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
• • 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
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • 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
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
  • G01N2410/04—Oxytocins; Vasopressins; Related peptides
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/50—Determining the risk of developing a disease

Definitions

• the invention relates to a method for risk stratification of acute coronary syndrome (ACS), in particular of acute myocardial infarction (AMI) and angina pectoris (AP), wherein a determination of provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II by means of a in vitro diagnosis is made. Furthermore, the invention relates to suitable combinations of biomarkers for in-vitro diagnostics.
• ACS acute coronary syndrome
• AMI acute myocardial infarction
• AP angina pectoris
• provasopressin provasopressin
• the invention relates to suitable combinations of biomarkers for in-vitro diagnostics.
• Risk stratification is becoming increasingly important in the area of heart disease, whether in symptomatic or asymptomatic patients. Especially in the field of acute coronary syndrome there is a high need for a suitable risk stratification. Risk stratification serves to locate patients with the worse prognosis, for the purpose of more intensive diagnostics and therapy / treatment with the aim of the most favorable course possible. Appropriate risk stratification should therefore entail effective treatments for acute coronary syndrome with percutaneous coronary interventions and newer drugs.
• biochemical markers - particularly the classics such as cardiac troponins, myoglobin and CK-MB mass - have been used to predict myocardial infarction (Katus, H.A.
• Copeptin (also: C-terminal proAVP) is described in WO 2006/018315 (BRAHMS AG) as a biomarker for the in-vitro diagnosis of AMI.
• a corresponding copeptin assay is described in Morgenthaler et al. (Nils G. Morgenthaler, Joachim Struck, Christine Alonso and Andreas Bergman, Assay for the Measurement of Copeptin, a Stable Peptide Derived from the Precursor of Vasopressin Clinical Chemistry 52: 112-119, 2006).
• Neurophysin has been previously described as a marker for nicotine uptake (Robinson AG, Isolation, assay, and secretion of individual human neurophysins J Clin Invest 1975; 55: 360-7), cancer and non-cancer associated SIADH (Syndrome of inappropriate ADH secretion) and nephrogenic diabetes insipidus (Pullan PT, Clappison BH, Johnston CI, Plasma vasopressin and human neurophysin in physiological and pathological states associated with changes in vasopressin secretion J Clin Endocrinol Metab 1979; 49: 580-7; North WG, LaRochelle FT , Jr., Melton J, Mills RC, Isolation and partial characterization of two human neurophysins: their use in the development of specific radioimmunoassays, J Clin Endocrinol Metab 1980; 51: 884-91).
• a disadvantage of the known diagnostic methods using the previously known markers is that early and complete detection of high-risk patients fails and therefore risk stratification is insufficient. It is therefore an object of the invention to develop a method for risk stratification of the acute coronary syndrome, which enables an improved detection of risk patients.
• Another object is therefore to provide a method for
• At least one marker or combination of markers has sufficient sensitivity and specificity in an in vitro diagnosis.
• provasopressin provasopressin
• inventive method a determination of the provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II by means of an in-vitro diagnosis
• provasopressin or fragments and partial peptides thereof, in particular copeptin or neurophysin II, have a high sensitivity and specificity for the diagnosis of acute coronary syndrome (see Examples and Figures).
• acute coronary syndrome encompasses various stages of coronary heart disease that are directly life-threatening, particularly in emergency medicine, such as acute myocardial infarction and / or angina pectoris and sudden cardiac death, in addition to acute myocardial infarction, which is classified according to WHO criteria ( WHO (1979): Nomenclature and criteria for diag- nosis of ischemic heart disease Report of the Joint International Society and Federation of Cardiology / World Health Organization task force on standardization of clinical nomenclature, Circulation 59 (3): 607- 609) is defined as an acute chest pain event lasting more than 20 minutes, associated with ST segment elevations and / or an increase in myocardial enzymes, the term unstable angina pectoris (AP) has been defined, which according to the invention can be read under "acute coronary syndrome" ( Hamm CW: Guidelines: Acute coronary syndrome
• the term "risk stratification” encompasses the finding of patients, in particular emergency patients and risk patients, with the worse prognosis, for the purpose of more intensive diagnostics and therapy / treatment of the acute coronary syndrome, in particular myocardial infarction, angina pectoris with the aim of enabling the most favorable course possible Risk stratification subsequently allows for an effective treatment procedure that is available in acute coronary syndrome with percutaneous coronary interventions and newer drugs.
• the invention also relates to the identification of patients at increased risk and / or an unfavorable prognosis of acute coronary syndrome, in particular myocarditis, angina pectoris in symptomatic and / or asymptomatic patients, especially emergency patients. Particularly in the case of emergency and / or intensive care medicine, secure stratification can take place by means of the method according to the invention.
• the method according to the invention therefore makes possible clinical decisions which lead to rapid therapeutic success and to avoid death traps.
• Such clinical decisions also include continuing treatment by drugs for the treatment or therapy of acute coronary syndrome, particularly myocardial infarction (AMI) and angina pectoris (AP).
• AMI myocardial infarction
• AP angina pectoris
• the invention also relates to a method for risk stratification of patients with an acute coronary syndrome for carrying out clinical decisions, such as further treatment and therapy by means of drugs, preferably in the time-critical intensive care or emergency medicine.
• the method according to the invention therefore relates to the therapy control of the acute coronary syndrome, in particular myocardial infarction (AMI) and angina pectoris (AP).
• AMI myocardial infarction
• AP angina pectoris
• the invention relates to a method for in-vitro diagnostics for the early or differential diagnosis or prognosis of myocardial infarction or angina pectoris, wherein a determination of the marker provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II a patient to be examined is performed.
• proAVP provasopressin
• copeptin or a fragment or a partial sequence thereof or neurophysin II or a fragment or a partial sequence thereof is particularly preferred.
• the invention relates to a method for
• the invention relates to a method for risk stratification of acute coronary syndrome or a method for in-vitro diagnostics for early or differential diagnosis or Prognosis of myocardial infarction or angina pectoris according to one of the above embodiments, wherein after onset of symptoms a cut-off (threshold) range of 10-30 pmol / L of the marker provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II, becomes significant (specific) for prognosis and / or risk stratification. Further preferred is a cut-off (threshold) of 10-20 pmol / L. From this basis, these inventive methods are advantageous sensitive.
• body fluid in particular blood
• body fluid is taken from the patient to be examined, optionally whole blood or serum or plasma obtained, and the diagnosis is made in vitro / ex vivo, ie outside the human or animal body.
• provasopressin proAVP
• fragments and partial peptides thereof in particular copeptin or neurophysin II
• a high sensitivity and specificity for the acute coronary syndrome, myocardial infarction and angina pectoris is achieved and based on the amount present in at least one patient sample the diagnosis or risk stratification respectively.
• the marker copeptin (stable fragment of proAVP or preprovasopressin) or a fragment or a partial sequence thereof is very particularly preferred.
• marker neurophysin stable fragment of proAVP or preprovasopressin
• marker neurophysin stable fragment of proAVP or preprovasopressin
• provasopressin is understood as meaning a human protein or polypeptide which can be obtained from the preprovasopressin and comprises amino acids 29-164 in the context of the preproasopeptic (see also WO2006 / 018315 and FIG.
• copeptin fragment: AS 126-164 (39AS: SEQ: ASDRSNATQL DGPAGALLLR LVQLAGAPEP FEPAQPDAY) or Neurophysin II (fragment: AS 32-124 of preprovasopressin (93AS: SEQ: AMSDLELRQC LPCGPGGKGR CFGPSICCAD ELGCFVGTAE ALRCQEENYL PSPCQSGQKA CGSGGRCAAF GVCCNDESCV TEPECREGFH RRA)
• these polypeptides of the invention may have post-translational modifications such as glycosylation, lipid (o) -identification or derivatizations.
• the invention relates to the diagnosis and / or risk stratification and / or early or
• AMI myocardial infarction
• AP angina pectoris
• provasopressin provasopressin
• the determination of provasopressin (proAVPP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II can additionally be carried out with further markers and preferably those which already indicate an acute coronary syndrome, in particular myocardial infarction or angina pectoris. Therefore, the invention relates to such an embodiment of the inventive method, wherein the determination is carried out in addition to at least one further marker selected from the group of inflammatory markers, cardiovascular markers, neurohormonal markers or ischemic markers on a patient to be examined.
• the inflammatory marker can consist of at least one marker from the group C-reactive protein (CRP), cytokines, such as TNF-alpha, interleukms, such as IL-6, procalcitonm (1-116, 3-116) and adhesion molecules, such as VCAM or ICAM and the cardiovascular marker, in particular necrosis of myocardial tissue indicating markers and blood pressure-influencing markers from at least one marker from the group creatine kinase, myoglobin, myeloperoxidase, natriuretic protein, in particular ANP (or ANF), proANP, NT proANP, BNP, proBNP, NT-proBNP or each a subsequence thereof, cardiac troponin, CRP.
• CRP C-reactive protein
• cytokines such as TNF-alpha
• interleukms such as IL-6
• adhesion molecules such as VCAM or ICAM
• the cardiovascular marker in particular
• pro-gastrin-releasing peptide proGRP
• pro-endothelin-1 pro-leptin
• pro-neuropeptide-Y pro-somatostatin
• pro-neuropeptide-YY pro Opiomelanocortin or pro-adrenomedullin (proADM) or each a subsequence thereof.
• the ischemic marker can be selected from at least one marker from the group troponin I and T, CK-MB.
• the neurohormonal marker may be at least one natriuretic protein, in particular ANP (or ANF), proANP, NT-proANP, BNP, proBNP, NT-proBNP or a subsequence of each.
• the invention relates to a particularly advantageous combination of biomarkers namely provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II with natriuretic proteins, in particular ANP (or ANF), proANP, NT-proANP, BNP , proBNP, NT-proBNP or a subsequence of each.
• provasopressin proAVP
• ANP or ANF
• proANP or ANF
• NT-proANP NT-proANP
• BNP proBNP
• NT-proBNP NT-proBNP
• the invention relates to a method for the in-vitro diagnosis of acute coronary syndrome, myocardial infarction or angina pectoris, wherein a determination of the marker provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II in combination with natriuretic proteins, in particular ANP (or ANF), proANP, NT-proANP, BNP, proBNP, NT-proBNP or one each
• Partial sequence thereof is performed on a patient to be examined.
• Particularly preferred is again a combination of neurophysin II, copeptin and BNP, proBNP, NT-proBNP, in particular copeptin and proBNP.
• the method according to the invention can be carried out as part of an in-vitro diagnosis by means of parallel or simultaneous determinations of the markers (eg multi-well plates with 96 or more wells), with determinations being made on at least one patient sample.
• the markers eg multi-well plates with 96 or more wells
• the method according to the invention and its determinations can be carried out in a diagnostic device by means of an automatic analyzer, in particular by means of a cryptor (http://www.kryptor.net/).
• the method according to the invention and its determinations can be carried out by means of a rapid test (for example, lateral-flow test), whether in single or multiparameter determination.
• a rapid test for example, lateral-flow test
• it is a self-test or a device that is suitable in emergency diagnostics.
• the invention relates to the use of provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II for risk stratification of acute coronary syndrome, myocardial infarction or angina pectoris and / or for in-vitro diagnosis for early or differential diagnosis or prognosis of myocardial infarction or Angina pectoris.
• provasopressin proAVP
• copeptin or neurophysin II for risk stratification of acute coronary syndrome, myocardial infarction or angina pectoris and / or for in-vitro diagnosis for early or differential diagnosis or prognosis of myocardial infarction or Angina pectoris.
• the invention relates to the use of provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II in combination with natriuretic proteins, in particular ANP (or ANF), proANP, NT-proANP, BNP, proBNP, NT-proBNP or a subsequence thereof, for the diagnosis and / or risk stratification of the acute coronary syndrome, myocardial infarction or angina pectoris.
• provasopressin proAVP
• ANP or ANF
• Another object is to provide a corresponding diagnostic device for carrying out the inventive method.
• such a diagnostic device in particular an array or assay (e.g.
• Immunoassay in the broadest sense an apparatus for carrying out the inventive method.
• the invention also relates to a kit for the risk stratification of the acute coronary syndrome, myocardial infarction and / or angina pectoris containing detection reagents for the determination of provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II, if appropriate, the above-mentioned further marker.
• detection reagents include e.g. Antibodies etc.
• the invention relates to a kit for the diagnosis and / or risk stratification of the acute coronary syndrome, myocardial infarction and / or angina pectoris containing detection reagents for the determination of provasopressin (proAVP) or fragments and partial peptides thereof, in particular
• provasopressin proAVP
• Copeptin or Neurophysin II in combination with natriuretic Proteins in particular ANP (or ANF), proANP, NT-proANP, BNP, proBNP, NT-proBNP or in each case a partial sequence thereof, if appropriate the above-mentioned further marker.
• detection reagents include, for example, antibodies, etc.
• MI myocardial infarction
• Events include the development of severe heart failure and / or death of the patient. Of the patients observed, 115 were without events (o.E.) and 16 with events (+ e.). The age medians of the groups were 63.8 and 64.5 years.
• the copeptin Increase, ie the time of blood collection for early diagnosis of minor importance (at the cut off 7.5 pmol / 1 eg 78.3 / 80.0 / 73% sensitivity at 0-2 / 2-4 / 4-6 hours, respectively ,
• NT-ProBNP has been isolated from Omland et al (Omland T, Persson A, Ng L, et al., N-terminal pro-B-type natriuretic peptide and long-term mortality in acute coronary syndrome., Circulation., 2002; 106: 2913-2918 ) determined with a luminescence immunoassay.
• Copeptin was determined by means of the Copeptm luminescence immunoassay from Brahms AG. The copeptin determination method is detailed in Morgenthaler NG et al Clin. Chem. 2006 Jan, 52 (1), 112-9. In summary: 50 ⁇ l of sample are pipetted into a tube coated with Copeptm antibody (AKI) and mixed with 200 ⁇ l of a solution of acne-tin ester-labeled anti-copeptin antibody (AK2) and incubated for 2 hours at room temperature. After removal of unbound (free) labeled antibody by washing with washing solution 4 times (Lumitest Waschlosung, Brahms AG), bound acridinium ester-labeled antibody was determined in a luminometer from Berthold.
• AKI Copeptm antibody
• AK2 acne-tin ester-labeled anti-copeptin antibody
• surv. Survive
• E. event (event): death, development of severe heart failure
• cutoff threshold in pmol / L
• Sens sensitivity
• Spef specificity
• o. without.
• Radioimmunoassay for Neurophysin Neurohypophyseal neurophysin was isolated and quantified. This was used to immunize rabbits and thus to obtain high-titer anti-neurophysin antisera. For the immunoassay, the highest-titer antiserum was used in a concentration of 1: 100,000. Purified neurophysin was radioiodinated by the chloramine T method and used as a tracer in the assay. Dilutions of purified neurophysin in normal horse serum served as standards.
• the assay was performed as follows: 50 ⁇ l of sample or standard were mixed with 100 ⁇ l of tracer (12,000 dpm per determination) and 100 ⁇ l of diluted anti-neurophysin Antiserum mixed and incubated at 4 0 C for 24 hours.
• the buffer used was: 100 mM sodium phosphate, pH 7.5, 0.1% BSA.
• Antibody-bound tracer was separated from free tracer by adding 60% ethanol and then centrifuged for 15 minutes at 4 ° C. and 5,000 g. The supernatant was discarded and the radioactivity remaining in the pellet was determined.
• the evaluation was carried out using the software Multicalc.
• the assay had an analytical detection limit of 22 pg / ml.
• the assay had a measurement range up to 400 pg / ml.
• the assay was used to measure plasma samples from different patients, as explained below.
• Myocardial infarction / prognosis Sixty-two patients with acute myocardial infarction were sampled and neurophysin was measured no later than 6 hours after onset of heart attack or on the second day after infarction. Patients were followed for 360 days. During this period, 58 patients had no adverse event, 8 died or were re-hospitalized for heart failure.
• Prognosis for Day 1 ( ⁇ 6 hours after the occurrence of the infarction): the best ⁇ Cut off value (defined as the largest product of sensitivity and specificity) for the prediction of mortality or rehospitalization by receiver operator characteristics analysis for heart failure : 777 pg / ml. At this cut-off value, the sensitivity of the prognosis was 62.5%, the specificity was 73%. The likelihood ratio of an adverse event at a cut-off value of 777 pg / ml was 2.3. Forecast for day 2:
• Receiver-Operator-Characteristics analysis determined the best cut-off value (defined as the largest product of sensitivity and specificity) for predicting mortality or rehospitalization for heart failure: 261 pg / ml. At this cut-off value, the sensitivity of the prognosis was 68.8%, the specificity was 73%. The likelihood ratio of an adverse event at a cut-off value of 261 pg / ml was 2.6.
• Figure 1 Figure 1:
• Copeptin values of patients after myocardial infarction Plasma samples were obtained from 131 patients at various times after myocardial infarction as indicated. In addition, Copeptin values of healthy are shown ("controls", 200 values) .Copeptin values of the groups are shown as box whiskers plots.
• FIG. 2 is a diagrammatic representation of FIG. 1
• Copeptin values of patients after heart attack Plasma specimens were recovered at various times after myocardial infarction, as indicated. The patients were grouped according to whether later death or re-hospitalization due to heart failure occurred ("event", 16
• Copeptin values of healthy are shown ("controls", 200 values). Copeptin values of the groups are shown as box whiskers plots.
• FIG. 3 is a diagrammatic representation of FIG. 3

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