WO2019025607A1 - Nouveau dosage de la fonction hdl - Google Patents

Nouveau dosage de la fonction hdl Download PDF

Info

Publication number
WO2019025607A1
WO2019025607A1 PCT/EP2018/071172 EP2018071172W WO2019025607A1 WO 2019025607 A1 WO2019025607 A1 WO 2019025607A1 EP 2018071172 W EP2018071172 W EP 2018071172W WO 2019025607 A1 WO2019025607 A1 WO 2019025607A1
Authority
WO
WIPO (PCT)
Prior art keywords
hdl
tgrl
subject
lipid
labeled
Prior art date
Application number
PCT/EP2018/071172
Other languages
English (en)
Inventor
Anatol Kontush
Original Assignee
Sorbonne Universite
Institut National De La Sante Et De La Recherche Medicale (Inserm)
Assistance Publique - Hopitaux De Paris
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sorbonne Universite, Institut National De La Sante Et De La Recherche Medicale (Inserm), Assistance Publique - Hopitaux De Paris filed Critical Sorbonne Universite
Publication of WO2019025607A1 publication Critical patent/WO2019025607A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • 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 present invention relates to a method for predicting or diagnosing a cardiovascular disease or a metabolic disease associated with cardiovascular disease by determining the capacity of high- density lipoprotein (HDL) to acquire surface lipids of triglyceride-rich lipoprotein (TG L) during lipolysis.
  • HDL high- density lipoprotein
  • the present invention also relates to a H DL-targeting compound for use in the prevention or the treatment of a subject which has been classified as being likely to respond by the method determining the capacity of H DL to acquire surface lipids of TGRL.
  • HDL plasma high-density lipoprotein
  • CV cardiovascular
  • H DL-cholesterol (H DL- C) concentration is present in all existing calculators of CV risk.
  • modifications of HDL-C levels using therapeutic approaches do not necessarily modify CV risk. It has therefore been suggested that it is not HDL-C itself that is causatively related to atheroprotection but rather some cardioprotective H DL function(s), which is (are) reflected by the simple measurement of HDL-C but cannot always be reliably estimated through this assay.
  • H DL function(s) cardioprotective H DL function(s)
  • HDL flux hypothesis involves in vitro measurement of cellular cholesterol efflux from lipid-loaded macrophages as a metric of CV risk (Khera AV et al. 2011; Rohatgi A et al. 2014). Promotion of cellular cholesterol efflux and RCT in vitro represents a therapeutic corollary of this hypothesis.
  • such method can be used for the stratification of patients to select those who may benefit from HDL-targeting therapy.
  • agents including CETP inhibitors (torcetrapib, dalcetrapib, evacetrapib) and formulations of nicotinic acid (niacin) developed to increase circulating HDL-C levels, beneficially target HDL metabolism and decrease cardiovascular disease, repeatedly failed in large-scaled clinical trials (Zakiev et al. 2017).
  • HDL can also serve as an acceptor for the surface constituents generated during lipolytic process, in particular by accepting free cholesterol and phospholipid from triglyceride-rich lipoprotein (Chajek et al. 1978, Redgrave et al. 1979, Tall et al. 1979).
  • the importance of HDL capacity to acquire the surface lipid constituent from triglyceride-rich lipoprotein for the evaluation of the risk of cardiovascular disease has never been considered.
  • the inventor herein proposes that the transfer of surface remnants of triglyceride-rich lipoproteins (TGRL) to HDL during their lipolysis by lipoprotein lipase (LPL) in the postprandial phase represents a main function of HDL-C and develops a method for determining presence or risk of developing a metabolic disease associated with cardiovascular disease by determining the capacity of HDL to acquire surface lipid of triglyceride-rich lipoproteins during in vitro lipolysis as a model of the postprandial phase.
  • TGRL triglyceride-rich lipoproteins
  • LPL lipoprotein lipase
  • the method can also be used for the stratification of patients with cardiovascular disease or metabolic disease associated with cardiovascular disease to select those who may benefit from HDL-targeting therapy via enhanced removal of surface lipid from TGRL during LPL- mediated lipolysis.
  • FIG. 1 A. Dose-dependence of Dil transfer from TGRL to HDL during LPL-induced lipolysis. HDL was isolated by density ultracentrifugation and incubated with Dil-labeled TGRL (30 mg TG/dl) and LPL (5 ⁇ ) at a final concentration of 2.5, 5, 10 and 20 mg protein/dL. B. Dose-dependence of Dil transfer from TGRL to apoB-depleted plasma during LPL-induced lipolysis.
  • ApoB-containing lipoproteins were precipitated from plasma by a mixture of phosphotungstic acid with Mg2+ and apoB-depleted plasma was incubated with Dil-labeled TGRL (30 mg TG/dl) and LPL (5 ⁇ ) at final dilutions of 40, 20, 10 and 5-fold. Fluorescence is expressed relative to Dil fluorescence in TGRL before incubations.
  • Figure 3 In vitro assay for determining the capacity of HDL (as apoB-depleted plasma) to acquire phospholipid (in the form of fluorescent Dil-PL): Phospholipid (PL) transfer to HDL in AMI patients is significantly decreased by -33% (p ⁇ 0.000001) relative to healthy normolipidemic controls.
  • Figure 4 Relationships of overall and CV mortality with plasma HDL-C levels and HDL capacity to acquire TopF and Dil from TGRL upon LPL-induced lipolysis. Sex-adjusted CV mortality was calculated using the data from the CANHEART Study (Ko, et al., J Am Coll Cardiol 2016; 68: 2073- 2083) and Copenhagen City Heart Study (CCHS) (Madsen, et al., Eur Heart J 2017; 38: 2478-2486) for the mean HDL-C levels observed in the five populations studied (AMI, T2D, high HDL-C, extremely high HDL-C, and controls) and plotted against the HDL-C levels (A), the mean HDL capacity to acquire TopF (C) and the mean HDL capacity to acquire Dil (D) in these populations. Relationships of HDL capacity to acquire TopF and Dil with HDL-C levels is also shown (B).
  • Figure 5 Relationship between increase in the transfer of free cholesterol (as TopF-cholesterol) from TGRL to HDL as a result of increasing HDL concentration and initial concentration of normolipidemic VLDL in the reaction mixture.
  • FIG. 6 Circulating HDL-C concentrations measured in high HDL-C (human Apo-I transgenic), low HDL-C (ApoA-l knock out) and control (wild-type) mouse models.
  • Figure 7 The capacity of HDL to acquire free cholesterol measured in high HDL-C (human Apo-I transgenic), low HDL-C (ApoA-l knock out) and control (wild-type) mouse models.
  • HDL was obtained by precipitation of apoB-containing lipoproteins as described above.
  • Figure 8 Postprandial aortic accumulation of [ 3 H]-cholesterol in vivo after a gavage of high HDL-C (human Apo-I transgenic animals), low HDL-C (ApoA-l knock out) and control (wild-type) mice with 100 ⁇ of [ 3 H]-cholesterol administrated with olive oil (100 ⁇ ). The animals were euthanized 2h after the gavage, their aortas removed and specific radioactivity (per tissue weight) measured.
  • Figure 9 Significant correlation between postprandial aortic accumulation of [ 3 H]-cholesterol in vivo measured in high HDL-C (human Apo-I transgenic), low HDL-C (ApoA-l knock out) and control (wild-type) mice, and capacity of HDL to acquire free cholesterol from TG L during LPL-mediated lipolysis in vitro evaluated in apoB-depleted plasma of these mice.
  • patient or “subject” means any human being or non-human mammal. Especially it is a man or woman, at any age.
  • patient is an individual at risk of cardiovascular disease or metabolic disease associated with cardiovascular disease.
  • the subject may be at increased risk of developing cardiovascular disease or a metabolic disease associated with cardiovascular disease.
  • An individual at risk is an individual who is considered more likely to develop a disease state such as a metabolic disease associated with cardiovascular disease or a cardiovascular disease than an individual who is not at risk.
  • An individual "at risk” may or may not have detectable symptoms indicative of the disease condition, and may or may not have displayed detectable disease prior to the treatment methods (e.g., therapeutic intervention).
  • At risk denotes that an individual has one or more so-called risk factors. An individual having one or more of these risk factors has a higher probability of developing one or more disease(s) or physiological condition(s) than an individual without these risk factor(s).
  • risk factors can include, but are not limited to, history of family members developing one or more diseases, related conditions, or pathologies, history of previous disease, age, sex, race, diet, presence of precursor disease, genetic (i.e., hereditary) considerations, and environmental exposure.
  • Bio sample refers to a sample from the subject.
  • biological sample include, but are not limited to, blood, plasma, serum, saliva, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, chorionic villi, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, semen, saliva and sweat.
  • suitable biological samples according to the invention include human biological matrices, urine, plasma, serum, and human lipoprotein fractions.
  • Lipoproteins are complex particles that have a central hydrophobic core of non-polar lipids, primarily cholesterol esters and triglycerides. This hydrophobic core is surrounded by a hydrophilic layer consisting of phospholipids, free cholesterol, and apolipoproteins. Plasma lipoproteins are divided into several classes based on size, lipid composition, and apolipoprotein composition (chylomicrons, chylomicrons remnants, very low-density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), high density lipoprotein (HDL) and Lp(a)).
  • VLDL very low-density lipoprotein
  • IDL intermediate density lipoprotein
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • TGRL Trosteaminoprotein-rich lipoproteins
  • TGRL can comprises chylomicrons, chylomicrons remnants, VLDL and/or IDL.
  • triglycerides contained in TGRL are hydrolyzed by apo-CII-dependent activation of lipoprotein lipase (LPL) present on the endothelial surface.
  • LPL removes triglycerides from VLDL and IDL in the same way as from chylomicrons and chylomicron remnants.
  • Chylomicrons are the largest (up to 1000 nm) and least dense (>0.95 g/ml) of the lipoproteins. They contain only 1-2% protein, 85-88% triglycerides, around 8% phospholipids, around 3% cholesterol esters and around 1% cholesterol. Chylomicrons contain several types of apolipoproteins including apo-AI, II & IV, apo-B48, apo-CI, II & III, apo-E and apo-H. Chylomicrons are produced for the purpose of transporting dietary triglycerides and cholesterol absorbed by intestinal epithelia. Chylomicron assembly originates in the intestinal mucosa. Excretion into the plasma is facilitated through the lymphatic system. In the plasma, chylomicrons acquire apoA-ll and apo-E from HDL.
  • Chylomicrons remnants are metabolic products of chylomicrons from which triglycerides have been selectively removed by the lipoprotein lipase. These chylomicrons remnants carry dietary lipids in the blood and are cholesterol-rich as compared to chylomicrons. Chylomicron remnants are typically several hundred nm in size but can be as small as 30-50 nm only. They contains apo- B48 and apo-E and have lost apo-CII.
  • VLDL Very Low density Lipoproteins
  • VLDL assembly in the liver involves the early association of lipids with apo-BlOO mediated by microsomal triglyceride transfer protein while apo-BlOO is translocated to the lumen of the endothelial reticulum (ER).
  • ER endothelial reticulum
  • IDL Intermediate Density Lipoproteins
  • HDL High Density Lipoproteins
  • apolipoproteins including primarily apo-AI, II & IV, apo-CI, II and III, apo-D and apo-E.
  • HDL contain approximately 35-55% protein, 3-15% triglycerides, 24-46% phospholipids, 15-30% cholesteryl esters and 2-10% cholesterol.
  • HDL are produced as a protein-rich particle in the liver and intestine and serve as a source of apo-CI & II and Apo-E proteins.
  • HDL particles accumulate cholesteryl esters by the esterification of cholesterol by lecithin-cholesterol acyl-transferase (LCAT).
  • LCAT is activated by apo-AI on HDL.
  • HDL can acquire cholesterol from cell membranes and can transfer cholesteryl esters to VLDL and LDL by cholesteryl ester transfer protein (CETP).
  • CETP cholesteryl ester transfer protein
  • HDL can return to the liver where cholesterol is removed in the process of reverse cholesterol transport, primarily via scavenger receptor Bl, serving as a scavenger to free cholesterol. The liver can then excrete excess cholesterol in the form of bile acids.
  • HDL concentration range from 2.0 to 3.0 g/L.
  • TG L triglyceride-rich lipoprotein
  • CM chylomicron
  • VLDL very low- density lipoprotein
  • LPL lipoprotein lipase
  • Core remnants core remnants
  • IDL IDL
  • Excess molecules of the surface monolayer are shred from the particles in a form of surface remnants; such molecules include surface apolipoproteins, phospholipid (PL) and free cholesterol (FC). Surface remnants are then predominantly transferred to HDL.
  • the inventor develops an efficient in vitro method for determining presence or risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease by determining the capacity of HDL to acquire surface lipid of triglyceride-rich lipoprotein.
  • HDL capacity to acquire surface lipids from TGRL is determined by measuring the transfer of surface lipids (also designated by lipids hereafter) from TGRL to HDL.
  • the present invention thus relates to an in vitro method for determining the capacity of HDL from a subject to acquire surface lipid of triglyceride-rich lipoprotein (TG L) comprising the steps of: i) providing a subject biological sample comprising HDL;
  • HDL is diluted to the final concentration of 2.5 to 20 mg protein/dl (for example when isolated by ultracentrifugation) and/or HDL is diluted 40- to 10-fold relative to its concentration in plasma (for example when isolated by apolipoprotein B precipitation), and TGRL is diluted to the final concentration of 5 to 100 mg triglyceride/dl;
  • step iv) adding lipoprotein lipase enzyme at a concentration comprised between 100 and 300 units/ml to the mixture obtained at step iii) and incubating between 30 to 180 minutes;
  • the sample may be fresh blood or stored blood or blood fractions.
  • the sample may be a blood sample expressly obtained for the assays of this invention or a blood sample obtained for another purpose which can be subsampled for use in accordance with the methods according to the invention.
  • said blood sample is a fasting blood sample.
  • the sample is derived form a cryopreserved sample.
  • the biological sample may be whole blood. Whole blood may be obtained from the subject using standard clinical procedures.
  • the biological sample may also be plasma, preferably fasting plasma. Plasma may be obtained from whole blood samples by centrifugation of anti-coagulated blood.
  • the biological sample may also be serum.
  • the sample may be pretreated as necessary by dilution in an appropriate buffer solution, concentrated if desired, or fractionated by any number of methods including but not limited to ultracentrifugation, fractionation by fast performance liquid chromatography (FPLC), or precipitation.
  • FPLC fast performance liquid chromatography
  • Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, at physiological to alkaline pH can be used.
  • the method is performed with HDL that has been isolated or purified from a biological sample, and preferably from subject or patient plasma.
  • the methods suitable for separating and/or purifying the different fractions of lipoprotein are well known by the person skilled in the art (see Schumaker & Puppioe, 1986).
  • Illustrative isolation methods include, but are not limited to ultracentrifugation, PEG precipitation, heparin MnCI2 precipitation, sodium phosphotungstate precipitation, dextran sulfate precipitation, gel filtration, fast protein liquid chromatography (FPLC) and immunoaffinity capture. Protocols for these and other HDL isolation methods are readily available.
  • HDL are isolated by heparin/MnCI2, dextran sulfate, sodium phosphotungstate-MgCI2, or PEG precipitation, preferably by phosphotungstate-MgCI2 precipitation.
  • HDL are isolated by ultracentrifugation on the appropriate density layer, knowing that density of HDL is from 1.063 to 1.210 g/mL.
  • HDL are isolated by ultracentrifugation at a density raised to 1.210 g/mL. The fractions isolated after ultracentrifugation should be further purified by dialysis.
  • the method of the invention comprises an additional step i'), conducted between step i) and step ii) or step ii) and iii), consisting in isolating HDL of said subject biological sample by precipitation.
  • the method of the invention comprises an additional step i'), conducted between step i) and step ii) or step ii) and iii), consisting in isolating HDL of said subject biological sample by ultracentrifugation.
  • the term “purified” when referring to HDL is intended to mean that the HDL represents at least 80% of lipoprotein on a mass basis of the composition comprising it. More preferably, the term “purified” indicates that lipoprotein represents by order of preference at least 85%, 90%, 92%, 95%, 97%, 98%, 99%, 100% on a mass basis of the composition.
  • Triglyceride rich lipoprotein comprises chylomicrons, chylomicrons remnants, VLDL and/or IDL.
  • chylomicron, chylomicron remnant, VLDL and/or IDL preferably chylomicron and VLDL or a mixture of both can be used in the method according to the present invention.
  • the sample may be fresh blood or stored blood or blood fractions.
  • said sample is normal or healthy plasma, preferably normolipidemic plasma.
  • said sample is subject or patient plasma, preferably said sample is the same plasma than subject plasma comprising HDL.
  • Illustrative isolation methods include, but are not limited to ultracentrifugation, precipitation, and immunoaffinity capture.
  • TGRL are isolated by ultracentrifugation.
  • TGRL are isolated by ultracentrifugation at a density raised to 1.019 g/ml.
  • the fractions isolated after ultracentrifugation can be further purified by dialysis.
  • TGRL are isolated by ultracentrifugation of a healthy normolipidemic human biological sample.
  • TGRL are isolated from said subject biological sample, preferably said sample is the same plasma than subject plasma comprising HDL.
  • synthetic VLDL or chylomicron can be used.
  • the method of the invention may be conducted with TGRL containing labeled or non-labeled lipids.
  • TGRL are TGRL containing labeled lipids.
  • the method according to the invention further comprises a contacting step of TGRL with HDL.
  • the contacting step comprises mixing HDL and TGRL.
  • HDL and TGRL can be diluted in suitable buffer.
  • suitable buffer include but are not limited to phosphate buffers, HEPES, MOPS, HEPPS, and Tris-acetate, glycine, etc.
  • HDL for example when isolated by ultracentrifugation, can be diluted to the final concentration of 1 to 50 mg protein/dl, preferably 2.5 to 20 mg protein/dl, more preferably 2.5 to 10 mg protein/dl, more preferably 4 mg protein/dl.
  • HDL isolated by apolipoprotein B precipitation can be diluted 40- to 10-fold relative to its concentration in plasma, more preferably 30-fold.
  • Labeled TGRL can be diluted to the final concentration of 5 to 100 mg triglyceride/dl, preferably 15 to 60 mg triglyceride/dl, preferably 20 to 40 mg triglyceride/dl, more preferably 30 mg triglyceride/dl.
  • the ratio HDL protein:TGRL triglyceride is comprised between 1:5 and 1:10.
  • LPL lipoprotein lipase
  • the method according to the present invention include the addition of LPL to the mixture of HDL and TGRL before measuring labeled lipid incorporation into HDL.
  • Lipoprotein lipase according to the invention is an enzyme capable of hydrolyzing triglycerides contained in TGRL. The catabolism of lipids results in the transfer of surface lipid from TGRL to HDL.
  • LPL may be obtained from animals, plants or microorganisms, or produced by genetic engineering techniques.
  • LPL is obtained from bovine milk (EC n°232- 669-1), from pseudomonas sp. (EC n°232-669-l) and from Burkholderia sp. (EC n° 232-669-1). Chemically modified LPL can also be used.
  • Examples of the chemically modified enzymes include enzymes that are modified with chemically modifying groups such as a group comprising polyethylene glycol or polypropylene glycol as a main component, a group having a copolymer of polypropylene glycol and polyethylene glycol, a group comprising a water-soluble polysaccharide, a sulfopropyl group, a sulfobutyl group, a polyurethane group and a group having the chelating function.
  • an enzyme modified with a group comprising polyethylene glycol as a main component examples of the water-soluble polysaccharides include dextran, pullulan and soluble starch.
  • the concentration of the LPL in the method according to the invention is preferably about 100 to 300 units/ml.
  • the concentration of the LPL is preferably adapted to the concentrations of TGRL and HDL; preferably, the ratio TGRL triglyceride (mg) : LPL activity unit is comprised between 1:300 and 1:1000.
  • Time incubation of LPL with labeled TGRL and HDL mixture is comprised between 5 to 180 minutes. Preferably between 30 to 180 minutes, preferably 120 minutes.
  • HDL capacity to acquire surface lipid from TGRL is determined by measuring the difference of quantity of lipid within HDL measured before step iii) and after step iv).
  • HDL can be isolated by precipitation or ultracentrifugation as described above.
  • the present invention thus relates to an in vitro method for determining the capacity of HDL from a subject to acquire surface lipids of triglyceride-rich lipoprotein (TGRL) further comprising the steps of: ii') measuring the quantity of lipids within HDL of subject between step i) and ii) or ii) and iii), iv') measuring the quantity of lipids within HDL between step iv) and step v) and wherein the quantity of lipids transferred from TGRL to HDL of subject of step v) is determined by measuring the difference of quantity of lipid within HDL measured in step ii') and iv').
  • ELISA radioimmunoassay
  • HPLC HPLC
  • FACS immuno- turbidometric assays
  • capillary electrophoresis capillary electrophoresis
  • two dimensional gel electrophoresis with or without immunodetection methods can be used as alternatives to NMR spectroscopy for measuring lipids.
  • Numerous methods are available for determination of cholesterol concentration, e.g., gravimetric, nephelometric, turbidimetric, or photometric methods, among others.
  • kits for quantitative colorimetric/fluorimetric cholesterol and cholesteryl esters determination may be used.
  • the concentrations of total and free cholesterol are determined, whereas the concentration of cholesteryl esters (esterified cholesterol) is calculated from the difference between these two concentrations.
  • Enzymatic determination of cholesterol concentration is specific and sensitive.
  • Illustrative, non-limitative, methods for determination of phospholipids concentration include commercially available assay kits for a quantitative colorimetric/fluorimetric phospholipid determination.
  • HDL capacity to acquire surface lipids from TGRL is determined by providing TGRL containing labeled lipids and measuring the transfer of labeled lipids from TGRL to HDL.
  • the present invention thus relates to an in vitro method for determining the capacity of HDL from a subject to acquire surface lipids of triglyceride-rich lipoprotein (TGRL) wherein said TGRL comprise labeled lipids and wherein the quantity of lipids transferred from TGRL to HDL of subject in step v) is determined by measuring the signal of labeled lipids within HDL of subject.
  • the present invention relates to an in vitro method for determining the capacity of HDL from a subject to acquire surface lipids of triglyceride-rich lipoprotein (TGRL) comprising the steps of: i) providing a subject biological sample comprising HDL;
  • HDL is diluted to the final concentration of 2.5 to 20 mg protein/dl (for example when isolated by ultracentrifugation) and/or HDL is diluted 40- to 10-fold relative to its concentration in plasma (for example when isolated by apolipoprotein B precipitation), and TGRL is diluted to the final concentration of 5 to 100 mg triglyceride/dl;
  • Labeled TGRL refers to TGRL comprising labeled lipids.
  • said labeled lipids are a labeled phospholipid and/or labeled free cholesterol, more preferably a labeled free cholesterol.
  • the labeled TGRL is obtained by contacting at least one labeled lipid with TGRL under conditions suitable for the binding of labeled lipid, preferably phospholipid or cholesterol, to TGRL to form a labeled TGRL. After being mixed, TGRL starts to absorb the labeled lipid.
  • the temperature conditions and the contact time in the step There is no particular limitation as to the temperature conditions and the contact time in the step.
  • the mixture of the sample and the labeled probe may be incubated at 20°C to 37°C, preferably 35°C to 37°C for 1 minute to 24 hours, preferably 1 hour to 4 hours. The mixture may be allowed to stand or may be stirred or shaken during incubation.
  • a detectable label refers to a molecule or moiety that can be detected, for example, by performing an assay known to those of skill in the art for its detection.
  • a detectable label may be, for example, (i) an isotopic label (e.g., a radioactive or heavy isotope, including, but not limited to, 2H, 3H, 13C, 14C, 15N, 31P, 32P, 35S, 67Ga, 99mTc (Tc-99m), lllln, 1231, 1251, 169Yb, and 186Re), (ii) an affinity label (e.g., an antibody or antibody fragment, an epitope, a ligand or a ligand-binding agent) (iii) and enzymatic label that produce detectable agents when contacted with a substrate (e.g., a horseradish peroxidase or a luciferase); (iv) a dye, (e.g., a colored, luminescent
  • Labeled lipid can be lipophilic dye.
  • Suitable lipophilic dye include fluorescently-tagged lipid anchors (e.g. fluorescently-labeled fatty acid analogs).
  • An example of labeled fatty acid analog is NBD-ceramide such as l-oleoyl-2- ⁇ 6-[(7-nitro-2-l,3-benzoxadiazol-4-yl)amino]hexanoyl ⁇ -s/i- glycero-3-phosphocholine.
  • Other exemplary lipophilic dyes include, without limitation, carboxyfluorescein, BODIPY dyes, or the Alexa Fluor series.
  • Such dyes are known by those skilled in the art and may be chosen from a group including, but not limited to lipophilic versions of fluorescent dyes including Alexa Fluor(R) 350, Alexa Fluor(R) 405, Alexa Fluor(R) 488, Alexa Fluor(R) 532, Alexa Fluor(R) 546, Alexa Fluor(R) 555, Alexa Fluor(R) 568, Alexa Fluor(R) 594, Alexa Fluor(R) 647, Alexa Fluor(R) 680, Alexa Fluor(R) 750, BODIPY(R) FL, Coumarin, Cy(R)3, Cy(R)5, Fluorescein (FITC), Oregon Green(R), Pacific Blue, Pacific Green, Pacific Orange, Tetramethylrhodamine (TRITC), Texas Red(R), DNA stains, DAPI, Propidium Iodide, SYTO(R) 9, SYTOX(R) Green, TO-PRO(R)-3, Qdot(R) probes, Q
  • labeled lipid is a labeled cholesterol.
  • Labeled cholesterol is a substance in which a label binds to a part of a molecule of cholesterol.
  • Fluorescent-labeled cholesterol and label including a fluorophore having a polar structure are known in the art.
  • the fluorescence-labeled cholesterol including a fluorophore having a polar structure is, for example, fluorescence-labeled cholesterol including a fluorophore having a boron-dipyrromethene skeleton, such as 23-(dipyrro-metheneboron-difluoride)-24-norcholesterol), (TopFluor Cholesterol, CAS No: 878557-19-8, available from Avanti Polar Lipids, Inc.) or a benzoxadiazole skeleton such as 25-[N-[(7-nitro-2-l,3-benzoxa diazole-4-yl)methyl] amino]-27-norcholesterol (25- NBD Cholesterol, CAS No: 105539-27-3, available from Avanti Polar Lipids, Inc.)
  • labeled lipid is a labeled phospholipid, preferably fluorescent phospholipid.
  • suitable label used can be fluorescent label-linked fatty acids include ADIFAB fatty acid indicators phospholipids with BODIPY dye-labeled acyl chains such as BODIPY glycerophospholipids, phospholipid with DPH-labeled acyl chain, phospholipids with NBD-labeled acyl chains, phospholipids with pyrene-labeled acyl chains, phospholipids with a fluorescent or biotinylated head group, LipidTOX phospholipid and neutral lipid stains.
  • BODIPY dye-labeled acyl chains such as BODIPY glycerophospholipids, phospholipid with DPH-labeled acyl chain, phospholipids with NBD-labeled acyl chains, phospholipids with pyrene-labeled acyl chains, phospholipids with a fluorescent or biotinylated head group, LipidTOX phospholipid and neutral lipid stains.
  • fluorescent phospholipid is selected from the group consisting of : l,l'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and l-oleoyl-2- ⁇ 6-[(7-nitro-2- l,3-benzoxadiazol-4-yl)amino]hexanoyl ⁇ -s/ glycero-3-phosphocholine.
  • fluorescent cholesterol is 23-(dipyrro-metheneboron difluoride)-24-norcholesterol.
  • labeled lipid can be isotopic labeled lipid which is identical to a corresponding unlabeled lipid but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.
  • isotopes that can be incorporated into compounds are hydrogen, carbon, nitrogen, fluorine such as 3H, 11C, 14C and 18F.
  • the signal of labeled lipid within HDL is determined by a quantitative measurement of labeled lipid within HDL, more particularly by detecting signal emitted by labeled lipid. Measurement of labeled lipid can be performed using any suitable technique known in the art.
  • the measuring step comprises quantitative measurement of fluorescent lipid within HDL.
  • Fluorescent lipid is excited by a light source and emits a signal which is detected by a fluorometric assay.
  • the following assays may be performed using any technique employing a device comprising a fluorimeter including: flow cytometry, microscopy, optical measurement of fluorescence, and combinations thereof.
  • methods herein utilize a fluorometric plate reader. These readers have, for example, a light source which is directed from above the plate and the resultant fluorescence is detected by a detector positioned either directly above the plate or at an angle above the plate.
  • isotopically labeled lipid molecules can be measured by various methods such as mass spectrometry, including but not limited to gas chromatography-mass spectrometry (GC-MS), isotope-ratio mass spectrometry, GC-isotope ratio-combustion-MS, GC- isotope ratio-pyrrolysis-MS, liquid chromatography-MS, electrospray ionization-MS, matrix assisted laser desorption-time of flight-MS, Fourier-transform-ion-cyclotron-resonance-MS, and cycloidal-MS.
  • mass spectrometry including but not limited to gas chromatography-mass spectrometry (GC-MS), isotope-ratio mass spectrometry, GC-isotope ratio-combustion-MS, GC- isotope ratio-pyrrolysis-MS, liquid chromatography-MS, electrospray ionization-MS, matrix assisted laser desorption-time of flight-MS, Fourier-transform-i
  • the present invention concerns an in vitro method for determining presence or risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease, in a subject comprising the steps of: i) determining the quantity of lipid transferred from TG L to HDL of a subject as described above; ii) comparing said quantity with a control value.
  • disorders related to metabolic diseases associated with cardiovascular disease include but are not limited to stroke, ischemic stroke, transient ischemic attack, myocardial infraction, angina pectoris, inflammatory disorder, V-related diseases and/or metabolic-related diseases: type 2 diabetes, metabolic syndrome, atherosclerosis, premature atherosclerosis, hyperlipidemia, especially hypercholesterolemia, familial hypercholesterolemia, familial combined hyperlipidemia, hypoalphalipoproteinemia, coronary heart disease, coronary artery disease, acute coronary syndrome, vascular and perivascular diseases, renovascular insufficiency, critical limb ischemia, rest pain, gangrene, restenosis, rheumatoid arthritis, dyslipidemic disorders, dyslipoproteinemia, high levels of low density lipoprotein cholesterol, high levels of very low density lipoprotein cholesterol, low levels of high density lipoproteins, high levels of lipoprotein Lp(a), high levels of apolipoprotein B, familial combined hyperlipidemia (FCH), lipoprotein lipa
  • cardiovascular disorders include but are not limited to: coronary artery disease (also known as coronary heart disease and ischemic heart disease), cardiomyopathy; hypertensive heart disease, heart failure, cardiac dysrhythmias, inflammatory heart disease, endocarditis, inflammatory cardiomegaly, myocarditis, valvular heart disease, cerebrovascular disease, stroke, ischemia, peripheral arterial disease, congenital heart disease (heart structure malformations existing at birth), rheumatic heart disease.
  • coronary artery disease also known as coronary heart disease and ischemic heart disease
  • cardiomyopathy hypertensive heart disease, heart failure, cardiac dysrhythmias, inflammatory heart disease, endocarditis, inflammatory cardiomegaly, myocarditis, valvular heart disease, cerebrovascular disease, stroke, ischemia, peripheral arterial disease, congenital heart disease (heart structure malformations existing at birth), rheumatic heart disease.
  • the method for determining presence or risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease thus comprises comparing the quantity of lipid transferred from TGRL to HDL of a subject with a control value.
  • control value refers to a standard value of quantity of lipid transferred from TGRL to HDL in healthy normolipidemic subjects or a population of healthy normolipidemic subjects; a normolipidemic subject is a healthy non-dislipidemic subject not having a dyslipidemia as defined by Fredrickson (Fredrickson DS, Lees RS. A system for phenotyping hyperlipoproteinemia. Circulation 1965;31:321-327).
  • the quantity of lipid transferred from TGRL to HDL can be determined by measuring the difference between the quantity of lipid within HDL before step iii) and after step iv).
  • the "control value” refers to a standard value of quantity of lipid transferred from TGRL to HDL in healthy normolipidemic subjects or a population of healthy normolipidemic subjects determining by measuring the difference between the quantity of lipid within HDL before step iii) and after step iv).
  • the method for determining presence or risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease comprises comparing the signal of labeled lipid within HDL in the patient with a control value.
  • the "control value” refers to a standard value of signal of labeled lipid within HDL in healthy normolipidemic subjects or a population of healthy normolipidemic subjects.
  • a decreased signal of the quantity of lipid transferred from TGRL to HDL in the subject sample compared to the control value is indicative of presence or risk of developing a metabolic disease associated with cardiovascular disease.
  • the terms “decreased quantity” or “decreased signal” refer to a significantly lower quantity, e.g. of more than 10%, preferably more than 20%, 30%, 40%, or 50%.
  • the in vitro method for determining presence or risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease, in a subject is such that:
  • said subject is a patient with acute myocardial infarction (AMI) and said lipid is free cholesterol, preferably labeled free cholesterol, or phospholipid, preferably labeled phospholipid;
  • AMI acute myocardial infarction
  • said subject is a patient with type 2 diabetes (T2D) and said lipid is free cholesterol, preferably labeled free cholesterol;
  • said subject is an individual with high HDL-C level (between 70 and 100 mg/dL) or very high HDL- C level (more than 100 mg/dL) and said lipid is free cholesterol, preferably labeled free cholesterol.
  • the method according to the invention can be associated with known methods for determining presence or risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease in a subject.
  • the method for determining presence or risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease in a subject further comprises steps of: measuring the quantity of LDLc, triglycerides, Lp(a), apoB in a biological sample of a subject and comparing the quantity of LDLc, triglycerides, Lp(a), apoB in the subject with a control value.
  • An increased quantity of LDLc, triglyceride, Lp(a) or ApoB in the subject biological sample compared to the control value is indicative of the presence of presence or risk of developing a metabolic disease associated with cardiovascular disease.
  • the term "increased signal" refers to a significantly higher quantity, e.g. of more than 10%, preferably more than 20%, 30%, 40%, or 50%.
  • the invention also provides a kit comprising TGRL containing labeled lipid, lipoprotein lipase and a control sample that is preferably a sample containing HDL obtained from an healthy normolipidemic subject.
  • said kit further comprises a Apo-B precipitant reagent such as sodium phosphotungstate precipitation agent to isolate HDL.
  • Said kit can further comprises suitable buffer.
  • buffers include but are not limited to phosphate buffers, HEPES, MOPS, HEPPS, and Tris-acetate, glycine, etc.
  • the kit is for determining the capacity of HDL to acquire surface lipid from TGRL in a subject.
  • the kit is for determining the presence or the risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease.
  • Control sample can be sample comprising HDL from healthy normolipidemic subjects or a population of healthy normolipidemic subjects.
  • control sample is plasma obtained from a healthy normolipidemic subject ("normolipidemic plasma").
  • the control sample allows to compare HDL capacity to acquire surface lipid from TGRL of a patient to a control value.
  • Non-limiting examples of one or more other kit components include instructions for use; vials, containers or other storage vessels containing each of the unit doses; delivery devices such as needles, catheters, syringes, tubing and the like; and/or packaging suitable for safely and conveniently storing and/or transporting the kit.
  • the present invention also encompasses a HDL-targeting compound for use in the prevention or the treatment of a subject which has been classified as presenting or as being at risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease with the method according to the present invention.
  • treatment refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease.
  • such term refers to the amelioration or eradication of a disease or symptoms associated with a disease.
  • this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.
  • HDL-targeting compound refers to compounds which raise HDL levels in circulation and/or improve or normalize HDL metabolism.
  • HDL-targeting compounds referring to compounds which raise HDL levels in circulation are also named HDL-raising compounds.
  • HDL targeting compounds raise HDL levels in circulation and/or improve or normalize HDL metabolism by either one of the following mechanisms: CETP inhibition/modulation, HDL mimetics, ApoA-l transcriptional regulators, PPAR agonism, LXR agonism, FXR agonist, HM74 agonism (niacin receptor) thyrotropin hormone receptor agonism, inhibitors of lipases and HDL catabolism, ApoA-l inducers, compounds which provide at least one of the HDL athero-protective activities such as compounds that would increase cellular lipid efflux (cholesterol and/or phospholipids), have antioxidant and antiinflammatory activities.
  • CETP inhibitors represent a novel class of drugs under development, which aim to improve and/or normalise lipoprotein metabolism via inhibition of
  • HDL mimetics constitute another class of perspective drugs that mimic native HDL particles and are composed of an apolipoprotein without or with a phospholipid, two major HDL components.
  • ApoA-l transcriptional regulators are compounds specifically enhancing hepatic production of ApoA-l at a transcriptional level.
  • HDL-targeting compound is selected from the group consisting of: niacin, fibrates, statins, Apo-AI mimetic peptides (e.g., APP018, D-4F, L-4F, 6F, 5A and ATI-5261), apoA-l transcriptional up-regulators (e.g., VX-208, Resverlogix), ACAT inhibitors (e.g., avasimibe; IC-976, Pfizer; MCC-147, Mitsubishi Pharma), CETP modulators (e.g., torcetrapib, evacetrapib, anacetrapib), HDL infusion compound (e.g. MDCO-216, CSL111, CSL112; CER-001) or combinations thereof.
  • Apo-AI mimetic peptides e.g., APP018, D-4F, L-4F, 6F, 5A and ATI-5261
  • apoA-l transcriptional up-regulators e.g.
  • HDL-targeting compound examples include niacin, fibrates, glitazone, dalcetrapib, anacetrapib, evacetrapib, torcetrapib, DEZ-001 (formerly known as TA- 8995, Mitsubishi Tanabe Pharma), ATH- 03 (Affris), DRL- 17822 (Dr.
  • the present invention also provides a pharmaceutical composition comprising an HDL-targeting compound as previously defined and a pharmaceutically acceptable excipient.
  • the present invention also relates to the pharmaceutical composition of the invention for use in the treatment of a subject which has been classified as presenting or as being at risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease with the method for determining the capacity of HDL from a subject to acquire surface lipid of TGRL according to the present invention.
  • HDL-targeting compound or a pharmaceutical composition of the invention for the manufacture of a medicament for use in the treatment of a subject which has been classified as presenting or as being at risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease with the method for determining the capacity of HDL from a subject to acquire surface lipid of TG L according to the present invention.
  • the pharmaceutically acceptable excipient is selected according to the route of administration and the nature of the active ingredient, e.g. a protein, a nucleic acid or a viral particle.
  • pharmaceutically acceptable means approved by a regulatory agency or recognized pharmacopeia such as European Pharmacopeia, for use in animals and/or humans.
  • excipient refers to a diluent, adjuvant, carrier, or vehicle with which the therapeutic agent is administered.
  • pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmacologically effective substance and can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use.
  • Possible pharmaceutical compositions include those suitable for oral, rectal, topical (including transdermal, buccal, sublingual, ocular instillation), intraocular (including intravitreal, intracameral, subretinal, suprachoroidal, periocular, subconjunctival) or parenteral (including subcutaneous, intramuscular, intraspinal, intravenous and intradermal) administration.
  • topical including transdermal, buccal, sublingual, ocular instillation
  • intraocular including intravitreal, intracameral, subretinal, suprachoroidal, periocular, subconjunctival
  • parenteral including subcutaneous, intramuscular, intraspinal, intravenous and intradermal
  • conventional excipient can be used according to techniques well known by those skilled in the art.
  • compositions according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration.
  • the amount of pharmaceutical composition of the invention to be administered has to be determined by standard procedure well known by those of ordinary skill in the art.
  • Physiological data of the patient e.g. age, size, and weight
  • the routes of administration and the disease to be treated have to be taken into account to determine the appropriate dosage.
  • the appropriate dosage of the pharmaceutical composition of the invention may also vary if it is used alone or in combination.
  • the present invention further concerns a method for treating cardiovascular disease or metabolic disease associated with cardiovascular disease in a subject comprising administering a of HDL-targeting compound wherein said subject has been classified as presenting or as being at risk of developing a cardiovascular disease or a metabolic disease associated with cardiovascular disease with the method for determining the capacity of HDL from a subject to acquire surface lipid of TGRL according to the invention.
  • a “therapeutically efficient amount” is intended an amount of the pharmaceutical composition administered to a subject that is sufficient to prevent or treat metabolic disease associated with cardiovascular disease or cardiovascular disease.
  • the present invention relates to a method of in vitro assessing the activity of a compound to be tested for improving the capacity of HDL from a subject to acquire surface lipid of triglyceride-rich lipoprotein (TGRL), said method comprising the steps of:
  • TGRL-protein lipoprotein-deficient serum
  • LPDS lipoprotein-deficient serum
  • the mixture was filtered with 0.8 ⁇ filter and the tube was covered with aluminium foil.
  • Dil Sigma, MW 933.87 g/mol
  • TopF TopF
  • BODIPY cholesterol BODIPY cholesterol
  • TGRL Triglycerides (TG) concentration (CTG) was measured in purified Dil-labeled TGRL or TopF-labeled TGRL by Microplate Reader (DYNEX TECHNOLOGIES) using a commercially available kit.
  • the volume of Dil-labeled TGRL or TopF-labeled TGRL in the reaction mixture (VTG) required to achieve the final concentration of 30mg TG/dL was calculated according to the formula VTG in TGRL 200/(CTG in TGRL/30).
  • Dil fluorescence in TGRL was measured at excitation and emission wavelengths of 525 nm and 570 nm respectively at TGRL-TG of 30mg/dl in a final volume of 200 ⁇ (normally about 300 fluounits).
  • TopF fluorescence in TGRL was measured at excitation and emission wavelengths of 500 nm and 525 nm respectively at TGRL-TG of 30mg/dl in a final volume of 200 ⁇ .
  • HDL is also named apo-B depleted plasma.
  • apo-B depleted plasma was prepared by adding apoB precipitant (mixture of phosphotungstic acid with Mg2+) to a reference plasma sample (normolipidemic human, EDTA; positive control) and to a plasma sample to be studied (EDTA) at a ratio of 1:10 by volume according to manufacturer's instructions which include incubation at room temperature for 10 min and centrifugation at 13 000 rpm for 30 min.
  • ApoB-depleted plasma is typically used at a final dilution of 30-fold v/v, which requires the volume of apoB-depleted plasma of 6.7 ⁇ to be added to the reaction mixture of 200 ⁇ total volume.
  • HDL was prepared by ultracentrifugation (XL-70 Ultracentrifuge (BECKMAN) or OptimaTM MAX-TL Ul Ultracentrifuge (BECKMAN COULTER)) of each plasma sample at a density raised to 1.21g/ml after removal by ultracentrifugation of all lipoproteins lighter than 1.063 g/ml.
  • Tris buffer 0.4 M, pH 8
  • Dil-labeled TGRL or TopF- labeled TGRL was added to the final concentration of 30 mg TG/dL followed by HDL to the final concentration of 4 mg protein/dL, or apoB-depleted plasma to the final dilution of 30-fold (6.7 ⁇ of apoB-depleted plasma).
  • Reference normolipidemic apoB-depleted plasma sample was added in each series of samples to be measured for data normalization.
  • LPL enzyme Lipoprotein Lipase from bovine milk (SIGMA, Lot SLBF4952V) was added on the tube wall. The tube content was mixed briefly for several seconds. All tubes were incubated in an air incubator (FIRLABO) for 2 hours at 37°C. Then, the tubes were put directly on ice to stop enzymatic reaction. 20 ⁇ of the HDL-C precipitant reagent (HDL-C Precipitation Reagent) was added to each tube. Tubes were incubated for 10 min at room temperature.
  • LPL enzyme Lipoprotein Lipase from bovine milk (SIGMA, Lot SLBF4952V) was added on the tube wall. The tube content was mixed briefly for several seconds. All tubes were incubated in an air incubator (FIRLABO) for 2 hours at 37°C. Then, the tubes were put directly on ice to stop enzymatic reaction. 20 ⁇ of the HDL-C precipitant reagent (HDL-C Precipitation Reagent) was added to each tube
  • Eppendorf tubes 1.5 ml Eppendorf tubes were prepared for each sample and plugs were removed from all of them. 500 ⁇ Eppendorf tubes with the samples were placed inside 1.5mL Eppendorf tubes without plugs. The tubes were centrifuged at 4°C for 10 min at a maximal speed (typically 13000 rpm).
  • a standard Dil-TGRL or TopF-TGRL sample was prepared by mixing 50 ⁇ Tris buffer (0.4 M, pH 8) with Dil-TGRL or TopF-TGRL at a final concentration of 30 mg TG/dL and with required volume of PBS in a final volume of 200 ⁇ . ⁇ of this sample were transferred in the black microplate for fluorescence reading and the microplate was read using the Gemini fluorescence reader with an excitation and emission wavelengths of 525 and 570 nm, respectively, for Dil-TGRL and 500 and 525 nm, respectively, for TopF-TGRL.
  • HDL was isolated by density ultracentrifugation and incubated with Dil-labeled TGRL or TopF- labeled TGRL (30 mg TG/dl) and LPL at final concentrations of 2.5, 5, 10, 20 mg protein/dl.
  • Apo-B containing lipoproteins were precipitated from plasma by a mixture of phosphotungstic acid with Mg2+.
  • Apo-B-depleted plasma was incubated with Dil-labeled TGRL or TopF-labeled TGRL (30 mg TG.dl) and 5 ⁇ of LPL at final dilutions of 40, 20, 10 and 5-fold.
  • Phospholipid (PL) transfer from lipolysed TGRL to ultracentrifuged HDL ( Figure 1A) or to apoB- depleted plasma (Figure IB) could be reliably observed using fluoremetrical detection (as Dil fluorescence). Similar dose-dependences were observed for cholesterol transfer (as TopF- cholesterol). The method was reproducible and could be employed for assay development.
  • the new assay of HDL function was first applied to a group of patients with acute myocardial infarction (AMI), an acute form of CV disease well known to involve low plasma levels of HDL-C.
  • AMI acute myocardial infarction
  • LDL-C concentrations did not differ between the groups, presumably reflecting statin treatment of a part of patients before the event.
  • Table 1 Age (years), lipid profile (mg/dl) and HDL capacity to acquire free cholesterol (FC; as TopF-cholesterol) and phospholipid (PL; as Dil-PL) upon TGRL lipolysis by LPL in patients with acute myocardial infarction (AMI) and healthy normolipidemic controls.
  • FC free cholesterol
  • PL phospholipid
  • the lipid-acquiring capacity of HDL is expressed as % of reference plasma. Means are shown for the each group followed p-values for the difference between the groups and standard deviations in each group.
  • T2D Type 2 diabetes
  • T2D Type 2 diabetes
  • Table 2 Age (years), lipid profile (mg/dl) and HDL capacity to acquire free cholesterol (FC; as TopF-cholesterol) and phospholipid (PL; as Dil-PL) upon TGRL lipolysis by LPL in patients with Type 2 diabetes (T2D) and healthy normolipidemic controls.
  • FC free cholesterol
  • PL phospholipid
  • T2D Type 2 diabetes
  • the lipid-acquiring capacity of HDL is expressed as % of reference plasma. Means are shown for the each group followed by p-values for the difference between the groups and standard deviations in each group.
  • the assay of HDL function was also applied to a group of subjects with high level of HDL-C.
  • the new assay of HDL function was further evaluated in terms of its capacity to predict the presence of CV disease, or CV disease risk factors - in other words, to discriminate between subjects without and with disease, or with risk factors for the disease.
  • HDL-C levels are firmly established to represent strong, independent and negative risk factor for CV disease and to be able to distinguish between subjects without and with CV disease.
  • CV risk factors primarily age, sex and LDL-C levels
  • circulating HDL-C concentrations ensure good prediction of the presence of CV disease.
  • a significant part of CV risk however still remains unaccounted using even the best established constellations of risk factors.
  • the HDL-C assay remains empirical, without clear mechanistic relevance linking it to underlying physiological processes.
  • the new assay of HDL function was therefore compared with the measurement of plasma HDL-C in terms of their capacity to predict the presence of AMI (an acute form of CV disease) and T2D (a CV risk factor).
  • the predictive accuracy of the assays was evaluated by logistic regression analysis which was either unadjusted or adjusted for established CV risk factors (age, sex and plasma LDL- C and TG concentrations).
  • the unadjusted logistic regression models involving HDL-C, HDL capacity to acquire FC and HDL capacity to acquire PL provided areas under the receiver- operating-characteristic curve (ROC) of 0.871, 0.819 and 0.881, respectively.
  • ROC receiver- operating-characteristic curve
  • the unadjusted logistic regression models involving H DL-C, HDL capacity to acquire FC and HDL capacity to acquire PL provided ROC areas of 0.809, 0.746 and 0.572, respectively.
  • HDL capacity to acquire FC was a more accurate predictor of T2D as compared to HDL-C, which was not significantly predictive of the disease after multiple adjustment.
  • Murine Models a) Assessment of HDL function in mouse models of dyslipidemia and atherosclerosis
  • apoA-l apolipoprotein A-l
  • hyperlipidemic mice transgenic for human apoA-l featuring high HDL levels.
  • ApoA-l knock-out, human apoA-l transgenic and wild-type mice were purchased from Jackson Labs. Blood was obtained from the tail vein, mixed with EDTA and centrifuged to obtain plasma. HDL was isolated from the plasma by apoB precipitation using a commercially available reagent and the capacity of HDL to acquire PL and FC was measured in vitro as described above.
  • mice were given 100 ⁇ of 3H-labelled cholesterol mixed with 100 ⁇ olive oil by oral gavage. Two hours later, mice were euthanized, organs and blood removed and their mass and radioactivity determined. HDL was isolated from plasma by apoB precipitation as described above. When all the mice received an oral gavage of 3H-cholesterol administrated together with 100 ⁇ of olive oil, apoA-l knock-out mice accumulated significantly less radioactivity (-68%) in the HDL fraction 2h after the gavage as compared to wild-type animals; no significant difference was observed between wild-type and apoA-l transgenic mice (Figure 8).
  • the data provide an in vitro evidence for the suitability of the method for the stratification of patients with metabolic disease associated with cardiovascular disease, or with cardiovascular disease itself, to select those who may specifically benefit from HDL-targeting therapy.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Endocrinology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé de prédiction ou de diagnostic d'une maladie cardiovasculaire ou d'une maladie métabolique associée à une maladie cardiovasculaire par détermination de la capacité de la lipoprotéine haute densité (HDL) à acquérir des lipides de surface de la lipoprotéine riche en triglycérides (TGRL) pendant la lipolyse. La présente invention concerne également un composé de ciblage de HDL destiné à être utilisé dans la prévention ou le traitement d'un sujet qui a été classé comme étant susceptible de répondre par le procédé déterminant la capacité de HDL à acquérir des lipides de surface de TGRL.
PCT/EP2018/071172 2017-08-04 2018-08-03 Nouveau dosage de la fonction hdl WO2019025607A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP17306042 2017-08-04
EP17306042.7 2017-08-04

Publications (1)

Publication Number Publication Date
WO2019025607A1 true WO2019025607A1 (fr) 2019-02-07

Family

ID=59649633

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2018/071172 WO2019025607A1 (fr) 2017-08-04 2018-08-03 Nouveau dosage de la fonction hdl

Country Status (1)

Country Link
WO (1) WO2019025607A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015131131A1 (fr) * 2014-02-28 2015-09-03 The Regents Of The University Of California Procédé fluorométrique biochimique à rendement élevé pour mesurer une activité d'oxydo-réduction de lipoprotéine à haute densité
WO2017007966A1 (fr) * 2015-07-07 2017-01-12 Ashmaig Mohmed E Méthodes de détermination d'un taux de phospholipides et de lipoprotéines de haute densité dans un échantillon

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015131131A1 (fr) * 2014-02-28 2015-09-03 The Regents Of The University Of California Procédé fluorométrique biochimique à rendement élevé pour mesurer une activité d'oxydo-réduction de lipoprotéine à haute densité
WO2017007966A1 (fr) * 2015-07-07 2017-01-12 Ashmaig Mohmed E Méthodes de détermination d'un taux de phospholipides et de lipoprotéines de haute densité dans un échantillon

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
AR TALL ET AL: "Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis", JOURNAL OF LIPID RESEARCH, 1 July 1985 (1985-07-01), UNITED STATES, pages 842, XP055413395, Retrieved from the Internet <URL:http://www.jlr.org/content/26/7/842.full.pdf> *
BERTRAND-PIERRE PERRET ET AL: "Free cholesterol distribution during in vitro lipolysis of rat plasma very low density lipoprotein: lack of a role for blood and heart cells", EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, vol. 13, no. 5, 1 October 1983 (1983-10-01), GB, pages 419 - 428, XP055413393, ISSN: 0014-2972, DOI: 10.1111/j.1365-2362.1983.tb00123.x *
CHAJEK T; EISENBERG S: "Very low density lipoprotein. Metabolism of phospholipids, cholesterol, and apolipoprotein C in the isolated perfused rat heart", J CLIN INVEST., vol. 61, no. 6, June 1978 (1978-06-01), pages 1654 - 65
CHAPMAN M JOHN ET AL: "Cholesteryl ester transfer protein: at the heart of the action of lipid-modulating therapy with statins, fibrates, niacin, and cholesteryl ester transfer protein inhibitors", EUROPEAN HEART JOURNAL (ONLINE), OXFORD UNIVERSITY PRESS, GB, US, NL, vol. 31, no. 2, 1 January 2010 (2010-01-01), pages 149 - 164, XP002638290, ISSN: 1522-9645, [retrieved on 20091012], DOI: 10.1093/EURHEARTJ/EHP399 *
FREDRICKSON DS; LEES RS: "A system for phenotyping hyperlipoproteinemia", CIRCULATION, vol. 31, 1965, pages 321 - 327
KHERA AV; CUCHEL M; DE LA LLERA-MOYA M; RODRIGUES A; BURKE MF; JAFRI K; FRENCH BC; PHILLIPS JA; MUCKSAVAGE ML; WILENSKY RL: "Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis", N ENGL J MED., vol. 364, no. 2, 13 January 2011 (2011-01-13), pages 127 - 35
KO DT; ALTER DA; GUO H; KOH M; LAU G; AUSTIN PC; BOOTH GL; HOGG W; JACKEVICIUS CA; LEE DS: "High-Density Lipoprotein Cholesterol and Cause-Specific Mortality in Individuals Without Previous Cardiovascular Conditions: The CANHEART Study", J AM COLL CARDIOL., vol. 68, no. 19, 8 November 2016 (2016-11-08), pages 2073 - 2083
KO ET AL., J AM COLL CARDIOL, vol. 68, 2016, pages 2073 - 2083
KO, D.T. ET AL.: "High-Density Lipoprotein Cholesterol and Cause-Specific Mortality in Individuals Without Previous Cardiovascular Conditions: The CANHEART Study", J AM COLL CARDIOL, vol. 68, 2016, pages 2073 - 2083
MADSEN ET AL., EUR HEART J, vol. 38, 2017, pages 2478 - 2486
MADSEN, C.M.; VARBO, A.; NORDESTGAARD, B.G.: "Extreme high high-density lipoprotein cholesterol is paradoxically associated with high mortality in men and women: two prospective cohort studies", EUR HEART J, vol. 38, 2017, pages 2478 - 2486
MARJA- RIITTA 'TASKINEN ET AL: "Catabolism of human very low density lipoproteins in vitro: a fluorescent phospholipid method for monitoring lipolysis", JOURNAL OF LIPID RESEARCH, vol. 22, 1 January 1981 (1981-01-01), pages 382 - 386, XP055413380 *
REDGRAVE TG; SMALL DM: "Quantitation of the transfer of surface phospholipid of chylomicrons to the high density lipoprotein fraction during the catabolism of chylomicrons in the rat", J CLIN INVEST., vol. 64, no. l, July 1979 (1979-07-01), pages 162 - 71
ROHATGI A ET AL.: "HDL cholesterol efflux capacity and incident cardiovascular events", N ENGL J MED., vol. 371, no. 25, 18 December 2014 (2014-12-18), pages 2383 - 93, XP055488506, DOI: doi:10.1056/NEJMoa1409065
SCHUMAKER VN; PUPPIONE DL: "Sequential flotation ultracentrifugation", METHODS ENZYMOL., vol. 128, 1986, pages 155 - 70
SMITS LP; KOOTTE RS; STROES ES: "Reversal of atherosclerosis with apolipoprotein A1: back to basics", ATHEROSCLEROSIS, vol. 232, no. 1, January 2014 (2014-01-01), pages 217 - 9, XP028548818, DOI: doi:10.1016/j.atherosclerosis.2013.08.010
TALL AR; ROBINSON LA: "Absence of liquid crystalline transitions of cholesterol esters in reconstituted low density lipoproteins", FEBS LETT., vol. 107, no. 1, 1 November 1979 (1979-11-01), pages 222 - 6, XP025569502, DOI: doi:10.1016/0014-5793(79)80500-1
TURNER S; VOOGT J; DAVIDSON M; GLASS A; KILLION S; DECARIS J; MOHAMMED H; MINEHIRA K; BOBAN D; MURPHY E: "Measurement of reverse cholesterol transport pathways in humans: in vivo rates of free cholesterol efflux, esterification, and excretion", J AM HEART ASSOC., vol. 1, no. 4, August 2012 (2012-08-01), pages e001826
WIEBE DA; SMITH SJ: "Six methods for isolating high-density lipoprotein compared, with use of the reference method for quantifying cholesterol in serum", CLIN CHEM., vol. 31, no. 5, May 1985 (1985-05-01), pages 746 - 50
ZAKIEV E; FENG M; SUKHORUKOV V; KONTUSH A: "HDL-Targeting Therapeutics: Past, Present and Future", CURR PHARM DES, vol. 23, no. 8, 2017, pages 1207 - 1215

Similar Documents

Publication Publication Date Title
Upadhyay Emerging risk biomarkers in cardiovascular diseases and disorders
Joven et al. Lipoprotein heterogeneity in end-stage renal disease
Kinpara et al. Lipoprotein (a)-cholesterol: a significant component of serum cholesterol
Untersteller et al. HDL functionality and cardiovascular outcome among nondialysis chronic kidney disease patients [S]
US10845371B2 (en) Soluble MANF in pancreatic beta-cell disorders
Cabezas et al. Postprandial apolipoprotein B100 and B48 metabolism in familial combined hyperlipidaemia before and after reduction of fasting plasma triglycerides
Zhang et al. Increased serum levels of β2-GPI-Lp (a) complexes and their association with premature atherosclerosis in patients with rheumatoid arthritis
WO2005080982A1 (fr) Procédé de distinction entre des anévrismes disséquants aigus de l&#39;aorte de type a et de type b et un infarctus du myocarde aigu et kit de différenciation
Picard et al. Tissue kallikrein–deficient mice display a defect in renal tubular calcium absorption
US20190282664A1 (en) Methods for stimulation of appetite and increase in weight by administration of asprosin
Rocha et al. Metabolism of triglyceride-rich lipoproteins and lipid transfer to high-density lipoprotein in young obese and normal-weight patients with polycystic ovary syndrome
JP3436444B2 (ja) 酸化ｈｄｌの測定法及びキット
Hayashi et al. Remarkable increase of apolipoprotein B48 level in diabetic patients with end-stage renal disease
WO2019025607A1 (fr) Nouveau dosage de la fonction hdl
Kawano et al. Pitavastatin decreases plasma preβ1-HDL concentration and might promote its disappearance rate in hypercholesterolemic patients
van Tienhoven-Wind et al. Pre-β-HDL formation relates to high-normal free thyroxine in type 2 diabetes mellitus
BR112019024966A2 (pt) Método para diagnosticar ou monitorar a função renal ou diagnosticar a disfunção renal
EP2668507A1 (fr) Méthode d&#39;évaluation du risque d&#39;un sujet de contracter une maladie cardiovasculaire
US20220118068A1 (en) Modulation of Proprotein Convertase Subtilisin/Kexin 9 Expression (PCSK9) with HSP 27 and/or HSP25
Ballout et al. Pediatric dyslipidemias: lipoprotein metabolism disorders in children
De Bruin et al. Proteolysis of human apolipoprotein B: effect on quantitative immunoturbidimetry
Ma Studies of the metabolism of lipoprotein (a) in humans
Brodeur et al. Dalcetrapib and anacetrapib increase apolipoprotein E-containing HDL in rabbits and humans
Ballout et al. Pediatric dyslipidemias: lipoprotein
US20110245340A1 (en) Screening Method for Identifying Patients at Risk of Adverse Hepatologic Events

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18746237

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18746237

Country of ref document: EP

Kind code of ref document: A1