EP1432425A1

EP1432425A1 - A method of reducing the "in vivo" cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins (hdl) and low density lipoproteins (ldl)

Info

Publication number: EP1432425A1
Application number: EP02764428A
Authority: EP
Inventors: P. Haydn Pritchard; Kishor M. Wasan
Original assignee: Forbes Medi-Tech Inc
Current assignee: Forbes Medi-Tech Inc
Priority date: 2001-09-26
Filing date: 2002-09-26
Publication date: 2004-06-30
Also published as: RU2004112556A; BR0212826A; JP2005507890A; CN1558766A; WO2003026672A1; CA2461136A1; NZ531938A

Abstract

A method of reducing the in vivo cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins (HDL) and low density lipoproteins (LDL) comprises administering a therapeutically effective amount of one or more compounds: formulae (I), (II), (III), wherein R is a sterol or stanol moiety R2 is derived from ascorbic acid and R3 is hydrogen or any metal, alkali earth metal, or alkali metal; and all salts thereof, whereby plasma HDL and LDL are controlled as a result of such administration.

Description

TITLE: A METHOD OF REDUCING THE IN VIVO CHOLESTEROL ESTER TRANSFER PROTEIN MEDIATED TRANSFER OF CHOLESTERYL ESTERS BETWEEN HIGH DENSITY LIPOPROTEINS (HDL) AND LOW DENSITY LIPOPROTEINS (LDL)

FIELD OF THE INVENTION

This present invention relates to methods of treating or preventing cardiovascular disease and its underlying conditions, including atherosclerosis, dyslipidemic conditions or disorders in animals, particularly humans.

BACKGROUND OF THE INVENTION

While recent advances in science and technology are helping to improve quality and add years to human life, the prevention of atherosclerosis, an underlying cause of cardiovascular disease ("CVD") has not been sufficiently addressed. Atherosclerosis is a degenerative process resulting from an interplay of inherited (genetic) factors and environmental factors such as diet and lifestyle. Research to date suggest that cholesterol may play a role in atherosclerosis by forming atherosclerotic plaques in blood vessels, ultimately cutting off blood supply to the heart muscle or alternatively to the brain or limbs, depending on the location of the plaque in the arterial tree (1 ,2). Overviews have indicated that a 1% reduction in an individual's total serum cholesterol yields a 2% reduction in risk of a coronary artery event (3). Statistically, a 10% decrease in average serum cholesterol (e.g. from 6.0 mmol/L to 5.3 mmol/L) may result in the prevention of 100,000 deaths in the United States annually (4). Accordingly, hyperlipidemic conditions associated with elevated concentrations of total cholesterol and low density lipoprotein (LDL) cholesterol are significant risk factors.

Studies also show that a low plasma concentration of high density lipoprotein (HDL) cholesterol is a significant risk factor for the development of atherosclerosis (5) and that high levels are protective. Lipoproteins are complexes of Iipids and proteins held together by non-covalent bonds. Each type of lipoprotein class has a characteristic mass, chemical composition, density and physiological role. Irrespective of density or particle size, circulating Iipids consist of a core of cholesteryl esters and triglycerides, and an envelope of phospholipids, free cholesterol and apolipoproteins. The apolipoproteins are involved in the assembly and secretion of the lipoprotein, provide structural integrity, activate lipoprotein-modifying enzymes, and are the ligand for a large assortment of receptors and membrane proteins. . Lipoprotein classes found in plasma include HDL, LDL, intermediate density lipoproteins (IDL) and very low density lipoproteins (VLDL).

Each type of lipoprotein has a characteristic apolipoprotein composition or ratio. The most prominent apolipoprotein in HDL is apolipoprotein-AI (apo-AI), which accounts for approximately 70% of the protein mass, with apo-AII accounting for another 20%. The ratio of apoA-l to apoA-ll may determine HDL functional and anti-atherogenic properties. Circulating HDL particles consist of a heterogeneous mixture of discoidal and spherical particles with a mass of 200 to 400 kilo-daltons and a diameter of 7 to 10 nm.

HDL is one of the major classes of lipoproteins that function in the transport of Iipids in plasma, and has multiple functions within the body, including reverse cholesterol transport, providing the cholesterol molecule substrate for bile acid synthesis, transport of clusterin, transport of paraoxanase, prevention of lipoprotein oxidation and selective uptake of cholesterol by adrenal cells. The major Iipids associated with HDL include cholesterol, cholesteryl ester, triglycerides, phospholipids and fatty acids To better understand how HDL is anti-atherogenic, a brief explanation of the atherosclerotic process is necessary. The atherosclerotic process begins when LDL becomes trapped within the vascular wall. Oxidation of this LDL results in the binding of monocvtes to the endothelial cells lining the vessel wall. These monocytes are activated and migrate into the endothelial space where they are transformed into macrophages, leading to further oxidation of the LDL. The oxidized LDL is taken up through the scavenger receptor on the macrophage, leading to the formation of foam cells. A fibrous cap is generated through the proliferation and migration of arterial smooth muscle cells, thus creating an atherosclerotic plaque.

HDL is essential for the transport of cholesterol from extra-hepatic tissues to the liver, where it is excreted into bile as free cholesterol or as bile acids that are formed from cholesterol. The process requires several steps. The first is the formation of nascent or pre-beta HDL particles in the liver and intestine. Excess cholesterol moves across cell membranes into the nascent HDL through the action of the ABCAI transporter. Lecithin cholesterol acyl transferase (LCAT) converts the cholesterol to cholesteryl ester and the subsequent conversion of nascent HDL to mature HDL. Esterified cholesterol is then transferred by cholesteryl ester transfer protein (CETP) from HDL to apolipoprotein-B containing lipoproteins, which are taken up by numerous receptors in the liver.¹ Nascent HDL is regenerated via hepatic triglyceride lipase and phospholipid transfer protein and the cycle continues. In addition to the cholesterol removed from peripheral cells, HDL accepts cholesterol from LDL and erythrocyte membranes. Another mechanism of reverse cholesterol transport may involve passive diffusion of cholesterol between cholesterol-poor membranes and HDL or other acceptor molecules.

HDL protects against the development of atherosclerosis both through its role in reverse cholesterol transport and possibly by impeding LDL oxidation. Several HDL- associated enzymes are involved in the process. Paroxonase (PON1), LCAT, and platelet activating factor acetylhydrolase (PAFAH) all participate by hydrolyzing phospholipid hydroperoxides generated during LDL oxidation and act in tandem to prevent the accumulation of oxidized lipid in LDL. These enzymes are responsible for the anti-oxidative and anti-inflammatory properties of HDL.

CETP is a plasma lipid transfer protein secreted by the liver and adipose tissues, which mediates the transfer and exchange of neutral Iipids and phospholipids (6). CETP exchanges cholesteryl esters of HDL with triglycerides of VLDL, IDL, and LDL. Since these lipoproteins are catabolized faster than HDL, CETP facilitates the clearance of HDL cholesteryl esters. This process results in decreased HDL size and protein content. Thus, CETP clearly plays a central role in reverse cholesterol transport by moving cholesterol from the periphery back to the liver for disposal.

Inhibition of CETP has been shown effectively to modify plasma HDL/LDL ratios (7) as movement of the cholesteryl ester from HDL to LDL has the effect of lowering HDL. Evidence of this effect is described by many in the field (8, 9,10). Other CETP inhibitors are described in detail by researchers (11-19).

Furthermore, there are innumerable patents and published applications disclosing various compounds allegedly useful to inhibit the action of CETP. These include: US Patent 5,519,001 which describes a 36 amino acid peptide derived form baboon Apo C-1 ; WO publication 98/35937; European Patent Application 796,846 which describes 2-aryl- substitiuted pyridines; European Patent Application 818,448 which describes tetrahydraquinoline derivatives; European Patent Application 818,197 which describes pyridines with fused heterocycles; PCT/US99/27946 (publication WO 00/38725) describes a combination therapy including known CETP inhibitors with ileal bile acid transport inhibitors; US Patent No. 5,932,587 which describes heterocyclic fused pyridines; and US Patent 5,948,435 which describes large liposomes.

The above noted references show continued and on-going work in trying to develop safe and effective lipid modulating compounds. It is an object of the present invention to obviate or mitigate the disadvantages and insufficiencies of safety and effectiveness known in the field.

SUMMARY OF THE INVENTION

The present invention provides a method of reducing the in vivo cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins (HDL) and low density lipoproteins (LDL) which comprises administering a therapeutically effective amount of one or more the following compounds:

i π in

wherein R is a sterol or stanol moiety, R2 is derived from ascorbic acid and R3 is hydrogen or any metal, alkali earth metal, or alkali metal; and all salts thereof, whereby plasma HDL and LDL are controlled as a result of such administration.

The present invention further comprises compositions for reducing the in vivo cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins and low density lipoproteins which comprises one or more of the compounds having the following formulae:

π in

wherein R is a sterol or stanol moiety, R2 is derived from ascorbic acid and R3 ^Λis hydrogen or any metal, alkali earth metal, or alkali metal; and all salts of these structures; and a pharmaceutically acceptable or food grade carrier therefore.

The present invention further provides a method of treating or preventing cardiovascular disease and its underlying conditions, including atherosclerosis, hyperiipidemic conditions, hypoalphalipoproteinemia and hypercholesterolemia, in an animal which comprises reducing the in vivo cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins (HDL) and low density lipoproteins (LDL), said reduction being accomplished by administering to the animal one or more sterol and/or stanol derivatives having the formulae noted above.

The compounds of the present invention have been found to be surprisingly effective in reducing the cholesterol ester transfer protein mediated transfer of cholesteryl esters between HDL and LDL. In particular, this results in an increase in serum HDL levels. Until now, there has been no appreciation of the regulatory effect of these compounds on CETP-mediated transfer of cholesterol. It is an object of the present invention to provide a superior method to control and manipulate, in vivo, the lipid content and composition of peripheral tissues, cells, membranes and extra-cellular regions. It is a further object of the present invention to provide compounds useful for such a method.

These effects and other significant advantages are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way the following non-limiting drawings in which:

Figure 1 is the chemical structure of one of the preferred compounds of the present invention: phytostanol-phosphoryl-ascorbate (also referred to as FM-VP4) and its sodium salt;

Figure 2 is a graph showing the CETP mediated transfer of [³H]CE from LDL to HDL in the presence or absence of deoxycholate, TP2 or FM-VP4 (one of the compounds of the present invention) wherein the CETP is purified. Data is presented as mean +/- standard deviation (n=4); *p>0.05 vs control; ND=below the detectable limit of assay;

Figure 3 is a is a graph showing the CETP mediated transfer of [³H]CE from LDL to HDL in the presence or absence of deoxycholate, TP2 or FM-VP4 (one of the compounds of the present invention) wherein the CETP is from a human plasma source. Data is presented as mean +/- standard deviation (n=4); *p>0.05 vs control; ND=below the detectable limit of assay; and

Figure 4 is a graph showing the CETP mediated transfer of [³H]CE from LDL to HDL in the presence of increasing concentrations of the compound of the present invention (FM- VP4) and comparing both purified and human plasma source CETP. Data is presented as mean +/- standard deviation (n=4); *p>0.05 vs FM-VP4.

PREFERRED EMBODIMENTS OF THE INVENTION

The following detailed description is provided to aid those skilled in the art in practising the present invention. However, this detailed description should not be construed so as to unduly limit the scope of the present invention. Modifications and variations to the embodiments discussed herein may be made by those with ordinary skill in the art without departing from the spirit or scope of the present invention.

According to the present invention, there is provided a method of reducing the in vivo cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins (HDL) and low density lipoproteins (LDL) which comprises administering a therapeutically effective amount of one or more the following compounds:

π in

wherein R is a sterol or stanol moiety R2 is derived from ascorbic acid and R3 is hydrogen or any metal, alkali earth metal, or alkali metal; and all salts thereof, whereby plasma HDL and LDL are controlled as a result of such administration. The term "therapeutically effective" is intended to qualify the amount of the compound(s) administered in order to achieve the goals of: a) inhibiting the CETP-mediated transfer of CE between HDL and LDL; and thereby b) increasing HDL levels; and c) preventing, reducing, eliminating or ameliorating a dyslipidemic condition or disorder; or d) preventing, reducing, eliminating or ameliorating hypercholesterolemia, hypoalphalipoproteinemia, toxic shock syndrome or the development of atherosclerotic lesions; or e) preventing, reducing, eliminating or ameliorating any condition, disease or disorder which has as its basis or which is exacerbated by a deficiency in plasma HDL, or excess of either LDL, VLDL, Lp(a), beta-VLDL, IDL or remnant lipoproteins.

In one preferred form, this method further comprises the step of periodically assaying plasma LDL levels (by techniques which are known and widely applied in the art) and modifying the amount and/or concentration of the compounds to be administered, or the administration method if required.

Using the method as described herein, the level of serum HDL is increased and the levels of atherogenic lipoproteins selected from the group consisting of LDL, VLDL, and IDL are effectively controlled. Modulating or controlling plasma lipoproteins provides for the prevention, reduction, elimination or amelioration of a number of conditions and disorders, including, but not limited to: cardiovascular disease and its underlying conditions, including atherosclerosis, dyslipidemic conditions or disorders, hypercholesterolemia, and hypoalphalipoproteinemia, development of atherosclerotic lesions and toxic shock syndrome. Many other conditions which have as their basis or which are exacerbated by a deficiency in plasma HDL, or excess of either LDL, VLDL, or IDL will also be assisted by the method of the present invention. The elements of the compounds will be described in more detail below. It should be noted that, throughout this disclosure, the terms "compound" "derivative", "structure" and "analogue" may be used interchangeably to describe the structures above which link both a sterol or stanol and ascorbic acid and which have been found to be effective in inhibiting the CETP-mediated transfer of CE between HDL and LDL.

Sterols/Stanols

As used herein, the term "sterol" includes all sterols without limitation, for example: sitosterol, campesterol, stigmasterol, brassicasterol (including dihydrobrassicasterol), desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, cholesterol and all natural or synthesized forms and derivatives thereof, including isomers. The term "stanol" refers to saturated or hydrogenated sterols including all natural or synthesized forms and derivatives thereof, and isomers. It is to be understood that modifications to the sterols and stanols i.e. to include side chains also falls within the purview of this invention. For example, the purview of this invention clearly includes 24 beta-ethylchlostanol, 24-alpha-ethyl-22-dehydrocholstanol. It is also to be understood that, when in doubt throughout the specification, and unless otherwise specified, the term "sterol" encompasses both sterol and stanol.

The sterols and stanols for use in forming derivatives in accordance with this invention may be procured from a variety of natural sources. For example, they may be obtained from the processing of plant oils (including aquatic plants) such as corn oil and other vegetable oils, wheat germ oil, soy extract, rice extract, rice bran, rapeseed oil, sunflower oil, sesame oil and fish (and other marine-source) oils. They may also be derived from fungi, for example ergosterol, or animals, for example cholesterol. Accordingly, the present invention is not to be limited to any one source of sterols. US Patent Serial No. 4,420,427 teaches the preparation of sterols from vegetable oil sludge using solvents such as methanol. Alternatively, phytosterols and phytostanols may be obtained from tall oil pitch or soap, by-products of forestry practises as described in US Patent Serial No.5,770,749, incorporated herein by reference.

In one preferred form, the derivative of the present invention is formed with naturally- derived or synthesized beta-sitosterol, campestanol, sitostanol and campesterol and each of these derivatives so formed may then be admixed a composition prior to delivery in various ratios. In another preferred form, the derivative of the present invention is formed with naturally-derived or synthesized sitostanol or with naturally derived or synthesized campestanol or mixtures thereof.

In a most preferred form, the sterol is in its saturated form and is sitostanol. ,

R2

R2 comprises ascorbic acid or any derivative thereof. What is achieved within the scope of the present invention is the creation of a new structure or compound wherein a sterol or stanol moiety is chemically linked to ascorbic acid. The union benefits and enhances the both parts of this new structure. The phytosterol moiety, formerly poorly soluble, becomes, as part of the new derivative, much more readily soluble in aqueous and non- aqueous media such as oils and fats. Accordingly, administration of the sterol becomes possible without any further enhancements to modify its delivery.

R3

R3 may be hydrogen or may convert the parent compound into a salt . The over-riding consideration in the selection of the appropriate salt is that they are acceptable pharmaceutically, nutraceutically or for use in foods, beverages and the like. iSuch salts must have an acceptable anion or cation. Within the scope of the present invention, suitable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, phosphoric, metaphosphorice, nitric, sulfonic and sulfuric acids and organic acids such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glyconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, succinic, toluenesulfonic, and tartaric.

Suitable base salts include ammonium salts, or any salt of a metal, alkali earth metal or alkali metal. Preferably, R3 is selected from one of: calcium, magnesium, manganese, copper, zinc, sodium, potassium and lithium. Most preferably, R3 is sodium.

In a most preferred form of the present invention, the compound is structure 1 noted above, the stanol is sitostanol and R3 is sodium.

Derivative Formation

The formation of the esters of the present invention and their salts is described in PCT/CAOO/00730, the entirety of which is incorporated herein by reference.

Combination with Cholesterol Transport Modulators

In a further embodiment, the derivatives of the present invention may be combined, prior to administration, co-adminstered or administered separately over a time interval with one or more agents or compounds which modulate cholesterol transport. The rationale for this combination is due to the fact that, as CETP increases the HDL level, there may be an increase in the amount of cholesterol being delivered from the peripheral tissues to the liver. This could result in down regulation of the LDL receptors and an increase in the production of apo B containing particles in the liver. Combination of the CETP-inhibitors of the present invention with compounds that inhibit cholesterol reabsorption from the intestine address this problem by maintaining a chemical composition gradient away from the liver thereby preventing increased hepatic lipoprotein synthesis. Suitable compounds which regulate cholesterol absorption include, but are not limited to: bile acid sequestrants, bile acid transporter inhibitors, phytosterols, phytostanols, modulators of ABC transporters, modulators of PPAR (alpha and gamma) and ezetimibe.

The combination of these cholesterol absorption inhibitors and the sterol derivatives of the present invention is synergistic and initiates and perpetuates the beneficial lipid- modulating effects.

Mechanism of Action

Although the precise mechanism of action of the compounds of the present invention on CETP is not certain, there are two possible explanations. These compounds may physically block the association of CETP and lipoproteins thereby preventing CE transfer. Alternatively, the compounds of the present invention may compete with CE for the 26 amino acid hydrophobic binding site of CETP near the -COOH terminus in a manner analogous to TP2 monoclonal antibody competitive binding. Either way, the effect is the same.

Methods of Use

The desired effect of inhibiting CETP-mediated transfer of CE between HDL and LDL may be achieved in a number of different ways. These compounds may be administered by any conventional means available for use in conjunction with pharmaceuticals, nutraceuticals, foods, beverages, and the like.

Without limiting the generality of the foregoing, the derivatives of the present invention may be admixed with various carriers or adjuvants to assist in direct administration or to assist in the incorporation of the composition into foods, beverages, nutraceuticals or pharmaceuticals. In order to appreciate the various possible vehicles of the delivery of the derivatives, the list below is provided. The amount or dose of the compound which is required to achieve the desired CETP-inhibiting effect will, of course, depend on a number of factors such as the particular compound chosen, the mode of administration and the preventing, reducing, eliminating or ameliorating a dyslipidemic condition or disorder.

1 ) Pharmaceutical Dosage Forms:

It is contemplated within the scope of the present invention that the compounds of the present invention may be incorporated into various conventional pharmaceutical preparations and dosage forms such as tablets (plain and coated) for use orally, bucally or lingually, capsules (hard and soft, gelatin, with or without additional coatings) powders, granules (including effervescent granules), pellets, microparticulates, solutions (such as micellar, syrups, elixirs and drops), lozenges, pastilles, ampoules, emulsions, microemulsions, ointments, creams, suppositories, gels, transdermal patches and modified release dosage forms together with customary excipients and/or diluents and stabilizers.

The derivatives of the present invention, adapted into the appropriate dosage form as described above may be administered to animals, including humans, orally, by injection (intravenously, subcutaneously, intra-peritoneally, intra-dermally or intra-muscularly), topically or in other ways.

Preferably, the compounds of the present invention are delivered in the form of phospholipid systems such as liposomes and other hydrated lipid phases, by physical inclusion. This inclusion refers to the entrapment of molecules without forming a covalent bond and is widely used to improve the solubility and subsequent dissolution of active ingredients.

Hydrated lipid systems, including liposomes, can be prepared using a variety of lipid and lipid mixtures, including phospholipids such as phosphatidylcholine (lecithin), phosphodiglyceride and sphingolipids, glycolipids, and the like. The Iipids may preferably be used in combination with a charge bearing substances such as charge-bearing phospholipids, fatty acids, and potassium and sodium salts thereof in order to stabilize the resultant lipid systems. A typical process of forming liposomes is as follows:

1 ) dispersion of lipid or Iipids and the compound of the present invention in an organic solvent (such as chloroform, dichloromethane, ether, ethanol or other alcohol, or a combination thereof). A charged species may be added to reduce subsequent aggregation during liposome formation. Antioxidants (such as ascorbyl palmitate, alpha- tocopherol, butylated hydroxytoluene and butylated hydroxyanisole) may also be added to protect any unsaturated Iipids, if present;

2) filtration of the mixture to remove minor insoluble components;

3) removal of solvents under conditions (pressure, temperature) to ensure no phase separation of the components occur;

4) hydration of the "dry" lipid mixture by exposure to an aqueous medium containing dissolved solutes, including buffer salts, chelating agents, cryoprotectorants and the like; and

5) reduction of liposome particle size and modification of the state of lamellarity by means of suitable techniques such as homogenization, extrusion etc..

Any procedure for generating and loading hydrated lipid with active ingredients, known to those skilled in the art, may be employed within the scope of this invention. For example, suitable processes for the preparation of liposomes are described in references 20 and 21 , both of which are incorporated herein by reference. Variations on these processes are described in US Patent Serial No. 5,096,629 which is also incorporated herein by reference.

US Patent Serial No. 4,508,703 (also incorporated herein by reference) describes a method of preparing liposomes by dissolving the amphiphillic lipidic constituent and the hydrophobic constituent to form a solution and thereafter atomizing the solution in a flow of gas to produce a pulverent mixture.

In another preferred form, the compounds of the present invention can be administered as cyclodextrin complexes. Cyclodextrins are a class of cyclic oligosaccharide molecules comprising glucopyranose sub-units and having a toroidal cylindrical spatial configuration. Commonly available members of this group comprise molecules containing six (alpha- cyclodextrin), seven (beta-cyclodextrin) and eight (gamma-cylcodextrin) glucopyranose molcules, with the polar (hydrophilic) hydroxyl groups oriented to the outside of the structure and the apolar (lipophilic) skeletal carbons and ethereal oxygens lining the interior cavity of the toroid. This cavity is capable of accomodating (hosting) the lipophilic moiety of an active ingredient (the guest molecule, here the derivative of the present invention ) by bonding in a non-covalent manner to form an inclusion complex.

The external hydroxyl substituents of the cyclodextrin molecule may be modified to form derivatives having improved solubility in aqueous media along with other desired enhancements, such as lowered toxicity, etc.. Examples of such derivatives are: alkylated derivatives such as 2,6-dimethyl-beta-cyclodextrin; hydroxyalkylated derivatives such as hydroxypropyl-beta-cyclodextrin; branched derivatives such as diglucosly-beta- cyclodextrin; sulfoalkyl derivatives such as sulfobutylether-beta-cyclodextrin; and carboxymethylated derivatives such as carboxymethyl-beta-cylcodextrin. Other types of chemical modifications, known to those in the art, are also included within the scope of this invention.

The cyclodextrin complex often confers properties of improved solubility, dispersability, stability (chemical, physical and microbiological), bioavailability and decreased toxicity on the guest molecule (here, the derivative of the present invention).

There are a number of ways known in the art to produce a cyclodextrin complex. Complexes may be produced, for example, by using the following basic methods: stirring the one or more derivatves into an aqueous or mixed aqueous-organic solution of the cyclodextrin, with or without heating; kneading, slurrying or mixing the cyclodextrin and the present composition in a suitable device with the addition of an appropriate quantity of aqueous, organic or mixed aqueous-organic liquid, with or without heating; or by physical admixture the cylcodextrin and the composition of the present invention using a suitable mixing device. Isolation of the inclusion complex so formed may be achieved by co- precipitation, filtration and drying; extrusion/ spheronisation and drying; subdivision of the moist mass and drying; spray drying; lyophilization or by other suitable techniques depending on the process used to form the cyclodextrin complex. A further optional step of mechanically grinding the isolated solid complex may be employed.

These cyclodextrin complexes further enhance the solubility and dissolution rate and increase the stability of the derivatives.

It is most preferred that the compounds of the present invention be administered in a program combining oral and intra-venous therapy. However, the precise modes of delivery in each case will depend upon the objectives of the administration protocol i.e. whether it be preventing, reducing, eliminating or ameliorating a dyslipidemic condition or disorder. In the case of existing conditions and disorders, it will depend upon the severity of the disorder, and as discussed above, the age, size and gender of the individual.

2) Foods/Beveraqes/Nutraceuticals:

In another form of the present invention, the derivatives of the present invention may be incorporated into foods, beverages and nutraceuticals, including, without limitation, the following:

1 ) Dairy Products -such as cheeses, butter, milk and other dairy beverages, spreads and dairy mixes, ice cream and yoghurt;

2) Fat-Based Products-such as margarines, spreads, mayonnaise, shortenings, cooking and frying oils and dressings; 3) Cereal-Based Products-comprising grains (for example, bread and pastas) whether these goods are cooked, baked or otherwise processed;

4) Confectioneries-such as chocolate, candies, chewing gum, desserts, non-dairy toppings (for example Cool Whip™), sorbets, icings and other fillings;

5) Beverages- whether alcoholic or non-alcoholic and including colas and other soft drinks, juice drinks, dietary supplement and meal replacement drinks such as those sold under the trade-marks Boost™ and Ensure™; and

6) Miscellaneous Products-including eggs and egg products, processed foods such as soups, pre-prepared pasta sauces, pre-formed meals and the like.

The derivatives of the present invention may be incorporated directly and without further modification into the food, nutraceutical or beverage by techniques such as mixing, infusion, injection, blending, dispersing, emulsifying, immersion, spraying and kneading. Alternatively, the derivatives may be applied directly onto a food or into a beverage by the consumer prior to ingestion. These are simple and economical modes of delivery.

EXAMPLES

The present invention is described by the following non-limiting examples:

Example 1

Objective: To determine if one of the compounds of the present invention, a water-soluble phvtostanol ascorbate (phvtostanol-phosphoryl-ascorbate) referred to also as FM-VP4, modifies CETP-mediated transfer of cholesteryl esters (CE) between HDL and LDL

Lipoprotein Separation:

Plasma was separated into HDL and LDL using density gradient ultracentrifugation, with sodium bromide to adjust plasma density.

Quantification of Plasma Lipids and Proteins:

Enzyme assay kits from Sigma™ were used to determine total and lipoprotein triglyceride, cholesterol and protein concentrations

Experimental design:

Experimental strategies involving the supplementation and inhibition of CETP were used to show the inhibition of CETP-mediated CE transfer between lipoproteins by one of the compounds of the present invention. To determine if phytostanol-phosphoryl-ascorbate modified CE transfer, [³H]cholesteryl oleate ([³H]CE) enriched LDL was co-incubated with increasing concentrations of phytostanol-phosphoryl-ascorbate (10-100uM) in T150 buffer (50uM Tris-HCI, 150mM NaCl, 0.02% sodium azide, 0.01% disodium EDTA), pH 7.4 which contained unlabeled HDL +/- purified (i.e. purified or human plasma source) CETP (1-2ug protein/ml) for 90 minutes at 37°. Following incubation, LDL was precipitated and the percent of CE transferred over 90 minutes was determined. TP2 (4ug protein/ml), a monoclonal antibody directed agianst CETP, and deoxycholate (50um), a known surface active agent, were additional treatment groups co-incubated with the radio-labeled LDL. TP2 was use to ensure that the CE transfer measure was CETP-mediated and deoxycholate was used to demonstrate that the observed effects of phytostanol- phosphoryl-ascorbate were not a non-specific surface active phenomenon

Statisical Anaylsis:

Differences in CETP-mediated CE transfer activity in the presence of different treatment groups was determined by a one-way analysis of variance (PCANOAV; Human Systems Dynamics). Critical differences were assessed by Neuman-Keuls posthoc tests. Differences were considered significant if p was <0.05. All data expressed as +/- standard deviation. Results:

Figure 2 clearly shows the efficacy of phytostanol-phosphoryl-ascorbate at both concentrations tested on the inhibition of purified CETP-mediated transfer of CE between LDL and HDL. At 100 uM, the compound reduced CE transfer to a level below that which was detectable using the assay. Figure 3 corroborates these results using human plasma, as opposed to purified, CETP. Similarly, Figure 4 confirms that the effect of FM- VP4 is via a CETP modulating mechanism, and not by some other means.

Conclusions:

Clearly, the compound inhibits CETP-mediated transfer of CE between LDL to HDL. Transfer of cholesteryl esters between LDL and HDL can occur only when mediated by CETP. Transfer of CE the opposite way, from HDL to LDL can be CETP mediated or can occur by diffusional transfer. For this reason, this study only assessed the unidirectional transfer from LDL to HDL and the inhibition of CETP activity thereon, to ensure that the results truly reflect the effects of the compound on CETP i.e. the diffusional variable was removed. Nonetheless, it is also the CETP-mediated transfer of CE from HDL (the anti- atherogenic lipoproteins) to LDL (the atherogenic lipoproteins) that is favourably inhibited by administration of the compounds of the present invention. TP2 is known specifically to bind CETP at the same location as the CE binding site and these results confirm blockage and inhibitions of CETP-mediated CE transfer.

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Claims

WE CLAIM:

1. A method of reducing the in vivo cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins (HDL) and low density lipoproteins (LDL) which comprises administering a therapeutically effective amount of one or more the following compounds:

π m

wherein R is a sterol or stanol moiety R2 is derived from ascorbic acid and R3 is hydrogen or any metal, alkali earth metal, or alkali metal; and all salts thereof, whereby plasma HDL and LDL are controlled as a result of such administration.

2. The method of claim 1 wherein the sterol is selected from the group consisting of sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, cholesterol and all natural or synthesized forms and derivatives thereof, including isomers.

3. The method of claim 1 wherein the stanol is selected from the group consisting of all saturated or hydrogenated sterols and all natural or synthesized forms and derivatives thereof, including isomers.

4. The method of claim 1 wherein the stanol is sitostanol.

5. The method of claim 1 wherein R3 is selected from the group consisting of calcium, magnesium, manganese, copper, zinc, sodium, potassium and lithium.

6. The method of claim 1 in which the level of serum HDL is increased as a result of such administration.

7. The method of claim 1 in which an atherogenic lipoprotein selected from the group consisting of LDL, VLDL, and IDL is controlled.

8 The method of claim 1 further comprising the step of periodically assaying plasma LDL levels and modifying the amount of the compounds to be administered, if required.

9. The method of claim 1 wherein the compounds are administered orally.

10. The method of claim 1 wherein the compounds are administered intra-venously.

11. The method of claim 1 wherein the compounds are combined prior to administration, co-adminstered or administered separately over a time interval with one or more agents which modulate cholesterol transport.

12. The method of claim 11 wherein the agent is selected from the group consisting of bile acid sequestrants, bile acid transporter inhibitors, phytosterols, phytostanols, modulators of ABC transporters, modulators of PPAR (alpha and gamma) and ezetimibe.

13. A composition for reducing the in vivo cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins and low density lipoproteins which comprises one or more of the structures having the following formulae:

π in

wherein R is a sterol or stanol moiety, R2 is derived from ascorbic acid and R3 s hydrogen or any metal, alkali earth metal, or alkali metal; and all salts of these structures; and a pharmaceutically acceptable carrier therefore.

14. The composition of claim 13 wherein the sterol is selected from the group consisting of sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, cholesterol and all natural or synthesized forms and derivatives thereof, including isomers.

15. The composition of claim 13 wherein the stanol is selected from the group consisting of all saturated or hydrogenated sterols and all natural or synthesized forms and derivatives thereof, including isomers.

16. The composition of claim 13 wherein the stanol is sitostanol.

17. The composition of claim 13 wherein R3 is selected from the group consisting of calcium, magnesium, manganese, copper, zinc, sodium, potassium and lithium.

18. The composition of claim 13 wherein the compound is structure 1 and the stanol is sitostanol.

19. The composition of claim 13 additionally comprising one or more agents or compounds which modulate cholesterol transport..

20. The composition of claim 13 wherein the agent is selected from the group consisting of bile acid sequestrants, bile acid transporter inhibitors, phytosterols, phytostanols, modulators of ABC transporters, modulators of PPAR (alpha and gamma) and ezetimibe.

21. A method of treating or preventing cardiovascular disease and its underlying conditions, including atherosclerosis, dyslipidemic conditions or disorders, hypercholesterolemia, and hypoalphalipoproteinemia in an animal which comprises reducing the in vivo cholesterol ester transfer protein mediated transfer of cholesteryl esters between high density lipoproteins (HDL) and low density lipoproteins (LDL), said reduction being accomplished by administering to the animal one or more sterol and/or stanol derivatives having the following formulae:

π in

wherein R is a sterol or stanol moiety, R2 is derived form ascorbic acid and R3 is hydrogen or any metal, alkali earth metal, or alkali metal; and all salts thereof.

22. The method of claim 21 wherein the animal is human.

23. The method of claim 21 wherein the sterol is selected from the group consisting of sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, cholesterol and all natural or synthesized forms and derivatives thereof, including isomers.

24. The method of claim 21 wherein the stanol is selected from the group consisting of all saturated or hydrogenated sterols and all natural or synthesized forms and derivatives thereof, including isomers.

25. The method of claim 21 wherein the stanol is sitostanol.

26. The method of claim 21 wherein R3 is selected from the group consisting of calcium, magnesium, manganese, copper, zinc.sodium, potassium and lithium.

27. The method of claim 21 wherein the compound is structure 1 and the stanol is sitostanol.