JP5306218B2 - Means and methods for optimization of diagnostic and therapeutic approaches in chronic arterial disease based on detection of troponin T and NT-proBNP - Google Patents

Abstract

The present invention relates to diagnostic means and methods. Specifically, the present invention encompasses a method of diagnosing the cause of cardiac necrosis in a subject comprising determining the amount of a cardiac Troponin and the amount of a BNP-type peptide in a sample of subject suffering from cardiac necrosis and co mparing the amount of the said cardiac Troponin and the amount of the said BNP-type peptide to reference amounts, whereby the cause of the cardiac necrosis is to be diagnosed. The present invention, further, relates to a method of determining whether a subject suffering from cardiac necrosis is susceptible for a therapy against initial heart failure and a method for determining whether a subject suffering from cardiac necrosis is susceptible for a therapy against coronary heart disease. Also encompassed are diagnostic uses, devices and kits.

Description

  The present invention relates to diagnostic means and methods. Specifically, the present invention is a method for diagnosing the cause of myocardial necrosis in a subject, measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis, and Comparing the amount of myocardial troponin and the amount of the BNP-type peptide with a reference amount, thereby diagnosing the cause of myocardial necrosis. The present invention further provides a method for determining whether a subject suffering from myocardial necrosis is susceptible to treatment for early heart failure and a method for determining whether a subject suffering from myocardial necrosis is susceptible to treatment for coronary heart disease About. Diagnostic uses, devices and kits are also encompassed.

  The purpose of modern medicine is to provide a personal or individual treatment regimen. They are treatment regimens that take into account the individual needs or risks of the patient. Individual or individual treatment regimens are taken into account even for emergency measures that require decisions on treatment regime candidates in a short period of time. Heart disease is a leading cause of morbidity and mortality in the Western Hemisphere. The disease can remain asymptomatic for long periods of time. However, acute cardiovascular events that cause cardiovascular disease, such as myocardial infarction, can have serious consequences.

  In particular, myocardial necrosis is a myocardial disease or disorder that often affects the function of the heart and often results in life-threatening myocardial infarction or progression of heart failure (ie, so-called progressive heart failure). Myocardial necrosis has a variety of causes and therefore requires different treatment regimens depending on the cause.

  Causes of myocardial necrosis include heart failure as well as coronary heart disease and cardiomyopathy. It should be understood that the applicable treatment is different for different causes. Specifically, in the case of heart failure, drug-based therapy appears promising, whereas coronary heart disease, such as stenosis of the coronary arteries, requires invasive therapy, such as angioplasty or bypass surgery ( For details, see Non-Patent Document 1.)

  Conventional diagnostic techniques for myocardial necrosis include pivotally electrocardiogram measurements. Recently, these conventional techniques have been further enhanced by the analysis of biomarkers, cardiac troponin I or troponin T. However, these diagnostic techniques cannot determine the cause of myocardial necrosis without additional differential diagnosis. Therefore, various different diagnostic measurements need to be performed later to determine the cause of myocardial necrosis. However, if such time-consuming diagnostic procedures are not possible or expensive, for example in an emergency patient, a personal treatment regimen cannot be determined with sufficient accuracy. As a result, many patients will receive treatment regimens that may be inadequate or have adverse side effects.

Gheorghiade 2006, Circulation 114: 1202-1213

  Therefore, there is a need for a diagnostic or prognostic tool that allows a fast and reliable diagnosis of the cause of myocardial necrosis to determine a suitable treatment regimen.

  The technical problem behind the present invention can be viewed as providing means and methods that meet the needs described above. The technical problem is solved by the embodiments characterized in the claims and below.

Thus, the present invention is a method for diagnosing the cause of myocardial necrosis in a subject comprising the following steps:
(A) measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis; and (b) comparing the amount of cardiac troponin and the amount of BNP-type peptide with a reference amount. A method comprising the step of diagnosing the cause of myocardial necrosis.

  The method of the present invention is preferably an in vitro method. It may further include steps in addition to those explicitly stated above. For example, the additional steps relate to sample pretreatment or evaluation of the results obtained by the method. The methods of the invention may be used for monitoring, confirmation, and subclassification of subjects in need of cardiac intervention. The method may be performed manually or supported by automation. Preferably, steps (a) and / or (b) may be assisted in whole or in part by automation, eg, measurement of step (a) or computer-implemented comparison of step (b) May be supported by suitable robots and sensor devices.

FIG. 1 is a linear regression analysis of NT-proBNP and troponin T in patients with ischemic cardiomyopathy. FIG. 2 is a linear regression analysis of NT-proBNP and troponin T in patients with coronary heart disease. FIG. 3 is a linear regression analysis of NT-proBNP and troponin T in a subgroup of patients suffering from 3-vessel disease. FIG. 4 is a linear regression analysis of NT-proBNP and troponin T in patients suffering from myocardial necrosis exposed to physical stress (bicycle exercise) and pharmacological stress. Thallium scintigraphy classifies patients into three different types of ischemia: no ischemia (n = 62 patients), reversible ischemia (n = 24 patients) and persistent ischemia (n = 57 patients) . Non-ischemic patients showed a weaker correlation between NT-proBNP and troponin T (R ² = 0,0025) than patients with reversible ischemia (R ² = 0,026). Maximum correlation could be observed in patients with persistent ischemia (R ² = 0,0791).

  As used herein, the term “diagnosis” refers to the identification of the cause of myocardial necrosis, ie the underlying disorder or disease symptom that causes obvious myocardial necrosis. As will be appreciated by those skilled in the art, such an assessment is usually not intended to be correct for all (ie 100%) subjects of a particular subject. However, the term requires that a statistically significant proportion of subjects can be identified (eg, a cohort in a cohort study). Whether a proportion is statistically significant is added by one skilled in the art using various well-known statistical evaluation tools such as confidence interval determination, p-value determination, student t-test, Mann-Whitney test, etc. Can be determined without any hassle. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of subjects in the population can be correctly identified by the methods of the invention.

  Diagnosis of the present invention includes monitoring, confirmation, subclassification and prediction of its associated disease, symptoms or risks. Monitoring relates to tracking the progress of an already diagnosed disease. Confirmation relates to the use of other indicators or markers to enhance or substantiate an already performed diagnosis. Subclassification relates to further clarifying the diagnosis according to different subclasses for the diagnosed disease, eg to clarifying according to the mild and severe forms of the disease.

  The term “cardiac necrosis” as used herein refers to myocardial necrotic cells or tissue. Such necrotic cells or tissue areas can be determined by electrocardiographic techniques. The reason is that necrotic cells or regions affect the electric field generated by the heart that is physiologically actuated, ie beating. Low blood pressure, elevated intraventricular pressure or coronary heart disease as well as cardiomyopathy are well known disorders that result in reduced myocardial perfusion resulting in myocardial necrosis. The cardiomyopathy of the present invention relates to disorders that affect the myocardium with mechanical or electrophysiological changes. In addition, the disorder may involve ventricular hypertrophy or dilation. Cardiomyopathy results, for example, from inflammatory diseases of the myocardium or is caused by toxic compounds (see also Maron 2006, Circulation 113: 1807-1816). It is well established that myocardial necrosis is associated with a deterioration in cardiac function. This disorder is also referred to as progressive heart failure and results in ischemic cardiomyopathy. Thus, it should be understood that the methods of the present invention can also be applied to diagnose the cause of ischemic cardiomyopathy, for example as a result of progressive heart failure or coronary heart disease.

  The cause of myocardial necrosis and hence progressive heart failure is both coronary heart disease, which reduces blood flow through the coronary vessels, resulting in hypoxic or ischemic regions in the myocardium. On the other hand, myocardial hypoxia or ischemia with progressive heart failure and thus myocardial necrosis may result from early heart failure. The initial heart failure referred to herein preferably results from elevated intraventricular pressure, hypotension or dilatative cardiac myopathy.

  The ischemia involved in myocardial necrosis as referred to herein is reversible or persistent (ie, permanent) ischemia. Persistent ischemia is a subject whose myocardium is only improperly supplied by blood and is therefore not subject to resting subjects, ie, physical or pharmacological stresses such as bicycle exercise or dipyridamol Is characterized by hypoxia. Reversible ischemia is characterized by an inadequate blood supply to the myocardium only when subjected to physical or pharmacological stress. As already mentioned above, myocardial necrosis may be the result of other causes and therefore is not necessarily accompanied by ischemia.

  As used herein, the term “coronary heart disease” includes coronary artery disease such as coronary stenosis, atherosclerosis or obstruction within the coronary vasculature such as thromboembolic obstruction.

  The term “subject” as used herein relates to an animal, preferably a mammal, more preferably a human. Preferably, the subject referred to in the above method is suffering from or associated with myocardial necrosis, i.e. suspected of suffering at least from myocardial necrosis.

  The term “sample” refers to a body fluid sample, isolated cell sample, or a sample from a tissue or organ. Body fluid samples can be obtained by well-known techniques, preferably including blood, plasma, serum or urine samples, more preferably blood, plasma or serum samples. A tissue or organ sample can be obtained from any tissue or organ, for example, by biopsy. Isolated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. Preferably, the cell sample, tissue sample or organ sample is obtained from a cell, tissue or organ that expresses or produces a peptide referred to herein.

  The term “cardiac Troponin” refers to all troponin isoforms expressed in heart cells and preferably subendocardial cells. These isoforms are well characterized in the art. For example, as described in Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493. Preferably, cardiac troponin refers to troponin T and / or troponin I, most preferably troponin T. It should be understood that troponin isoforms can be measured in the method of the invention together, ie simultaneously or sequentially, or individually, ie without measuring any other isoform. The amino acid sequences of human troponin T and human troponin I are disclosed in Anderson, supra and Ferrieres 1998, Clinical Chemistry, 44: 487-493.

  The term “cardiac troponin” also encompasses the specific troponins mentioned above, ie preferably variants of troponin T or troponin I. Such variants have at least the same basic biological and immunological properties as the specific cardiac troponin. In particular, they are identical if they are detectable by the same specific assay referred to herein, eg, an ELISA assay using a polyclonal or monoclonal antibody that specifically recognizes the cardiac troponin. Share biological and immunological properties. Furthermore, the variants referred to in the present invention have different amino acid sequences due to at least one amino acid substitution, deletion and / or addition, and the amino acid sequence of the variant is still the amino sequence of a particular troponin And preferably it is at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical. Let's go. Variants may be allelic variants or any other species-specific homolog, paralogs, or ortholog. Furthermore, the variants referred to herein include as long as the particular cardiac troponin or fragment of the above type of variant has the basic immunological and biological properties referred to above. ,included. Such a fragment may be, for example, a degradation product of troponin. Further included are variants which differ due to post-translational modifications such as phosphorylation or myristylation.

  The term “brain natriuretic peptide (BNP) -type peptide” relates to pre-proBNP, proBNP, NT-proBNP, and BNP and variants thereof having the same predicted ability (eg, Bowen, 1996, Circulation 93: 1946 See -1950). Specifically, the above-mentioned brain natriuretic peptide prepropeptide (having a length of 134 amino acids) contains a short signal peptide, which is cleaved by the enzyme to release the propeptide (108 amino acids). The propeptide is further cleaved into an N-terminal propeptide (NT-propeptide, 76 amino acids) and an active hormone (32 amino acids).

  BNP is metabolized in the blood, but NT-proBNP circulates in the blood as an intact molecule and is directly eliminated by the kidney. NTproBNP has an in vivo half-life of 120 minutes longer than BNP. The half-life of BNP is 20 minutes (Smith 2000, J Endocrinol. 167: 239-46.). Pre-analysis is more robust with NT-proBNP, which allows samples to be easily transported to a central laboratory (Mueller 2004, Clin Chem Lab Med 42: 942-4.). Blood samples can be stored at room temperature for several days, or can be mailed or shipped without recovery loss. In contrast, storage of BNP at room temperature or 4 ° C. for 48 hours results in a concentration loss of at least 20% (Mueller (supra); Wu 2004, Clin Chem 50: 867-73.). Therefore, depending on the time course or desired properties, it may be advantageous to measure the active or inactive form of the BNP-type peptide.

  The most preferred BNP-type peptide referred to herein is human NT-proBNP. As briefly discussed above, the human NT-proBNP referred to in the present invention is preferably a polypeptide comprising a 76 amino acid length corresponding to the N-terminal portion of the human NT-proBNP molecule. The structures of human BNP and NT-proBNP have already been reported in detail in the prior art, for example in WO 02/089657, WO 02/083913 or Bonow (supra). Preferably, the human NT-proBNP used in the present invention is the human NT-proBNP disclosed in EP 0 648 228 B1. These prior art documents are hereby incorporated by reference into the specific sequence of NT-proBNP and variants thereof disclosed therein. NT-proBNP referred to in the present invention further encompasses allelic and other variants of said specific sequence of human NT-proBNP disclosed above. Specifically, mutations at the amino acid level that are at least 60% identical to human NT-proBNP, more preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical Body polypeptides are envisioned. Proteolytic degradation products still recognized by diagnostic tools or by ligands for the respective full-length peptides are also assumed to be substantially similar. Variant polypeptides having amino acid deletions, substitutions, and / or additions compared to the amino acid sequence of human NT-proBNP are also encompassed so long as the polypeptide has NT-proBNP properties. The NT-proBNP properties referred to herein are immunological and / or biological properties. Preferably, the NT-proBNP variant has immunological properties (ie, epitope composition) equivalent to NT-proBNP. Hence, the variant is recognized by the above-mentioned means or ligands used for measuring the amount of natriuretic peptide. Biological and / or immunological NT-proBNP properties are described in Karl et al. (Karl 1999, Scand J Clin Invest 59: 177-181), Yeo et al. (Yeo 2003, Clinica Chimica Acta 338: 107-115). ). Variants also include post-translationally modified peptides such as glycosylated peptides. Furthermore, a “variant” of the present invention is also a peptide or polypeptide that has been modified after collection of the sample, for example by covalent or non-covalent binding of a label to the peptide, in particular a radioactive or fluorescent label.

  Measurement of the amount of peptide or polypeptide referred to herein preferably relates to measuring the amount or concentration semi-quantitatively or quantitatively. Measurements can be made directly or indirectly. A direct measurement relates to the determination of the amount or concentration of a peptide or polypeptide based on a signal obtained from the peptide or polypeptide itself and whose intensity directly correlates with the number of molecules of the peptide present in the sample. Such a signal (sometimes referred to herein as an intensity signal) can be obtained, for example, by measuring the intensity value of a particular physical or chemical property of the peptide or polypeptide. For indirect measurements, measurement of signals obtained from secondary components (ie, components that are not peptides or polypeptides themselves) or biological readout systems such as measurable cellular responses, ligands, labels, or enzymatic reaction products. Is included.

In the present invention, measurement of the amount of peptide or polypeptide can be accomplished by all known means for measuring the amount of peptide in a sample. Such means include immunoassay devices and methods that utilize labeled molecules in various sandwich assays, competitive assays, or other assay formats. The assay generates a signal indicating the presence or absence of the peptide or polypeptide. Moreover, the signal intensity can preferably be directly or indirectly correlated (eg, inversely proportional) to the amount of polypeptide present in the sample. Additional suitable methods include measuring physical or chemical properties unique to the peptide or polypeptide, such as its exact molecular weight or NMR spectrum. The method preferably includes a biosensor, an optical device in combination with an immunoassay, a biochip, an analytical device such as a mass spectrometer, NMR analyzer, or chromatography device. In addition, methods include microplate ELISA-based methods, fully automated or robotic immunoassays (eg available with Elecsys ^™ analyzers), CBA (enzymatic cobalt binding assays (eg available with Roche-Hitachi ^™ analyzers)), and latex. Agglutination assays (eg, available with Roche-Hitachi ^™ analyzer) are included.

  Preferably, measurement of the amount of peptide or polypeptide comprises: (a) contacting a cell capable of inducing a cellular response whose intensity is indicative of the amount of peptide or polypeptide with said peptide or polypeptide for an appropriate time; (b) cell Measuring the response. For measuring the cellular response, the sample or treated sample is preferably added to the cell culture and the internal or external cellular response is measured. A cellular response includes measurable expression of a reporter gene or secretion of a substance, such as a peptide, polypeptide, or small molecule. The expression or substance produces an intensity signal that correlates with the amount of peptide or polypeptide.

  Also preferably, measuring the amount of peptide or polypeptide comprises measuring a specific intensity signal obtained from the peptide or polypeptide in the sample. As noted above, such a signal may be an m / z variable specific to the peptide or polypeptide observed in a mass spectrum or a signal intensity observed in an NMR spectrum specific to the peptide or polypeptide. .

Measurement of the amount of peptide or polypeptide preferably comprises (a) contacting the peptide with a specific ligand, (b) (optionally) removing unbound ligand, (c) measuring the amount of bound ligand. including. The bound ligand generates an intensity signal. The bonds of the present invention include both covalent bonds and non-covalent bonds. The ligand of the invention can be any compound that binds to a peptide or polypeptide described herein, such as a peptide, polypeptide, nucleic acid, or small molecule. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides, such as receptors or binding partners of peptides or polypeptides and fragments thereof comprising a binding domain for the peptide, and aptamers, such as nucleic acids or peptide aptamers. Methods for preparing such ligands are well known in the art. For example, identification and production of suitable antibodies or aptamers are also provided by commercial sources. Those skilled in the art are familiar with methods to develop derivatives of such ligands with high affinity or specificity. For example, random mutations can be introduced into nucleic acids, peptides or polypeptides. The binding of these derivatives can then be examined according to screening procedures known in the art, such as phage display. The antibodies referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof capable of binding to an antigen or hapten, such as Fv, Fab and F (ab) ₂ fragments. The present invention also includes single chain antibodies and humanized hybrid antibodies (wherein the amino acid sequence of a non-human donor antibody exhibiting the desired antigen specificity is combined with the sequence of a human acceptor antibody). The donor sequence typically includes at least antigen binding amino acid residues of the donor, but may also include other structurally and / or functionally important amino acid residues of the donor antibody as well. Such hybrids can be prepared by several methods well known in the art. Preferably, the ligand or substance specifically binds to a peptide or polypeptide. Specific binding of the invention means that the ligand or substance should not substantially bind ("cross-react") with another peptide, polypeptide or substance present in the sample to be analyzed. To do. Preferably, the specific binding peptide or polypeptide is at least 3 times higher affinity than any other corresponding peptide or polypeptide, more preferably at least 10 times higher affinity, even more preferably at least 50 times higher affinity. Should be combined. Non-specific binding is allowed if it can still be clearly discriminated and measured, for example according to its size in a Western blot or by its relatively high abundance in the sample. Ligand binding can be measured by any method known in the art. Preferably, the method is semi-quantitative or quantitative. A suitable method is described below.

  First, ligand binding can be measured directly, for example by NMR or surface plasmon resonance.

  Second, if the ligand further serves as a substrate for the enzymatic activity of the peptide or polypeptide of interest, the enzymatic reaction product can be measured (eg, the amount of cleaved substrate is measured, eg, by Western blot) The amount of protease can be determined. Alternatively, the ligand may exhibit enzymatic properties per se, by contacting the “ligand / peptide or polypeptide” complex or the ligand to which the peptide or polypeptide is bound, respectively, with a suitable substrate and generating an intensity signal. Detection may be possible. In measuring enzyme reaction products, the amount of substrate is preferably saturated. Prior to the reaction, the substrate may be labeled with a detectable label. Preferably, the sample is contacted with the substrate for an appropriate time. Appropriate time refers to the time required to produce a detectable, preferably measurable amount of product. Instead of measuring the amount of product, the time required for appearance of a given (eg detectable) amount of product can be measured.

Third, the ligand can be coupled either covalently or non-covalently to a label that allows detection and measurement of the ligand. Labeling can be done by direct or indirect methods. Direct labeling involves coupling the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding of a secondary ligand to a primary ligand (either covalently or non-covalently). The secondary ligand should bind specifically to the primary ligand. The secondary ligand may be coupled with a suitable label and / or may be the target (receptor) of a tertiary ligand that binds to the secondary ligand. Often the signal is increased by the use of secondary, tertiary or even higher order ligands. Suitable secondary and higher order ligands include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). One or more tags known in the art may be used to “tag” the ligand or substrate. Such tags may then be targets for higher order ligands. Suitable tags include biotin, digoxigenin, His tag, glutathione-S-transferase, FLAG, GFP, myc tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, preferably the tag is located at the N-terminus and / or C-terminus. A suitable label is any label detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzyme activity labels, radioactive labels, magnetic labels (eg “magnetic beads” (including paramagnetic and superparamagnetic labels)), and A fluorescent label is included. Enzymatic activity labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and derivatives thereof. Suitable substrates for detection include diaminobenzidine (DAB), 3,3′-5,5′-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl -Phosphoric acid (available as ready-made stock from Roche Diagnostics), CDP-Star ^™ (Amersham Biosciences), ECF ^™ (Amersham Biosciences). The combination of a suitable enzyme and substrate results in a color reaction product, fluorescence or chemiluminescence that can be measured according to methods known in the art (eg, using a light-sensitive film or a suitable camera system). For the measurement of enzymatic reactions, the above criteria apply in the same way. Typical fluorescent labels include fluorescent proteins (eg, GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dye (eg, Alexa 568). Additional fluorescent labels are available from, for example, Molecular Probes (Oregon). The use of quantum dots as fluorescent labels is also considered. Typical radioactive labels include ³⁵ S, ¹²⁵ I, ³² P, ³³ P, and the like. The radioactive label can be detected by any method known and appropriate, such as a light-sensitive film or a phosphor imager. Suitable measurement methods of the present invention include precipitation (particularly immunoprecipitation), electrochemiluminescence (electrogenerated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immunoassay, electrochemiluminescence. Includes sandwich immunoassay (ECLIA), dissociation enhanced lanthanide fluorescence immunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetry, turbidimetry, latex-accelerated turbidimetry or turbidimetry, or solid phase immunoassay . Additional methods known in the art (eg, gel electrophoresis, two-dimensional gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and mass spectrometry) can be used alone or as described above. Alternatively, it can be used in combination with other detection methods.

  The amount of peptide or polypeptide may also preferably be measured as follows: (a) a solid support comprising the peptide or polypeptide ligand specified above with a sample comprising said peptide or polypeptide; And (b) measuring the amount of peptide or polypeptide bound to the support. A ligand, preferably selected from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, is preferably present in a fixed form on a solid support. Materials for producing solid supports are well known in the art and include, among others, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, Includes nitrocellulose strips, membranes, sheets, duracytes, reaction tray wells and walls, plastic tubes, and the like. The ligand or substance may be bound to a number of different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. For purposes of the present invention, the nature of the carrier may be soluble or insoluble. Suitable methods for attaching / immobilizing the ligand are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like. It is also contemplated to use a “suspension array” as an array of the present invention (Nolan 2002, Trends Biotechnol. 20 (1): 9-12). In such suspension arrays, carriers such as microbeads or microspheres are present in suspension. The array consists of different microbeads or microspheres, optionally labeled, carrying different ligands. Methods for producing such arrays, for example based on solid phase chemistry and photosensitive protecting groups, are generally known (US 5,744,305).

  As used herein, the term “amount” refers to the absolute amount of a polypeptide or peptide, the relative amount or concentration of the polypeptide or peptide, as well as any value or parameter that can be correlated or derived therefrom. Include. Such values or parameters include intensity signal values obtained from all specific physical or chemical properties obtained from the peptide by direct measurement, such as intensity values in mass spectra or NMR spectra. In addition, all values or parameters obtained by indirect measurements specified elsewhere in this description, such as response levels determined from biological reading systems in response to peptides or specific binding ligands. Intensity signals to be included are included. It should be understood that values correlating to the quantities or parameters described above may be obtained by all standard mathematical operations.

  As used herein, the term “comparison” refers to comparing the amount of peptide or polypeptide contained in a sample to be analyzed with the amount of a suitable reference source specified elsewhere in this description. Include. It should be understood that the comparisons used herein represent comparisons of corresponding parameters or values. For example, the absolute amount is compared to the absolute reference amount and the concentration is compared to the reference concentration, or the intensity signal obtained from the test sample is compared to the same type of intensity signal of the reference sample. The comparison referred to in step (b) of the method of the invention may be performed manually or computer assisted. In a computer-aided comparison, the measured quantity value may be compared to a value corresponding to a suitable reference stored in a database by a computer program. The computer program can further evaluate the comparison results, i.e. automatically provide the desired evaluation in a suitable output format. Based on the comparison between the amount measured in step (a) and the reference amount, the cause of myocardial necrosis can be identified. Thus, the reference amount allows the identification of subjects belonging to the group of subjects whose differences or similarities compared are suffering from myocardial necrosis caused by either early heart failure or coronary heart disease Should be selected to do.

  Thus, as used herein, the term “reference amount” refers to the amount of polypeptide that allows the identification of a subject suffering from myocardial necrosis caused by either early heart failure or coronary heart disease. Represents. Thus, from (i) a subject known to suffer from myocardial necrosis caused by early heart failure or (ii) from a subject known to suffer from myocardial necrosis caused by coronary heart disease A reference can be derived. Furthermore, the reference quantity preferably defines a threshold value. A suitable reference amount or threshold amount can be determined by the method of the present invention from the reference sample to be analyzed together with the test sample, ie simultaneously or subsequently. A preferred reference amount useful as a threshold can be derived from the upper limit of normal (ULN), ie, the upper limit of the physiological amount found in a population of subjects (eg, patients enrolled in a clinical trial). The ULN of a given subject population can be measured by various well-known techniques. A preferred technique is to measure the median population for the amount of peptide or polypeptide being measured by the method of the invention. The ULN of cardiac troponin and especially troponin T referred to herein preferably varies in the range of 0.001 ng / mL to 0.01 ng / mL. More preferably, the ULN of the cardiac troponin is 0.001 ng / mL. The ULN of BNP-type peptides and particularly NT-proBNP referred to herein varies preferably in the range of 100-150 pg / mL. More preferably, the ULN of the BNP-type peptide is 125 pg / mL.

  In principle, an increase in the amount of BNP-type peptide with an increase in the amount of cardiac troponin in the sample of the subject indicates myocardial necrosis caused by early heart failure, while in the absence of an increase in the amount of BNP-type peptide Has been found to indicate coronary heart disease as a cause of myocardial necrosis. Based on this finding, the cause of myocardial necrosis can be determined by the method of the present invention.

  Advantageously, the invention provides that a combination of cardiac troponin as a biomarker, such as troponin T and a BNP-type peptide, such as NT-proBNP, is sufficient to resolve the cause of myocardial necrosis in a reliable and efficient manner. Has been found in the research that underlies it. As mentioned above, the techniques currently used to resolve the cause of myocardial necrosis require a lot of time and cost. Furthermore, the technology cannot usually be implemented in a portable system. However, the method of the present invention allows diagnostics that are fast, reliable and require low cost, and can be realized even in portable assays, such as test stripes. The method is therefore particularly well suited for the diagnosis of emergency patients. Furthermore, the method of the present invention can be performed on a sample of a subject, thus dramatically reducing the risk of producing adverse side effects that are at least unavoidable with invasive prior art. Thanks to the findings of the present invention, a treatment suitable for the subject can be reliably selected. Serious side effects caused by wrong patient treatment can be avoided. As described in detail below, treatment of diseases or disorders involving myocardial necrosis varies.

  It should be understood that in a preferred embodiment of the method of the invention, the reference amount is the upper limit of normal (ULN) for cardiac troponin and BNP-type peptides.

  In a further preferred embodiment of the method of the invention, an increase in cardiac troponin level and an increase in BNP-type peptide level indicate myocardial necrosis caused by early heart failure.

  Also, in a preferred embodiment of the method of the present invention, an increase in the amount of cardiac troponin and an amount of BNP-type peptide that has not increased indicates myocardial necrosis caused by coronary heart disease.

  The definitions and explanations of the terms apply to the following methods mutatis mutandis, unless otherwise specified.

The present invention is also a method for determining whether a subject suffering from myocardial necrosis is susceptible to treatment for early heart failure, comprising the following steps:
(A) measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis; and (b) comparing the amount of cardiac troponin and the amount of BNP-type peptide with a reference amount, thereby It relates to a method comprising determining whether a subject is susceptible to treatment for early heart failure.

  As used herein, the phrase “determining whether a subject suffering from myocardial necrosis is susceptible to treatment for early heart failure” assesses whether the subject is susceptible to treatment for early heart failure. Means. As will be appreciated by those skilled in the art, such an assessment is usually not intended to be correct for all (ie 100%) subjects of a particular subject. However, the term requires that a statistically significant proportion of subjects can be identified (eg, a cohort in a cohort study). Whether a ratio is statistically significant can be determined without additional hassle by one skilled in the art using various well-known statistical evaluation tools. Additional details regarding the statistical evaluation can be found elsewhere in the specification.

  The term “treatment for early heart failure” encompasses drug-based treatment for heart failure as well as other treatments. More preferably, the treatment for early heart failure is drug-based therapy, preferably ACE inhibitor administration, β-blocker administration, digital AT1 and / or AT2 blocker administration, inotropic drugs administration, Selected from the group consisting of assistive device based therapy, interventional therapy (either cardiac resynchronization therapy (CRT) or based on cardioverter defibrillator (ICD) implantation and heart transplantation). More preferably, the drug is an ACE inhibitor, preferably captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, or trandolapril, AT-1 receptor blockade An agent, preferably candesartan, losartan, or valsartan, a β-receptor blocker, preferably bisoprolol, carvedilol, metoprolol or succinate, or an aldosterone antagonist, preferably spironolacton or eplerenone is there. Additional details regarding the treatment can be found in Heart Disease, 2005, 7th Edition, Eds. Braunwald, Elsevier Sounders. See Figures 24G-1 to 24G-7.

  In a preferred embodiment of the method of the invention described above, the reference amount is the upper limit of normal (ULN) for cardiac troponin and BNP-type peptides.

  Further, in a preferred embodiment of the method of the present invention, an increase in cardiac troponin level and an increase in BNP-type peptide level indicate that the subject is susceptible to treatment for early heart failure.

  Further, in a preferred embodiment, the method of the invention comprises a supplemental diagnostic technique selected from the group consisting of echocardiography, computed tomography, NMR based diagnostics, electrocardiography, thorax x-ray diagnostics and spirometry. May further be included. Additional details regarding the treatment can be found in Heart Disease, 2005, 7th Edition, Eds. Braunwald, Elsevier Sounders. See Figures 24G-1 to 24G-7.

The present invention also provides a method for determining whether a subject suffering from myocardial necrosis is susceptible to treatment for coronary heart disease comprising the following steps:
(A) measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis; and (b) comparing the amount of cardiac troponin and the amount of BNP-type peptide with a reference amount, thereby Including the step of determining whether the subject is susceptible to treatment for coronary heart disease.

  As used herein, the phrase “determining whether a subject suffering from myocardial necrosis is susceptible to treatment for coronary heart disease” refers to whether the subject is sensitive to treatment for coronary heart disease. Means to evaluate. As will also be appreciated by those skilled in the art, such an assessment is usually not intended to be correct for all (ie 100%) subjects of a particular subject. However, the term requires that a statistically significant proportion of subjects can be identified (eg, a cohort in a cohort study). Whether a ratio is statistically significant can be determined without additional hassle by one skilled in the art using various well-known statistical evaluation tools. Additional details regarding the statistical evaluation can be found elsewhere in the specification.

  The term “treatment for coronary heart disease” includes drug-based as well as invasive treatment for coronary heart disease. More preferably, the treatment for coronary heart disease is selected from the group consisting of administration of an inhibitor of platelet aggregation, preferably aspirin or clopidogrel, and cardiac intervention. Preferred cardiac interventions of the present invention include percutaneous coronary angioplasty, percutaneous transluminal for the purpose of restoring blood flow, vessel patency, plaque stabilization, and / or reducing intracoronary thrombus load. Specially selected from the group consisting of coronary balloon angioplasty, laser angioplasty, coronary stenting, bypass grafting or endovascular techniques.

  In a preferred embodiment of the method of the invention described above, the reference amount is the upper limit of normal (ULN) for cardiac troponin and BNP-type peptides.

  Further, in a preferred embodiment of the method of the present invention, an increase in cardiac troponin levels and a non-increased BNP-type peptide level indicate that the subject is susceptible to treatment for coronary heart disease.

  Further, in a preferred embodiment, the method of the present invention is selected from the group consisting of angiography, echocardiography, computed tomography, NMR based diagnostics, electrocardiography, thorax x-ray diagnostics and spirometry. Additional diagnostic techniques may be included.

  In principle, the present invention is for diagnosing the cause of myocardial necrosis in a subject, determining whether a subject suffering from myocardial necrosis is susceptible to treatment for early heart failure, or subject suffering from myocardial necrosis. Includes the use of cardiac troponin and BNP-type peptides to produce a diagnostic composition for determining whether the body is susceptible to treatment for coronary heart disease.

Furthermore, the present invention is also a method for monitoring the success of treatment for coronary heart disease, comprising the following steps:
(A) measuring the amount of cardiac troponin in at least first and second samples of a subject subjected to the treatment, wherein the first sample is obtained in advance, and the second sample is obtained after the treatment is started; And (b) comparing the amounts obtained from the first and second samples, thereby reducing the amount of cardiac troponin in the second sample relative to the first sample. A method including a step where success is indicated.

  Preferably, the BNP-type peptide is further measured in the first and second samples. The reduction in the amount of the BNP-type peptide further indicates the success of the applied treatment regimen.

Furthermore, the present invention is a method for monitoring the success of treatment for early heart failure, comprising the following steps:
(A) measuring the amount of cardiac troponin and BNP-type peptide in at least first and second samples of a subject subjected to the treatment, wherein the first sample is obtained in advance, and the second sample is the treatment (B) comparing the amounts obtained from the first and second samples, thereby comparing the amount of cardiac troponin in the second sample relative to the first sample; And a step that shows success if the amount of BNP-type peptide is reduced.

  The term “monitoring” as used herein relates to tracking the course of effects produced by a treatment regimen on the amount of NT-proBNP and / or cardiac troponin. It should be understood that the amount needs to be measured at least prior and after the start of the treatment regimen. However, the amount may be measured at other stages.

  As used herein, the term “success” refers to at least amelioration of a disease, ie, prognosis, symptoms or clinical signs associated with coronary heart disease or early heart failure.

  The present invention is also a diagnostic device suitable for performing any one of the methods of the present invention, and means for measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis And a device comprising means for preparing the amount relative to a reference amount.

  The term “device” as used herein relates to a system of means including at least the means described above operatively coupled to each other to allow prediction. Preferred means for measuring the amount of cardiac troponin and BNP-type peptide and means for performing the comparison are disclosed above in connection with the method of the present invention. The method of connecting the means in an operating manner depends on the type of means included in the device. For example, if means for automatically measuring the amount of peptide is applied, the data obtained by the automatically actuating means can be processed, for example by a computer program, in order to obtain the desired result. Preferably, in such cases, the means are included in a single device. Thus, the device may comprise an analysis unit for measuring the amount of peptide or polypeptide in the applied sample and a computer unit for processing the obtained data for evaluation. The computer unit preferably includes a stored reference amount and a database containing the values as described elsewhere herein, as well as a measured amount of the polypeptide and a reference amount stored in the database. Includes a computer-implemented algorithm for performing the comparison. Computer implementation as used herein refers to computer readable program code that may be securely contained in a computer unit. Alternatively, where a means such as a test strip is used to measure the amount of peptide or polypeptide, the means for comparison includes a control strip or table that assigns the measured quantity to a reference quantity. The test strip is preferably coupled with a ligand that specifically binds to a peptide or polypeptide referred to herein. The strip or device preferably includes means for detecting binding of the peptide or polypeptide to the ligand. Preferred detection means are disclosed in connection with embodiments relating to the method of the invention above. In such a case, the means are operably linked in that the user of the system matches the measurement result with its diagnostic or prognostic value based on instructions and interpretations provided in the manual. The means may exist as a separate device in such embodiments and is preferably packaged as a kit. Those skilled in the art understand how to connect means without additional hassle. Preferred devices are those that can be applied without the specific knowledge of a professional clinician, such as test strips or electronic devices that only require sample loading. The results can be obtained as raw data output that requires clinician interpretation. Preferably, however, the output of the device is processed, ie, evaluated, raw data that does not require a clinician for interpretation. Additional preferred devices are analytical units / devices (eg biosensors coupled with ligands that specifically recognize natriuretic peptides, arrays, solid supports, plasmon surface resonance devices, NMR spectrometers, mass spectrometers, etc.) And / or an evaluation unit / device of the method of the invention as mentioned above.

  Finally, the present invention is a diagnostic kit suitable for performing any one of the methods of the present invention, for measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis. And a kit comprising means for comparing said amount with a reference amount.

  The term “kit” as used herein refers to a collection of the aforementioned means, which means are preferably provided separately or in a single container. The container also preferably contains instructions for performing the method of the present invention.

  All references cited in this specification are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically described herein.

  The following examples merely illustrate the invention. They should be construed as not limiting the scope of the invention in any way.

Example 1: NT-proBNP and troponin T in patients with coronary heart disease
NT-proBNP and sensitive troponin T were measured in blood samples from a total of 235 patients with stable coronary heart disease. NT-proBNP was measured using an Elecsys ^™ assay and troponin T was assayed by a sensitive troponin T test purchased from Roche Diagnostics. Patients were subjected to detailed cardiological investigations including echocardiography and coronary angioplasty. Coronary heart disease was subdivided into 1 branch lesion, 2 branch lesion, or 3 branch lesion. According to it, more than 50% stenosis should occur per blood vessel.

  The two biomarkers were analyzed by statistical linear regression analysis. As shown in FIG. 2, a correlation between NT-proBNP and high-sensitivity troponin T was found, and the correlation R value was 0.0667. Patients with increased troponin T levels and normal NT-proBNP show substantial stenosis when examined by angiography, while patients with elevated NT-proBNP levels and elevated troponin T are examined by angiography Also did not show such substantial stenosis. Similar results were obtained for a subgroup of patients suffering from 3-branch lesions (see FIG. 3).

  59 patients with ischemic cardiomyopathy were examined for biomarkers as described above. Similarly, a correlation between NT-proBNP and troponin T was found with an R value of 0.013 (see FIG. 1).

Example 2: NT-proBNP and troponin T in patients with myocardial necrosis examined by thallium scintigraphy
A total of 143 patients who underwent thallium scintigraphy for suspicion of significant coronary artery disease were studied.

  Patients received a bicycle exercise test using a standard exercise protocol, or a pharmacological study with a 42.72 mg (± 7.61) dose of dipyridamol using a standard protocol . Assignment to dynamic or pharmacological exercise tests was left to the judgment of the cardiologist. The protocol was approved by the Heidelberg University Ethics Committee and all patients participated after consenting.

  Single-photon emission computed tomography myocardial perfusion (SPECT) imaging was performed as follows: thallium was administered during peak stress and imaging was performed immediately thereafter. After 4 hours, repeated imaging was performed. A 17 segment myocardial model was used for semi-quantitative analysis. Two cardiologists who were unaware of the biomarker results classified the images as having no perfusion defects, only reversible perfusion defects, and having established perfusion defects. Patients with a combination of reversible, partially reversible, and established perfusion defects were excluded from the main analysis and evaluated separately.

  Blood samples were obtained immediately before, immediately after, and 4 hours after the stress test. The third day of the next day (redistribution scan) collected the last blood sample of all patients (n = 20). Blood samples were placed on ice and processed within 30 minutes. Plasma aliquots were stored at −80 ° C. and thawed before analysis.

  Statistical analysis was performed as follows: plasma concentrations of cardiac troponin T and NT-proBNP with corresponding quartile ranges, or mean values with corresponding standard errors. The baseline characteristics of patient groups were compared using Mann-Whitney U test or Student t test for continuous variables and chi-square test for categorical variables. The normal distribution was examined using the Kolmogorov-Smirnov test. In all analyses, values of P <0.05 were considered statistically significant. All statistical analyzes were performed using SPSS software package version 12.01 (SPSS Inc, Chicago, Ill, USA).

The results are shown in the table below and in FIG.

Claims (17)

A method for detecting whether the cause of myocardial necrosis in a subject is early heart failure resulting from elevated intraventricular pressure, hypotension or dilated cardiomyopathy or coronary heart disease, comprising the following steps:
(A) measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis; and (b) comparing the amount of cardiac troponin and the amount of BNP-type peptide with a reference amount. ,
(I) an increase in cardiac troponin level and an increase in BNP-type peptide level indicate myocardial necrosis caused by early heart failure;
(Ii) Increased amount of cardiac troponin and not increased amount of BNP-type peptide indicate myocardial necrosis caused by coronary heart disease,
The above method.   The method of claim 1, wherein the reference amount is a normal upper limit (ULN) for cardiac troponin and BNP-type peptides. A method for providing data for use in determining treatment for early heart failure resulting from elevated intraventricular pressure, hypotension or dilated cardiomyopathy in a subject suffering from myocardial necrosis comprising the following steps:
(A) measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis; and (b) comparing the amount of cardiac troponin and the amount of BNP-type peptide with a reference amount. The method as described above, wherein an increase in the amount of cardiac troponin and an increase in the amount of BNP-type peptide indicate that the subject is susceptible to treatment for early heart failure.   4. The method of claim 3, wherein the reference amount is an upper limit of normal (ULN) for cardiac troponin and BNP-type peptides.   Treatment of early heart failure comprises ACE inhibitor administration, β-blocker administration, digital AT1 and / or AT2 blocker administration, inotropic drug administration, support device based treatment and heart transplantation The method according to claim 3 or 4, wherein the method is selected from: A method for providing data for use in determining treatment for coronary heart disease in a subject suffering from myocardial necrosis comprising the following steps:
(A) measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis; and (b) comparing the amount of cardiac troponin and the amount of BNP-type peptide with a reference amount. The method as described above, wherein the amount of cardiac troponin and the amount of BNP-type peptide not increased indicate that the subject is susceptible to treatment for coronary heart disease.   7. The method of claim 6, wherein the reference amount is an upper limit of normal (ULN) for cardiac troponin and BNP-type peptides.   The treatment for coronary heart disease is selected from the group consisting of administration of an inhibitor of platelet aggregation, administration of aspirin or clopidogrel, and cardiac intervention including balloon angioplasty, stent implantation or bypass surgery. The method described in 1.   9. The method according to any one of claims 1 to 8, wherein the cardiac troponin is troponin T.   The method according to any one of claims 1 to 9, wherein the BNP-type peptide is NT-proBNP.   The method of any one of claims 1 to 10, wherein the subject is a human.   A diagnostic composition for diagnosing the cause of myocardial necrosis in a subject, comprising means for measuring the amount of cardiac troponin and BNP-type peptide, (i) increase in cardiac troponin level and amount of BNP-type peptide Increases in myocardial necrosis caused by early heart failure resulting from elevated intraventricular pressure, hypotension or dilated cardiomyopathy, (ii) increased myocardial troponin levels and not increased BNP-type peptide levels The said diagnostic composition containing the instruction | indication which shows the myocardial necrosis caused by the heart disease.   A diagnostic composition for determining whether a subject suffering from myocardial necrosis is susceptible to treatment for early heart failure resulting from elevated intraventricular pressure, hypotension or dilated cardiomyopathy, comprising a cardiac troponin and a BNP-type peptide A diagnostic composition as described above, comprising a means for measuring the amount of phospholipids together with instructions indicating that an increase in cardiac troponin levels and an increase in BNP-type peptide levels are susceptible to treatment for early heart failure.   A diagnostic composition for determining whether a subject suffering from myocardial necrosis is susceptible to treatment for coronary heart disease, comprising means for measuring the amount of cardiac troponin and BNP-type peptide, A diagnostic composition as described above, wherein the amount of BNP-type peptide that is increased and not increased, together with instructions indicating that the subject is susceptible to treatment for coronary heart disease.   A diagnostic device for carrying out the method according to any one of claims 1 to 11, for measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis. 12. The device comprising: means and means for comparing the amount with a reference amount; and instructions for performing the method of any one of claims 1-11.   A diagnostic kit for carrying out the method according to any one of claims 1 to 11, for measuring the amount of cardiac troponin and the amount of BNP-type peptide in a sample of a subject suffering from myocardial necrosis. 12. The kit comprising: means and means for comparing the amount to a reference amount; and instructions for performing the method of any one of claims 1-11. A method for providing data for use in monitoring the success of treatment for early heart failure resulting from elevated intraventricular pressure, hypotension or dilated cardiomyopathy, comprising the following steps:
(A) measuring the amount of cardiac troponin and BNP-type peptide in at least first and second samples of a subject subjected to treatment, wherein the first sample is obtained in advance, and the second sample (B) comparing the amounts obtained from the first and second samples, whereby the amount of cardiac troponin in the second sample relative to the first sample is obtained. A method as described above, comprising the step of indicating success if reduced and the amount of BNP-type peptide is reduced.

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