WO2012104411A1 - Marquage des lipoprotéines, procédés pour suivre le transport/l'évolution des lipoprotéines et pour dépister les troubles dyslipidémiques - Google Patents

Marquage des lipoprotéines, procédés pour suivre le transport/l'évolution des lipoprotéines et pour dépister les troubles dyslipidémiques Download PDF

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WO2012104411A1
WO2012104411A1 PCT/EP2012/051846 EP2012051846W WO2012104411A1 WO 2012104411 A1 WO2012104411 A1 WO 2012104411A1 EP 2012051846 W EP2012051846 W EP 2012051846W WO 2012104411 A1 WO2012104411 A1 WO 2012104411A1
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cholesterol
pyrene
general formula
hdl
lipoproteins
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PCT/EP2012/051846
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Xavier Collet
Gérald GAIBELET
François TERCE
Stéphane ORLOWSKI
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/001—Pyrene dyes
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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

Definitions

  • the invention relates to lipoproteins labelled with a fluorescent analogue of cholesterol, and their use for determining the presence of dyslipidemia disorders in a subject and for tracking in vivo the transport and/or outcome of lipoproteins which naturally comprise cholesterol.
  • Cholesterol is a particularly important molecule for mammals since it is an essential constituent of mammalian cell membranes, it is implied in several biochemical pathways including synthesis of vitamin D and of the steroid hormones, and it is the precursor of bile salts, which are necessary for intestinal absorption of fat molecules as well as the fat- soluble vitamins, vitamin A, vitamin D, vitamin E, and vitamin K.
  • Cholesterol is an isoprenoid molecule with 4 hydrocarbon rings and a hydrocarbon side chain at C 17. It contains 27 carbon atoms and possesses a hydroxyl group at C3. In spite of the hydroxyl group at C3, cholesterol is a very hydrophobic molecule.
  • VLDL very-low-density lipoprotein
  • IDL intermediate-density lipoprotein
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • Lipoproteins are complex globular particles formed of i) polar lipids (phospholipids and free cholesterol), which are located on the outer part of the lipoprotein with their charged groups pointing out towards the water molecules, ii) hydrophobic lipids (esterified cholesterol and triglycerides), which are located in the core of the lipoprotein particle, iii) apolipoproteins (which span the region between the central core and the outer envelope, and have part of their structure exposed at the surface), and iv) enzymes (e.g. lecithin cholesterol acyltransferase, phospholipase A2 and paraoxonase).
  • polar lipids phospholipids and free cholesterol
  • hydrophobic lipids esteerified cholesterol and triglycerides
  • apolipoproteins which span the region between the central core and the outer envelope, and have part of their structure exposed at the surface
  • enzymes e.g. lecithin cholesterol acyltransferas
  • Chylomicrons transport dietary triglycerides (hereinafter abbreviated "TG") and cholesterol from within the enterocytes through lymphatic vessels into the blood circulation.
  • TG dietary triglycerides
  • glycerol fatty acids and glycerol which are taken up by adipocytes and muscle cells for energy use or storage.
  • Cholesterol-rich chylomicron remnants then go to the liver to be cleared.
  • VLDL, IDL, LDL and HDL are implied in the endogenous (non dietary) lipid metabolism.
  • VLDL which contain apoprotein B-100 (apoB), are synthesized in the liver, and transport TGs and cholesterol to peripheral tissues. VLDL is the way the liver exports excess TGs derived from plasma free fatty acids (hereinafter abbreviated "FFA”) and chylomicron remnants.
  • FFA plasma free fatty acids
  • IDL are the products of endothelial lipoprotein lipase (hereinafter abbreviated "LPL") processing of VLDL and chylomicrons.
  • LPL endothelial lipoprotein lipase
  • IDL are cholesterol-rich VLDL and chylomicron remnants that are either cleared by the liver, or metabolized by hepatic lipase into LDL, which retains apoB.
  • LDL are the products of VLDL and IDL metabolism. They are the most cholesterol- rich of all lipoproteins. About 40 to 60% of all LDL are cleared by the liver in a process mediated by apo B and hepatic LDL receptors. The rest is taken up by either hepatic non- LDL or nonhepatic LDL receptors. Hepatic LDL receptors are down-regulated by delivery of cholesterol to the liver by chylomicrons and by increased dietary saturated fat; they can be up-regulated by decreased dietary fat and cholesterol. There are 2 forms of LDL: large, buoyant, and small, dense LDL. Small, dense LDL is especially rich in cholesterol esters, associated with metabolic disturbances such as hypertriglyceridemia and insulin resistance, and especially atherogenic. The increased atherogenicity of small, dense LDL derives from less efficient hepatic LDL receptor binding, leading to prolonged circulation and exposure to endothelium and increased oxidation.
  • HDL are initially cholesterol-free lipoproteins that are synthesized in both enterocytes and the liver.
  • HDL metabolism is complex, but HDL's overall role is to obtain cholesterol from peripheral tissues and other lipoproteins and transport it to where it is needed most— other cells, other lipoproteins, and the liver (for clearance). Its overall effect is anti-atherogenic.
  • Efflux of free cholesterol from cells is mediated by ATP-binding cassette transporter A1 (ABCA1 ), which combines with apoprotein A-l (apoA-l) to produce nascent HDL.
  • Free cholesterol in nascent HDL is then esterified by the enzyme lecithin- cholesterol acyl transferase (hereinafter abbreviated "LCAT”), producing mature HDL.
  • ABCA1 ATP-binding cassette transporter A1
  • LCAT lecithin- cholesterol acyl transferase
  • HDL fraction contains several sub-fractions like HDL 2 , HDL 3 , VHDL, and like nascent HDL particles such as pre ⁇ -HDL, pre ⁇ 2 -HDL and pre ⁇ 3 -HDL.
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • Dyslipidemias were traditionally classified by patterns of elevation in lipids and lipoproteins (Frederickson classification), as shown in the Table I below.
  • hypolipidemia which is defined as a total cholesterol under 120 mg/dL ( ⁇ 3.1 mmol/L) and/or LDL-cholesterol under 50 mg/dL ( ⁇ 1 .3 mmol/L).
  • Diseases associated with hypolipidemia are for instance hyperthyroidism, chronic infections and other inflammatory states and cancers.
  • hypolipidemia is also considered as a dyslipidemia.
  • High plasma cholesterol concentrations, especially in the low-density lipoprotein (LDL) fraction, are associated with an increased risk for the development of atheromatous lesions an/or atherosclerosis.
  • Atherosclerosis is a prerequisite for the majority of cases of coronary heart disease, this disease being one of the most prevalent cause of death in industrial countries, as well as for cerebral vascular accidents ("strokes").
  • Atherosclerosis is a longstanding process wherein lipids, such as cholesterol derived from lipoproteins, are taken up and accumulate in vessel wall macrophages. Cholesterol-loaded macrophages, so called foam cells because of their microscopically visible opalescent lipid content, form fatty streaks in the endothelium of the vessel. Due to the interaction with inflammatory blood cells, an atherosclerotic plaque develops which subsequently can block the vessel or can rupture and subsequently block capillaries at other places, e.g., the heart or the brain.
  • lipids such as cholesterol derived from lipoproteins
  • Atherosclerosis can express itself in a number of cardiovascular diseases such as angina pectoris, myocardial infarction, transient ischemic attacks, cerebrovascular accidents, peripheral artery disease.
  • Oxidative stress is believed to be a potential important risk factor for premature atherosclerosis and cardiovascular diseases since it mediates the formation of proinflammatory and proatherogenic oxidized LDL (hereinafter abbreviated "OxLDL"). Further, it was shown that macrophages in culture take up OxLDL much more rapidly than they take up native LDL, which would allow in vivo progressive accumulation of cholesterol to the point of foam cell generation.
  • OxLDL proinflammatory and proatherogenic oxidized LDL
  • LDL is a vehicle for cholesterol supply to peripheral cells (and, therefore, to macrophages)
  • HDL plays a key role in the process of reverse cholesterol transport (abbreviated by "RCT”), in which it promotes the efflux of excess cholesterol by collecting it (in particular from macrophages) and transporting it from peripheral tissues to the liver for biliary excretion.
  • RCT reverse cholesterol transport
  • HDL protects LDL from oxidative damage and reduces the level of proatherogenic oxidized LDL.
  • ApoA-l the major HDL apolipoprotein
  • Other apolipoprotein and enzymatic components of HDL like apoE, apoJ, apoA-ll and apoA-IV and LCAT, possess or contribute to anti-oxidative properties of HDL.
  • oxidized form of HDL has been reported to have reduced beneficial properties and is converted into a cytotoxic particle, suggesting that oxidized HDL may also contribute to the genesis of coronary artery diseases.
  • OxLDL OxLDL-linked immunosorbent assay
  • the two main fluorescence detection methods of lipoproteins in use today involve either protein marker dyes or lipophilic dyes. While the former is composed of covalent fluorochrome-apolipoprotein conjugates, the latter is composed of dyes noncovalently bound with lipids.
  • DASP for N,N- dipentadecylaminostyrylpyridinium iodide
  • the probes currently used for detecting or quantifying lipoproteins can neither be used in vivo for investigating the transport and metabolism and/or catabolism of lipoproteins, in particular the reverse cholesterol transport of HDL, nor for investigating metabolism and/or catabolism of lipoproteins in cell culture.
  • lipophilic dyes currently used in the art is not covalently bound to cholesterol of the lipoproteins, in vivo or cell culture investigations are impossible since such dyes cannot remain bound to cholesterol during lipoproteins remodelling process which occurs in vivo.
  • esterified cholesterol is transfered in vivo between serum lipoproteins, in particular from HDL to apoB-containing lipoproteins (LDL, IDL, VLDL, chylomicrons) by the action of CETP (herein abbreviation of "cholesterol ester transfer protein"). Consequently, in vivo study of the outcome of a chosen type of labelled lipoprotein is not reliable when using 3 H- radiolabeled cholesterol.
  • - X represents either an Oxygen atom or a CH 2 group
  • - n is an integer from 2 to 10. is particularly suitable for in vivo studies. Indeed, cholesterol-pyrene of the invention is stably integrated into the monolayer of the lipoproteins, so that it remains bound to cholesterol ex vivo and in vivo, even during lipoproteins remodelling process which occurs in vivo.
  • cholesterol-pyrene of general formula (I) is not esterified by the enzyme LCAT. This feature is of particular interest for in vivo studies since transfer of the cholesterol-pyrene between serum lipoproteins, in particular from HDL to apoB-containing lipoproteins (LDL, IDL, VLDL, chylomicrons) which normally occurs in vivo by the action of CETP is prevented.
  • LCAT apoB-containing lipoproteins
  • Cholesterol-pyrene of general formula (I) was previously described as a probe which has propensity for inserting into biological membranes, such as liposomes, extracted cell membranes and cell membranes of living cells.
  • This fluorescent analogue of cholesterol has been used for characterizing the collective organization of the membrane constituents, in particular to monitor the effect on the membranes of agonists or antagonists of G-protein-coupled membrane receptors ("GPCR").
  • GPCR G-protein-coupled membrane receptors
  • WO 2006/100388 The methods embodied in WO 2006/100388 are conducted with ⁇ -cyclodextrin as a transporter. Further, WO 2006/100388 is silent concerning lipoproteins and the potential use of cholesterol-pyrene of general formula (I) for labelling lipoproteins.
  • lipoproteins labelled with it for instance labelled HDL, LDL and VLDL, can be readily detected and quantified by spectrofluorometry when a radiation of excitation wavelength of 330 to 350 nm is applied to a sample which comprises such labelled lipoproteins.
  • the inventors also showed that the fluorescence emission spectrum of labelled lipoproteins provides information regarding structural and functional properties of the surface of lipoproteins (oxidation state of lipoproteins, ).
  • the lipoproteins of the sample to be analysed can be readily and easily characterized.
  • lipoproteins labelled with it can be reliably detected and quantified, normal and abnormal lipoproteins can be differentiated from each other, dyslipidemia, lipid metabolic disorders and related diseases can be diagnosed, and in vivo the transport and outcome of lipoproteins can be investigated.
  • the present invention is directed to an isolated lipoprotein which comprises cholesterol and cholesterol- rene of general formula (I):
  • - X represents either an Oxygen atom or a CH 2 group
  • n is an integer from 2 to 10
  • n is an integer from 4 to 8
  • n is an integer from 4 to 6
  • still more preferably n is an integer from 2 to 8
  • still more preferably n is an integer from 2 to 6,
  • n is an integer from 2 to 6
  • n is an integer from 2 to 4 or n is 2.
  • the cholesterol-pyrene of general formula (I) comprised in said lipoprotein has the following formula:
  • lipoprotein is intended to mean a lipoprotein which naturally comprises cholesterol.
  • a lipoprotein is preferably selected from the group consisting of chylomicron, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL).
  • HDL is preferably chosen from the group consisting of HDL subtractions, for instance HDL 2 , HDL 3 , VHDL, pre ⁇ -HDL, pre-p 2 -HDL and pre-p 3 -HDL
  • the lipoprotein of the invention does not contain a "transporter” (e.g. ⁇ - cyclodextrin) intended to exchange cholesterol of biological membranes.
  • a "transporter” e.g. ⁇ - cyclodextrin
  • - X represents either an Oxygen atom or a CH 2 group
  • - n is an integer from 2 to 10
  • - X represents either an Oxygen atom or a CH 2 group
  • n is an integer from 2 to 10
  • n is an integer from 4 to 8
  • n is an integer from 4 to 6
  • n is an integer from 2 to 8
  • n is an integer from 2 to
  • n is an integer from 2 to 4 or n is 2;
  • the cholesterol-pyrene of general formula (I) comprised in said li oprotein has the following formula:
  • lipoproteins to be labelled are comprised in a biological sample selected from the group consisting of serum, plasma, purified HDL composition, purified subtractions of HDL like HDL 2 , HDL 3 , VHDL, pre ⁇ -HDL, pre-p 2 -HDL, pre-p 3 -HDL, purified LDL composition, purified IDL composition, purified VLDL composition and purified chylomicron composition.
  • the term “purified”, when referring to lipoprotein HDL, LDL, IDL, VLDL, chylomicron composition, and purified subtractions of HDL, is intended to mean that the lipoprotein is substantially free of other type of lipoproteins which naturally comprise cholesterol and substantially free of contaminating serum components.
  • the term “purified” indicates that the lipoprotein represents at least 80% of lipoproteins 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%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% on a mass basis of the composition.
  • HDL 2 , HDL 3 , VHDL, pre ⁇ -HDL, pre-p 2 -HDL, pre-p 3 -HDL), is from 1 .063 to 1 .210 g/ml (for instance density of HDL 2 is from 1 .063 to 1 .125 g/ml, density of HDL 3 is from 1 .125 to 1 .210 g/ml, and density of VHDL is from 1 .216 to 1 .256).
  • the fractions isolated after ultracentrifugation can optionally be further purified by dialysis.
  • the biological sample is brought into contact with cholesterol-pyrene of general formula (I) preferably at a temperature of about 20 to about 37°C, more preferably about 25 to about 37°C, advantageously at a temperature near 37°C (that is between 36.5 and 37.5, inclusive).
  • the mean of contacting the biological sample with cholesterol-pyrene can be simple incubation with free cholesterol-pyrene molecule, or with the use of any possible "transporter".
  • a "transporter” is any molecule facilitating cholesterol-pyrene transfer in the aqueous medium, such as cyclodextrins, liposomes, dextrans, albumin.
  • incubating such a procedure will be quoted "incubating”.
  • the period of time where the biological sample is brought into contact with cholesterol-pyrene of general formula (I) is preferably from 6 hours to 80 hours, still preferably from 14 hours to 72 hours, still preferably from 24 hours to 60 hours, still preferably from 24 hours to 48 hours, more preferably from 36 to 48, and advantageously about 48 hours.
  • the ex vivo method for labelling lipoproteins of the invention further comprises a step after the period of time where the biological sample is brought into contact with cholesterol-pyrene of general formula (I), consisting of separating the lipoproteins labelled by cholesterol-pyrene of general formula (I) from the free cholesterol-pyrene molecule of general formula (I) which did not bind to said lipoprotein.
  • This step of separation of labelled lipoproteins from free cholesterol-pyrene can be performed by ultracentrifugation and/or dialysis and/or FPLC with a gel filtration column.
  • the present invention is also relative to a method for detecting and/or quantifying lipoproteins in a biological sample, said method comprises the following steps:
  • a biological sample preferably said sample likely comprises lipoproteins which naturally comprise cholesterol, with cholesterol-pyrene of general formula (I):
  • - X represents either an Oxygen atom or a CH 2 group
  • - n is an integer from 2 to 10;
  • lipoproteins labelled with said cholesterol-pyrene of general formula (I) ii) separating said lipoproteins which naturally comprise cholesterol and which are labelled by cholesterol-pyrene of general formula (I) from the free cholesterol- pyrene molecule of general formula (I);
  • the method is carried out without using a "transporter” (e.g. ⁇ -cyclodextrin) intended to exchange cholesterol with the cholesterol-pyrene.
  • a "transporter” e.g. ⁇ -cyclodextrin
  • Step i) is preferably conducted at a temperature of about 20 to about 37 °C, more preferably about 25 to about 37 °C, advantageously at a temperature near 37 °C (that is between 36.5 and 37.5, inclusive), during 6 hours to 80 hours, preferably 14 hours to 72 hours, still preferably from 24 hours to 60 hours, still preferably from 24 hours to 48 hours, more preferably from 36 to 48, and advantageously about 48 hours.
  • n is an integer from 2 to 10, preferably n is an integer from 4 to 8, still preferably n is an integer from 4 to 6, still more preferably n is an integer from 2 to 8, still more preferably n is an integer from 2 to 6, advantageously n is an integer from 2 to 4 or n is 2.
  • the cholesterol-pyrene used in step i) has the following formula:
  • the biological sample to be analysed is serum or plasma.
  • the method comprises an additional step i') either before i) or between step ii) and iii), said step i') consists of sorting all or part of the lipoprotein fractions HDL, LDL, IDL, VLDL, chylomicron and/or HDL subtractions, preferably HDL subtractions are HDL 2 , HDL 3 , VHDL, pre ⁇ HDL, pre ⁇ 2 -HDL and pre ⁇ 3 -HDL, and step iv) consists of measuring the intensity of fluorescence emitted by each of lipoprotein fraction or subtraction which has been sorted out.
  • step i') is carried out by using chromatography.
  • the measurement of the fluorescence radiation emitted by labelled lipoproteins of step iv) is preferably performed at an emission wavelength of 350 to 650 nm, still preferably between 360 and 650nm, more preferably between 360 and 600 nm.
  • step i') can be carried out by a variety of procedures known in the art including, but not limited to, chromatography and centrifugation.
  • the intensity of fluorescence measured in step iv) provides a measure of the proportion of cholesterol-pyrene of the invention that is integrated into lipoproteins.
  • the value obtained in step iv) is applied to a standard calibration curve showing a relationship between concentration of the lipoproteins which are likely comprised in the biological sample (i.e. HDL, LDL and VLDL) and an intensity of fluorescence.
  • the standard calibration curve used in the present method can be obtained by measuring a set of standard solutions, each solution containing a known amount of the labelled lipoprotein(s) which is (are) likely comprised in the biological sample.
  • the inventors showed that the fluorescence emission spectrum of labelled lipoproteins gives information in relation to the oxidation state of said lipoproteins.
  • Another object of the present invention relates to an ex vivo method for assessing oxidation state of a lipoprotein which naturally comprises cholesterol, comprising the following steps:
  • - X represents either an Oxygen atom or a CH 2 group
  • n is an integer from 2 to 10, preferably n is an integer from 4 to 8, still preferably n is an integer from 4 to 6, still more preferably n is an integer from 2 to 8, still more preferably n is an integer from 2 to 6, advantageously n is an integer from 2 to 4 or n is 2;
  • step iv) comparing the fluorescence emission spectrum determined in step iv) to the fluorescence emission spectrum of a lipoprotein of the same type which is non oxidized and which is labelled by cholesterol-pyrene of general formula (I), difference between fluorescence emission spectrum of step iv) and the fluorescence emission spectrum of a lipoprotein of the same type which is non oxidized is indicative of a different state of oxidation.
  • the method is carried out without using a "transporter"
  • Step i) is preferably carried out at a temperature of about 20 to about 37°C, more preferably about 25 to about 37 °C, advantageously at a temperature near 37 °C (that is between 36.5 and 37.5, inclusive), during 6 hours to 80 hours, preferably 14 hours to 72 hours, still preferably from 24 hours to 60 hours, still preferably from 24 hours to 48 hours, more preferably from 36 to 48, and advantageously about 48 hours.
  • the cholesterol-pyrene of general formula (I) used in step i) has the following formula:
  • the lipoprotein of which oxidation state is assessed is selected from the group consisting of HDL (or HDL subtractions HDL 2 , HDL 3 , VHDL, pre ⁇ -HDL, pre-p 2 - HDL and pre ⁇ 3 -HDL), LDL, IDL, VLDL, chylomicron or mixtures thereof. More preferably, the lipoprotein of which the state of oxidation is assessed is LDL or HDL or HDL subtractions.
  • the non oxidized lipoprotein from which the fluorescence emission spectrum is used in step v) comes from a healthy subject, e.g. a subject who does not have abnormally oxidized lipoproteins (considering that any lipoprotein oxidation is abnormal).
  • the fluorescence emission spectrum of the lipoprotein labelled by cholesterol-pyrene of general formula (I) is determined between 350 and 650 nm.
  • a fluorescence emission spectrum typical of oxidized LDL labelled by cholesterol-pyrene of general formula (I) presents an inversion of the relative intensities of the emission peaks at about 380 and 400 nm, along with an increase of the shoulder at about 420 nm, as well as a growth of the excimer peak around 460 nm.
  • the amount of oxidized lipoproteins can be determined by comparing the intensity of fluorescence measured at a determined emission wavelength, for instance at 380 or 400 nm or preferentially at the excimer peak around 460 nm, with a standard calibration curve showing a relationship between concentration of the same type of oxidized lipoprotein(s) and an intensity of fluorescence.
  • the present invention also relates to a method, preferably ex vivo, for determining the presence of dyslipidemia disorder in a subject.
  • said method comprises the following steps:
  • a biological sample preferably said sample likely comprises lipoproteins which naturally comprise cholesterol with cholesterol-pyrene of general formula (I :
  • - X represents either an Oxygen atom or a CH 2 group
  • - n is an integer from 2 to 10, preferably n is an integer from 4 to 8, still preferably n is an integer from 4 to 6, still more preferably n is an integer from 2 to 8, still more preferably n is an integer from 2 to 6, advantageously n is an integer from 2 to 4 or n is 2;
  • lipoproteins labelled with said cholesterol-pyrene of general formula (I) ii) separating lipoproteins which naturally comprise cholesterol and which is labelled with cholesterol-pyrene of general formula (I) from the free cholesterol- pyrene molecule of general formula (I);
  • step iii) applying to lipoproteins which naturally comprise cholesterol and which are labelled by cholesterol-pyrene of general formula (I) of step ii) a radiation of excitation wavelength of 330 to 350 nm, preferably of 335 to 345;
  • step iv) comparing the fluorescence emission spectrum determined in step iv) to the fluorescence emission spectrum of a same biological sample obtained from an healthy subject which was submitted to step ii) to iv), difference between fluorescence emission spectrum of step iv) and the fluorescence emission spectrum from a same biological sample from an healthy subject which was submitted to step ii) to iv) is indicative of dyslipidemia.
  • said method comprises the following steps:
  • - X represents either an Oxygen atom or a CH 2 group
  • - n is an integer from 2 to 10; said lipoproteins have been previously separated from the free cholesterol-pyrene molecule of general formula (I), and optionally from contaminating serum components.
  • step iii) comparing the fluorescence emission spectrum determined in step ii) to the fluorescence emission spectrum of the same type of lipoproteins obtained from an healthy subject contacted beforehand with a cholesterol-pyrene of general formula (I), difference between fluorescence emission spectrum determined in step ii) and the fluorescence emission spectrum of the same type of lipoproteins from an healthy subject is indicative of dyslipidemia.
  • the method according to the second embodiment may comprise, before step i), the step consisting of administering said subject with cholesterol-pyrene of general formula (I).
  • lipoproteins of step i) represent by order of preference at least 80%
  • n is an integer from 2 to 10, preferably n is an integer from 4 to 8, still preferably n is an integer from 4 to 6, still more preferably n is an integer from 2 to 8, still more preferably n is an integer from 2 to 6, advantageously n is an integer from 2 to 4 or n is 2.
  • Step i) is preferably carried out at a temperature of about 20 to about 37°C, more preferably about 25 to about 37 °C, advantageously at a temperature near 37 °C (that is between 36.5 and 37.5, inclusive), during 6 hours to 80 hours, preferably 14 hours to 72 hours, still preferably from 24 hours to 60 hours, still preferably from 24 hours to 48 hours, more preferably from 36 to 48, and advantageously about 48 hours.
  • the cholesterol-pyrene of general formula (I) used in both embodiments of the method has the following formula:
  • the biological sample is preferably selected from the group consisting of serum, plasma, purified HDL, purified sub fractions of HDL like HDL 2 , HDL 3 , VHDL, pre ⁇ -HDL, purified LDL, purified IDL, purified VLDL and purified chylomicron.
  • the fluorescence emission spectrum of the lipoprotein labelled by cholesterol-pyrene of general formula (I) is determined between 350 and 650 nm, between 360 and 600 nm.
  • the biological sample is serum or plasma
  • emission wavelengths between about 440 and about 500 nm (excimer peak) and/or around 420 nm.
  • hypertriglyceridemia as well as hypercholesterolemia (although in a lower extent), appear to be correlated with a decrease of the excimer peaks (between about 440 and 500 nm).
  • hyperlipemia that is both high triglyceride and cholesterol levels
  • the decrease of the excimer peaks appears to be cumulative.
  • the shoulder at 420 nm follows the same tendencies, with smaller variations.
  • hypercholesterolemia appears correlated with an increased excimer peak (between about 440 and 500 nm), but hyperlipemia does not show significant alteration with respect to an healthy donor, indicating that isolated hypertriglyceridemia could have an inverse effect.
  • the method for determining the presence of dyslipidemia disorder of the invention is also useful for identifying a subject as having a predisposition to a lipid metabolism associated disease, said lipid metabolism associated disease preferably selected from the group consisting of heart coronary disease (angina pectoris, myocardial infarction), transient ischemic attacks, cerebrovascular accidents, peripheral artery disease.
  • both embodiments of the method are carried out without using a "transporter” (e.g. ⁇ -cyclodextrin) intended to exchange cholesterol with the cholesterol- pyrene.
  • a "transporter” e.g. ⁇ -cyclodextrin
  • the inventors have shown that the cholesterol-pyrene of the invention is particularly suitable as a marker for tracking in vivo the transport and outcome of a chosen lipoprotein.
  • the present invention also relates to method for tracking in vivo the transport and outcome of lipoproteins which naturally comprise cholesterol.
  • This method may be carried out i) by systemically administering to a mammal a lipoprotein labelled with cholesterol-pyrene of the invention, and/or ii) by orally administering (possibly simultaneously with lipophilic nutrients) to a mammal the cholesterol-pyrene of the invention, and/or by infusion (IV or IP) of pre-labelled circulating or stem cells.
  • the method is carried out without using a "transporter” (e.g. ⁇ - cyclodextrin) intended to exchange cholesterol with the cholesterol-pyrene.
  • the method for tracking in vivo the transport and outcome of lipoproteins which naturally comprise cholesterol may comprise the steps consisting of:
  • cholesterol-pyrene of general formula (I) b) - assessing excretion of cholesterol-pyrene of general formula (I) by measuring cholesterol-pyrene of general formula (I) in the bile and/or feces from a mammal administered beforehand with a least one lipoprotein which naturally comprises cholesterol and which is labelled with cholesterol-pyrene of general formula (I); wherein cholesterol- rene of general formula (I) is:
  • - X represents either an Oxygen atom or a CH 2 group
  • - n is an integer from 2 to 10; and wherein measuring incorporated cholesterol-pyrene of general formula (I) in cells and/or tissues, and/or measuring cholesterol-pyrene of general formula (I) in the bile and/or feces are indicative of the transport and outcome of lipoproteins.
  • the method may comprise, before step a), the step consisting of administering said mammal with at least one lipoprotein which naturally comprises cholesterol and which is labelled with cholesterol-pyrene of general formula (I).
  • the at least one lipoprotein which naturally comprises cholesterol and which is labelled with cholesterol-pyrene of general formula (I) is preferably selected from the group consisting of HDL, subtractions of HDL like HDL 2 , HDL 3 , VHDL, pre ⁇ HDL, pre ⁇ 2 - HDL, pre-p 3 -HDL, LDL, IDL, VLDL and chylomicron.
  • the way of administration of the at least one lipoprotein labelled with cholesterol-pyrene of general formula (I) to the mammal is selected from: intravenous route, intraperitoneal route, or by infusion (IV or IP) of pre-labelled circulating or stem cells.
  • the at least one lipoprotein labelled with a cholesterol-pyrene of the invention administered to the mammal is the HDL fraction or a subtraction of HDL like HDL 2 , HDL 3 , VHDL, pre ⁇ -HDL, pre ⁇ 2 - HDL, pre-p 3 -HDL
  • the method for tracking in vivo the transport and outcome of lipoproteins which naturally comprise cholesterol preferably selected from the group consisting of, LDL, IDL, VLDL, chylomicron, HDL and subtractions of HDL like HDL 2 , HDL 3 , VHDL, pre ⁇ -HDL, pre ⁇ 2 -HDL, pre ⁇ 3 -HDL, the method may also comprise the steps consisting of:
  • cholesterol-pyrene of general formula (I) b- assessing excretion of cholesterol-pyrene of general formula (I) by measuring cholesterol-pyrene of general formula (I) in the bile and/or feces from a mammal orally administered beforehand with cholesterol-pyrene of general formula (I); wherein cholesterol-pyrene of general formula (I) is:
  • - n is an integer from 2 to 10;
  • measuring incorporated cholesterol-pyrene of general formula (I) in cells and/or tissues, and/or measuring cholesterol-pyrene of general formula (I) in the bile and/or feces are indicative of the transport and outcome of lipoproteins.
  • the method may comprise, before step a), the step consisting of administering orally said mammal with cholesterol-pyrene of general formula (I).
  • the mammal is preferably selected from the group consisting of human and non human mammals, for instance mouse, rat, rabbit, hamster, dog, pig or primates.
  • cholesterol-pyrene of general formula (I) used is such as n is an integer from 2 to 10, preferably n is an integer from 4 to 8, still preferably n is an integer from 4 to 6, still more preferably n is an integer from 2 to 8, still more preferably n is an integer from 2 to 6, advantageously n is an integer from 2 to 4 or n is 2.
  • the cholesterol-pyrene of general formula (I) used in step i) has the following formula:
  • Measurement of the incorporation of cholesterol-pyrene of general formula (I) in cells and/or tissues can be performed either by histological examination of cells/tissues harvested from the mammal administered beforehand i) with a least one lipoprotein labelled with cholesterol-pyrene of general formula (I), and/or ii) with cholesterol-pyrene of general formula (I) (when the mammal is a non human mammal, preferably after the non human mammal being sacrified), or by in situ imagery on the mammal, preferably anesthetized.
  • the mammal used in the method was treated prior to be used in the method, or is treated when the method is carried out, with a compound which is supposed to have an effect on the cholesterol metabolism (e.g. statins, ezetimib). It could be of particular interest to use non human mammal ("animal models") with phenotype similar to that of human with well characterized disease.
  • ABCA1 knock-out mice have macrophage-specific deficiency of enzyme ABCA1 (an important cellular protein that facilitates efflux of cellular cholesterol to lipid-poor apoA-l as the preferred acceptor).
  • ABCA1 knock-out mice have an extremely low HDL-cholesterol phenotype similar to that of humans with Tangier disease, and present significantly increased atherosclerosis. These mice were disclosed by Aiello RJ. et al. (Arterioscler. Thromb. Vase. Biol., 23:972-980, 2003).
  • ABCG1 knock-out mice which have marked macrophage cholesterol accumulation (ABCG1 , as well as ABCG4, are enzymes which were reported to mediate net mass efflux of cellular cholesterol to mature HDL but not to lipid-poor apoA-l). These mice were disclosed by Kennedy MA. et al., (Cell Metabolism, 1 :121 -131 , 2005).
  • SR-BI for "Scavenger Receptor class B, type I"; the HDL receptor
  • SR-BI seems to play a role in mediating cellular cholesterol efflux to mature HDL.
  • SR-BI knock-out mice have increased HDL-cholesterol levels (probably because of the key role of hepatic SR-BI in HDL catabolism). Further, these mice have increased atherosclerosis (Trigatti et al., Biochem. Soc. Trans., 32:1 16-120, 2004).
  • mice deleted for NPC1 L1 (Altman et al., Science, 303: 1201 -4,
  • non human mammals described above which comprise lipoproteins which naturally comprise cholesterol and which are labelled with cholesterol- pyrene of general formula (I) are also part of the invention, and therefore represent another object of the present invention.
  • the invention is also drawn to a method for screening for a compound which alters cholesterol metabolism.
  • This method comprises the steps of:
  • step a) determining whether said candidate compound alters cholesterol metabolism by comparing incorporation of cholesterol-pyrene in cells and/or tissues, and/or excretion of cholesterol-pyrene which happens in the non human mammal of step a) to incorporation of cholesterol-pyrene in cells and/or tissues and/or excretion of cholesterol-pyrene which happens in a control non human mammal which was not administered with said candidate compound.
  • the method is carried out without using a "transporter” (e.g. ⁇ -cyclodextrin) intended to exchange cholesterol with the cholesterol-pyrene.
  • a "transporter” e.g. ⁇ -cyclodextrin
  • the term “has an effect on the cholesterol metabolism” is intended to mean that the compound increases or decreases the cholesterol metabolism, for instance that it decreases the level of LDL present in the body, or it increases the level of HDL present in the body, or both.
  • a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier may be manufactured.
  • the pharmaceutical composition may be tested in order to confirm its efficacy.
  • the method for screening according to the invention is of particular interest in anti- atherosclerosis drug development since it allows identification of compounds which have in vivo (in a non human animal model) an effect on HDL metabolism regulation and on reverse cholesterol transport.
  • This accumulation of cholesterol, essentially in esterified form, is due to various factors such as an increase of influx from the lipoproteins LDL and HDL, and of synthesis and of esterification, as well as decrease of the efflux (Tosi & Tugnoli, Clin. Chim. Acta., 359(12): 27-45, 2005). Further, it is well known that LDL receptors and SR-BI (the HDL receptor) are commonly over-expressed by tumor cells.
  • the invention is relative to a method for assessing tumor growth.
  • This method comprises a step of:
  • cholesterol-pyrene of general formula (I) is:
  • - X represents either an Oxygen atom or a CH 2 group
  • - n is an integer from 2 to 10;
  • the method may comprise a step consisting of administering said mammal with at least one lipoprotein which naturally comprises cholesterol and which is labelled with cholesterol-pyrene of general formula (I), and/or with cholesterol-pyrene of general formula (I), as appropriate, before the step of measuring.
  • the mammal is preferably selected from the group consisting of human and non human mammals, for instance mouse, rat, rabbit, hamster, dog, pig, cat, horse or primates.
  • the present invention is also relative to a method for screening for a compound which inhibits growth of tumors.
  • This method comprises the steps of:
  • cholesterol-pyrene of general formula (I) is:
  • - X represents either an Oxygen atom or a CH 2 group
  • - n is an integer from 2 to 10;
  • the non-human animal suffering from cancer and having tumors can be an animal grafted with tumors, for instance grafted with human tumors.
  • the non-human animal is preferably selected from the group consisting of mouse, rat and rabbit.
  • the non-human animal is immunodepressed in order to avoid rejection of the grafted tumors (e.g. xenograft model of a tumor).
  • the non-human animal used in the methods is Nude mouse or Nude rat for studying xenogenic tumors; this is not necessary for the development of syngenic tumors.
  • Experimental tumors can be obtained on a non- human animal by infusing (intravenous, intraperitoneous, subcutaneous) tumorigenic cells (from in vitro cell culture), or grafting a tumor fragment (from in vivo passaged tumor), or inducing a tumor development by irradiation or by treatment with a chemical carcinogen or by oncogenic viral infection.
  • the non-human animal can also bear a spontaneous tumor obtained from a veterinary clinical recruitment.
  • the progression of tumors growth can be assessed by measuring incorporation of cholesterol-pyrene of general formula (I) in tumor tissues and/or tumor cells of said non human mammal suffering from cancer at different time following administration of the at least one lipoprotein which naturally comprises cholesterol labelled with cholesterol- pyrene of general formula (I) and/or administration of the candidate compound.
  • the at least one lipoprotein which naturally comprises cholesterol labelled with cholesterol- pyrene of general formula (I) and/or the candidate compound can be administrated several time during the period of time when the method is conducted.
  • cholesterol-pyrene of general formula (I) used is such as n is an integer from 2 to 10, preferably n is an integer from 4 to 8, still preferably n is an integer from 4 to 6, still more preferably n is an integer from 2 to 8, still more preferably n is an integer from 2 to 6, advantageously n is an integer from 2 to 4 or n is 2.
  • the cholesterol-pyrene of general formula (I) used in step i) has the followin formula:
  • the at least one lipoprotein which naturally comprises cholesterol is preferably selected from the group consisting of LDL, IDL, VLDL, chylomicron, HDL, and subtractions of HDL like HDL 2 , HDL 3 , VHDL, pre ⁇ -HDL, pre-p 2 -HDL, pre-p 3 -HDL
  • Measure of the incorporation of cholesterol-pyrene of general formula (I) in cells and/or tissues can be performed either by histological analysis on cells/tissues harvested on the mammal administered beforehand with a least one lipoprotein labelled with cholesterol-pyrene of general formula (I) (when the mammal is a non human mammal, preferably after the non human mammal being sacrified), or by in situ imagery on the mammal anesthetized.
  • the methods are carried out without using a "transporter” (e.g. ⁇ -cyclodextrin) intended to exchange cholesterol of biological membranes with the cholesterol-pyrene.
  • a "transporter” e.g. ⁇ -cyclodextrin
  • FIG. 1 illustrates the HPLC profiles of the pyrene fluorescence emission at 385 nm (A) and of the protein absorption at 280 nm (B) after incubation of bovine serum at 20% with 5 ⁇ of cholesterol-pyrene of the invention (hereinafter abbreviated "Chol-Pyr").
  • FIG. 2 illustrates liquid chromatography (on sepharose® column) profiles of the pyrene fluorescence emission at 385 nm after a 4h and 24h incubation at room temperature of purified HDL (A) or LDL (B) of human origin with Chol-Pyr at 5% of free cholesterol and resuspension in PBS after dialysis. Results obtained after 4h incubation at room temperature are represented by solid lines, whereas results obtained after 24h incubation at room temperature are represented by dashed lines.
  • FIG. 4 illustrates the emission fluorescence spectrum of purified HDL from either a healthy donor (A) or from patients presenting a global hyperlipemia (B), or an hypercholesterolemia (C), after labelling by 10 ⁇ Chol-Pyr and dialysis.
  • FIG. 5 illustrates the emission fluorescence spectrum of purified LDL of human origin, either native (A) or oxidized by CuS0 4 during 4 (B) or 8 hours (C), after labelling by 5 ⁇ Chol-Pyr and dialysis.
  • Adequate amount of 21 -methylpyrenyl-cholesterol (Chol-Pyr) is prepared from a stock solution in chloroform/methanol by evaporation and solubilization in ethanol.
  • Chol-Pyr is then added at the desired final concentration (with ⁇ 1 % v/v ethanol) to a sample of total serum (from human or bovin origin, either native, ca 40 mg/ml, or 5 to 10- fold diluted) or of purified lipoproteins (of human origin, in the range 1 -7 mg/ml, kept in the KBr solution which had been used to prepare the fraction by ultracentrifugation on the appropriated density layer).
  • the incubation is realized under gentle stirring and protected from light, during 48 hours at 37°C. Then, unbound Chol-Pyr is relieved after serum incubation by dialysis (overnight, on a membrane with a cutoff at 10 kDa), or after purified lipoproteins incubation by ultracentrifugation on a KBr density layer appropriated for its collection and/or by dialysis (overnight, on a membrane with a cutoff at 10 kDa). Labelled sample is kept at 4 ⁇ .
  • Example II Evidence of lipoprotein fluorescent stable labelling
  • Example III Spectral analysis of Chol-Pyr labelled lipoproteins from dyslipidemic patients
  • hypercholesterolemia appears correlated with an increased excimer peak (around 460 nm), but hyperlipemia does not show significant alteration with respect to the healthy donor, indicating that isolated hypertriglyceridemia could have an inverse effect, as observed considering total serum.
  • Example IV Effect of in vitro lipoprotein oxidation on the fluorescence emission of the cholesterol probe

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Abstract

Cette invention concerne des lipoprotéines marquées avec un analogue fluorescent du cholestérol, et leur utilisation pour déterminer la présence de troubles dyslipidémiques chez un sujet et pour suivre in vivo le transport et/ou l'évolution des lipoprotéines qui comprennent naturellement le cholestérol.
PCT/EP2012/051846 2011-02-03 2012-02-03 Marquage des lipoprotéines, procédés pour suivre le transport/l'évolution des lipoprotéines et pour dépister les troubles dyslipidémiques WO2012104411A1 (fr)

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EP3009844A1 (fr) 2014-10-16 2016-04-20 Sysmex Corporation Procédé de mesure de la capacité de lipoprotéine à accepter le cholestérol et trousse de réactifs
JP2016080685A (ja) * 2014-10-16 2016-05-16 シスメックス株式会社 リポタンパク質のコレステロール取り込み能を測定する方法及び試薬キット
WO2016194825A1 (fr) * 2015-05-29 2016-12-08 シスメックス株式会社 Méthode et kit de réactifs pour mesurer la capacité d'absorption de cholestérol de lipoprotéines
CN110333359A (zh) * 2019-07-11 2019-10-15 郭威莉 一种分析不同大小hdl促胆固醇流出能力的方法及应用

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EP3425406A1 (fr) * 2014-10-16 2019-01-09 Sysmex Corporation Procédé de mesure de la capacité de lipoprotéine à accepter le cholestérol et trousse de réactifs
JP2016080685A (ja) * 2014-10-16 2016-05-16 シスメックス株式会社 リポタンパク質のコレステロール取り込み能を測定する方法及び試薬キット
US10436806B2 (en) 2014-10-16 2019-10-08 Sysmex Corporation Method of measuring lipoprotein's capacity to accept cholesterol
EP3009844A1 (fr) 2014-10-16 2016-04-20 Sysmex Corporation Procédé de mesure de la capacité de lipoprotéine à accepter le cholestérol et trousse de réactifs
JPWO2016194825A1 (ja) * 2015-05-29 2017-07-13 シスメックス株式会社 リポタンパク質のコレステロール取り込み能を測定する方法及び試薬キット
CN108369225A (zh) * 2015-05-29 2018-08-03 希森美康株式会社 测定脂蛋白质的胆甾醇负载能力的方法及试剂盒
AU2016272252B2 (en) * 2015-05-29 2017-09-28 Sysmex Corporation Method for measuring cholesterol uptake capacity of lipoproteins and reagent kit thereof
AU2017268606B2 (en) * 2015-05-29 2019-03-14 Sysmex Corporation Method for measuring cholesterol uptake capacity of lipoproteins and reagent kit thereof
WO2016194825A1 (fr) * 2015-05-29 2016-12-08 シスメックス株式会社 Méthode et kit de réactifs pour mesurer la capacité d'absorption de cholestérol de lipoprotéines
CN108369225B (zh) * 2015-05-29 2019-10-11 希森美康株式会社 测定脂蛋白质的胆甾醇负载能力的方法及试剂盒
CN110596365A (zh) * 2015-05-29 2019-12-20 希森美康株式会社 测定脂蛋白质的胆甾醇负载能力的方法及试剂盒
EP3623811A1 (fr) 2015-05-29 2020-03-18 Sysmex Corporation Procédé pour mesurer la capacité d'absorption de cholestérol de lipoprotéines
US11486875B2 (en) 2015-05-29 2022-11-01 Sysmex Corporation Method for measuring cholesterol uptake capacity of lipoproteins
CN110596365B (zh) * 2015-05-29 2022-11-15 希森美康株式会社 测定脂蛋白质的胆甾醇负载能力的方法及试剂盒
CN110333359A (zh) * 2019-07-11 2019-10-15 郭威莉 一种分析不同大小hdl促胆固醇流出能力的方法及应用

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