally one or more other marker useful in assessing an individual's risk of developing a myocardial disorder, and correlating the value or the values obtained to the individual's risk of developing a myocardial disorder is described.
• myocardial disease Despite significant advances in therapy, myocardial disease (CVD) remains the single most common cause of morbidity and mortality in the developed world. Thus, prevention of myocardial disorders such as heart failure, myocardial infarction and stroke is an area of major public health importance.
• CVD myocardial disease
• risk factors for future myocardial disorders have been described and are currently in wide clinical use in the detection of individuals at high risk.
• screening tests include for example evaluations of total cholesterol level, of LDL cholesterol level, of HDL cholesterol level and the level of C-reactive protein.
• a large number of myocardial disorders occur in individuals with apparently low to moderate risk profiles, and the diagnostic options to identify such patients is still limited.
• Individuals diagnosed as suffering from an underlying coronary heart disease can be divided into individuals showing no clinical symptoms and those which appear with breathlessness and/or chest pain.
• the latter group can be divided into individuals having stable angina pectoris (SAP) and those with acute coronary syndromes (ACS).
• ACS patients can show unstable angina pectoris (UAP), or these individuals have already suffered from a myocardial infarction (MI).
• MI myocardial infarction
• LVD left ventricular dysfunction
• CHF congestive heart failure
• the heart is a unique organ. This is also true for the heart tissue and many of its constituents.
• the detection of such cardiac specific markers forms the basis of diagnostic means in the fields of myocardial infarction and congestive heart failure.
• an acute MI can be diagnosed with high sensitivity and at high specificity by measuring the level of a cardiac troponin in the circulation. Severe and acute stages of congestive heart failure can be diagnosed by measuring e.g. brain derived natriuretic peptide (BNP) or its N-terminal propeptide (NT-proBNP).
• BNP brain derived natriuretic peptide
• NT-proBNP N-terminal propeptide
• antibodies to a cardiac troponin can be used as a diagnostic marker, especially in the field of myocardial disorders.
• Antibodies to a cardiac troponin either alone or optionally in combination with one or more other marker of cardiovascular risk are valuable in the assessment of an individual's risk of developing a myocardial disorder.
• This invention describes new diagnostic tests that determine and utilize the presence, absence or level of antibodies to a cardiac troponin in the assessment of a myocardial disorder.
• the present invention relates to method aiding in the assessment of an individual's risk of developing a myocardial disorder, comprising the steps of a) measuring in vitro antibodies to a cardiac troponin and optionally one or more other marker useful in assessing an individual's risk of developing a myocardial disorder, and b) correlating the value or the values obtained in (a) to the individual's risk of developing a myocardial disorder
• new tests broadly include (1) the assessment of risk of a future myocardial disorder such as for example myocardial infarction and congestive heart failure, and (2) the determination of the likelihood that certain individuals will benefit to a greater or lesser extent from the use of certain treatments designed to prevent and/or treat a myocardial disorder.
• the present invention also discloses a kit comprising a cardiac troponin and auxiliary reagents appropriate for measurement of antibodies to said cardiac troponin.
• the present invention relates to the use of antibodies to a cardiac troponin as a diagnostic marker.
• the skilled artisan is aware of different methods that may be used in the determination of antibodies as present in an individual's sample.
• the diagnostic field in which antibodies as present in a patient's sample are determined is called serology.
• the detection of an antibody comprised in a patient's sample is for example very important in the diagnosis of an infectious disease or of an autoimmune disease.
• DCM Dilated cardiomyopathy
• the inventors of the present invention have now surprisingly found that the presence and/or level of antibodies against a cardiac troponin as determined in a patient's sample is of diagnostic utility. They can e.g. be used as a marker in the assessment of an individual's risk of developing a myocardial disorder.
• a marker means one marker or more than one marker.
• an antibody and “antibodies” to a cardiac troponin is considered interchangeable, because, as the skilled artisan will appreciate it is always many antibodies which are detected.
• a "cardiac troponin” is a troponin that is present in heart tissue and not present at all or not present to any relevant extend in tissue other than heart. By way of example for human beings two cardiac specific troponins have been described.
• troponin T human cardiac specific troponins
• troponin I human cardiac specific troponins
• Troponin T has a molecular weight of about 37.000 Da.
• the troponin T isoform that is found in cardiac tissue (cTnT) is sufficiently divergent from skeletal muscle TnT to allow for the production of antibodies that distinguish both these TnT isoforms.
• TnT is considered a marker of acute myocardial damage; cf. Katus, H.A., et al., J. MoI. Cell. Cardiol. 21 (1989) 1349-1353; Hamm, C.W., et al., N. Engl. J. Med. 327 (1992) 146-150; Ohman, E.M., et al., N. Engl. J. Med. 335 (1996) 1333- 1341; Christenson, R.H., et al., Clin. Chem. 44 (1998) 494-501; and EP 0 394 819.
• Troponin I is a 25 IcDa inhibitory element of the troponin complex, found in muscle tissue. TnI binds to actin in the absence of Ca 2+ , inhibiting the ATPase activity of actomyosin.
• the TnI isoform that is found in cardiac tissue (cTnl) is
• cTnl 40% divergent from skeletal muscle TnI, allowing both isoforms to be immunologically distinguished.
• the normal plasma concentration of cTnl is ⁇ 0.1 ng/ml (4 pM).
• cTnl is released into the bloodstream following cardiac cell death; thus, the plasma cTnl concentration is elevated in patients with acute myocardial infarction (Benamer, H., et al., Am. J. Cardiol. 82 (1998) 845-850).
• the present invention relates to a method aiding in the assessment of an individual's risk of developing a myocardial disorder, comprising: a) measuring in vitro antibodies to a cardiac troponin and optionally one or more other marker useful in assessing an individual's risk of developing a myocardial disorder, and b) correlating the value or the values obtained in (a) to the individual's risk of developing a myocardial disorder.
• the method according to the present invention will "aid in the assessment" of an individual's risk of developing a myocardial disorder.
• no biochemical marker is diagnostic with 100% specificity and at the same time 100% sensitivity for a given disease. Rather, biochemical markers are used to assess with a certain likelihood or predictive value the presence, absence or severity of a disease. Therefore, in routine clinical diagnosis various clinical symptoms and biological markers are generally considered together in the diagnosis, treatment, and management of the underlying disease.
• the measurement of antibodies to a cardiac troponin will aid the physician in his task of establishing the correct diagnosis or prognosis. The final diagnosis or prognosis is always made by the physician.
• myocardial disorder or “myocardial disorders” relate to a group of disorders affecting the heart muscle.
• a preferred group of myocardial disorders consists of atherosclerosis, congestive heart failure, acute coronary syndrome including myocardial infarction and unstable angina.
• the myocardial disorder assessed in a method according to the present invention is selected from the group consisting of congestive heart failure and acute coronary syndrome.
• myocardial disorder in the sense of the present invention relates to the graft rejection in patients after heart transplantation.
• the present invention relates to a method aiding in the assessment of the risk for overt heart failure for a patient with myocardial infarction, comprising: a) measuring in vitro antibodies to a cardiac troponin and optionally one or more other marker useful in assessing an individual's risk of developing a myocardial disorder, and b) correlating the value or the values obtained in (a) to the individual's risk of developing overt heart failure.
• the patient with MI may have suffered from heart failure before the MI occurred in this case the method according to the present invention may aid in assessing the risk of disease deterioration.
• stages A and B the individuals at risk of developing heart failure are found, whereas stages C and D represent the groups of patients showing signs and symptoms of heart failure. Details for defining the different stages A through D as given in the above reference are hereby included.
• the disease is clinically asymptomatic in the compensatory and early decompensatory phases (completely asymptomatic in stage stage A and with structural heart disease but no signs and symptoms of HF in stage B, cf. the ACC/AHA practice guidelines).
• Outward signs of the disease (such as shortness of breath) do not appear until well into the decompensatory phase (i.e., stages C and D according to the ACC/AHA guidelines).
• Current diagnosis is based on the outward symptoms of patients in stages C and D.
• Overt heart failure in the sense of the present invention is heart failure in the stages C or D as defined by the ACC/AHA guidelines.
• the antibody to a cardiac troponin is measured "in vitro". This means that a sample is obtained from an individual for diagnostic purposes. This sample is used for one or several in vitro investigations and not for treatment of said individual. Preferred samples are cardiac tissue biopsy, whole blood, plasma, or serum, especially preferred are plasma and serum.
• the cardiac troponin antigen is directly or indirectly bound to a solid phase. Usually the sample is diluted in a sample buffer. The solid phase bound antigen is incubated with the (diluted) sample under investigation. Incubation is performed under conditions permissive for binding of an antibody comprised in the sample under investigation to the solid phase bound antigen. The antibody attached to the solid phase bound antigen is detected by appropriate means.
• antibody detection systems according to the double antigen bridge format, e.g., described in US 4,945,042, are used.
• the same assay principle can be used to detect antibodies to a cardiac troponin.
• the immunoassays according to this bridge concept require the use of an antigen directly or indirectly bound to a solid phase and of the same or a cross-reactive readily soluble antigen that is directly or indirectly detectable.
• the antibodies under investigation, if present, form a bridge between the solid phase bound antigen and the labeled detection antigen. Only if the two antigens are bridged by specific antibodies - e.g., by antibodies to a cardiac troponin - a signal is generated which is correlated to the concentration of antibodies present in the sample.
• the cardiac troponin antigen used in a method according to the present invention in one preferred embodiment comprises troponin I and troponin T. It is also preferred to set up assays for the detection of antibodies to either troponin I, or troponin T, respectively. In the latter assays each cardiac troponin is individually used as an antigen. In a preferred embodiment the antibodies measured in a method according to the present invention are antibodies to troponin I.
• a troponin from skeletal muscle is added to the sample buffer in order to enhance specificity of antibody binding to a cardiac troponin by blocking unspecific antibodies, i.e. antibodies cross-reacting between a muscle and a cardiac troponin.
• test result may be recorded in qualitative and in quantitative terms.
• the invention involves comparing the level of marker for the individual with a predetermined value.
• the predetermined value can take a variety of forms. It can be single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as where the risk in one defined group is double the risk in another defined group. It can be a range, for example, where the tested population is divided equally (or unequally) into groups, such as-a low-risk group, a medium-risk group and a high-risk group, or into quadrants, the lowest quadrant being individuals with the lowest risk and the highest quadrant being individuals with the highest risk.
• the predetermined value can depend upon the particular population selected. For example, an apparently healthy population will have a different v normal v range of markers than will a population the members of which have had a prior myocardial disorder. Accordingly, the predetermined values selected may take into account the category in which an individual falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
• a positive result may e.g. be recorded if the antibodies measured are above a predetermined threshold level.
• a predetermined threshold level usually is set to the 90 %- percentile or to the 95%-percentile of a healthy control population.
• a threshold level at the 95%-percentile of a healthy control population is preferred when practicing this invention. Quantitative values can easily be correlated to a disease state by methods that need not to be explained to the man skilled in the art.
• the autoantibodies or briefly antibodies to a cardiac troponin may be of different immunoglobin classes.
• first antibodies of the immunoglobin class M (IgM) are formed.
• the first humoral immune response in form of IgM is followed by a second humoral immune response, reflected by a more or less pronounced formation of antibodies of the immunoglobin class G (IgG).
• IgG immunoglobin class G
• the antibodies to a cardiac troponin as present in a patient's sample may comprise antibodies of the IgG as well as of the IgM class of immunoglobin. It may well be that different classes of antibodies to a cardiac troponin are indicative for different subsets of patients.
• the method according to the present invention is based on antibodies to a cardiac troponin that are of both the immunoglobin classes G, and M.
• a high titer in antibodies to a cardiac troponin may be considered indicative for a higher risk of further cardiac complications.
• the method according to the present invention is based on the detection of antibodies to a cardiac troponin that are of immunoglobin class M (IgM).
• IgM immunoglobin class M
• a high titer in IgM antibodies may be considered indicative for a more recent necrotic event at the heart muscle.
• a high titer of IgM antibodies may indicate a treatment more suited for acute events at the heart.
• the method according to the present invention is based on antibodies to a cardiac troponin that are of immunoglobin class G (IgG).
• IgG immunoglobin class G
• a high titer in IgG antibodies may be considered indicative for at least one necrotic event at the heart muscle in the past. Such event in the past has most likely occurred at least four weeks before the sample has been obtained.
• a high titer of IgG antibodies may also indicate a severe and/or several necrotic episodes and may point to a mode of a treatment more suited for past and/or chronic events at the heart.
• said individual can be stratified for one or more modes of therapeutic treatment.
• These can be selected from antibodies (monoclonal antibodies, polyclonal antibodies), small molecules, pharmacologically active compounds, i.e. antiinflammatory and lipid-lowering drugs (e.g. statins), thrombolytic drugs (e.g. platelet antagonists), fibrinolytic drugs (e.g. heparin), revascularization therapy (e.g. PCTI (percutaneous therapeutic intervention), balloon dilatation, stenting, by- pass surgery).
• pharmacologically active compounds i.e. antiinflammatory and lipid-lowering drugs (e.g. statins), thrombolytic drugs (e.g. platelet antagonists), fibrinolytic drugs (e.g. heparin), revascularization therapy (e.g. PCTI (percutaneous therapeutic intervention), balloon dilatation, stenting, by- pass surgery).
• Agents for reducing the risk of a myocardial disorder include those selected from the group consisting of anti-inflammatory agents, anti-thrombotic agents and/or fibrinolytic agents, anti-platelet agents, lipid reducing agents, direct thrombin inhibitors, and glycoprotein II b/IIIa receptor inhibitors and agents that bind to cellular adhesion molecules and inhibit the ability of white blood cells to attach to such molecules (e.g. anti-cellular adhesion molecule antibodies).
• Anti-inflammatory agents include Alclofenac; Alclometasone Dipropionate;
• Clobetasol Propionate Clobetasone Butyrate; Clopirac; Cloticasone Propionate;
• Fenclorac Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin
• Piconol Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole
• Meclofenamic Acid Meclorisone Dibutyrate
• Mefenamic Acid Meclorisone Dibutyrate
• Mefenamic Acid Meclorisone Dibutyrate
• Mefenamic Acid Meclorisone Dibutyrate
• Mefenamic Acid Meclorisone Dibutyrate
• Mefenamic Acid Meclorisone Dibutyrate
• Mefenamic Acid Meclorisone Dibutyrate
• Prodolic Acid Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit;
• Salcolex Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate;
• Tebufelone Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide;
• Tetrydamine Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium;
• Triclonide Triflumidate; Zidometacin; Glucocorticoids; Zomepirac Sodium.
• Anti-thrombotic and/or fibrinolytic agents include Plasminogen (to plasmin via interactions of prekallikrein, kininogens, Factors XII, XIIIa, plasminogen proactivator, and tissue plasminogen activator [TPA]) Streptokinase; Urokinase: Anisoylated Plasminogen-Streptokinase Activator Complex; Pro-Urokinase; (Pro- UK); rTPA (alteplase or activase; r denotes recombinant), rPro-UK; Abbokinase; Eminase; Sreptase Anagrelide Hydrochloride; Bivalirudin; Dalteparin Sodium; Danaparoid Sodium; Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin Sodium; Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; retaplase
• Anti-platelet agents include Clopridogrel; Sulfinpyrazone; Aspirin; Dipyridamole; Clofibrate; Pyridinol Carbamate; PGE; Glucagon; Antiserotonin drugs; Caffeine; Theophyllin Pentoxifyllin; Ticlopidine; Anagrelide.
• Lipid reducing agents include gemfibrozil, cholystyramine, colestipol, nicotinic acid, probucol lovastatin, fluvastatin, simvastatin, atorvastatin, pravastatin, cirivastatin.
• Direct thrombin inhibitors include hirudin, hirugen, hirulog, agatroban, PPACK, thrombin aptamers.
• Glycoprotein Ilb/IIIa receptor Inhibitors are both antibodies and non-antibodies, and include but are not limited to ReoPro (abciximab), lamifiban, tirofiban.
• One preferred agent which may be used to reduce the risk of a future cardiac disorder in an individual testing positive for antibodies to a cardiac troponin is aspirin.
• the method according to the present invention also may permit a therapeutic treatment monitoring of the individual which is treated by said regimen.
• antibodies to a cardiac troponin have a predictive value independent of other markers used in assessing an individual's risk of developing a myocardial disorder.
• the invention relates to a method aiding in the assessment of an individual's risk of developing a myocardial disorder, comprising: a) measuring in vitro antibodies to a cardiac troponin and one or more other marker useful in assessing an individual's risk of developing a myocardial disorder, and b) correlating the values obtained in (a) to the individual's risk of developing a myocardial disorder.
• the one or more additional marker used together with an antibody to a cardiac troponin may be considered to be part of a marker panel for assessing an individual's risk of developing a myocardial disorder, i.e., a series of markers appropriate to further refine the risk assessment.
• the total number of markers in such marker panel is preferably less than 20 markers, more preferred less than 15 markers, also preferred are less than 10 markers, with 8 or less markers being even more preferred.
• marker panels for assessing an individual's risk of developing a myocardial disorder comprising 2, 3, 4, 5, or 6 markers in addition an antibody to a cardiac troponin.
• the one or more other marker will be selected from the group consisting of a cardiac troponin, a natriuretic peptide or a natriuretic peptide-related marker, an inflammation marker, D-dimer, cholesterol, homocysteine, adiponectin, sCD40L, myeloperoxidase, and ischemia modified albumin.
• a “marker” is a molecule or feature whose absence, presence or level can be correlated to a status of interest, e.g., to a disease.
• the natriuretic peptide preferably is selected from A-type natriuretic peptide (ANP) and/or B-type natriuretic peptide (BNP).
• related marker refers to one or more polypeptide fragments having at least 10, 12, 15 or 20 contiguous amino acids of a particular marker or its biosynthetic parent. Preferably said fragment is an immunologically detectable fragment as present in the circulation.
• a natriuretic peptide related marker preferably is either an ANP-related or a BNP-related marker.
• BNP B-type natriuretic peptide
• BNP M o 8 Human BNP is derived by proteolysis of a 108 amino acid precursor molecule, referred to hereinafter as BNP M o 8 .
• Mature BNP, or "the BNP natriuretic peptide," or “BNP-32” or simply "BNP” is a 32 amino acid molecule representing amino acids 77-108 of this precursor, which may also be referred to as BNP 77 . 108 .
• the remaining residues 1-76 of the BNP precursor molecule are known in the art as N- terminal proBNP (NT-proBNP).
• BNP MO8 is synthesized as part of an even larger precursor, the pre-pro-BNP, having 134 amino acids in total of which the N-terminal 26 represent the "pre- "sequence.
• Mature BNP itself may be used as an additional marker in the present invention.
• the prepro-BNP, BNP I-108 and NT-proBNP molecules all represent BNP-related markers that may be measured either as surrogates for mature BNP or as markers in and of themselves.
• one or more fragments of these molecules including BNP-related polypeptides or markers selected from the group consisting Of BNP 77 -I 06 , BNP 79-106 , BNP 76 -I 07 , BNP 69 -I 08 , BNP 79 . 108 , BNP 80 -I 08 , BNP 81 . 108 , BNP 83 .
• natriuretic peptide fragments may comprise one or more oxidizable methionines, the oxidation of which to methionine sulfoxide or methionine sulfone produces additional BNP- related markers. See, e.g., US Application Serial No. 10/419,059, filed Apr. 17, 2003, which is hereby incorporated by reference in its entirety.
• markers described herein below are synthesized as larger precursor molecules, which are then processed to provide the mature molecule or marker; and/or are present in circulation in the form of fragments and/or a marker molecule carrying secondary modifications.
• a "related markers" to each of the markers described herein below may be identified and used in an analogous fashion to that described above for BNP.
• A-type natriuretic peptide (ANP) A-type natriuretic peptide (ANP)
• A-type natriuretic peptide (also referred to as atrial natriuretic peptide or cardiodilatin Forssmann, W.-G., et al., Histochem. Cell Biol. 110 (1998) 335-357) is a 28 amino acid peptide that is synthesized, stored, and released by atrial myocytes in response to atrial distension, angiotensin II stimulation, endothelin, and sympathetic stimulation (beta-adrenoceptor mediated).
• ANP is synthesized as a precursor molecule (pro-ANP) that is converted to an active form, ANP, by proteolytic cleavage and also forming N-terminal ANP (1-98).
• N-terminal ANP and ANP have been reported to increase in patients exhibiting atrial fibrillation and heart failure (Rossi, A., et al., J. Am. Coll. Cardiol. 35 (2000) 1256-1262). As the skilled artisan will recognize, however, because of its relationship to ANP, the concentration of N-terminal ANP molecule can also provide diagnostic or prognostic information in patients.
• the phrases "marker related to ANP” or "ANP related peptide” refer to any polypeptide of at least 10, 12, 15 or 20 contiguous amino acids that originates from the pro-ANP molecule (1-126), other than the 28- amino acid ANP molecule itself. Proteolytic degradation of ANP and of peptides related to ANP have also been described in the literature and these naturally occurring proteolytic fragments are also encompassed it the term "ANP related peptides.”
• the two cardiac specific troponins i.e. troponin I, and troponin T, respectively, have been exemplified above.
• troponin I troponin I
• troponin T troponin T
• the cardiac troponin used as a further marker or analyte in a marker panel is the molecule itself.
• cardiac troponin I and/or cardiac troponin T are measured according to state of the art procedures and the values measured are combined with the result of a measurement for antibodies to a cardiac troponin and used in the assessment of a cardiac disorder.
• the presence of both of antibodies to a cardiac troponin and of a cardiac troponin may be further indicative for a recurring disease with acute coronary complications, like ACS.
• Preferred inflammation markers for use in a marker panel according to the present invention together with antibodies to a cardiac troponin are markers of acute inflammation and so-called proximal inflammatory markers.
• Acute inflammatory markers known to the person skilled in the art include C- reactive protein (CRP), fibrinogen, D-dimer, serum amyloid A (SAA), pregnancy- associated polypeptide A (PAPP-A), intercellular adhesion molecules (e.g.
• Preferred markers of acute inflammation for use in a method according to the present invention are CRP, fibrinogen, D-dimer and SAA, of which CRP and D- dimer are more preferably used.
• CRP C-reactive protein
• CRP C-reactive protein
• CRP synthesis is induced by IL-6, and indirectly by IL-I, since IL-I can trigger the synthesis of IL-6 by Kupffer cells in the hepatic sinusoids.
• the normal plasma concentration of CRP is ⁇ 3 ⁇ g/ml (30 nM) in 90% of the healthy population, and ⁇ 10 ⁇ g/ml (100 nM) in 99% of healthy individuals.
• Plasma CRP concentrations can, e.g. be measured by homogeneous assay formats or ELISA.
• C-reactive protein is considered a marker for ongoing systemic inflammation.
• CRP can be measured with very high sensitivity and CRP-values in the range of between 1 and 3 mg/1 of blood can be reliably detected.
• a measurement in that range is called a measurement of high- sensitive CRP or hs-CRP, which also is preferably used in a method according to the present invention.
• Fibrinogen also called Factor I
• Fibrinogen is a 340 kD protein encoded on chromosome 4 and synthesized by hepatocytes. It is composed of two identical subunits, each containing three dissimilar polypeptide chains (alphaA, betaB, gammaG) which are linked by disulphide bonds. Thrombin cleaves fibrinopeptides A and B from fibrinogen, resulting in the formation of strands of insoluble fibrin monomer which consists of three paired alpha, beta and gamma chains.
• Dysfibrinogenaemia is a condition associated with production of structurally abnormal fibrinogen. More than 250 structural variants have been described which are associated with a bleeding tendency (Ebert, R.
• fibrinogen F., CRC Press, Boca Raton, 1991. Most of these variants exhibit impaired thrombin-catalyzed release of fibrinopeptides, or impaired fibrin polymerization. Some variants of fibrinogen are associated with a thrombotic tendency rather than a bleeding tendency, and this has been attributed to impaired binding of plasminogen or tissue plasminogen activator to the abnormal fibrinogen molecule. Elevated levels of fibrinogen may be indicative for an ongoing infection or inflammation.
• D-dimer is a crosslinked fibrin degradation product with an approximate molecular mass of 200 kDa.
• the normal plasma concentration of D-dimer is ⁇ 150 ng/ml (750 pM).
• the plasma concentration of D-dimer is elevated in patients with acute myocardial infarction and unstable angina, but not in stable angina (Hoffmeister, H.M., et al., Circulation 91 (1995) 2520-2527).
• the plasma concentration of D- dimer also will be elevated during any condition associated with coagulation and fibrinolysis activation, including stroke, surgery, atherosclerosis, trauma, and thrombotic thrombocytopenic purpura.
• D-dimer is released into the bloodstream immediately following proteolytic clot dissolution by plasmin.
• the plasma concentration of D-dimer can exceed 2 ⁇ g/ml in patients with unstable angina
• Plasma D-dimer is a specific marker of fibrinolysis and indicates the presence of a prothrombotic state associated with acute myocardial infarction and unstable angina.
• Proximal inflammatory markers are macromolecules situated upstream, i.e. close to or at the ethiopathogenetic origin of the disease event. In particular, they are produced at the site of the coronary heart lesion, preferably at the site of an arterial plaque. Proximal inflammatory markers are in particular associated with the risk that plaques already present in an individual will undergo inflammation, or growth, and with the probability of plaque rupture and thrombus formation.
• Proximal inflammatory markers are known to the person skilled in the art, and non-limiting examples include pregnancy-associated polypeptide A (PAPP-A), matrix metalloproteinases (MMPs, e.g. MMP-I, -2, -3, -4, -5, -6, -7, -9, -10, -11, - 12) and lipoprotein-associated phospholipase A2 (Lp-PLA2).
• PAPP-A pregnancy-associated polypeptide A
• MMPs matrix metalloproteinases
• Lp-PLA2 lipoprotein-associated phospholipase A2
• the preferred proximal inflammatory markers are PAPP-A, MMP-9 and Lp-PLA2.
• the most preferred proximal inflammatory markers are PAPP-A and Lp-PLA2, in particular PAPP-A.
• the pregnancy-associated plasma protein-A belongs to the metzincin superfamily of zinc metalloproteinases. The molecular weight of PAPP-A is 187 kDa.
• Human pregnancy associated plasma protein A PAPP-A cleaves insulin- like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and IGF-II.
• IGFBP-4 insulin-like growth factor binding protein-4
• PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system.
• a recent study shows that PAPP-A may also be a new candidate marker of acute coronary syndromes (Bayes-Genis, A., N. Engl. J. Med. 345 (2001) 1022-1029). The data in this study showed that PAPP-A levels are significantly elevated in patients with unstable angina or acute myocardial infarction when compared to patients with stable angina and control subjects.
• Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 )
• Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) is a 50 kDa, Ca-insensitive lipase which is produced predominantly by macrophages. This enzyme resides mainly on low density lipoprotein (LDL) in human plasma. It is distinct from secretory phospholipase A 2 (sPLA 2 ). The levels Of Lp-PLA 2 are not affected by acute systemic inflammatory conditions. Clinical studies have demonstrated that Lp-PLA 2
• PLA 2 is related to atherosclerosis. Elevated plasma levels have been also found to correlate with CHD and ischemic stroke risk. In pre-clinical animal studies, inhibition of the enzyme attenuates the inflammatory process and slows down atherosclerotic disease progression.
• the concentration of circulating total homocysteine is a sensitive marker of inadequate folate and vitamin B 12 status. Elevated homocysteine concentrations are associated with an increased risk for vascular disease. Reference ranges (5th and 95th percentiles) for the total homocysteine concentration have been recently determined (Selhub, J., et al, Ann. Intern. Med. 131 (1999) 331-339). A high total homocysteine concentration was defined as one that exceeded the sex-specific 95th percentile for the reference sample.
• Reference ranges for serum total homocysteine concentration are age-dependent; these ranges are 4.3 to 9.9 micromole/L for male participants and 3.3 to 7.2 micromole/L for female participants 12 to 19 years of age and from 5.9 to 15.3 micromole/L for men and 4.9 to 11.6 micromole/L for women 60 years of age or older.
• a high homocysteine concentration was defined as at least 11.4 micromole/L for male participants and at least 10.4 micromole/L for female participants.
• Adiponectin is a protein of 226 amino acids which is produced mainly by adipocytes. The level of adiponectin appears to reflect insulin sensitivity and to link fat storage and arteriosclerosis. With regard to clinical utility several different intended uses are in discussion and/or under investigation. US 6,461,821 describes and claims the use of adiponectin as a marker for arthrosclerosis.
• the sCD40L has been supposed to be a marker of inflammation (Aukrust, P., et al.,
• sCD40L has been described as a systemic marker of inflammation. Recently sCD40L has also been discussed and described as a candidate marker for myocardial disorders. Heeschen, C, et al., N. Engl. J. Med. 348 (2003) 1104-1111, indicate that sCD40L might be used as a marker in acute coronary syndrome.
• sCD40L is in particular associated with platelet activation, platelet aggregation and thrombus propagation, representative of the risks that plaque having already become vulnerable will rupture, resulting in reversible vascular occlusion (UAP) or irreversible vascular occlusion (AMI) which may lead to left ventricular dysfunction (LVD), congestive heart failure (CHF) and death.
• UAP reversible vascular occlusion
• AMI irreversible vascular occlusion
• LPD left ventricular dysfunction
• CHF congestive heart failure
• Cholesterol at the same time is a steroid, a lipid, and an alcohol. It is found in the cell membranes of all body tissues, and transported in the blood plasma of all animals. Most cholesterol is not dietary in origin, it is synthesized internally. Cholesterol plays a central role in many biochemical processes, but is best known for its association with myocardial disease. Cholesterol travels through the blood in vesicles wherein it is attached to a protein. This cholesterol-protein package is called a lipoprotein. Lipoproteins are either high density or low density, depending on how much protein they have in relation to fat. Lipoproteins with more protein than fat are called high-density lipoproteins (HDL).
• HDL high-density lipoproteins
• LDL low-density lipoproteins
• High-density lipoprotein cholesterol is sometimes called "good” cholesterol.
• HDL cholesterol helps to remove LDL cholesterol from the body by binding with it in the bloodstream and carrying it back to the liver for disposal. A high level of HDL cholesterol appears to lower your risk of developing heart disease and stroke.
• Low-density lipoprotein cholesterol is sometimes called "bad” cholesterol.
• LDL cholesterol collects inside the walls of the arteries and often contributes to the plaque formation. LDL cholesterol is calculated from the total cholesterol, HDL, and triglyceride levels.
• LDL cholesterol or the ratio of HDL to LDL is determined and used as part of a marker panel in order to assess an individual's risk of developing a myocardial disorder.
• Myeloperoxidase is a lysosomal enzyme that is found in white blood cells, neutrophils.
• Myeloperoxidase is an enzyme that uses hydrogen peroxide to convert chloride to hypochlorous acid. The produced hypochlorous acid reacts with and destroys bacteria.
• neutrophils are also causing tissue damage.
• Myeloperoxidase is also produced when arteries are inflamed and have rupture-prone fatty deposits.
• Myeloperoxidase is considered a promising cardiac marker. By measuring the myeloperoxidase level in blood it is possible to predict whether a person is in risk of heart attack or death in the following six months (Baldus S., et al., Circulation 108 (2003) 1440-1445).
• Placenta growth factor (PlGF)
• Placenta growth factor is a polypeptide growth factor and a member of the platelet- derived growth factor family but more related to vascular endothelial growth factor (VEGF).
• PlGF-I acts only as a very weak mitogen for some endothelial cell types and as a potent chemoattractant for monocytes. The physiological function in vivo is still controversy. In several reports it was shown not to be a potent mitogen for endotehlial cells and not angiogenic in vivo by using different assays. Very recently it was shown by one investigator, that PlGF-I from cell culture supernatants was angiogenic in the CAM assay and in the rabbit cornea assay. Two different proteins can be generated by differential splicing of the human
• PlGF gene PlGF-I (131 aa native chain) and P1GF-2 (152 aa native chain). Both mitogens are secretable proteins, but P1GF-2 can bind to heparin with high affinity.
• PlGF-I is a homodimer, but preparations of PlGF show some heterogeneity on SDS gels depending of the varying degrees of glycosylation. All dimeric forms posses a similar biological profile. There is good evidence that heterodimeric molecules between VEGF and PlGF exists and that they are biological active.
• a protein related of PlGF is VEGF with about 53% homology.
• ischemia modified albumin ischemia modified albumin
• ACB test is a quantitative assay that measures ischemia-modified albumin (IMA) in human serum.
• IMA ischemia-modified albumin
• cobalt added to serum does not bind to the NH 2 terminus of IMA, leaving more free cobalt to react with dithiothreitol and form a darker color in samples from patients with ischemia.
• the assay is available on a variety of clinical chemistry platforms.
• IMA may be considered as an additional marker to be included into a marker panel for assessment of an individual's risk of developing a myocardial disorder.
• the method according to the present invention in a preferred embodiment is practiced in the investigation of apparently healthy individuals.
• "Apparently healthy" means individuals who have not previously had or at not aware of a previous adverse cardiovascular event such as a myocardial infarction.
• the diagnostic question is the relative risk of developing a myocardial disorder in the future.
• the relative risk is given in comparison to healthy controls.
• healthy controls are matched for age and other covariates.
• Marker values may be combined by any appropriate state of the art mathematical method.
• Well-known mathematical methods for correlating a marker combination to a disease or to the risk of developing a disease employ methods like, Discriminant analysis (DA) (i.e. linear-, quadratic-, regularized-DA), Kernel Methods (i.e. SVM), Nonparametric Methods (i.e. k-Nearest-Neighbor Classifiers),
• DA Discriminant analysis
• SVM Kernel Methods
• Nonparametric Methods i.e. k-Nearest-Neighbor Classifiers
• PLS Partial Least Squares
• Tree-Based Methods i.e. Logic Regression, CART, Random Forest Methods, Boosting/Bagging Methods
• Generalized Linear Models i.e. Logistic Regression
• Principal Components based Methods i.e. SIMCA
• Generalized Additive Models Fuzzy Logic based Methods, Neural Networks and Genetic Algorithms based Methods.
• DA i.e. Linear-, Quadratic-, Regularized Discriminant Analysis
• Kernel Methods i.e. SVM
• markers are no longer independent but form a marker panel.
• ROC receiver-operating characteristics
• the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease, respectively.
• the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as
• Each point on the ROC plot represents a sensitivity/ 1 -specificity pair corresponding to a particular decision threshold.
• a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
• the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
• ROC-values range between 1.0
• the present invention relates to a method for improving the assessment of an individual's risk of developing a myocardial disorder by measuring in a sample the concentration of an antibody to a cardiac troponin and the level of cardiac troponin and correlating the concentrations determined to the risk of developing a myocardial disorder.
• the present invention relates to a method for improving the assessment of an individual's risk of developing a myocardial disorder by measuring in a sample the concentration of an antibody to a cardiac troponin and the level of cholesterol and correlating the concentrations determined to the risk of developing a myocardial disorder.
• the present invention relates to a method for improving the assessment of an individual's risk of developing a myocardial disorder by measuring in a sample the concentration of at an antibody to a cardiac troponin and the level of CRP and correlating the concentrations determined to the risk of developing a myocardial disorder.
• the present invention relates to a method for improving the assessment of an individual's risk of developing a myocardial disorder by measuring in a sample the concentration of at an antibody to a cardiac troponin and the level of natriuretic peptide or a natriuretic peptide-related marker and correlating the concentrations determined to the risk of developing a myocardial disorder.
• kits or assays which are specific for, and have appropriate sensitivity with respect to antibodies to a cardiac troponin.
• a preferred kit accordingly to the present invention comprises a cardiac troponin and auxiliary reagents appropriate for measurement of antibodies to said cardiac troponin.
• the invention provides methods for evaluating the likelihood that an individual will benefit from treatment with an agent for reducing risk of a future myocardial disorder. This method may have important implications for patient treatment and also for clinical development of new therapeutics. Physicians select therapeutic regimens for patient treatment based upon the expected net benefit to the patient. The net benefit is derived from the risk to benefit ratio.
• the present invention may permit selection of individuals who are more likely to benefit by intervention, thereby aiding the physician in selecting a therapeutic regimen. This might include using drugs with a higher risk profile where the likelihood of expected benefit has increased.
• clinical investigators desire to select for clinical trials a population with a high likelihood of obtaining a net benefit. The present invention can help clinical investigators select such individuals.
• a cardiac troponin may be released into the circulation during a surgical intervention at the heart. This may be specially the case for patients undergoing surgery for heart transplantation.
• the release of a cardiac troponin during cardiac surgery may or may not trigger the formation of autoantibodies.
• Anti-troponin autoantibodies, however, once induced may well have a negative impact on the patient and may e.g. become relevant in rejection of the transplanted heart.
• autoantibodies to a cardiac troponin will be of aid in the follow-up of patients after heart surgery, especially and preferably in the follow-up of heart transplantations.
• Heart transplant patients that develop anti-troponin antibodies may require additional or different treatment as compared to patients not testing positive for such autoantibodies.
• the following assay can be used: Wells of a microtiter plate are first coated with a mouse monoclonal antibody to cardiac troponin T or I. In a second step the corresponding antigen, either cardiac troponin T or I is bound to the antibodies. By incubating an appropriately diluted serum sample with the bound troponin antigen the serum antibodies capable of binding to the troponin in the well will bind thereto. The bound serum antibodies can then be detected by an appropriate detection antibody, e.g. an anti-human IgG peroxidase conjugate. The skilled artisan is familiar with appropriate blocking and washing steps.
• an appropriate detection antibody e.g. an anti-human IgG peroxidase conjugate.
• Wells were washed with PBS/Tw as above. Human sera were diluted 1:20 and further down in steps of 2 in sample diluent. Duplicates of 100 ⁇ l diluted serum per well were incubated at RT for 90 min in both test wells as well as control wells, respectively. Wells were washed with PBS/Tw as above. Per well 100 ⁇ l of detection antibody (Horseradish Peroxidase (HRP) conjugated anti-human IgG Monoclonal Antibody- BD Pharmingen product-no. 555788) diluted 1:10,000 in sample diluent were then added to each well and incubated for one hour, followed by washing as described above.
• detection antibody HRP conjugated anti-human IgG Monoclonal Antibody- BD Pharmingen product-no. 555788
• Peroxidase activity bound to the wells was detected by use of 100 ⁇ l/well Blue Star TMB-HRP-Substrate (Diarect AG, product-no. DIA91000) as recommended by the supplier. Reaction was stopped after 45 min by adding 100 ⁇ l/well of 0.3 M H 2 SO 4 J.T. Baker order number 6057. Extinction was recorded 450 nm using 550 nm as a reference wave length by SLT Spectra II, Tecan.
• LVEF left ventricular ejection fraction

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