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WO2006102979A2 - Marqueurs d'inflammation et combinaisons de ces marqueurs en tant que marqueurs biochimiques pour des maladies cardio-vasculaires - Google Patents

Marqueurs d'inflammation et combinaisons de ces marqueurs en tant que marqueurs biochimiques pour des maladies cardio-vasculaires Download PDF

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WO2006102979A2
WO2006102979A2 PCT/EP2006/002230 EP2006002230W WO2006102979A2 WO 2006102979 A2 WO2006102979 A2 WO 2006102979A2 EP 2006002230 W EP2006002230 W EP 2006002230W WO 2006102979 A2 WO2006102979 A2 WO 2006102979A2
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Prior art keywords
timp
adma
caspase
stnfr
svcam
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PCT/EP2006/002230
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German (de)
English (en)
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WO2006102979A3 (fr
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Stefan Blankenberg
Hans J. Rupprecht
Christoph Bickel
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Johannes Gutenberg-Universität Mainz
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Priority claimed from DE200510014752 external-priority patent/DE102005014752A1/de
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Publication of WO2006102979A2 publication Critical patent/WO2006102979A2/fr
Publication of WO2006102979A3 publication Critical patent/WO2006102979A3/fr

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    • 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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • 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

  • the invention relates to novel markers of vascular inflammation and combinations thereof as tools for diagnosis and their prognosis in patients with cardiovascular diseases.
  • the markers also serve as tools that facilitate the selection of drugs for the treatment of such diseases, and finally as a starting point for the treatment of cardiovascular diseases.
  • the invention relates to the creation of an individual risk profile of negative events associated with the progression of arteriosclerosis.
  • Thrombus formation in the coronary vasculature is the triggering event of unstable coronary heart disease.
  • the central role of platelet activation in patients with unstable coronary heart disease is further enhanced by thromboxane and prostaglandin metabolites released by the platelets. Therefore, platelet activation is a general therapeutic target. So far, such therapies have involved the use of aspirin, tienopyridines and a direct glycoprotein IIb / IIIa inhibitor.
  • MI myocardial infarction
  • the ECG has only limited prognostic relevance in this respect, as significant abnormalities are rare and their detection is less sensitive and specific (Kaul P, Fu Y, Chang WC, et al., J Am Coli Cardiol 2001; 38: 64-71 and Savonitto S, Ardissino D, Granger CB, et al JAMA 1999; 281: 707-13). Therefore, markers of necrosis of myocardial cells, especially cardiac troponins, have become valuable tools in the evaluation of patients with acute coronary syndromes (Hamm CW, Braunwald E. Circulation 2000, 102: 118-22).
  • troponins are not actively involved in the pathophysiology of acute coronary syndromes, but are rather a surrogate marker of fragile thrombus formation (Lindahl B, Diderholm E, Lagerqvist B, Venge P, Wallentin L. J Am Coli Cardio, 2001; : 979-86, Heeschen C, van den Brand MJ, Hamm CW, Simoon ML. Circulation 1999; 100: 1509-14; Benamer H, Steg PG, Benessiano J, et al. Am Heart J 1999; 137: 815-20).
  • Inflammatory markers that determine the activity of the disease could be important additional information for diagnostic and therapeutic stratification in patients with acute coronary syndromes.
  • the specific therapeutic inhibition of cytokines that are essential for plaque stability may be a new treatment strategy for patients with unstable and stable coronary heart disease.
  • markers there are many different molecular markers in the art which may be useful in the diagnosis of cardiovascular disease. Examples of such markers include: Pregnancy-associated plasma protein A (PAPP-A); C-reactive protein (CRP); hs-CRP; placental growth factor (PlGF); Interleukin-18 (IL-18 / IL-18b); Brain natriuretic peptide (BNP); NT-per brain natriuretic peptide (NT-pro NP); sCD40L, cTnI / T, IL-10, ICAM-I, VCAM-I, E-selectin, P-selectin, IL-6, VEGF, serum amyloid A (SAA), CKMB, MPO, ILI-RA, TAFl, soluble Fibrin, MCP-I, Tissue Factor (TF), MMP-9, bFGF, PCM and VEGF-A.
  • PAPP-A Pregnancy-associated plasma protein A
  • CRP C-reactive protein
  • the endothelium plays an important role in the regulation of vascular tone.
  • One factor of great importance for the maintenance of endothelial homeostasis is nitric oxide.
  • the substrate for endothelial nitric oxide synthesis is the amino acid L-arginine.
  • the endothelium is also capable of producing methylated amino acids, such as asymmetric dimethylarginine (ADMA), which are capable of being competitive with L-arginine as a substrate for endothelial nitric oxide synthase, resulting in endothelial dysfunction (for a summary, see Cooke JP, 2000).
  • ADMA asymmetric dimethylarginine
  • Resistin is a cytokine expressed by adipocytes.
  • the main function is antagonizing the insulin effect and impairing glucose tolerance.
  • MMPs tissue-matrix metalloproteinases
  • Several studies have shown that extracellular matrix degradation by tissue-matrix metalloproteinases (MMPs), particularly MMP-9, is involved in the pathogenesis of a wide range of cardiovascular diseases, including arteriosclerosis, restenosis, cardiomyopathy, congestive heart failure, myocardial infarction and aortic aneurysm (Dollery , Benjamin, 2001). Levels of circulating MMP-9 are associated with future cardiovascular events (Blankenberg, 2003).
  • TIMP-I tissue inhibitors of metalloproteinases
  • TIMP-I endogenous tissue inhibitors of metalloproteinases
  • TIMP-I is one of the best characterized TIMPs that binds to activated MMPs with high affinity.
  • TIMP-I appears to play an important role in the regulation of left ventricular structure and systolic function (Li, 1998, Timms, 2002) and plasma TIMP-I levels are elevated in coronary disease (Hirohata, 1997).
  • total TIMP-I was associated with significant cardiovascular risk factors (Sundstrom, 2004).
  • the direct mechanistic role of MMPs and TIMPs has not been fully established in the post-MI remodeling process.
  • Caspases have functions in apoptotic signaling pathways.
  • the primary role of caspase-1 is the regulation of inflammatory processes.
  • co-localization of caspase-1 with apoptotic cells in advanced human plaques was reported.
  • focal apoptosis of macrophages with extensive expression of caspase-1 was detected and associated with sudden cardiac death.
  • TnT is of particular value for the diagnosis and the Prediction of MI (see above).
  • Inflammatory markers such as CRP are valuable for the diagnosis and prediction of inflammation, which can lead to plaque rupture and MI.
  • cardiovascular risk stratification is currently performed using classical risk factors and various risk calculators (Framingham / PROCAM). Recently, it has been increasingly discussed to what extent highly sensitive C-reactive protein (CRP) as a marker of inflammation enables more accurate risk stratification.
  • CRP C-reactive protein
  • the association between different markers of inflammation (CRP, interleukin-6 or soluble intercellular adhesion molecule-1) and future cardiovascular risk has been confirmed in several studies.
  • the known solutions ie the determination of the cardiovascular risk by means of traditional risk factors, have less than 50% sensitivity and specificity.
  • ECG and troponin as well as CK value determinations do not provide 100% diagnostic accuracy for the acute coronary syndrome.
  • the object of the present invention is to provide a method by which the possibility of risk stratification of coronary heart disease is improved by the determination of specific individual markers and / or a specific "marker panel" consisting of biomarkers of inflammation be extended.
  • the attending physician should be better able than before to be able to select suitable measures in order to positively influence the patient, to prevent a disadvantageous event or at least to lessen its severity for the affected patient.
  • the object of the present invention is achieved by a method for the analysis of samples in connection with acute cardiovascular diseases.
  • the process according to the invention comprises the steps of:
  • c optionally, determining the concentration of at least one further marker selected from sTNFR-1, troponin T (TnT), IL-6, sILI-RA, CRP, soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1, soluble E-selectin , Matrix metalloproteinase-9, CD40, lipoprotein-associated phospholipase A2 and other inflammatory markers, and (d) Comparison of the results obtained for the sample to be tested / s with reference values and / or the values from reference samples.
  • TnT troponin T
  • IL-6 intercellular adhesion molecule-1
  • soluble vascular adhesion molecule-1 soluble E-selectin
  • Matrix metalloproteinase-9 Matrix metalloproteinase-9
  • CD40 lipoprotein-associated phospholipase A2 and other inflammatory markers
  • This invention contemplates that various markers of inflammation (soluble tumor necrosis factor - receptor 1 and 2 (sTNFR-1 and sTNFR-2), soluble interleukin-1 receptor antagonist (sIL1-RA), interleukin-18 (IL-18 ), soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1, soluble E-selectin, matrix metalloproteinase-9, interleukin-6 (IL-6), fibrinogen, ADMA, caspase-1, TIMP-I, resistin, sVCAM- 1 and C-reactive protein (CRP)) allow predicting the cardiovascular risk.
  • soluble tumor necrosis factor - receptor 1 and 2 soluble interleukin-1 receptor antagonist
  • IL-18 interleukin-18
  • soluble intercellular adhesion molecule-1 soluble vascular adhesion molecule-1
  • E-selectin matrix metalloproteinase-9
  • IL-6 interleukin-6
  • fibrinogen ADMA
  • caspase-1 TIMP-
  • markers have the potential to diagnose an acute coronary syndrome;
  • markers as well as a specially designed "marker cluster”, can be used to estimate the risk of suffering a fatal or non-fatal myocardial infarction, which extends the risk assessment currently available with classical risk factors.
  • acute phase reactants C-reactive protein, fibrinogen and interleukin-6
  • pro-inflammatory cytokines and soluble receptors pro-inflammatory cytokines and soluble receptors
  • soluble adhesion molecules intercellular adhesion molecules
  • a variety of inflammatory biomarkers are associated with cardiovascular prognosis. Particularly high is an inflammatory value consisting of the parallel determination of 8 biomarkers of inflammation (TIMP-I, ADMA, Resistin, caspase-1, sTNFR-2, IL-18, sVCAM-1, fibrinogen) with the risk of a future cardiovascular event associated.
  • TIMP-I 8 biomarkers of inflammation
  • an inflammatory marker panel is described as a strong and independent predictor of cardiovascular risk. Inflammatory vascular wall processes are causally involved in destabilizing arteriosclerotic plaques and enhancing thrombus formation. These results have triggered the search for circulating biochemical markers that reflect inflammatory activity within the vascular wall.
  • C-reactive protein In addition to fibrinogen, the first inflammatory biomarker associated with future cardiovascular risk, C-reactive protein has been the most widely studied. However, its clinical usefulness has recently been doubted.
  • Other (pro) inflammatory markers such as interleukin-6, soluble adhesion molecules, interleukin-18 or lipoprotein-associated phospholipase A2 are also potential emerging tools for cardiovascular risk prediction.
  • the inventors analyzed several circulating inflammatory biomarkers in a large cohort of patients with coronary heart disease.
  • the inventors selected among others acute phase reactants (C-reactive protein, fibrinogen and interleukin-6), proinflammatory cytokines and soluble receptors (tumor necrosis factor receptors 1 and 2, soluble interleukin-1 receptor antagonist and interleukin-18), soluble adhesion molecules (intercellular Adhesion molecule-1, vascular adhesion molecule-1, soluble E-selectin) and matrix metalloproteinase-9.
  • acute phase reactants C-reactive protein, fibrinogen and interleukin-6
  • proinflammatory cytokines and soluble receptors tumor necrosis factor receptors 1 and 2, soluble interleukin-1 receptor antagonist and interleukin-18
  • soluble adhesion molecules intercellular Adhesion molecule-1, vascular adhesion molecule-1, soluble E-selectin
  • matrix metalloproteinase-9 matrix metalloproteinase-9.
  • C-reactive protein (CRP) and hsCRP are markers of systemic inflammation, troponin cTnl / T as a marker of necrosis; the pregnancy-associated plasma protein A (PAPP-A) as a marker for macrophage activation; IL-10 (interleukin 10) as marker for the inflammatory balance, sCD40L as marker for the thrombo-inflammatory activation, MPO (myeloperoxidase) as marker for oxidative stress, placental growth factor (PlGF) as marker for vascular inflammation and the markers brain natriuretic Peptide (BNP) and NT-pro brain natriuretic peptide (NT-proNP) as markers of neurohumoral activation and ischemia.
  • C-reactive protein (CRP) and hsCRP are markers of systemic inflammation, troponin cTnl / T as a marker of necrosis; the pregnancy-associated plasma protein A (PAPP-A) as a marker for macrophage activ
  • interleukin-18 IL-18 / IL-18b
  • ICAM-1 interleukin-1
  • VCAN-I E-selectin
  • P-selectin IL-6
  • VEGF serum amyloid A
  • SAA serum amyloid A
  • CKMB CKMB
  • IL-I-RA IL-I-RA
  • TAF-I soluble fibrin
  • MCP-I 5 Tissue Factor (TF) MCP-I 5 Tissue Factor (TF)
  • bFGF bFGF
  • PCM VEGF-A
  • the sample to be examined and / or the reference sample originate from a mammal, in particular from humans.
  • the sample to be examined and / or the reference sample is selected from the group consisting of peripheral blood or fractions thereof and cell culture suspensions or fractions thereof. It is furthermore preferred that the examining sample and / or reference sample is blood serum or blood plasma. Peripheral whole blood is particularly preferred as the sample to be examined and / or the reference sample.
  • the sample to be examined and / or the reference sample can be pretreated, wherein the peripheral blood z.
  • a anticoagulant especially heparin, is added.
  • the analyzed markers and combinations thereof are selected from:
  • Particularly preferred according to the invention is then a method wherein the analyzed markers and combinations thereof are selected from fibrinogen + sTNFR-2 + IL-18 + sVCAM-1 and fibrinogen + sTNFR-2 + IL-18 + ADMA + caspase-1 + TIMP-I + Resistin + sVCAM-1,
  • the method used according to the invention for determining the concentration of the markers analyzed can be selected from any suitable method known to the person skilled in the art for the detection of proteins in biological samples.
  • the specific procedure is within the scope of the present.
  • the invention is not important as long as the method is sensitive enough to undercut the detection limit required for an accurate determination of the concentrations of the markers.
  • Suitable methods are usually based on the binding of a label to the label to be detected and the subsequent detection of this label.
  • the bond may be covalent or noncovalent and / or directly or indirectly.
  • Suitable measuring methods according to the present invention include e.g. For example, electrochemiluminescence. Turbidimetry, nephelometry and latex enhanced turbidimetry or nephelometry may also be used.
  • the concentration is determined by means of an immunological method by means of molecules binding to the marker.
  • examples of such methods are enzyme-linked immunosorbent assay (ELISA), sandwich enzyme immunoassay or solid phase immunoassay.
  • ELISA enzyme-linked immunosorbent assay
  • the molecules that bind to the markers are selected from the group consisting of anti-marker antibodies or parts thereof and marker receptors or parts thereof. These molecules can be selected from a very wide variety of molecules specific for the markers. It is preferred that the molecules that bind to the markers are selected from the group consisting of antibodies directed specifically against markers or against portions thereof, or portions or fragments thereof and a marker receptor or portions thereof or an integrin. Particularly preferred is a method according to the invention, wherein the antibodies, parts or fragments thereof include polyclonal antibodies, monoclonal antibodies, Fab fragments, scFv antibodies and diabodies.
  • components of the method may be bound to a solid phase, thus, the molecules that bind to a marker may be in solution or matrix immobilized.
  • matrices a variety of materials known to those skilled in the art may be used, such as resin matrices and / or conventional column matrices.
  • Particular preference is furthermore given to a method according to the invention in which the molecules binding to a marker are coupled to one or more detection molecules from the group consisting of fluorescein thioisocyanate, phycoerythrin, enzymes (for example horseradish peroxidase) and magnetic bead.
  • the molecules binding to the markers can be detected with an antibody to which one or more detection molecules are coupled. It is thus an indirect proof of the binding of the molecule.
  • two-stage detections are well known to those skilled in the art, for example, from the anti-antibody detection technique.
  • immunocytological methods can be used to analyze the sample. All methods are suitable which allow a specific determination based on the marker / molecule interaction. Preferred are methods selected from the group consisting of sandwich enzyme immunoassay, ELISA and solid phase immunoassays. The results obtained for the sample to be examined are usually compared with a reference sample. Which sample can serve as a reference sample will depend, in particular, on the type of sample being tested and the medical history of the individual from which the sample to be tested is derived. Preferred is a method according to the invention, in which the reference sample originates from one or the mean of several mammals in which a cardiovascular disease has been excluded. But this does not necessarily have to be the case if z. B. the progression of a disease is to be determined, an "old" sample of the same patient can be used as a reference sample. It is obvious to the person skilled in the art which samples are suitable as a reference sample for the method according to the invention.
  • the acute cardiovascular diseases to be diagnosed and / or prognosticated and / or their therapy may be selected from the group consisting of unstable angina, myocardial infarction, acute heart syndrome, coronary artery disease and cardiac insufficiency.
  • the method according to the invention is suitable and can still be used for other acute cardiac disease states.
  • a further aspect of the present invention relates to a diagnostic kit, wherein the kit comprises means for carrying out the method according to the invention, possibly together with further components and / or auxiliaries.
  • Such agents are preferably at least one antibody for detection of marker and means for subsequent quantification of the markers.
  • the kit may also contain other components and / or enzymes for carrying out the methods of the present invention, e.g. B. Instructions for use for interpreting the results of the test with regard to the risk profile of the patient and appropriate countermeasures and therapy suggestions.
  • Another aspect of the present invention thus relates to the use of the method according to the invention for the diagnosis and / or prognosis of acute cardiovascular diseases and / or for the monitoring of their therapy. This is done by the quantitative and critical determination of markers. On the basis of the risk profile which can then be created, appropriate countermeasures can then be carried out by the attending physician in order to positively influence the patient and to prevent or at least reduce the severity of the adverse event for the affected patient.
  • Such a therapy according to the invention z.
  • the administration of statins or inhibitors of the glycoprotein IIb / III receptor in particular abciximab include.
  • those skilled in the art will be aware of other possible therapies to treat cardiovascular diseases that may occur according to conventional schemes.
  • an anti-inflammatory agent may further be administered.
  • these agents came to be selected from non-steroid or steroid anti-inflammatory agents, which may include eg: alclofenac; alclometasone; dipropionate; Algestonacetonide; Alpha-amylase; Amcinafal; amcinafide; Amfenac sodium; Amiprilosehydrochlorid; anakinra; Anirolac; anitrazafen; apazone; Balsalazide disodium; bendazac; benoxaprofen; Benzydamine hydrochloride; bromelain; broperamole; Budesonide; carprofen; cicloprofen; Cintazon; Cliprofen; clobetasol propionate; clobetasone; clopirac; Cloticasonpropionat; Cormethasonacetat; cortodoxone; deflazacort; desonide; deoxymethasone; Dex
  • diagnosis refers to the verification of whether an individual has suffered from a particular cardiovascular event.
  • prognosis refers to predicting the likelihood (in%) of whether an individual will suffer from a particular cardiovascular event.
  • stratification of therapy refers to the determination of the appropriate therapeutic treatment for the cardiovascular event that will occur or has occurred.
  • monitoring therapy refers to the control and, optionally, discontinuation of the therapeutic treatment of an individual.
  • therapeutic treatment includes any treatment that may or may not include the pathophysiological condition of an individual and includes, for example: As the administration of pharmaceuticals and surgical treatment (eg., Balloon dilatation).
  • tumor necrosis factor- ⁇ and interleukin-1 are difficult to measure in the circulation because of their short half-life.
  • the increase in soluble tumor necrosis factor receptor and soluble interleukin-1 receptor antagonist are closely associated with tumor necrosis factor- ⁇ and interleukin-1, respectively, and show stable basal serum levels.
  • soluble tumor necrosis factor receptor 2 and soluble interleukin-1 receptor antagonist counteract potentially deleterious effects, endogenous production does not appear to be sufficient to prevent the negative effects of primary cytokines produced locally (26).
  • the soluble receptors seem to be better markers of the activity of the disease than the cytokines themselves (27).
  • the present invention demonstrates a strong association of soluble interleukin-1 receptor antagonist with cardiovascular risk in univariate analysis, however, the effect was no longer significant when several inflammatory markers were considered simultaneously. This may be due to the fact that soluble interleukin-1 receptor antagonist levels strongly correlate with soluble tumor necrosis factor receptor 2.
  • interleukin-18 expression another member of the interleukin-1 family has been identified in human arteriosclerotic plaques, and inhibition of interleukin-18 has been shown to be a beneficial effect on the progression of plaque formation and composition in animal models.
  • the association of baseline interleukin-18 levels with future cardiovascular death has recently been demonstrated in atherosclerotic examination in both stable angina pectoris and acute coronary syndrome, and was further confirmed in the cohort of initially healthy subjects in the PRIME study.
  • interleukin-18 levels are weakly correlated with other inflammatory markers, suggesting that interleukin-18 represents a rather distinct pathophysiological pathway.
  • the present invention recognizes the significant and independent role of fibrinogen, sTNFR-2, IL-18, ADMA, caspase-1, TIMP-I, resistin and sVCAM-1 as platelet activators for the diagnostic and therapeutic Risk stratification documented.
  • Figure 1 shows the pairwise Pearson correlation coefficients between inflammatory markers in patients with stable angina pectoris and those with acute coronary syndrome (log or square root transformations were used to normalize the distributions).
  • Figure 2 shows the hazard ratios of future cardiovascular events by increasing thirds of inflammatory markers. Hazard ratios were adjusted for age, sex, body mass index, smoking status, history of high pressure or diabetes, HDL cholesterol, statin therapy and number of stenotic vessels.
  • Figure 3 the Kaplan-Meier survival curves for cardiovascular events by inflammatory value in patients with stable angina (Panel A) and acute coronary syndrome (Panel B).
  • FIG. 4 shows the difference in the Akaike Information Criterion (AIC) between the model based on the 4-marker value (surrounded) and those based on the best 1-marker, 2-marker and 3-marker values (left) and the 5 Marker value (right).
  • AIC Akaike Information Criterion
  • Figures 5 and 6 show the Kaplan-Meier curves for event-free survival according to thirds of TIMP-1 levels.
  • FIG. 7 shows the predictive strength of TIMP-I in the context of the new biological markers CRP and BNP.
  • Figure 8 shows the Kaplan-Meier curves for event-free survival according to thirds of ADMA Spiegel.
  • FIG. 9 shows the predictive strength of TIMP-I in the context of the new biological markers CRP, BNP and creatinine.
  • the inventors first examined which outcomes of eleven inflammatory markers - some analyzed for the first time - are independently associated with future cardiovascular risk and whether the simultaneous identification of multiple inflammatory markers improved the ability to detect high-risk patients beyond the classic risk factors and single-marker determination.
  • An inflammatory value was next calculated by combining the 4 markers selected by stepwise analysis.
  • the value of an individual was defined as the number of markers in the upper third of the distribution.
  • the Cox model based on this 4-marker value was further compared for information with models based on values having a smaller or larger number of markers.
  • the Akaike Information Criterion (AIC) (19) was used, and the model with the minimum AIC (minAIC) was considered to have the best empirical support (20).
  • a model with an AIC difference ⁇ 2 with the minAIC was considered equivalent to the minAIC model, whereas a model with an AIC difference> 4 was considered different from the minAIC model (20, 21).
  • Figure 2 shows the hazard ratios of cardiovascular event, adjusted for classical risk factors, according to the increasing thirds of each marker.
  • the model included the classic risk factors and soluble tumor necrosis factor receptor 2, interleukin-18, soluble interleukin-1 receptor antagonist, soluble vascular adhesion molecule-1, matrix metalloproteinase-9 and fibrinogen as the representative of the cluster of acute phase reactants.
  • the inventors generated an inflammatory score that summarized the upper thirds of soluble tumor necrosis factor receptor-2, soluble vascular adhesion molecule-1, interleukin-18, and fibrinogen.
  • the value of an individual was defined as the number of markers in the upper third. The value varied from 0 to 4, with 24.9% (26.3%), 34.3% (35.4%), 26.6% (23.7%), 10.9% (11%) and 3.3% (3.6%) of patients with stable angina (acute coronary syndrome) who had 0, 1, 2, 3, and 4 markers in the upper third, respectively.
  • the inventors compared the AIC value of the Cox model based on the 4-marker value with those of the best 1 markers. , 2-marker and 3-marker values (FIG. 4).
  • the minAIC was obtained for the 4-marker value (on 0) and was considered to have the best empirical support. Values that had a lower number of markers had worse empirical support (AIC difference> 4), whereas the best model based on a 5 marker was not superior to the 4-marker value in terms of information (AIC difference ⁇ 2).
  • the inventors examined whether the inflammatory value added additional information beyond that provided by traditional risk factors. The addition of the inflammatory value to conventional risk factors increased the area under the receiver operating characteristics curve from 0.63 to 0.68,
  • Event (n 1057)
  • Event (n 235)
  • Triglycerides mg / dl 166.4 + 3.3 179.7 + 7.1 0.01
  • Categorical variables are expressed as percent of patients, continuous variables are given as age- and sex-adjusted means + standard deviation. * The relationship between cardiovascular output and each variable (categorical or continuous) was determined by Cox regression analysis, adjusted for age and gender. F The ejection fraction was determined in 1083 patients.
  • ACE angiotensin converting enzyme
  • CAD coronary artery disease
  • MI myocardial infarction
  • a log or * square root transform was used to normalize the distribution of each marker, and antilog or squared means (95% confidence interval) are given.
  • sTNF-R1 soluble tumor necrosis receptor 1 receptor (receptor 2)
  • IL-6, IL-18 Interleukin-6 or 18 sIL-IRa: soluble IL-1 receptor antagonist sICAM-1: soluble intercellular adhesion molecule-1 sV CAM-I: soluble vascular cell adhesion molecule-1
  • MMP-9 Matrix metalloproteinase-9 hs-CRP: highly sensitive C reactive protein
  • Resistin (ng / ml) was determined in 1194 patients with documented CHD at the time of study enrollment. Patients presented with stable angina pectoris (AP), troponin negative unstable AP, subacute and acute myocardial infarction (MI). During the observation period of 3.2 ⁇ 0.9 years, follow-up (f / u) was performed in 1171 patients (98.1%).
  • Plasma concentration of the resistin (ng / ml)
  • Resistin is highly predictive of the risk of acute coronary syndrome in patients with chest pain and is moderately associated with mortality risk.
  • Model 1 is not controlled; Model 2 is controlled by age and sex; Model 3 is additionally controlled for BMI, history of hypertension, diabetes, smoking, HDL
  • Model 4 additionally adjusted to multiple vascular disease, statin and beta
  • Model 5 is made by forward-probability variable selection from all
  • Serum TIMP-I was determined using the ELISA technique according to the manufacturer's instructions (Human, Biotrak, ELISA System, Amersham Biosciences, USA) at a range of 3.1-50 ng / ml. The test had a variation coefficient of between 8.9 - 11.4% within the test and between 13.1 - 15.25% variation between the test coefficients.
  • Plasma BNP was determined using a fluorescence immunoassay (Biosite Diagnostics Inc., San Diego, California). The indicated detection limit is less than 5 pg / ml. The test showed a coefficient of variation of close to 10% between tests and a yield of 100% of added peptide was found. C-reactive protein and lipid serum levels were determined as in Example 1 and by standard methods.
  • Table 6 provides baseline characteristics of the 75 study participants who subsequently died for cardiovascular reasons and 1870 who had no future cardiovascular event.
  • the left ventricular ejection fraction as determined by angiography was significantly higher in patients without cardiovascular death (P ⁇ 0.001).
  • Diabetes mellitus % 0.0001 no 84.0 62.7
  • Triglycerides mg / dl 131 (96-184) 149 (102-179) 0.34
  • the TIMP-I level was normally distributed among the study participants. It ranged from 6.0 to 2667 ng / ml, with a mean (+ SD) of 699 + 225, a median of 676 and an interquartile range of 561 to 803 ng / ml.
  • the baseline level of TIMP-I activity was significantly higher between those who died of cardiovascular causes than those who did not (820 vs 692 ng / ml; P ⁇ 0.0001).
  • BNP (P ⁇ 0.0001), CRP (P ⁇ 0.0001) and creatinine (P ⁇ 0.0001) were significantly increased in patients with future cardiovascular death.
  • Table 7 each represents the mean and median levels of inflammatory markers, such as TIMP-I, CRP and BNP, according to the prevalence of traditional risk factors and clinical variables.
  • TIMP-I was significantly higher among patients with diabetes, hypertension and severe vascular disease. Patients receiving statin medication had lower TIMP-I levels.
  • TIMP-I levels correlated inversely with the smoking history.
  • Figure 5 shows the Kaplan-Meier curves for event-free survival, according to thirds of TIMP-I levels.
  • the unadjusted rate of cardiovascular death was highest in patients within the upper third of TIMP-I levels (P log rank ⁇ 0.0001).
  • P log rank 0.002
  • Table 8 shows the hazard ratios for cardiovascular death associated with thirds of TIMP-I levels.
  • Asymmetric dimethylarginine was assayed from serum samples by competitive enzyme-linked immunosorbent assay (DLD Diagnostika GmbH, Hamburg, Germany) at a range of 0.1 ⁇ mol / 1 to 5.0 ⁇ mol / l. The detection specificity is 0.05 ⁇ mol / l. The cross-reactivity with methylarginines is low (arginine ⁇ 0.02 percent, A ⁇ monomethyl-L-arginine 1.0 percent, symmetrical dimethylarginine 1.2 percent). Plasma BNP was determined using a fluorescence immunoassay (Biosite Diagnostics Inc., San Diego, California). The indicated detection limit is less than 5 pg / ml. The test showed a coefficient of variation of close to 10% between tests and a yield of 100% of added peptide was found. Serum C-reactive protein and lipid levels were determined as in Example 1 and by standard methods.
  • a log transformation was performed and the hazard ratio applied per one standard deviation increment.
  • the hazard ratios for future coronary event according to thirds of asymmetric dimethylarginine were estimated by Cox regression models adjusted for potential confounders. Three discontinued models were constructed. The inventors focused first on age and sex and secondly on other traditional and clinical risk factors. The final model included clinical and therapeutic variables as well as C-reactive protein and B-type natriuretic peptide. Furthermore, the strengths of asymmetric dimethylarginine, B-type natriuretic peptide, C-reactive protein and creatinine for cardiovascular risk prediction were compared.
  • Cox predicative models were calculated for the upper third of each variable, set on cardiovascular confounders (model 2), and additionally for all three markers simultaneously (model 3). The hazard ratios and 95 percent confidence intervals are given.
  • areas under the receiver operating curves were used for the combined analysis of traditional risk factors (Model 1) and individual biomarkers. Determination, as well as simultaneous determination of all four markers in addition to classical risk factors calculated. The P values are two-sided; Values of p ⁇ 0.05 were considered significant. All calculations were compared using the SPSS version 11.05 program.
  • the median age of the study participants was 61.0 ⁇ 9.8 years, 79.1 percent were male.
  • the concentration of asymmetric dimethylarginine was slightly distorted among the study participants. It ranged from 0.07 to 7.49 with a mean of 0.68 and a median of 0.63 ⁇ mol / l with a 25 ⁇ 115 interquartile range of 0.53 / 0.74 ⁇ mol / l.
  • the baseline concentration of asymmetric dimethylarginine was significantly higher between those who died of cardiovascular causes than those who did not.
  • Plasma ADMA (umol / 1) 0.63 (0.53 / 0.74) 0.70 (0.60 / 0.82) 0.001
  • Plasma C-Reactive Protein 2.98 (1.40 / 7.43) 5.62 (2.51 / 15.43) 0.001
  • Table 10 provides the association of a standard deviation increment in log asymmetric dimethylarginine values with future cardiovascular event.
  • the increase in the asymmetric dimethylarginine level was associated with an increased risk of cardiovascular death (increase of 42 percent for asymmetric dimethylarginine for every increment of 1 standard deviation in log transformed asymmetric dimethylarginine values), coronary event and stroke.
  • these associations remained independently significant.
  • the primary endpoint of the study was death for cardiovascular reasons and non-fatal coronary event.
  • Figure 8 provides the Kaplan-Meier curves for event-free survival in one third of the asymmetric dimethylarginine level.
  • the unadjusted cardiovascular event rate increased in a stepwise fashion over the rising thirds of baseline asymmetric dimethylarginine levels (P ⁇ 0.001).
  • the inventors performed a series of Cox predictive models (Table 11). In an age and gender adjusted model, individuals in the upper third showed a 2.47 increase (95 percent confidence interval 1.51 to 4.03) in cardiovascular risk, compared to those in the first third. Further adjustment to clinical and therapeutic variables as well as C-reactive protein, creatinine, and B-type natriuretic peptide did not alter the hazard ratio associated with asymmetric dimethylarginine concentration.

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Abstract

L'invention concerne de nouveaux marqueurs de l'inflammation vasculaire et des combinaisons de ces marqueurs en tant qu'outils pour le diagnostic et le pronostic chez des patients souffrant de maladies cardio-vasculaires. Ces marqueurs servent en outre comme outils facilitant la sélection de principes actifs pour le traitement de telles maladies, ainsi que comme point de départ pour le traitement de maladies cardio-vasculaires. L'invention concerne en outre la création d'un profil de risque individuel d'événements négatifs qui sont liés au développement de l'artériosclérose.
PCT/EP2006/002230 2005-03-31 2006-03-10 Marqueurs d'inflammation et combinaisons de ces marqueurs en tant que marqueurs biochimiques pour des maladies cardio-vasculaires WO2006102979A2 (fr)

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EP2319924A4 (fr) * 2008-08-15 2012-01-25 Fujikura Kasei Kk Marqueur polypeptidique pour le diagnostic de l'artériosclérose, procédé de détection de l'artériosclérose à l'aide du marqueur ou similaire, et trousse de diagnostic de l'artériosclérose
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CN110763839A (zh) * 2019-10-31 2020-02-07 安徽大千生物工程有限公司 一种timp-i胶乳增强比浊法检测试剂盒及其制备使用方法
CN116773825A (zh) * 2022-08-19 2023-09-19 天津云检医学检验所有限公司 诊断急性川崎病的血液生物标志物和方法

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2147115A4 (fr) * 2007-04-16 2010-05-05 Cardibioindice/cardibioscore et utilité d'un protéome salivaire dans des diagnostics cardiovasculaires
EP2319924A4 (fr) * 2008-08-15 2012-01-25 Fujikura Kasei Kk Marqueur polypeptidique pour le diagnostic de l'artériosclérose, procédé de détection de l'artériosclérose à l'aide du marqueur ou similaire, et trousse de diagnostic de l'artériosclérose
US9366681B2 (en) 2008-08-15 2016-06-14 Fujikura Kasei Co., Ltd. Polypeptide marker for diagnosis of arteriosclerosis, method for detection of arteriosclerosis by using the maker or the like, and kit for diagnosis of arteriosclerosis
CN102792161A (zh) * 2009-08-28 2012-11-21 阿斯图特医药公司 肾损伤和肾衰竭的诊断及预后的方法及组合物
CN102792161B (zh) * 2009-08-28 2014-11-12 阿斯图特医药公司 肾损伤和肾衰竭的诊断及预后的方法及组合物
CN102175873A (zh) * 2011-01-11 2011-09-07 江苏迈迪基因生物科技有限公司 心脑血管疾病蛋白标志物的联合并行检测方法及其诊断试剂盒
CN102680697A (zh) * 2011-03-10 2012-09-19 北京吉奥众艺科技有限公司 检测肌钙蛋白i的试剂盒及其制备和使用方法
CN102680697B (zh) * 2011-03-10 2015-11-25 王迎峰 检测肌钙蛋白i的试剂盒及其制备和使用方法
CN110763839A (zh) * 2019-10-31 2020-02-07 安徽大千生物工程有限公司 一种timp-i胶乳增强比浊法检测试剂盒及其制备使用方法
CN116773825A (zh) * 2022-08-19 2023-09-19 天津云检医学检验所有限公司 诊断急性川崎病的血液生物标志物和方法
CN116773825B (zh) * 2022-08-19 2023-12-01 天津云检医学检验所有限公司 诊断急性川崎病的血液生物标志物和方法

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