WO2012104411A1

WO2012104411A1 - Lipoproteins labelling, methods for tracking transport/outcome of lipoproteins and for determining dyslipidemia disorders

Info

Publication number: WO2012104411A1
Application number: PCT/EP2012/051846
Authority: WO
Inventors: Xavier Collet; Gérald GAIBELET; François TERCE; Stéphane ORLOWSKI
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2011-02-03
Filing date: 2012-02-03
Publication date: 2012-08-09

Abstract

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.

Description

LIPOPROTEINS LABELLING, METHODS FOR TRACKING TRANSPORT/OUTCOME OF LIPOPROTEINS AND FOR DETERMINING DYSLIPIDEMIA DISORDERS

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.

Because cholesterol, as well as other lipids such as triglycerides, are insoluble in aqueous medium, such as blood, mechanisms exist to transport them to their various destinations. Thus, five classes of lipoproteins, in order of increasing density, chylomicron, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), are implied in the transport of cholesterol and triglycerides through the blood.

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).

These five lipoproteins also play a key role in the lipid metabolism.

Chylomicrons transport dietary triglycerides (hereinafter abbreviated "TG") and cholesterol from within the enterocytes through lymphatic vessels into the blood circulation. In the capillaries of adipose and muscle tissues, most of the TG of chylomicrons are converted to 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.

IDL are the products of endothelial lipoprotein lipase (hereinafter abbreviated "LPL") processing of VLDL and chylomicrons. 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. Blood HDL levels may not completely represent reverse cholesterol transport. HDL fraction contains several sub-fractions like HDL₂, HDL₃, VHDL, and like nascent HDL particles such as pre^-HDL, pre^₂-HDL and pre^₃-HDL.

Because of its crucial role in the mammalian body, mammals are able to synthesize cholesterol completely de novo, so that in principle mammals are independent from dietary intake of cholesterol. However, since food generally comprises cholesterol, cholesterol is also derived from the diet after being absorbed in the mammalian intestine. Apart from its essential role in the body, cholesterol can also pose a potential threat on human health when lipid metabolism is not properly regulated.

The quantification of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) is currently one of the most important clinical measurements for characterizing metabolic syndrome, such as dyslipidemia (which is traditionally defined as an elevation of plasma cholesterol, triglycerides or both, or a low HDL level) which is a major risk factor for cardiovascular diseases.

Dyslipidemias were traditionally classified by patterns of elevation in lipids and lipoproteins (Frederickson classification), as shown in the Table I below.

Table I

There is also a number of secondary causes for high cholesterol, among which diabetes mellitus and syndrome X, kidney disease (nephrotic syndrome) and hypothyroidism.

Improper regulation of lipid metabolism can also result in 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. In the context of the present invention, 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").

The development of 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.

Atherosclerosis can express itself in a number of cardiovascular diseases such as angina pectoris, myocardial infarction, transient ischemic attacks, cerebrovascular accidents, peripheral artery disease.

Recent studies have revealed additional factors that may be more strongly associated with the coronary heart disease than simple measurement of LDL or HDL levels, such as small dense (sd) LDL particles and oxidized LDL or HDL particles.

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.

On the contrary, epidemiological and experimental studies demonstrated the protective effects of high HDL level against development of atherosclerosis.

It was generally believed that the direction of cholesterol transport in the body is the underlying mechanism for these correlations. LDL is a vehicle for cholesterol supply to peripheral cells (and, therefore, to macrophages), whereas 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.

Further, protective effect of HDL could be in part due to antioxidative and antiinflammatory properties of HDL. Indeed, it was shown that HDL protects LDL from oxidative damage and reduces the level of proatherogenic oxidized LDL. ApoA-l, the major HDL apolipoprotein, may play a central role in HDL-mediated anti-oxidative activity. 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. However, 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.

Thus oxidized LDLs and HDLs have become a focus of attention as a new arteriosclerosis and cardiovascular diseases risk factor.

To date, oxidized LDLs and HDLs are difficult to detect and to quantify. Currently several methods using enzyme-linked immunosorbent assay (ELISA) or fluorescent probes are available to detect OxLDL.

However, slight variations in the different ELISA methods result in substantial differences in the results obtained. Currently, immunoassays used in detection of OxLDL involve three different types of antibodies, i.e. an antibody turned against a modified apoB, an antibody anti-oxidized phospholipid and an antibody specific for a circulating protein which after being oxidized aggregates to LDL. These methods are expensive, mainly because of the cost of the antibodies, and are not always reliable (Itabe and Ueda, J. Atheroscler. Thromb., 14:1 -1 1 , 2007).

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.

Recently, a direct and simple fluorescence detection method for oxidized lipoproteins based on the use of a lipopholic dye called DASP (for N,N- dipentadecylaminostyrylpyridinium iodide) was developed (Ikeda et al, Anal. Chem., 82:1 128-32, 2010). This method has been demonstrated as being suitable for quantifying oxidized lipoproteins as well as for distinguishing LDL and HDL from a subject having dyslipidemia and LDL and HDL from healthy normal subjects.

Nonetheless, the probes currently used for detecting or quantifying lipoproteins, as well as the probes used for assessing the structure or content of lipoproteins (such as abnormal oxidation or abnormal triglyceride an/or cholesterol contents) 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.

Indeed, because labelling by protein marker dyes is specific of the apolipoproteins of lipoproteins, cholesterol is not labelled and therefore the outcome of cholesterol comprised in lipoproteins can not be studied. Further, given that 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.

However, in vivo studies, in particular studies of the different processes implied in reverse cholesterol transport of HDL, are of seminal importance for understanding of normal or pathologic regulation of lipoproteins metabolism pathway.

At the present time, methods for studying reverse cholesterol transport are conducted either by injecting macrophages beforehand loaded ex vivo with cholesterol labelled with cholesterol tracer (generally ³H-radiolabelled cholesterol) into mice, or by injecting HDL comprising ³H-radiolabelled cholesterol. Because these methods use radiolabeled materials, they have to be conducted with particular caution and the radioactive wastes need to be properly treated, stored and disposed off. Further, another drawback of these methods lies in that ³H-radiolabelled cholesterol of lipoproteins is esterified in vivo by the enzyme LCAT which catalyzes the transfer of 2-acyl chain from lecithin to unesterified cholesterol to generate esterified cholesterol. Then, 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 ³H- radiolabeled cholesterol.

Investigating for a probe usable for carrying out ex vivo and in vivo studies which does not suffer from the drawbacks above recited, the inventors have unexpectedly observed that a previously described fluorescent analogue of cholesterol called cholesterol-pyrene of general formula I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- 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.

Further, the inventors showed that 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.

It is therefore an advantage of this invention to provide a suitable marker for tracking in vivo the transport and outcome of a chosen lipoprotein, for instance HDL for investigating reverse cholesterol transport.

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"). However, it seems that incorporation of cholesterol-pyrene of general formula (I) must preferably be carried out using a "transporter" intended to exchange cholesterol of biological membranes with the cholesterol-pyrene of general formula (I) (WO 2006/100388). The methods embodied in WO 2006/100388 are conducted with β-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.

Further, taking advantage of the fact that cholesterol-pyrene of general formula (I) is stably integrated in lipoproteins peripheral lipid monolayer, the inventors showed that 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, ...).

Further, by comparing the fluorescence emission spectrum of a biological sample which comprises lipoproteins labelled by cholesterol-pyrene of general formula (I) with fluorescence emission spectrum of a normal and abnormal (that is oxidized or of increased density or containing altered amounts of TG and/or cholesterol) lipoproteins labelled by cholesterol-pyrene, the lipoproteins of the sample to be analysed can be readily and easily characterized.

Because the properties of cholesterol-pyrene, 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.

Cholesterol-pyrene labelled lipoprotein

Accordingly, the present invention is directed to an isolated lipoprotein which comprises cholesterol and cholesterol- rene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- 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. Advantageously, the cholesterol-pyrene of general formula (I) comprised in said lipoprotein has the following formula:

In the present invention, "lipoprotein" is intended to mean a lipoprotein which naturally comprises cholesterol. Such 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 HDL₂, HDL₃, VHDL, pre^-HDL, pre-p₂-HDL and pre-p₃-HDL

Preferably, the lipoprotein of the invention does not contain a "transporter" (e.g. β- cyclodextrin) intended to exchange cholesterol of biological membranes.

Method for labelling lipoproteins

The inventors unex ectedly found that cholesterol-pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10,

can be stably integrated into the peripheral monolayer of the lipoproteins ex vivo (as well as in vivo) without need of a "transporter" (e.g. β-cyclodextrin) intended to exchange cholesterol of biological membranes with the cholesterol-pyrene, and thus provides a probe suitable for labelling lipoproteins.

Consequently, it is another object of the present invention to provide a simple ex vivo method for labelling lipoproteins which naturally comprise cholesterol, said method comprising the step consisting of contacting said lipoproteins with cholesterol-pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- 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;

whereby labelled lipoproteins are obtained.

In a particularly preferred embodiment, the cholesterol-pyrene of general formula (I) comprised in said li oprotein has the following formula:

Preferably, 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 HDL₂, HDL₃, VHDL, pre^-HDL, pre-p₂-HDL, pre-p₃-HDL, purified LDL composition, purified IDL composition, purified VLDL composition and purified chylomicron composition.

In the context of the present invention 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. Preferably, 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.

The methods suitable for separating and/or purifying the different fractions of lipoproteins are well known by the person skilled in the art (see Schumaker & Puppione, Methods in Enzymology 128, 155-170 (1986)). For instance, one can use ultracentrifugation on the appropriated density layer, knowing that density of chylomicron is inferior to 0.93 g/ml, density of VLDL is from 0.93 to 1 .006 g/ml, density of IDL is from 1 .006 to 1 .019 g/ml, density of LDL is from 1 .019 to 1 .063 g/ml, density of the different sub-fractions of HDL (e.g. HDL₂, HDL₃, VHDL, pre^-HDL, pre-p₂-HDL, pre-p₃-HDL), is from 1 .063 to 1 .210 g/ml (for instance density of HDL₂ is from 1 .063 to 1 .125 g/ml, density of HDL₃ 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". In the context of the present invention, a "transporter" is any molecule facilitating cholesterol-pyrene transfer in the aqueous medium, such as cyclodextrins, liposomes, dextrans, albumin. Hereafter, 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.

In a particular embodiment, 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. Method for quantifying lipoproteins in a biological sample

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:

i) incubating a biological sample, preferably said sample likely comprises lipoproteins which naturally comprise cholesterol, with cholesterol-pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10;

to produce 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);

iii) applying to said lipoproteins which naturally comprise cholesterol and which are labelled by cholesterol-pyrene of general formula (I) a radiation of excitation wavelength of 330 to 350 nm, preferably of 335 to 345;

iv) measuring the intensity of fluorescence emitted by lipoproteins of the biological sample;

v) determining the quantity of said lipoproteins from the intensity of fluorescence measured.

In a preferred embodiment, the method is carried out without using a "transporter" (e.g. β-cyclodextrin) intended to exchange cholesterol with the cholesterol-pyrene.

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. Preferably, in cholesterol-pyrene of general formula (I) used in step i) of the method, 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.

In a particularly preferred embodiment, the cholesterol-pyrene used in step i) has the following formula:

In a preferred embodiment of the method for detecting and/or quantifying lipoproteins according to the invention, the biological sample to be analysed is serum or plasma.

In this preferred embodiment, 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 HDL₂, HDL₃, 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.

Methods for sorting out the different lipoprotein fractions are well known by one skilled in the art and include chromatography method and centrifugation. Preferably, 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.

Preferably, 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. In order to determine the quantity of lipoproteins in step v), 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.

Method for assessing oxidation state of a lipoprotein which naturally

comprise cholesterol

The inventors showed that the fluorescence emission spectrum of labelled lipoproteins gives information in relation to the oxidation state of said lipoproteins.

Consequently, 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:

i) incubating said lipo rotein with cholesterol-pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- 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;

to produce lipoproteins labelled with said cholesterol-pyrene of general formula (I); ii) separating said lipoprotein which naturally comprises cholesterol and which is labelled by cholesterol-pyrene of general formula (I) from the free cholesterol- pyrene molecule of general formula (I);

iii) applying to said lipoprotein which naturally comprises cholesterol and which is labelled by cholesterol-pyrene of general formula (I) a radiation of excitation wavelength of 330 to 350 nm, preferably of 335 to 345; iv) determining the fluorescence emission spectrum of said lipoprotein labelled by cholesterol-pyrene of general formula (I), preferably between 350 and 650 nm, still preferably 360 and 650 nm and more preferably between 360 and 600 nm;

v) 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.

In a preferred embodiment, the method is carried out without using a "transporter"

(e.g. β-cyclodextrin) intended to exchange cholesterol with the cholesterol-pyrene.

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.

In a particularly preferred embodiment, the cholesterol-pyrene of general formula (I) used in step i) has the following formula:

Preferably, the lipoprotein of which oxidation state is assessed is selected from the group consisting of HDL (or HDL subtractions HDL₂, HDL₃, VHDL, pre^-HDL, pre-p₂- 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). In an embodiment of the method, the fluorescence emission spectrum of the lipoprotein labelled by cholesterol-pyrene of general formula (I) is determined between 350 and 650 nm. In particular, 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.

Optionally, 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.

Method for determining the presence of dyslipidemia disorders in a subject The inventors showed that plasma sample from a patient having dyslipidemia, once labelled by cholesterol-pyrene of the invention, presents fluorescence emission spectrum which differs from the one of a healthy subject. Further, different types of dyslipidemia can be differentiated from each other since fluorescence emission spectrum is typical for each type of dyslipidemia.

Thus the present invention also relates to a method, preferably ex vivo, for determining the presence of dyslipidemia disorder in a subject.

In a first embodiment, said method comprises the following steps:

i) incubating a biological sample, preferably said sample likely comprises lipoproteins which naturally comprise cholesterol with cholesterol-pyrene of general formula (I :

wherein - X represents either an Oxygen atom or a CH₂ group; and - 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;

to produce 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);

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;

iv) determining the fluorescence emission spectrum of said lipoprotein labelled by cholesterol-pyrene of general formula (I);

v) 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.

In a second embodiment, said method comprises the following steps:

i) applying a radiation of excitation wavelength of 330 to 350 nm, preferably of 335 to 345, to lipoproteins which naturally comprise cholesterol from a biological sample from a subject administered beforehand with cholesterol-pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- 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.

ii) determining the fluorescence emission spectrum of said lipoprotein;

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.

Optionally, 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).

Preferably, lipoproteins of step i) represent by order of preference at least 80%,

85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% on a mass basis of the composition.

Preferably, in cholesterol-pyrene of general formula (I), 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.

In a particularly preferred embodiment, 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 HDL₂, HDL₃, VHDL, pre^-HDL,

purified LDL, purified IDL, purified VLDL and purified chylomicron.

Typically, 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.

When the biological sample is serum or plasma, particular attention should be given to emission wavelengths between about 440 and about 500 nm (excimer peak) and/or around 420 nm. Indeed, 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). Further, in case of 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.

When the biological sample is purified HDL (obtained by LDL precipitation) of human origin, 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.

It is well-known that high plasma cholesterol concentrations, in particular in the low- density lipoprotein (LDL) fraction, and that low concentration of high-density lipoprotein (HDL), are associated with an increased risk for the development of atheromatous lesions, which results in late stage in atherosclerosis.

Since atherosclerosis is a precondition for artery disease, like heart coronary disease (angina pectoris, myocardial infarction), transient ischemic attacks, cerebrovascular accidents, peripheral artery disease, 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.

Preferably, both embodiments of the method are carried out without using a "transporter" (e.g. β-cyclodextrin) intended to exchange cholesterol with the cholesterol- pyrene. Method for tracking in vivo the transport and outcome of lipoproteins

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.

Accordingly, 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. Preferably, the method is carried out without using a "transporter" (e.g. β- 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, preferably selected from the group consisting of HDL or subtractions of HDL like HDL₂, HDL₃, VHDL, pre^-HDL, pre-p₂-HDL, pre-p₃-HDL, LDL, IDL, VLDL and chylomicron, may comprise the steps consisting of:

a) - measuring incorporation of cholesterol-pyrene of general formula (I) in cells and/or tissues of a mammal administered beforehand with at least one lipoprotein which naturally comprises cholesterol and which is labelled with cholesterol-pyrene of general formula (I), by measuring fluorescence, and/or

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:

wherein - X represents either an Oxygen atom or a CH₂ group; and

- 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.

Optionally, 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 HDL₂, HDL₃, VHDL, pre^ HDL, pre^₂- HDL, pre-p₃-HDL, LDL, IDL, VLDL and chylomicron.

Preferably, 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.

When HDL reverse cholesterol transport is to be investigated, 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 HDL₂, HDL₃, VHDL, pre^-HDL, pre^₂- HDL, pre-p₃-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 HDL₂, HDL₃, VHDL, pre^-HDL, pre^₂-HDL, pre^₃-HDL, the method may also comprise the steps consisting of:

a- measuring incorporation of cholesterol-pyrene of general formula (I) in cells and/or tissues of a mammal orally administered beforehand with cholesterol- pyrene of general formula (I), and/or

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:

wherein - X represent a CH₂ group; and

- 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.

Optionally, the method may comprise, before step a), the step consisting of administering orally said mammal with cholesterol-pyrene of general formula (I). In the method according to the invention 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.

Preferably, in the method for tracking in vivo the transport and outcome of lipoproteins of the invention, 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.

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.

Further, in an embodiment of any of the methods for tracking in vivo the transport and outcome of lipoproteins which naturally comprise cholesterol as described above, 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.

For instance, one can use:

- ABCA1 knock-out mice. These 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) knock-out mice. These mice were disclosed by Rigotti et al., (P.N.A.S., 94:12610-12615, 1997) (see also Trigatti and Rigotti, Int. J. Tissue React. 22:29-37, 2000). 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).

One can also use mice deleted for NPC1 L1 (Altman et al., Science, 303: 1201 -4,

2004), for ApoE (Plump et al., Cell, 71 : 343-53, 1992) or for the LDL receptor (Ishibashi et a/., J. Clin. Invest., 92: 883-93, 1993), or for both (Van Dijk et al., Arterioscl. Thromb. Vase. Biol., 19(12): 2945-51 , 1999). One can also use transgenic animals overexpressing either one of these proteins. All these genetically engineered animals can present global alterations, or tissue-specific alterations thanks to the Cre-Lox model.

It is to be noted that the 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.

Method for screening for a compound which has an effect on the cholesterol metabolism

The invention is also drawn to a method for screening for a compound which alters cholesterol metabolism.

This method comprises the steps of:

a) Administering to a non human mammal such as described above a candidate compound;

b) carrying out the method for tracking in vivo the transport and outcome of lipoproteins of the invention as described above;

c) 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.

In the context of the invention, 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.

Once a compound which decreases the level of LDL and/or increases the level of HDL, has been identified, 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.

Method for assessing tumors growth

It is reported from long time that some cancerous tumor tissues present an accumulation of cholesterol (Tosi & Tugnoli, Clin. Chim. Acta., 359(12): 27-45, 2005; Brown, Clin. Exp. Pharmacol. Physiol., 34:135-141 , 2007].

This phenomenon is mainly reported for leukemia, and tumors from prostate, colon and breast (Banker et al., Blood, 104(6):1816-1824, 2004; Hager et al, Curr. Opin. Clin. Nutr. Metab. Care, 9(4):379-395, 2006).

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.

It is an object of the invention to take benefit of cholesterol accumulation in cancerous tumor tissues/cells as a mean to assess the growth of tumors, and to screen for a compound which inhibits growth of tumors.

Thus, the invention is relative to a method for assessing tumor growth.

This method comprises a step of:

- measuring incorporation of cholesterol-pyrene of general formula (I) in tumor tissues and/or tumor cells of a mammal suffering from cancer administered beforehand i) with at least one lipoprotein which naturally comprises cholesterol, wherein said at least one lipoprotein is labelled with cholesterol-pyrene of general formula (I), and/or ii) with cholesterol-pyrene of general formula (I);

wherein cholesterol-pyrene of general formula (I) is:

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10; and

wherein measuring incorporated cholesterol-pyrene of general formula (I) in tumor tissues and/or tumor cells is indicative of the tumor growth.

Optionally, 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.

In the method according to the invention 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:

- administering a candidate compound to a non-human animal suffering from cancer and having tumors;

- administering i) at least one lipoprotein which naturally comprises cholesterol, wherein said at least one lipoprotein which naturally comprises cholesterol is labelled with cholesterol-pyrene of general formula (I), and/or ii) with cholesterol-pyrene of general formula (I)

- determining whether the administration of said candidate compound inhibits growth of tumors present in said non-human animal suffering from cancer administered with the at least one lipoprotein which naturally comprises cholesterol labelled with cholesterol- pyrene of general formula (I), 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;

wherein cholesterol-pyrene of general formula (I) is:

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10; and

wherein decrease in incorporation of cholesterol-pyrene of general formula (I) in tumor tissues and/or tumor cells is indicative that said candidate compound inhibits the tumor growth.

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. Advantageously, the non-human animal is immunodepressed in order to avoid rejection of the grafted tumors (e.g. xenograft model of a tumor). Typically, 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.

When suitable (for instance when the method is conducted for several days), 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.

Preferably, in the method for assessing tumor growth and the method for screening for a compound which inhibits growth of tumors according to the invention, 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.

In a particularly preferred embodiment, the cholesterol-pyrene of general formula (I) used in step i) has the followin formula:

Further, 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 HDL₂, HDL₃, VHDL, pre^-HDL, pre-p₂-HDL, pre-p₃-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.

In a preferred embodiment, the methods are carried out without using a "transporter" (e.g. β-cyclodextrin) intended to exchange cholesterol of biological membranes with the cholesterol-pyrene.

The present invention will be further illustrated by the additional description and drawings which follow, which refer to examples illustrating the stable labelling of lipoproteins with the cholesterol-pyrene of general formula (I) and the spectral analysis of labelled lipoproteins. It should be understood however that these examples are given only by way of illustration of the invention and do not constitute in anyway a limitation thereof. FIGURES

- Figure 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 5μΜ of cholesterol-pyrene of the invention (hereinafter abbreviated "Chol-Pyr").

- Figure 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.

- Figure 3 illustrates the emission fluorescence spectrum of serum from either:

- a healthy donor (A) (TG= 1 .27 g/l ; TC= 2.17 g/l);

- a patient presenting an hypercholesterolemia (B) (TG= 0.99 g/l ; TC= 2.51 g/l);

- a patient presenting an hypertriglyceridemia (C) (TG= 1 .87 g/l ; TC= 2.03 g/l); or

- a patient presenting global hyperlipemia (D) (increased TC and TG (TG= 2.14 g/l ; TC= 2.54 g/l);

after labelling by 10 μΜ Chol-Pyr and dialysis.

- Figure 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 μΜ Chol-Pyr and dialysis.

- Figure 5 illustrates the emission fluorescence spectrum of purified LDL of human origin, either native (A) or oxidized by CuS0₄ during 4 (B) or 8 hours (C), after labelling by 5 μΜ Chol-Pyr and dialysis. EXAMPLES

Example I. Labelling procedure

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).

For labelling total serum, 5-10 μΜ Chol-Pyr were used, while for labelling purified lipoprotein fractions, either the fixed concentration of 10 μΜ, or the fixed ratio of 5% to the free cholesterol contained in the lipoproteins were used, this amount being initially determined, and typically corresponds to 20-50 μΜ for HDL and 130-300 μΜ for LDL.

For optimal labelling, 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

After an incubation of fetal calf serum at 20% (in culture medium Ham F12) with

Chol-Pyr 5 μΜ, simultaneous measurements of fluorescence of the pyrene group (Exc =

335 nm / Em = 385-400 nm; see Figure 1 .A) and of absorbance of proteins (OD at 280 nm; Figure 1 .B), in the outflow of a molecular sieve sepharose® column.

These results show that the fluorescent cholesterol probe is associated with the respective peaks corresponding to VLDL, LDL, HDL and albumin, the latter being largely the weakest when compared to the protein amounts.

The same HPLC analysis performed on purified lipoproteins after their incubation with Chol-Pyr (at 5% of free cholesterol), and resuspension in PBS after dialysis, shows a time-dependent selective labelling of HDL and LDL, as illustrated respectively in Figure

2.A and 2.B. Indeed, labelling of HDL and LDL with Chol-Pyr increases between 4 hours

(see solid lines) and 24 hours (see dashed lines)

Example III. Spectral analysis of Chol-Pyr labelled lipoproteins from dyslipidemic patients

After labelling by 10 μΜ Chol-Pyr of total serum of human origin, coming either from a healthy donor or from patients presenting various forms of dyslipidemia (either enhanced cholesterolemia or triglyceridemia, or both), and subsequent dialysis, the fluorescence emission spectrum (360-600 nm range) was analyzed. The result of this analysis is illustrated in Figure 3. As shown in Figure 3, hypertriglyceridemia appears correlated with a decreased excimer peak (around 460 nm), as well as hypercholesterolemia (although in a lower extent), and both effects appears additive in case of hyperlipemia. The shoulder at 420 nm follows the same tendencies, with smaller variations.

After labelling by 10 μΜ Chol-Pyr of purified HDL (obtained by LDL precipitation) of human origin, coming either from a healthy donor or from patients presenting various forms of dyslipidemia, and subsequent dialysis, the fluorescence emission spectrum (360- 600 nm range) was analyzed. The result of this analysis is illustrated in Figure 4.

As shown in Figure 4, 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

After treatment of a purified LDL fraction of human origin by Cu²⁺ ions at 5 μΜ during 4 or 8 hours, and subsequent labelling by 5 μΜ Chol-Pyr and dialysis, the fluorescence emission spectrum (360-600 nm range) was analyzed.

As shown in Figure 5, in one hand, an inversion of the relative intensities of the emission peaks at 380 and 400 nm when LDL is oxidized, along with an increase of the shoulder at 420 nm, independently of the oxidation duration were observed, and in the other hand, a progressive growth of the excimer peak, around 460 nm, with increasing oxidation duration was observed.

Claims

1 . An isolated lipoprotein which comprises cholesterol and cholesterol-pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10.

2. The lipoprotein according to claim 1 , wherein said cholesterol-pyrene of general formula (I) has the following formula:

3. The lipoprotein according to claim 1 or 2, wherein said lipoprotein is selected from the group consisting of: chylomicron, very-low-density lipoprotein (VLDL), intermediate- density lipoprotein (IDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and HDL subtractions HDL₂, HDL₃, VHDL, pre^ HDL, pre-p₂-HDL and pre-p₃-HDL .

4. An ex vivo method for labelling lipoproteins which naturally comprise cholesterol, said method comprising the step consisting of contacting said lipoproteins with cholesterol- pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10;

whereby labelled lipoproteins are obtained.

5. The method for labelling lipoproteins according to claim 4, wherein said lipoproteins are comprised in a biological sample selected from the group consisting of serum, plasma, purified HDL, purified subtractions of HDL, HDL₂, HDL₃, VHDL, pre^ HDL, pre-p₂-HDL, pre^₃-HDL, purified LDL, purified IDL, purified VLDL and purified chylomicron.

6. A method for quantifying lipoproteins in a biological sample, comprising the following steps:

i) incubating a biolo ical sample with cholesterol-pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10;

to produce 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); iii) applying to said lipoproteins which naturally comprise cholesterol and which are labelled by cholesterol-pyrene of general formula (I) a radiation of excitation wavelength of 330 to 350 nm;

7. The method according to claim 6, wherein said biological sample is serum or plasma, and wherein said method comprises an additional step either before step i) or between step ii) and iii), of:

i') sorting all or part of the lipoprotein fractions HDL, LDL, IDL, VLDL, chylomicron and/or HDL subtractions, preferably HDL subtractions are HDL₂, HDL₃, VHDL, pre- βι-HDL, pre-p₂-HDL and pre-p₃-HDL; and

wherein step iv) consists of measuring the intensity of fluorescence emitted by each lipoprotein fraction or subtraction.

8. An ex vivo method for assessing oxidation state of a lipoprotein which naturally comprises cholesterol, comprising the following steps:

i) incubating said lipoprotein which naturally comprises cholesterol with cholesterol-pyrene of eneral formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10;

to produce lipoproteins labelled with said cholesterol-pyrene of general formula (I); ii) separating said lipoprotein which naturally comprises cholesterol and which is labelled by cholesterol-pyrene of general formula (I) from the free cholesterol- pyrene molecule of general formula (I); iii) applying to said lipoprotein which naturally comprises cholesterol and which is labelled by cholesterol-pyrene of general formula (I) a radiation of excitation wavelength of 330 to 350 nm;

v) 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 the 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.

9. An ex vivo method for determining the presence of dyslipidemia disorder in a subject comprising the following steps:

i) incubating a biological sample of a subject with cholesterol-pyrene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10;

to produce lipoproteins labelled with said cholesterol-pyrene of general formula (I) ii) separating lipoproteins which naturally comprise cholesterol and which are labelled with cholesterol-pyrene of general formula (I) from the free cholesterol- pyrene molecule of general formula (I);

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;

iv) determining the fluorescence emission spectrum of said lipoproteins labelled by cholesterol-pyrene of general formula (I); v) comparing said 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 is indicative of dyslipidemia.

10. An ex vivo method for determining the presence of dyslipidemia disorder in a subject comprising the following steps:

i) applying a radiation of excitation wavelength of 330 to 350 nm to lipoproteins which naturally comprise cholesterol obtained from a subject administered beforehand with cholesterol- rene of general formula (I):

ii) determining the fluorescence emission spectrum of said lipoproteins; 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.

1 1 . The method according to any one of claims 9 or 10, wherein said biological sample is selected from the group consisting of serum, plasma, purified HDL, purified LDL, purified IDL, purified VLDL purified chylomicron and purified subtraction of HDL, subtraction of HDL are preferably selected from the group consisting of HDL₂, HDL₃, VHDL, pre^-HDL, pre-p₂-HDL and pre-p₃-HDL.

12. A non human mammal comprising lipoproteins which naturally comprise cholesterol, wherein said lipoproteins are labelled with cholesterol- rene of general formula (I):

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10.

13. A method for tracking in vivo the transport and outcome of lipoproteins which naturally comprise cholesterol, wherein said method comprises the steps consisting of:

- measuring incorporation of cholesterol-pyrene of general formula (I) in cells and/or tissues of a mammal administered beforehand with a least one lipoprotein which naturally comprises cholesterol, wherein said at least one lipoprotein is labelled with cholesterol- pyrene of general formula (I), by measuring fluorescence, and/or

- 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 at least one lipoprotein which naturally comprises cholesterol, wherein said at least one lipoprotein is labelled with cholesterol-pyrene of general formula (I);

wherein cholesterol-pyrene of eneral formula (I) is:

wherein 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.

14. A method for tracking in vivo the transport and outcome of lipoproteins which naturally comprise cholesterol, wherein said method comprises the steps consisting of:

- measuring incorporation of cholesterol-pyrene of general formula (I) in cells and/or tissues of a mammal orally administered beforehand with cholesterol-pyrene of general formula (I), and/or

- 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 eneral formula (I) is:

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10; and

15. Method for assessing tumor growth which comprises a step of:

wherein cholesterol-pyrene of general formula (I) is:

wherein - X represents either an Oxygen atom or a CH₂ group; and

- n is an integer from 2 to 10; and