WO2011038786A1 - Combinaison de facteurs de risque cardiovasculaire de masse spla2 de type iia et oxpl/apob pour le diagnostic/pronostic d'une maladie/d'un événement cardiovasculaire - Google Patents

Combinaison de facteurs de risque cardiovasculaire de masse spla2 de type iia et oxpl/apob pour le diagnostic/pronostic d'une maladie/d'un événement cardiovasculaire Download PDF

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WO2011038786A1
WO2011038786A1 PCT/EP2009/066604 EP2009066604W WO2011038786A1 WO 2011038786 A1 WO2011038786 A1 WO 2011038786A1 EP 2009066604 W EP2009066604 W EP 2009066604W WO 2011038786 A1 WO2011038786 A1 WO 2011038786A1
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WIPO (PCT)
Prior art keywords
oxpl
apob
cardiovascular
risk
disease
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PCT/EP2009/066604
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English (en)
Inventor
Ziad Mallat
Alain Tedgui
Sotirios Tsimikas
Joseph Witztum
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INSERM (Institut National de la Santé et de la Recherche Médicale)
The Regents Of The University Of California
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Priority claimed from PCT/EP2009/062852 external-priority patent/WO2010037860A1/fr
Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), The Regents Of The University Of California filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority to US13/498,208 priority Critical patent/US8383355B2/en
Priority to EP09768373A priority patent/EP2483692A1/fr
Publication of WO2011038786A1 publication Critical patent/WO2011038786A1/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
    • 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
    • G01N2333/775—Apolipopeptides
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • 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

Definitions

  • the invention relates to the use of a combination of sPLA2 type IIA mass and OxPL/apoB cardiovascular risk factors for the diagnosis/prognosis of a cardiovascular disease/event or for the monitoring of a cardiovascular disease.
  • a key problem in treating vascular diseases is proper diagnosis. Often the first sign of the disease is sudden death. For example, approximately half of all individuals who die of coronary artery disease die suddenly, Furthermore, for 40-60% of the patients who are eventually diagnosed as having coronary artery disease, myocardial infarction is the first presentation of the disease. Unfortunately, approximately 40% of those initial events go unnoticed by the patient. Because of our limited ability to provide early and accurate diagnosis followed by aggressive treatment, cardiovascular diseases (CD) remain the primary cause of morbidity and mortality worldwide. Patients with CD represent a heterogeneous group of individuals, with a disease that progresses at different rates and in distinctly different patterns. Despite appropriate evidence-based treatments for patients with CD, recurrence and mortality rates remain high. Also, the full benefits of primary prevention are unrealized due to our inability to accurately identify those patients who would benefit from aggressive risk reduction.
  • CD cardiovascular diseases
  • biomarkers such as the lipid and inflammatory markers, have been shown to predict outcome and response to therapy in patients with CD and some are utilized as important risk factors for developing atherosclerotic disease.
  • One object of the invention is a method of identifying a subject having or at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising:
  • OxPL/apoB oxidized phospholipids on apolipoprotein B-100 particles
  • sPLA2 type IIA mass and OxPL/apoB being indicative of having or a risk of having or developing a cardiovascular disease and/or cardiovascular event.
  • said combined value of sPLA2 type IIA mass and OxPL/apoB is compared to a reference value.
  • said method further comprises measuring at least one cardiovascular risk factor selected in the group of Framingham Risk Score (FRS), CRP, IgM IC of apoBlOO or IgM MDA-LDL, Lp-PLA2 and sPLA2 activity.
  • FRS Framingham Risk Score
  • CRP CRP
  • IgM IC of apoBlOO or IgM MDA-LDL Lp-PLA2 and sPLA2 activity.
  • said method is for identifying a subject having or at risk of having or developing a cardiovascular disease and/or a cardiovascular event, said cardiovascular disease and/or cardiovascular event being Metabolic Syndrome, Syndrome X, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina pectoris, stroke, diseases of the aorta and its branches (such as aortic stenosis, thrombosis or aortic aneurysm), peripheral artery disease, peripheral vascular disease, cerebrovascular disease, and any acute ischemic cardiovascular event.
  • Another object of the invention is a method as described here above, for monitoring the efficacy of a treatment for a cardiovascular disease.
  • Another object of the invention is a kit for identifying whether a subject has or is at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising:
  • said kit further comprises means for measuring at least one cardiovascular risk factor selected in the group of Framingham Risk Score (FRS), CRP, IgM IC of apoB 100 or IgM MDA-LDL, Lp-PLA2 and sPLA2 activity.
  • FRS Framingham Risk Score
  • Cardiovascular disease or "arterio vascular disease” as defined herein is a general term used to classify numerous conditions affecting the heart, heart valves, blood, and vasculature of the body and encompasses any disease affecting the heart or blood vessels, including, but not limited to, Metabolic Syndrome, Syndrome X, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina pectoris, stroke, diseases of the aorta and its branches (such as aortic stenosis, thrombosis or aortic aneurysm), peripheral artery disease, peripheral vascular disease, cerebrovascular disease, and including, without limitation, any acute ischemic cardiovascular event.
  • Arteriovascular disease as used herein is meant to most commonly refer to the ischemic or pro-ischemic disease, rather than generally to-non- ischemic disease.
  • Cardiovascular event is used interchangeably herein with the term “cardiac event”, “acute arteriovascular event”, or “Arteriovascular event” and refers to sudden cardiac death, acute coronary syndromes such as, but not limited to, plaque rupture, myocardial infarction, unstable angina, as well as non-cardiac acute arteriovascular events such as blood clots of the leg, aneurysms, stroke and other arteriovascular ischemic events where arteriovascular blood flow and oxygenation is interrupted.
  • atherosclerosis and “atherothrombosis” refer to a complex arteriovascular inflammatory disease that develops in response to multiple stimuli and cardiovascular risk factors, and is associated with systemic inflammation.
  • Cells involved in the atherosclerotic process include vascular (endothelial and smooth muscle) cells, monocytes/macrophages, lymphocytes (T, B, NKT), dendritic cells, mast cells and platelets. They secrete or are stimulated by soluble factors including peptides, glycoproteins, proteases and a set of cytokines. They are involved in the perpetuation of the inflammatory response, the progression and the destabilization of atherosclerosis, including plaque erosion, rupture and thrombosis.
  • CAD coronary artery disease
  • coronary arteries the coronary arteries
  • CAD coronary artery disease
  • ACS acute coronary syndromes
  • myocardial infarction heart attack
  • angina stable and unstable
  • CVD cardiovascular disease
  • Cerebrovascular disease is an arteriovascular disease in the blood vessels that feed oxygen-rich blood to the face and brain, such as atherosclerosis and atherothrombosis. This term is often used to describe “hardening" of the carotid arteries, which supply the brain with blood. It is a common comorbid disease with CAD and/or PAD. It is also referred to as an ischemic disease, or a disease that causes a lack of blood flow.
  • CVD encompasses disease states such as "cerebrovascular ischemia,” “acute cerebral infarction,” “stroke,” “ischemic stroke,” “hemorrhagic stroke,” “aneurysm,” “mild cognitive impairment (MCI)” and “transient ischemic attacks” (TIA).
  • Ischemic CVD is believed to closely relate to CAD and PAD; non-ischemic CVD may have multiple pathophysiologies.
  • An estimated 5 million Americans are the survivors of past diagnosed acute CVD events, with an estimated 700 thousand acute CVD events occurring each year.
  • subjects deemed to be at low risk or no risk of CVD based on clinical assessments of traditional arteriovascular disease risk factors, or without symptoms such as TIAs, MCI or severe headache, may still be at risk for an acute CVD event.
  • PAD peripheral artery disease
  • PAD peripheral artery disease
  • Claudication can be defined as pain or discomfort in the muscles of the legs occurring due to a decreased amount of blood flowing to a muscle from narrowing of the peripheral arteries, producing ischemia and often arterial occlusion, causing skeletal muscle and limb necrosis.
  • PAD Pain, tightness, cramping, tiredness or weakness is often experienced as a result of claudication.
  • PAD not only causes the hemodynamic alterations common in CAD, but also results in metabolic changes in skeletal muscle.
  • PAD has progressed to severe chronic and acute peripheral arterial occlusion, surgery and limb amputation often become the sole therapeutic options.
  • PAD is widely considered to be an underdiagnosed disease, with the majority of confirmed diagnoses occurring only after symptoms are manifested, or only with other arteriovascular disease, and irreversible arteriovascular damage due to such ischemic events has already occurred.
  • Cardiovascular Risk Factor encompasses one or more biomarker whose level is changed in subjects having a cardiovascular disease or predisposed to developing a cardiovascular disease, or at risk of a cardiovascular event.
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the conversion to arteriovascular events, and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion.
  • “Risk evaluation,” or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to an arteriovascular condition or to one at risk of developing an arteriovascular event, or from at risk of an arteriovascular event to a more stable arteriovascular condition.
  • Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of arteriovascular disease, such as coronary calcium scores, other imaging or treadmill scores, passive or provocative tesing results, arteriovasculature percentage stenosis or occlusion and other measurements of plaque burden and activity, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to arteriovascular disease and events, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for an arteriovascular event.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk for arteriovascular events.
  • the present invention may be used so as to discriminate those at risk for developing an arteriovascular event from those having arteriovascular disease, or those having arteriovascular disease from normal.
  • sample in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. As used herein "blood” includes whole blood, plasma, serum, circulating cells, constituents, or any derivative of blood.
  • “Clinical parameters or indicia” encompasses all non-sample or non-analyte bio markers of subject health status or other characteristics, such as, without limitation, age (Age), ethnicity (RACE), gender (Sex), diastolic blood pressure (DBP) and systolic blood pressure (SBP), family history (FamHX), height (HT), weight (WT), waist (Waist) and hip (Hip) circumference, body-mass index (BMI), as well as others such as Type I or Type II Diabetes Mellitus or Gestational Diabetes Mellitus (DM or GDM, collectively referred to here as Diabetes), and resting heart rate.
  • a "subject" in the context of the present invention is preferably a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of arteriovascular disease or arteriovascular events.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed or identified as having arteriovascular disease or an arteriovascular event, and optionally has already undergone, or is undergoing, a therapeutic intervention for the arteriovascular disease or arteriovascular event. Alternatively, a subject can also be one who has not been previously diagnosed as having arteriovascular disease.
  • a subject can be one who exhibits one or more risk factors for arteriovascular disease, or a subject who does not exhibit arteriovascular risk factors, or a subject who is asymptomatic for arteriovascular disease or arteriovascular events.
  • a subject can also be one who is suffering from or at risk of developing arteriovascular disease or an arteriovascular event.
  • a “formula,” “algorithm,” or “model” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called “parameters”) and calculates an output value, sometimes referred to as an "index” or “index value.”
  • Parameters continuous or categorical inputs
  • Non- limiting examples of “formulas” include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value transformations and normalizations (including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, or ethnicity), rules and guidelines, statistical classification models, and neural networks trained on historical populations.
  • Cardiovascular Risk Factor Of particular use in combining Cardiovascular Risk Factor and other biomarkers are linear and non-linear equations and statistical classification analyses to determine the relationship between levels of Cardiovascular Risk Factor detected in a subject sample and the subject's risk of cardiovascular disease.
  • structural and synactic statistical classification algorithms, and methods of risk index construction utilizing pattern recognition features, including established techniques such as cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, and Hidden Markov Models, among others.
  • PCA Principal Components Analysis
  • LogReg Logistic Regression
  • LDA Linear Discrimin
  • “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's non-analyte clinical parameters.
  • biomolecules may be used interchangeably. These terms are meant to be interpreted broadly, and generally encompass polypeptides, peptides, oligosaccharides, polysaccharides, oligopeptides, proteins, oligonucleotides, and polynucleotides. Oligonucleotides and polynucleotides include, for example, DNA and RNA, e.g., in the form of aptamers.
  • Biomolecules also include organic compounds, organometallic compounds, salts of organic and organometallic compounds, saccharides, amino acids, and nucleotides, lipids, carbohydrates, drugs, steroids, lectins, vitamins, minerals, metabolites, cofactors, and coenzymes. Biomolecules further include derivatives of the molecules described. For example, derivatives of biomolecules include lipid and glycosylation derivatives of oligopeptides, polypeptides, peptides, and proteins, such as antibodies. Further examples of derivatives of biomolecules include lipid derivatives of oligosaccharides and polysaccharides, e.g., lipopolysaccharides. The Invention
  • One object of the invention is a method of identifying a subject having or at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising:
  • OxPL/apoB oxidized phospholipids on apolipoprotein B-100 particles
  • the subject may be a substantially healthy subject, which means that the subject has not been previously diagnosed or identified as having or suffering from a cardiovascular disease, or that has not developed a cardiovascular event.
  • the subject may also be one that is asymptomatic for the cardiovascular disease.
  • an "asymptomatic" subject refers to a subject that do not exhibit the traditional symptoms of a cardiovascular disease or event, including, but not limited to, chest pain and shortness of breath for CAD, claudication for PAD, and TIAS, MCI and severe headache for CVD.
  • the subject may be one that is at risk of having or developing a cardiovascular disease or cardiovascular event, as defined by clinical and biological indicia such as for example: age, gender, LDL concentration, HDL concentration, triglyceride concentration, blood pressure, body mass index, CRP concentration, coronary calcium score, waist circumference, tobacco smoking status, previous history of cardiovascular disease, family history of cardiovascular disease, heart rate, fasting insulin concentration, fasting glucose concentration, diabetes status, and use of high blood pressure medication.
  • clinical and biological indicia such as for example: age, gender, LDL concentration, HDL concentration, triglyceride concentration, blood pressure, body mass index, CRP concentration, coronary calcium score, waist circumference, tobacco smoking status, previous history of cardiovascular disease, family history of cardiovascular disease, heart rate, fasting insulin concentration, fasting glucose concentration, diabetes status, and use of high blood pressure medication.
  • the subject may be one that has been previously diagnosed or identified for a cardiovascular disease or cardiovascular event, such as for example chronic ischemic disorders without myocardial necrosis (for example stable or effort angina pectoris), acute ischemic disorders without myocardial necrosis (for example unstable angina pectoris), ischemic disorders with myocardial necrosis (for example ST segment evaluation myocardial infarction or non-ST segment elevation myocardial infarction).
  • chronic ischemic disorders without myocardial necrosis for example stable or effort angina pectoris
  • acute ischemic disorders without myocardial necrosis for example unstable angina pectoris
  • ischemic disorders with myocardial necrosis for example ST segment evaluation myocardial infarction or non-ST segment elevation myocardial infarction.
  • Tissue ischemia is often defined in relative terms and occurs when the needs in oxygen exceed the delivery of oxygen to tissues. There is an imbalance between tissue (myocardial for example) oxygen demands and supply. This condition of oxygen deprivation may be accompanied by inadequate removal of metabolites consequent to reduced perfusion.
  • Myocardial ischemia can be diagnosed clinically (chest pain for example), biologically (increase in myeloperoxidase activity for example), metabolically, using scintigraphy, by analyzing regional wall motion disorders or by use of an electrocardiogram (typical modifications of the ST segment, upper or lower ST segment deviation, typical changes in T wave such as T wave inversion or steep symmetric or high amplitude positive T waves).
  • Silent ischemia is typically diagnosed using scintigraphy or a 24h electrocardiogram recording.
  • Stable and effort angina is typically manifested by a chest pain during exercise and slowly recovers at rest. It usually reflects tissue ischemia during exercise.
  • Unstable angina is a recent increase in the frequency and/or severity of stable angina, a first episode of angina, or an angina at rest.
  • Myocardial necrosis is typically diagnosed by an increase in myocardial enzymes (for example troponin I, troponin T, CPK) in the circulating blood.
  • myocardial enzymes for example troponin I, troponin T, CPK
  • the subject may be one that who shows an improvement in cardiovascular risk factors as a result of treatments and/or therapies for cardiovascular diseases.
  • improvements include a reduction in body mass index, a reduction in total cholesterol, a reduction in LDL levels, an increase in HDLC levels, a reduction in systolic and/or diastolic blood pressure, or other aforementioned risk factor or combinations thereof.
  • no onset of ischemic symptom has been diagnosed in the subject.
  • Myocardial ischemia can be diagnosed clinically (chest pain for example), biologically (increase in myeloperoxidase activity for example), metabolically using scintigraphy, by analysing regional wall motion disorders or by use of an electrocardiogram (typical modifications of the ST segment, upper or lower ST segment deviation, typical changes in T wave such as T wave insertion or steep symmetric or high amplitude positive T waves).
  • the sample used to measure sPLA2 type IIA mass and OxPL/apoB and optionally other cardiovascular risk factors is a blood sample, whole blood, plasma or serum.
  • the measure of sPLA2 type IIA mass can be performed by a variety of techniques.
  • the methods may comprise contacting the sample with a bio molecule capable of selectively interacting with sPLA2 type IIA in the sample.
  • the biomolecules are antibodies, such as, for example, monoclonal antibodies or even aptamers as above described.
  • the aforementioned assays generally involve the binding of the biomolecule (ie. antibody or aptamer) to a solid support.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • the concentration of sPLA type IIA may be measured by using standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays include, but are not limited to, agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation.
  • An exemplary biochemical test for identifying specific proteins employs a standardized test format, such as ELISA test, although the information provided herein may apply to the development of other biochemical or diagnostic tests and is not limited to the development of an ELISA test (see, e.g., Molecular Immunology: A Textbook, edited by Atassi et al. Marcel Dekker Inc., New York and Basel 1984, for a description of ELISA tests). It is understood that commercial assay enzyme-linked immunosorbant assay (ELISA) kits for various plasma constituents are available. Therefore ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize sPLA2 type IIA.
  • a sample containing or suspected of containing said sPLA2 type IIA is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • Measuring the concentration of sPLA2 type IIA may also include separation of the compounds: centrifugation based on the compound's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the compound's affinity for the particular solid-phase that is used.
  • said one or two biomarkers proteins may be identified based on the known "separation profile" e. g., retention time, for that compound and measured using standard techniques.
  • the separated compounds may be detected and measured by, for example, a mass spectrometer.
  • levels of immunoreactive sPLA2 type IIA in a sample may be measured by an immunometric assay on the basis of a double-antibody "sandwich” technique, with a monoclonal antibody specific for sPLA2 type IIA (Cayman Chemical Company, Ann Arbor, Michigan).
  • the antibody has no cross-reactivity with the other types of sPLA2 such as sPLA2 type I, IV, or type V sPLA2.
  • said means for measuring sPLA2 type IIA mass are for example:
  • sPLA 2 type IIA mass may be measured as previously described in Mallat Z. et al 2005 (Mallat Z., Steg P, Benessiano J et al. Circulating secretory phospholipase A2 activity predicts recurrent events in patients with severe acute coronary syndromes. J Am Coll Cardiol. 2005;46: 1249-1257) or in Mallat Z. et al. (2007) (Mallat Z, Benessiano J, Simon T et al. Circulating secretory phospholipase A2 activity and risk of incident coronary events in healthy men and women: The EPIC-NORFOLK Study. Arterioscler Thromb Vase Biol. 2007;27: 1177-1183).
  • the level of OxPL and the level of apoB in the sample obtained from the subject are measured with two or more different biomolecules.
  • the first bio molecule specifically interacts with OxPL and the second bio molecule specifically interacts with apoB.
  • the biomolecules are antibodies, such as, for example, monoclonal antibodies or even aptamers as above described.
  • the antibody that interacts with OxPL may be, for example, E06 or DLH3.
  • the biomolecules are antigens.
  • the biomolecules are immobilized to form an array comprising a first set of a plurality of the first biomolecule and a second set of a plurality of the second biomolecule.
  • Exemplary oxidized phospholipid include oxidized forms of l-palmitoyl-2- arachidonoyl-sn-glycero-3-phos-phorylcholine (Ox-PAPC), l-palmitoyl-2-oxovaleroyl-sn- glycero-3-phosphoryl-choline (POVPC), 1 -palmitoyl-2-glutaroyl-sn-glycero-3- phosphorylcholine (PGPC), 1 -palmitoyl-2-epoxyisoprostane-sn-glycero-3- phosphorylcholine (PEIPC), oxidized l-stearoyl-2-arachidonoyl-sn-glycero-3- phosphorylcholin-e (Ox-SAPC), 1 -stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC, l-stearoyl-2-glutaroyl-sn
  • an immunoassay can be performed either by first capturing the LDL on a microtiter well by use of an antibody that binds both oxidized and nonoxidized LDL (e. g. anti-apoB), and then detection of the OxLDL by a labelled E06 antibody.
  • E06 antibody can be bound to the bottom of the microtiter well and the amount of OxLDL bound determined by the use of labeled anti-LDL antibody.
  • OxLDL could also be used to coat the microtiter wells and various concentrations of patient sera, putatively containing OxLDL, could be mixed with a constant, limiting amount of labeled (e. g. biotinylated) E06 or T15 to compete for binding to the OxLDL on the plate.
  • labeled e. g. biotinylated
  • T15 a constant, limiting amount of labeled (e. g. biotinylated) E06 or T15 to compete for binding to the OxLDL on the plate.
  • a standard curve could be developed using PC as a competing agent.
  • a parallel set of reactions can be run using PC as a source of competing agent rather than patient sera.
  • the PC can be used alone, or linked to a carrier protein, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • the measurements obtained for sPLA2 type II A mass and OxPL/apoB and optionally other cardiovascular risk factors are combined in statistical analysis, wherein the combined value of sPLA2 type IIA mass and OxPL/apoB and optionally other cardiovascular risk factors is indicative of having or a risk of having or developing a cardiovascular disease and/or cardiovascular event.
  • a positive result is assumed if a sample is positive for at least one of the markers investigated. This may be for example the case when diagnosing an infectious disease, like AIDS. Frequently, however, a combination of risk factors is evaluated.
  • the measurements obtained for sPLA2 type IIA mass and OxPL/apoB and optionally other cardiovascular risk factors are mathematically combined and the combined value is correlated to the underlying diagnostic/prognostic question.
  • Risk factor measurements may be combined by any appropriate state of the art mathematical method.
  • Well-known mathematical methods for correlating a marker combination to a disease employ methods like, Discriminant analysis (DA) (i.e. linear-, quadratic-, regularized-DA), Kernel Methods (i.e.
  • Nonparametric Methods i.e. k-Nearest-Neighbor Classifiers
  • PLS Partial Least Squares
  • Tree-Based Methods i.e. Logic Regression, CART, Random Forest Methods, Boosting/Bagging Methods
  • Generalized Linear Models i.e. Logistic Regression
  • Principal Components based Methods i.e. SIMCA
  • Generalized Additive Models Fuzzy Logic based Methods, Neural Networks and Genetic Algorithms based Methods.
  • the skilled artisan will have no problem in selecting an appropriate method to evaluate a marker combination of the present invention.
  • the method used in correlating the risk factors combination of the invention to the risk of having or being at risk of having a cardiovascular disease or cardiovascular event is selected from DA (i.e. Linear-, Quadratic-, Regularized Discriminant Analysis), Kernel Methods (i.e. SVM), Nonparametric Methods (i.e. k- Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-Based Methods (i.e. Logic Regression, CART, Random Forest Methods, Boosting Methods), or Generalized Linear Models (i.e. Logistic Regression).
  • DA i.e. Linear-, Quadratic-, Regularized Discriminant Analysis
  • Kernel Methods i.e. SVM
  • Nonparametric Methods i.e. k- Nearest-Neighbor Classifiers
  • PLS Partial Least Squares
  • Tree-Based Methods i.e. Logic Regression, CART, Random Forest Methods, Boosting Methods
  • an optimized multivariate cut-off for the underlying combination of risk factors is used to discriminate state A from state B, e.g. diseased from substantially healthy.
  • the risk factors are no longer independent but form a risk factor panel.
  • Combining the measurements of sPLA2 type IIA mass and OxPL/apoB does significantly improve the diagnostic/prognostic accuracy for cardiovascular disease and/or cardiovascular event as compared to substantially healthy subjects or as compared to subjects which have been diagnosed for a cardiovascular disease or event.
  • the statistical analysis of the measurements of sPLA2 type IIA mass and OxPL/apoB and optionally other cardiovascular risk factors is based on the determination of odds ratios (OR) using standard procedures.
  • An odds ratio is calculated by dividing the odds in the test group by the odds in the control group.
  • the odds of an event are calculated as the number of events divided by the number of non-events. If the odds of an event are greater than one the event is more likely to happen than not (the odds of an event that is certain to happen are infinite); if the odds are less than one the chances are that the event won't happen (the odds of an impossible event are zero).
  • the strength of association is reported as odds ratios (OR) (with 95% lower (LCL) and upper (UCL) confidence limit), indicating the factor by which the risk of having a disease or being at risk of having or developing a disease is increased (0R>1).
  • the 95% confidence interval (95% CI) is the range of numerical values in which we can be confident (to a computed probability, here 95%) that the population value being estimated will be found.
  • Confidence intervals indicate the strength of evidence; where confidence intervals are wide, they indicate less precise estimates of effect. The larger the trial's sample size, the larger the number of outcome events and the greater becomes the confidence that the true relative risk reduction is close to the value stated. Thus the confidence intervals get narrower and "precision" is increased.
  • the lower boundary of the confidence interval, or lower confidence limit should be >1 if the 0R>1, or the upper boundary of the confidence interval should be ⁇ 1 if the 0R ⁇ 1.
  • ROC receiver-operating characteristics
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds.
  • sensitivity or the true-positive fraction [defined as (number of true- positive test results)/(number of true-positive + number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • false-positive fraction or 1 - specificity [defined as (number of false-positive results)/(number of true-negative + number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/1 -specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
  • the combined value of sPLA2 type II A mass and OxPL/apoB and optionally other risk factors is compared to a reference value.
  • the reference value may be an index value or may be derived from one ore more risk prediction algorithms or computed indices for the cardiovascular disease and/or cardiovascular event.
  • a reference value can be relative to a number or value derived from population studies, including without limitation, such subjects having similar body mass index, total cholesterol levels, LDL/HDL levels, systolic or diastolic blood pressure, subjects of the same or similar age range, subjects in the same or similar ethnic group, subjects having family histories of atherosclerosis, atherothrombosis, or CAD, PAD, or CVD, or relative to the starting sample of a subject undergoing treatment for an arteriovascular disease, such as atherosclerosis, atherothrombosis, CAD, PAD, or CVD.
  • Such reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of arteriovascular disease, such as but not limited to, algorithms reported in the Framingham Study, NCEP/ATP III, among others. Cardiovascular Risk Factor reference value can also be constructed and used using algorithms and other methods of statistical and structural classification.
  • the reference value is derived from the combination of OxPL/apoB and sPLA2 type II A mass and optionally others cardiovascular risk factors in a control sample derived from one or more subjects who are substantially healthy as defined here above.
  • Such subjects who are substantially healthy lack traditional risk factors for a cardiovascular disease: for example, those subjects have a serum cholesterol level less than 200 mg/dl, systolic blood pressure less than or equal to 120 mm Hg, diastolic blood pressure less than or equal to 80 mm Hg, non-current smoker, no history of diagnosed diabetes, no previously diagnosed acute coronary syndrome or hypertension, among other aforementioned other risk factors, or can be verified by another invasive or non-invasive diagnostic test of cardiovascular disease known in the art, such as but not limited to, electrocardiogram (ECG), carotid B-mode ultrasound (for intima-medial thickness measurement), electron beam computed tomography (EBCT), coronary calcium scoring, multi-slice high resolution computed tomography, nuclear magnetic resonance, stress exercise testing
  • ECG electro
  • such subjects are monitored and/or periodically retested for a diagnostically relevant period of time ("longitudinal studies") following such test to verify continued absence from cardiovascular disease or acute cardiovascular events (disease or event free survival).
  • Such period of time may be one year, two years, two to five years, five years, five to ten years, ten years, or ten or more years from the initial testing date for determination of the reference value.
  • retrospective measurement of OxPL/apoB and sPLA2 type IIA mass levels in properly banked historical subject samples may be used in establishing these reference values, thus shortening the study time required, presuming the subjects have been appropriately followed during the intervening period through the intended horizon of the product claim.
  • a reference value can also be derived from the combination of OxPL/apoB and sPLA2 type IIA mass and optionally other cardiovascular risk factors in a sample derived from one or more subject who (1) has been previously diagnosed or identified for a cardiovascular disease or cardiovascular event by one of the above invasive or non-invasive techniques, or who has suffered from an cardiovascular event or plaque rupture, and (2) has not experienced a recurrent cardiovascular event.
  • a reference value can also be derived from the combination of OxPL/apoB and sPLA2 type IIA mass and optionally other cardiovascular risk factors in a sample derived from one or more subject who is at high risk for developing a cardiovascular event, or who is at high risk for developing an atherosclerotic or atherothrombotic plaque rupture.
  • a reference value can also be derived from the combination of OxPL/apoB and sPLA2 type IIA mass and optionally other cardiovascular risk factors in a sample derived from one or more subject who shows an improvement in cardiovascular risk factors as a result of treatments and/or therapies for cardiovascular diseases.
  • Such improvements include a reduction in body mass index, a reduction in total cholesterol, a reduction in LDL levels, an increase in HDLC levels, a reduction in systolic and/or diastolic blood pressure, or other aforementioned risk factor or combinations thereof.
  • the reference value is an index value or a baseline value.
  • An index value or baseline value is derived from one or more subjects who do not have a cardiovascular disease, such as atherosclerosis, atherothrombosis, CAD, PAD, or CVD, or subjects who are asymptomatic for a cardiovascular disease.
  • a baseline value can also be derived from a subject who has shown an improvement in cardiovascular risk factors (as a result of cardiovascular treatments or therapies. Such improvements include, without limitation, a reduction in body mass index, a reduction in total cholesterol, a reduction in LDL levels, an increase in HDLC levels, a reduction in systolic and/or diastolic blood pressure, or combinations thereof.
  • the method of the invention comprises combining sPLA2 type IIA mass and OxPL/apoB with clinical and biological indicia such as age, a history of hypertension, diabetes, myocardial infarction, heart failure, coronary angiography or angioplasty, Killip class, ST-segment deviation, coronary revascularization (angioplasty or coronary artery bypass surgery), and creatinine.
  • the method of the invention comprises:
  • OxPL/apoB oxidized phospholipids on apolipoprotein B-100 particles
  • FRS Risk Score
  • CRP CRP
  • IgM IC IgM Immune Complexes
  • IgM MDA- LDL IgM Malondialdehyde LDL
  • Lp-PLA2 activity Lp-PLA2 activity and sPLA2 activity
  • FRS is calculated using a previously reported algorithm, which takes into account age, sex, total cholesterol, HDL-C, systolic and diastolic blood pressure, smoking and the presence of diabetes (Wilson P.W.et al, Circulation. 1998;97:1837-1847, which is incorporated by reference).
  • CRP can be measured by methods known in the art, such as a the method described in Arima et al (Arterioscler Thromb Vase Biol. 2008 Jul;28(7): 1385-91) and Ridker et al. (New England Journal of Medecine, 2000, 342:836- 843), which are incorporated by reference.
  • IgM IC of apoBlOO and IgM MDA-LDL can be measured by methods known in the art such as according to Tsimikas et al (2004, Circulation, 110: 1406-1412 and 2003 J. Am. Coll. Cardiol. 41 :360-370), which are incorporated by reference.
  • Lp-PLA-2 activity can be measured by methods known in the art such as according to Kiechl et al. (2007, Atherioscler. Thromb. Vase. Biol. 27: 1788-1795), which is incorporated by reference.
  • Lp-PLA2 activity may be measured using a commercially available kit (Azwell Inc) based on the method of Kosaka et al (2000 Clin. Chim. Acta 296: 151-161, which is incorporated by reference). LpPLA2 activity may also be measured by the method described in WO2005074604, which is incorporated by reference.
  • sPLA2 activity is measured in said sample.
  • the measure of sPLA2 activity can be performed by a fluorimetric assay according to Radvanyi et al. (1989 Anal Biochem 177: 103-9) as modified by Pernas et al. (1991 Biochem Biophys Res Commun 178: 1298-1305), all incorporated by reference.
  • the following assay is used.
  • the l-hexadecanoyl-2- (l-pyrenedecanoyl)-sn-glycero-3-phosphomethanol sodium salt (Interchim, Montlucon, France) is used as a substrate for sPLA2.
  • the following assay is used for automated measurement.
  • the 1 -hexadecanoyl-2-(l -pyrenedecanoyl)-sn-glycero-3- phosphomethanol sodium salt (Interchim, Montlucon, France) is used as a substrate for sPLA2.
  • the hydrolysis of this substrate by sPLA2 yields 1-pyrenedecanoic acid, which emits fluorescence at 405 nm.
  • 1 nmol of fluorescent substrate in 0.2 ml of buffer substrate (10 mM Tris-HCL pH 8.7, 0.1 % albumin, 10 mM CaCI2) was automatically distributed in Black Maxisorp microtitration plate (96 wells).
  • a two-point procedure was used to measure the corrected fluorescence intensity of each sample and to evaluate the enzymatic activity (expressed in nmol/min/ml). All samples were tested in duplicate.
  • the hydrolysis of the substrate in the absence of serum is used as negative control and deduced from PLA2 activity. All samples are tested in duplicate. Unless otherwise mentioned, all the numerical values given herein for serum sPLA2 activity are measured according to the above defined assay for automated measurement.
  • the phospholipase that can be used to perform the assay is a secretory phospholipase or a phospholipase with a known activity and preferably a bee venom phospholipase.
  • sPLA2 activity may be also determined by a process based on a fluorimetric assay comprising contacting a biological sample containing said sPLA2 and taken from said patient, with a substrate at a concentration from 1 nM to 15 nM, the serum sample volume being from 5 ⁇ 1 to 50 ⁇ and the substrate volume being from ⁇ to 300 ⁇ 1, at a temperature range from about 15°C to about 40°C and preferably 30°C.
  • the phospholipase used could be a phospholipase from bee venom or snake venom like Naja venom, preferably bee venom. It could be a recombinant phospholipase from any species.
  • This assay is described in example 2 of WO2008/015546, which is incorporated by reference. The advantage of this method is the small sample volume of substrate used and the thermostating, providing a higher precision and sensitivity.
  • a variant of the automated fluorimetric measurement as defined above can be used, which enables to alleviate imprecision which might result from a nonspecific increase in fluorescence intensity due to other factors in the sample, thus interfering with the measure of sPLA2 activity.
  • This method only differs from the above- defined automated fluorimetric measurement method in that the following formula is used for determining sPLA2 activity:
  • - A represents sPLA2 activity expressed in nmol/min/ml
  • - s represents the quantity of substrate expressed in nmol (usually 1 nmol in a volume of 200 ⁇ of working solution);
  • - V represents the sample volume expressed in ml (usually from 0.30 to 0.50 ml);
  • Fmax - Fmin represents the difference between the maximal fluorescence signal at the end of the reaction in the presence of PLA2 from bee venom and the negative control;
  • the method of the invention comprises the combination of sPLA2 type IIA mass, OxPL/apoB and FRS measurements.
  • the method of the invention comprises sPLA2 type IIA mass, OxPL/apoB, FRS and CRP measurements. In another embodiment, the method of the invention comprises sPLA2 type IIA mass, OxPL/apoB, FRS and sPLA2 activity measurements. According to the invention, the method as described here above is for identifying whether a subject has or is at risk of having or developing a cardiovascular disease and/or a cardiovascular event.
  • said cardiovascular disease and/or cardiovascular event is Metabolic Syndrome, Syndrome X, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina pectoris, stroke, diseases of the aorta and its branches (such as aortic thrombosis or aortic aneurysm), peripheral artery disease, peripheral vascular disease, cerebrovascular disease, and any acute ischemic cardiovascular event.
  • subjects identified as having, or being at increased risk of developing a cardiovascular disease or cardiovascular event are chosen to receive a therapeutic regimen to slow the progression of a cardiovascular disease, or decrease or prevent the risk of developing a cardiovascular disease or a cardiovascular event.
  • the method as described here above is for monitoring a cardiovascular disease or event in a subject in need thereof, said cardiovascular disease or event being Metabolic Syndrome, Syndrome X, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina pectoris, stroke, diseases of the aorta and its branches (such as aortic thrombosis or aortic aneurysm), peripheral artery disease, peripheral vascular disease, cerebrovascular disease, and any acute ischemic cardiovascular event.
  • the method as described here above is for assessing the progression of a cardiovascular disease in a subject in need thereof.
  • the method as described here above is for monitoring the effectiveness of a treatment for a cardiovascular disease.
  • the efficacy of the treatment will be reflected by changes in the measurements of the cardiovascular risk factors. If a treatment has a desired effect, the measurements and thus the combined value of the cardiovascular risk factors will be lower compared to the measurements and combined value obtained before the treatment.
  • the method as described here above is for selecting a treatment regimen for a subject diagnosed with or at risk for a cardiovascular disease.
  • Another object of the invention is a kit for identifying whether a subject has or is at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising:
  • said kit further comprises means for combining the measurements in order to obtain a combined value.
  • Said means for combining the measurements of the cardiovascular risk factors are algorithms allowing statistical analysis such as DA (i.e. Linear-, Quadratic-, Regularized Discriminant Analysis), Kernel Methods (i.e. SVM), Nonparametric Methods (i.e. k- Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-Based Methods (i.e. Logic Regression, CART, Random Forest Methods, Boosting Methods), or Generalized Linear Models (i.e. Logistic Regression).
  • DA i.e. Linear-, Quadratic-, Regularized Discriminant Analysis
  • Kernel Methods i.e. SVM
  • Nonparametric Methods i.e. k- Nearest-Neighbor Classifiers
  • PLS Partial Least Squares
  • Tree-Based Methods i.e. Logic Regression, CART, Random Forest Methods, Boosting Methods
  • Generalized Linear Models i.e. Log
  • said means for measuring sPLA2 type IIA mass are antibodies that interact specifically with sPLA2 type IIA and
  • said means for measuring OxPL/apoB are
  • OxPL such as for example E06, T15 or DLH3
  • said control OxPL sample may be a sample containing a phosphorylcholine (PC).
  • PC phosphorylcholine
  • the PC can be used alone, or linked to a carrier protein, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • said kit may further comprise means for measuring at least one cardiovascular risk factor selected in the group of Framingham Risk Score (FRS), CRP, IgM IC of apoBlOO or IgM MDA-LDL and LpPLA-2 or sPLA2 activity.
  • FSS Framingham Risk Score
  • CRP CRP
  • IgM IC of apoBlOO or IgM MDA-LDL LpPLA-2 or sPLA2 activity.
  • the Framingham Risk Score is determined by method known in the art, such as the method described in Wilson PW et al. (Circulation. 1998 May 12;97(18):1837-47) and DAgostino et al. (JAMA: The Journal of the American Medical Association. 2001;286: 180-187), which are incorporated by reference.
  • said means for measuring CRP are for example:
  • said means for measuring IgM IC of apoBlOO o are for example:
  • said means for measuring IgM MDA-LDL are for example:
  • said means for measuring Lp-PLA-2 are for example:
  • said means may further comprises an antibody that interacts specifically with Lp-PLA2 such as for example the monoclonal antibody 2C10, 4B4, B200, B501, 90D1E, 90E3A, 90E6C, 90G1 ID, or 90F2D.
  • Lp-PLA2 such as for example the monoclonal antibody 2C10, 4B4, B200, B501, 90D1E, 90E3A, 90E6C, 90G1 ID, or 90F2D.
  • said compound which reduces active thiol(s) and said substrate converted to a free thiol product in the presence of enzymatically active Lp-PLA2 are described in WO2005074604, which is incorporated by reference.
  • said means for measuring sPLA2 activity are described in WO2005074604, which is incorporated by reference.
  • the compound liable to be hydrolyzed by sPLA2 is a natural or non natural substrate of the enzyme.
  • the hydrolysis products are not quantifiable by themselves, compounds which can react with these products and which yield quantifiable compounds can be used, such a method is an indirect quantification.
  • the compound liable to be hydro lysed by sPLA2 is a phospholipid or a phospholipid analogue comprising a fiuorogenic or a chromogenic moiety.
  • said phospholipid is a glycerophospho lipid which is substituted in position 2 by a fluorescent acyl; such as l-hexadecanoyl-2-(l-pyrenedecanoyl)-sn-glycero-3- phosphomethanol or the fluorescent acyl 1-pyerenedecanoyl, the substrate for horseradish peroxidase being for example 3,3',5,5' tetramethyl-benzidine (TMB).
  • TMB 3,3',5,5' tetramethyl-benzidine
  • Fluorescent acyls liable to be used according to the invention are for example acyls substituted by fluorescent groups well known in the art, such as pyrene or fiuoresceine.
  • radioactive glycerophospho lipids can be used, such as glycerophospho lipids substituted in position 2 by radioactive acyls or radioactive phosphatidyl ethanolamine.
  • control sPLA2 activity sample comprises for example bee venom sPLA2.
  • the invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
  • Figure 1 Baseline characteristics of study participants.
  • Figure 2 Unadjusted odds ratios of incident coronary artery disease during follow up according to combined tertiles of sPLA2 activity or Lp-PLA2 activity and OxPL/apoB.
  • Figure 3 Adjusted odds ratios of incident coronary artery disease during follow up according to combined tertiles of sPLA2 activity or Lp-PLA2 activity and OxPL/apoB.
  • Figure 4 Area under ROC for FRS and combination with at least one of
  • OxPL/apoB Lp-PLA2 activity, sPLA2 activity and CRP.
  • Figure 5 Odds ratios for CAD based on tertiles of OxPL/apoB and sPLA2 activity, sPLA2 type IIA mass and Lp-PLA2.
  • the tertile cutoffs for OxPL/apoB are ⁇ 1150 RLU, 1151-2249 RLU and >2249
  • Figure 6 Area under ROC for FRS and combination with at least one of OxPL/apoB, Lp-PLA2 activity, sPLA2 activity and CRP.
  • Figure 7 Relationship between tertile groups of OxPL/apoB ( ⁇ 1150, 1151-2249 and >2249 RLU, (A)), sPLA 2 activity ( ⁇ 4.05, 4.05-4.83 and >4.83 nmol/min per ml; (B)), sPLA2 type IIA mass ( ⁇ 6.80, 6.80-11.29 and >11.19 mg/dl; (C)), Lp-PLA 2 activity ( ⁇ 44.05, 44.05-56.23 and >56.23 nmol/min per ml; (D)), and future CAD risk within each Framingham Risk Score Group. Framingham Risk Score was calculated as low risk ( ⁇ 10% risk of events over 10 years), moderate risk (10% to 20%), and high risk (>20%>). The p- values in the figure represent comparison of each tertile of the respective biomarkers with the lowest tertile in the low FRS category of each biomarker.
  • CAD was defined as codes 410 to 414 according to the International Classification of Diseases 9th revision. These codes encompass the clinical spectrum of CAD such as unstable angina, stable angina, and myocardial infarction. Controls were participants who remained free of CAD events during follow-up. Data on non-cardiac events has not been collected. Two controls were matched to each case by sex, age (within 5 years) and time of enrolment (within 3 months). The study was approved by the Norwich Health Authority Ethics Committee, and all participants provided written informed consent.
  • OxPL/apoB was measured by chemiluminescent ELISA, as described previously in detail, using the murine monoclonal antibody E06, which binds to the phosphocholine head group of oxidized but not native phospholipids (Tsimikas S, Brilakis ES, Miller ER et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease. N Engl J Med. 2005;353:46-57; Tsimikas S, Witztum JL, Miller ER et al.
  • OxPL/apoB assay does not measure the totality of OxPL present in plasma or OxPL not detected by E06.
  • Serum sPLA 2 activity was measured by a selective fluorometric assay by using fluorescent substrate 1 -hexadecanoyl-2-( 1 -pyrenedecanoyl)-sn-glycero-3 phosphomethanol, sodium salt (Interchim, Montlucon, France), as previously described (Mallat Z, Steg P, Benessiano J et al. Circulating secretory phospholipase A2 activity predicts recurrent events in patients with severe acute coronary syndromes. J Am Coll Cardiol. 2005;46: 1249-1257; Mallat Z, Benessiano J, Simon T et al.
  • Circulating secretory phospholipase A2 activity and risk of incident coronary events in healthy men and women The EPIC-NORFOLK Study. Arterioscler Thromb Vase Biol. 2007;27: 1177-1183). The minimum detectable activity was 0.10 nmol/min per ml. sPLA 2 type IIA mass was measured as previously described (Mallat Z, Steg P, Benessiano J et al. Circulating secretory phospholipase A2 activity predicts recurrent events in patients with severe acute coronary syndromes. J Am Coll Cardiol. 2005;46: 1249-1257; Mallat Z, Benessiano J, Simon T et al. Circulating secretory phospholipase A2 activity and risk of incident coronary events in healthy men and women: The EPIC-NORFOLK Study. Arterioscler Thromb Vase Biol. 2007;27: 1177-1183).
  • Lp-PLA 2 activity was measured in duplicate from EDTA plasma stored at -80°C by the trichloroacetic acid precipitation procedure in 96-well plates as described previously (Blankenberg S, Stengel D, Rupprecht HJ et al. Plasma PAF-acetylhydrolase in patients with coronary artery disease: results of a cross-sectional analysis. Journal of Lipid Research. 2003;44: 1381-1386.).
  • Lp(a) levels were measured as previously described with monoclonal antibody LPA4 (Tsimikas S, Lau HK, Han KR et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109:3164- 3170).
  • Myeloperoxidase mass and high sensitivity C-reactive protein were measured as previously described (Meuwese MC, Stroes ES, Hazen SL et al. Serum myeloperoxidase levels are associated with the future risk of coronary artery disease in apparently healthy individuals: the EPIC-Norfolk Prospective Population Study. J Am Coll Cardiol. 2007;50: 159-165 ;Boekholdt SM, hack CE, Sandhu MS et al. C-reactive protein levels and coronary artery disease incidence and mortality in apparently healthy men and women: the EPIC-Norfolk prospective population study 1993-2003. Atherosclerosis. 2006;187:415-422).
  • Analyses took into account the matching for sex, age and enrollment time, and adjusted for diabetes mellitus, smoking (never, previous, current), systolic blood pressure, LDL-C and HDL-C. Analyses were also performed that took into account the matching variables and adjusted for the Framingham Risk Score (FRS). 29 Analyses were performed for men and women separately, and in addition for sexes pooled, additionally adjusting for sex. Additional analyses were performed according to tertiles of OxPL/apoB, Lp(a), sPLA 2 mass and activity, Lp-PLA 2 activity and myeloperoxidase mass. Tertiles provided the best balance between discrimination and statistical power.
  • EXAMPLE 2 COMBINED MEASUREMENTS OF SPLA2 TYPE IIA MASS AND OXPL/APOB TO ASSESS THE RISK OF INCIDENT CAD

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Abstract

La présente invention concerne un procédé d'identification d'un sujet qui présente ou risque de présenter ou de développer une maladie cardiovasculaire et/ou un événement cardiovasculaire. Ledit procédé consiste : - à mesurer, dans un prélèvement obtenu dudit sujet, au moins deux facteurs de risque cardiovasculaire : a) la masse sPLA2 de type IIA et b) les phospholipides oxydés sur les particules d'apolipoprotéine B-100 (OxPL/apoB), - à combiner lesdites mesures, la valeur combinée de la masse sPLA2 de type IIA et d'OxPL/apoB indiquant si le sujet présente ou risque de présenter ou de développer une maladie cardiovasculaire et/ou un événement cardiovasculaire.
PCT/EP2009/066604 2009-10-02 2009-12-08 Combinaison de facteurs de risque cardiovasculaire de masse spla2 de type iia et oxpl/apob pour le diagnostic/pronostic d'une maladie/d'un événement cardiovasculaire WO2011038786A1 (fr)

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EP09768373A EP2483692A1 (fr) 2009-10-02 2009-12-08 Combinaison de facteurs de risque cardiovasculaire de masse spla2 de type iia et oxpl/apob pour le diagnostic/pronostic d'une maladie/d'un événement cardiovasculaire

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WO2013185214A1 (fr) * 2012-06-11 2013-12-19 Universite Laval Pla2g7/lp-pla2 en tant que biomarqueur et cible thérapeutique dans la prévention et le traitement d'une maladie de valve aortique calcifiée
WO2014131034A2 (fr) * 2013-02-25 2014-08-28 The Regents Of The University Of California Anticorps contre des phospholipides oxydés
WO2014131034A3 (fr) * 2013-02-25 2014-10-16 The Regents Of The University Of California Anticorps contre des phospholipides oxydés
US11008382B2 (en) 2013-02-25 2021-05-18 The Regents Of The University Of California Antibodies to oxidized phospholipids
US11008381B2 (en) 2013-02-25 2021-05-18 The Regents Of The University Of California Antibodies to oxidized phospholipids
US11655288B2 (en) 2013-02-25 2023-05-23 The Regents Of The University Of California Antibodies to oxidized phospholipids

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