WO2000015201A2 - Compositions comprising one or more phytosterols, phytostanols or mixtures of both and one or more alpha, beta, delta, or gamma tocotrienols or derivatives thereof and use of the compositions in treating or preventing cardiovascular disease, its underlying conditions and other disorders - Google Patents

Abstract

A composition and/or novel structural analogue for use in preventing and treating cardiovascular disease, its underlying conditions and other disorders comprises one or more phytosterols, phytostanols or mixtures of both, and one or more tocotrienols or derivatives thereof.

Description

COMPOSITIONS COMPRISING ONE OR MORE PHYTOSTEROLS, PHYTOSTANOLS OR MIXTURES OF BOTH AND ONE OR MORE ALPHA, BETA, DELTA, OR GAMMA TOCOTRIENOLS OR DERIVATIVES THEREOF AND USE OF THE COMPOSITIONS IN TREATING OR PREVENTING CARDIOVASCULAR DISEASE, ITS UNDERLYING CONDITIONS AND OTHER DISORDERS.

FIELD OF THE INVENTION

This present invention relates to the field of preventing and treating cardiovascular disease and other disorders using phytosterol-based compositions or derivatives

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, the underlying cause of cardiovascular disease ("CVD") has not been sufficiently addressed. 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 a person'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).

Sterols are naturally occurring triterpeπoids that perform many critical cellular functions. Phytosterols such as campesterol, stigmasterol and beta-sitosterol in plants, ergosterol in fungi and cholesterol in animals are each primary components of cellular and sub-cellular membranes in their respective cell types. The dietary source of phytosterols in humans comes from plant materials i.e. vegetables and plant oils. The estimated daily phytosterol content in the conventional western-type diet is approximately 60-80 milligrams in contrast to a vegetarian diet which would provide about 500 milligrams per day.

Phytosterols have received a great deal of attention due to their ability to decrease serum cholesterol levels when fed to a number of mammalian species, including humans. While the precise mechanism of action remains largely unknown, the relationship between cholesterol and phytosterols is apparently due in part to the similarities between the respective chemical structures (the differences occurring in the side chains of the molecules). It is assumed that phytosterols displace cholesterol from the micellar phase and thereby reduce its absorption or possibly compete with receptor and/or carrier sites in the cholesterol absorption process.

Over forty years ago, Eli Lilly marketed a sterol preparation from tall oil and later from soybean oil called Cytellin™ which was found to lower serum cholesterol by about 9% according to one report (5). Various subsequent researchers have explored the effects of sitosterol preparations on plasma lipid and lipoprotein concentrations (6) and the effects of sitosterol and campesterol from soybean and tall oil sources on serum cholesterols (7). A composition of phytosterols which has been found to be highly effective in lowering serum cholesterol is disclosed in US Patent Serial No. 5,770,749 to Kutney et al.and comprises no more than 70% by weight beta-sitosterol, at least 10% by weight campesterol and stigmastanol. It is hypothesized in this patent application (which has already issued to patent in some countries) that there may be some form of synergy between the constituent phytosterols.

It is an object of the present invention to optimize the effects of phytosterols on atherosclerosis and other disorders.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a composition suitable for use alone or for incorporation into foods, beverages, pharmaceuticals, nutraceuticals and the like which comprises one or more phytosterols or phytostanols or mixtures of both and one^or more alpha, beta, delta or gamma tocotrienols or derivatives thereof.

In a second aspect, the present invention provides novel analogues, preferably ethers of phytosterols and/or stanols and alpha, beta, delta or gamma tocotrienols, or derivatives thereof, for use alone or for incorporation into foods, beverages, pharmaceuticals, nutraceuticals and the like.

The present invention further comprises foods, beverages, pharmaceuticals, nutraceuticals and the like which comprise a composition of one or more phytosterols, phytostanols or mixtures of both and one or more alpha, beta, delta or gamma tocotrienols or derivatives thereof or a new structural analogue of phytosterols and/or stanols and alpha, beta, delta or gamma tocotrienols.

The present invention further comprises a method of treating or preventing CVD and its underlying conditions including atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, and related diseases such as Type II diabetes, as well as other diseases that include oxidative damage as part of the underlying disease process such as dementia, aging, and cancer by administering to the animal either a composition or novel structural analogue which comprises one or more phytosterols, phytostanols or mixtures of both and one or more alpha, beta, delta or gamma tocotrienols or derivatives thereof.

The composition and novel structural analogues of the present invention have marked advantages over the phytosterol/stanol compositions previously known and described in the art. In particular and quite surprisingly, it has been found that there is an additive or svnergistic effect between the phytosterol/stanol component and the tocotrienol component of the composition or analogue on the absorption, metabolism and excretion of cholesterol. These effects and other advantages are described in more detail below. PREFERRED EMBODIMENTS OF THE INVENTION

According to one aspect of the present invention, there are provided compositions suitable for use alone or for incorporation into foods, beverages, pharmaceuticals, nutraceuticals and the like which comprise one or more phytosterols, phytostanols or mixtures of both and one or more alpha, beta, delta or gamma tocotrienols or derivatives thereof. According to another aspect of the present invention, there are provided novel structural analogues, preferably ethers, comprising phytosterols or phytostanols and alpha, beta, delta or gamma tocotrienols. The compositions and analogues have been found to have significant effects on the prevention and treatment of CVD, its underlying disorders such as atherosclerosis and hypercholesterolemia and other disorders. In order to understand the possible mechanism of the synergy between the phytosterol/stanol component and the tocotrienol component of the compositions/analogues, it is necessary first to outline what is known about phytosterols and their effects on cholesterol metabolism and likewise, what is known about tocotrienols.

While the precise mechanism of action is unclear, it is known that phytosterols have a beneficial effect on cholesterol homeostasis (absorption, transport, tissue distribution and excretion) in humans.

The intestine and the liver are the primary organs of cholesterol homeostasis in humans. The absorption of dietary cholesterol begins with lipids from the intestine. .Cholesterol and fatty acids are then esterified in intestinal mucosal cells to form non-polar products and arranged with apoprotein to form chylomicrons. Chylomicrons enter the general circulation via the lymphatic system and are hydrolysed by adipose tissue or hepatic lipoprotein lipase into free fatty acids and monoglycerides. The dietary cholesterol transported in chylomicrons is delivered almost entirely to the liver as part of chylomicron remnants which are then processed by hepatocyte cholesterol-7 alpha-hydroxylase into bile acids or excreted unmetabolized. Conversely, phytosterols are not endogenously synthesized in the animal body, therefore, are derived solely from the diet (originating from plants and edible oils) entering the body only via intestinal absorption. Within the intestine, cholesterol absorption is preferred over phytosterols absorption in mammals. For healthy humans, the absorption rate of phytosterols is usually less than 5% of dietary levels which is considerably lower than that of cholesterol which is over 40% (8 and 9). Thus approximately 95% of dietary phytosterols enter the colon. Only 0.3 to 1.7 mg/dl of phytosterols are found in human serum under normal conditions compared with daily dietary intakes of 160 to 360 mg\day but plasma levels have been shown to increase up to two-fold by dietary supplementation (10, 11 and 12). In summary, phytosterol serum levels are low due to poor phytosterol intestinal absorption and rapid biliary elimination.

Phytosterols have two classes of known effects. One effect, referred to here as the "extrinsic" effect is the inhibition of cholesterol absorption from the intestine. Phytosterols compete with cholesterol for the enterocyte shuttle transport from the gut lumen to the lymph or plasma. This transport requires intra-cellular re-assemblance of cholesterol and triglyceride rich microparticle complexes with apoprotein B ("apo B"). In the enterocytes, phytosterols compete with cholesterol for apo B forming more lipophilic, apolar apo B complexes which cause shuttle inhibition and decrease lymphatic cholesterol content. It is also probable that phytosterois/stanols also compete with cholesterol for micellar binding by bile salts. If this is true, it may decrease the presentation of absorbable cholesterol at sites of lumenal absorption and increase the levels of phytosterois/stanols available at these same sites.

The other class of effects are referred to here as "systemic effects." These effects refer primarily to the actions of absorbed phytosterols on the body but may also include effects that are secondary to inhibition of cholesterol absorption. One systemic effect is the stimulation of cholesterol synthesis that results from a feedback response to reduced cholesterol absorption after administration of phytosterols (24, 25. Another "systemic" effect is the reduction of atherosclerotic plaque formation. The mixture of phytosterols and phytostanols described in U.S. Patent Serial No. 5,770,749 are effective in blocking the formation of atherosclerotic plaque in the apo E deficient mouse. Reductions of plaque formation of 50-60% are typically seen after 12-18 weeks of treatment with phytosterols. While the precise mechanism is unknown, it is likely that the reduction in plaque formation results from a combination of the lowering of LDL cholesterol and systemic activities associated with phytosterols including anti- oxidative effects, antiplatelet aggregation, anti-inflammatory activity and estrogenic activity.

Phytosterols are known to have a number of other effects in the capacity of anti- inflammatories, estrogenic agents and anti-coagulatories. Beta-sitosterol has been shown to have anti-inflammatory activity as potent as that of oxyphenbutazone (12a). The cellular mechanism involved in the pathogenesis of atherosclerosis may be similar to those in the inflammatory response. Many of the processes are activated by the arachidonic acid cascade. The estrogenic activity of beta-sitosterol has been shown in rats (12b and 12c). It has been demonstrated that estrogen replacement therapy affords protection against coronary artery disease (12d). With respect to anti-coagulant properties, it has been shown that beta-sitosterol may be protective against thrombosis formation, the precipitating event in heart attacks. Apparently beta-sitosterol stimulates the formation of plasminogen activator in cultured endothelial cells from a bovine carotid artery (12e). Furthermore, beta-sitosterol inhibits platelet aggregation induced by arachidonic acid, ADP and platelet activating factor (12f). In the apo-E deficient mouse model, administration of the phytosterol composition described in US Patent Serial No. 5,770,749 significantly reduced plasma fibrinogen levels

Tocotrienols are a series of farnesylated benzopyran compounds related to but distinct from tocopherols. By way of background, when vitamin E was first being isolated, the term tocopherol was used to denote the initial four compounds sharing a similar structure. These first compounds were designated alpha, beta, delta and gamma tocopherol. Extensive research was then conducted on the beneficial role of vitamin E in reducing the risk of developing heart disease and other debilitating illnesses. Subsequently, a distinct but related group of compounds denoted alpha, beta, delta and gamma tocotrienols was discovered, the Greek letter prefix designating the degree and placement of methyl substitution on the chroman ring. Tocotrienols are less widely distributed in nature than tocopherols and as such have not been as extensively studied. Initially, it was discovered that cereal grains such as barley had a benefical effect on lipid levels in vivo. The active ingedient, based on in vivo and in vitro studies was later found to be alpha- tocotrienol (12g ). Subsequently, a US Patent issued to The Wisconsin Alumni Research Foundation claiming the use of aipha-tocotrienol in lowering lipids: US Patent Serial No.4,603,142 to Qureshi et al.

Each tocotrienol comprises two primary parts: a complex chroman ring structure and a long unsaturated isoprenoid sidechain. Tocotrienols, in contrast to tocopherols do not have saturated side chains.

Some studies over the past five to ten years have suggested that tocotrienols do have a role as powerful anti-oxidants exhibiting anti-cancer and cholesterol-lowering properties in their own right (13, 14). In fact, these beneficial effects of tocotrienols may be greater than those exhibited by tocopherols (15).

It is likely that the mechanism by which tocotrienols exert these beneficial effects is three fold:

1 ) tocotrienols decrease cholesterol synthesis in the liver;

2) tocotrienols decrease the formation of atherosclerotic plaque;

3) tocotrienols, in an anti-oxidant capacity, scavenge free radicals.

With respect to cholesterol lowering, tocotrienols suppress the activity of the cholesterol synthesis rate-limiting enzyme, 3-hydroxy-3-methyl glutaryl coenzyme-A ("HMG-CoA") reductase. (15a). Accordingly, tocotrienols decrease the liver's capacity to manufacture cholesterol. In one double-blind crossover study, serum concentrations of total cholesterol and thromboxane (a potent inducer of platelet aggregation and vasoconstriction) decreased significantly in only those subjects given a natural, palm- derived tocotrienol mix (16). Some of the test subjects had their cholesterol lowered by 31% in four weeks when administered just 200mg of tocotrienol daily. This same study concluded that gamma tocotrienol had the most potent cholesterol-lowering effects of all tocotrienols. With respect to the decreased formation of atherosclerotic plaque, it is likely that tocotrienols reduce the oxidation of low density lipoprotein ("LDL") thereby decreasing macrophage cholesterol accumulation at potential sites of atherosclerotic plaque. One of the features of atherogenesis is the accumulation of cholesterol in the macrophages resident in the arterial walls (17). Macrophages take up oxidized LDL ("ox-LDL") via a scavenger receptor, which is not regulated by cellular cholesterol content, at an enhanced rate in comparison the native, non-oxidized LDL. This results in cellular cholesterol accumulation and foam cell formation (18 and 19). LDL oxidation involves lipid peroxidation, aldehyde formation protein fragmentation and consumption of its vitamin E. Tocotrienols somehow block this oxidative process.

Like tocopherols, tocotrienols exhibit superior anti-oxidative activity (20). Active oxygen species are known to play pivotal roles in the genesis of atherosclerotic plaques, thrombotic episodes, ischemic damage, cancer, aging, dementia and inflammatory conditions. Tocotrienols administered to rats induced with a potent liver cancer agent greatly ameliorating the impact of the agent. Cell damage to the liver was significant in the untreated group as compared to the tocotrienol-treated group (21).

US Patent Serial Nos.5,217,992 and 5,348,974 both to Wright et al., disclose the use of purified tocotrienol, gamma-tocotrienol and delta-tocotrienol in reducing hypercholesterolemia, hyperlipidemia and thromboembolotic disorders in mammals. In particular, it was found that the activities of gamma-tocotrienol and beta-tocotrienol in lowering the risk factors for CVD were greater than the activity of alpha-tocotrienol.

Synergy of Action

In the method of the present invention, CVD, its underlying disorders such as atheroscerosis and hypercholesterolemia and many other oxidative disorders including Alzheimer's disease, cancer, hypertension, inflammatory disorders and Diabetes Type II are treated and/or prevented by administering to an individual a composition or an analogue comprising one or more phytosterols, phytostanols or mixtures of both and one or more alpha, beta, delta or gamma tocotrienols. The beneficial effect of such combined administration on these disorders is greater than would have been expected. In other words, a synergistic effect, heretofore unappreciated, between the components of this composition/analogue has been found. The exact mechanism by which this synergy operates is unclear, although it is suspected that it is at least partially due to the different mechanisms by which phytosterois/stanols and tocotrienols effect the extrinsic and systemic cholesterol pathways i.e. the absorption, catabolism and excretion of cholesterol.

Although the present invention is not limited to any particular mode of action, it is believed that the systemic effects of the tocotrienols perpetuate and enhance the extrinsic effects of the phytosterols and/or phytostanols. As tocotrienols suppress HMG-CoA reductase activity, thereby inhibiting cholesterol biosynthesis and decreasing LDL cholesterol levels, billiary cholesterol secretion is decreased. Concomittantly, the phytosterol/cholesterol intestinal ratio increases which decreases cholesterol absorption and increases phytosterol and/or phytostanol transport via the enterocyte shuttle mechanism. In other words, as less cholesterol is synthesised and secreted, phytosterols and/or phytostanols have less competition at the enterocyte shuttle and are absorbed more readily. Once absorbed, phytosterols and/or phytostanols exert their effects systemically as previously described. Tocotrienols not only exhibit cholesterol lowering and other related effects but synergistically enhance these same effects of phytosterols and/or phytostanols.

A second mechanism that contributes to synergy arises from the difference in the mechanisms of action of phytosterols and tocotrienols in lowering serum cholesterol. The phytosterols act primarily by blocking cholesterol absorption from the intestine. This effect results in a compensatory increase in cholesterol synthesis in the liver which partially offsets the benefit of reduced cholesterol absorption (24, 25). Tocotrienols by blocking HMG-Co A reductase mediated cholesterol synthesis prevent the feed-back stimulated increase in cholesterol synthesis while at the same provide a further reduction in circulating cholesterol levels by blocking cholesterol synthesis. The result is a greater reduction of serum cholesterol than will occur with either phytosterols or tocotrienols alone. At the level of plaque formation, a number of complementary effects occur. Both tocotrienols and phytosterols have anti-oxidative activity and both have anti-platelet aggregation activity. Additionally, phytosterols exhibit anti-inflammatory and estrogenic activity. The tocotrienols by enhancing absorption of phytosterols enhance such activity. The combination of tocotrienols and phytosterols causes a beneficial shift of all of the known factors contributing to the development of atherosclerotic plaque and heart disease and these include: a greater reduction of LDL cholesterol, reduced oxidation of LDL cholesterol, reduced inflammatory response, reduced platelet aggregation and estrogenic activity.

Phytosterols/Phytostanols

As used herein, the term "phytosterol" includes all phytosterols without limitation, for example: sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol and all natural or synthesized forms and derivatives thereof, including isomers. The term "phytostanol" includes all saturated or hydrogenated phytosterols and all natural or synthesized forms and derivatives thereof, including isomers. It is to be understood that modifications to the phytosterols and phytostanols i.e. to include side chains also falls within the purview of this invention. It is also to be understood that this invention is not limited to any particular combination of phytosterols and/or phytostanols forming a composition. In other words, any phytosterol or phytostanol alone or in combination with other phytosterols and phytostanols in varying ratios as required depending on the nature of the ultimate formulation may incorporated with the tocotrienol component. For example, the composition described in PCT/CA95/00555 which comprises no more than 70% by weight beta-sitosterol, at least 10% by weight campesterol and stigmastanol may be used within the scope of the present invention.

The phytosterols and phytostanols for use in 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, sesame oil and fish oils. Without limiting the generality of the foregoing, it is to be understood that there are other sources of phytosterols and phytostanols such as marine animals from which the composition of the present invention may be prepared. 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.

Optionally, the phytosterol or phytostanol may be esterified prior to formation of the composition described herein. This esterification step renders the phytosterols and/or phytostanols more soluble in fats and oils which may, in some instances, facilitate the incorporation of the composition into various delivery systems.

To form phytosterol and/or phytostanol esters, one or more suitable aliphatic acids or their esters with low boiling alcohols are condensed with the phytosterol and/or phytostanol. A wide variety of aliphatic acids or their esters may be used successfully within the scope of the present invention and include all aliphatic acids consisting of one or more alkyl chains with one or more terminal carboxyl groups. These aliphatic acids may be natural or synthetic and are represented by the following chemical formulae:

a) R1-COOH (monocarboxylic acid) wherein:

R1 is an unbranched saturated alky group, represented by CH3-, CH3CH2- or CH3(CH2)nCH2- where n=3-25; or

R1 is a branched saturated alkyl group represented by CnH2n+1 -where n=1-25 is the number of carbon atoms contained in the group R1 ; the branching typically refers, but is not limited to one or more methyl group side chains (branches); or R1 is an unbranched or branched unsaturated alkyl group, represented by the formula CnH2n-2m+1 , where n=1-25 is the number of carbon atoms in R1 and m=degree of unsaturation; or b) HOOC-R2-COOH is a dicarboxylic acid wherein:

R2 is an unbranched saturated alkyl group, represented by -CH2-, or -CH2CH2-, or -CH2(CH2)nCH2 where n=3-25; or

R2 is a branched saturated alkyl group represented by -CnH2n- where n=1-25 is the number of carbon atoms contained in the group R2; the branching typically refers, but is not limited to, one or more methyl group side chains (branches); or R2 is an unbranched or branched unsaturated alkyl group, represented by the formula CnH2n-2m, where n=1 -25 is the number of carbon atoms in R2 and m=degree of unsaturation; or

c) a tricarboxylic acid represented by the formula:

HOOC R3 COOH

COOH wherein, in this formula:

R3 is a branched saturated alkyl group represented by -CnH2n-1 - where n=1 -25 is the number of carbon atoms contained in the group R3; the branching typically refers, but is not limited to, one or more methyl group side chains (branches); or R3 is a branched unsaturated alkyl group, represented by CnH2n-2m-1 - wherein n=1-25 is the number of carbon atoms in R3 and m= the degree of unsaturation; or

d) a mono-, di-, or tricarboxylic acid as defined above, which may contain one, two or three hydroxyl groups in the molecule.

In a preferred form, the aliphatic acid is either a straight-chain or branched unsaturated or saturated fatty acid selected, inter alia, from the following list: valeric acid, isovaleric acid, sorbic acid, isocaproic acid, lauric acid, myristic acid, palmitic acid, stearic acid, caproic acid, ascorbic acid, arachidic acid, behenic acid, hexacosanoic acid, octacosanoic acid, pentadecanoic acid, erucic acid, linoleic acid, linolenic acid, arachidonic acid, acetic acid, citric acid, tartaric acid, palmitoleic acid and oleic acid. The most preferable fatty acids within the scope of the present invention are linoleic acid, linolenic acid, erucic acid and arachidonic acid which may be obtained from natural sources such as safflower oil, sunflower oil, olive oil and corn oil (linoleic acid), safflower oil, sunflower oil, olive oil and jojoba oil (linolenic acid and arachidonic acid) and rapeseed oil (erucic acid).

To form a phytosterol ester in accordance with the present invention, the selected phytosterol and/or stanol and aliphatic acid or its ester with volatile alcohol are mixed together under reaction conditions to permit condensation of the phytosterol and/or stanol with the aliphatic acid to produce an ester. A most preferred method of preparing these esters which is widely used in the edible fat and oil industry is described in US Patent Serial No. 5,502,045 (which is incorporated herein by reference). As no substances other than the free phytosterol, a fatty acid ester or mixture thereof and an interesterification catalyst like sodium ethylate are used, the technique is highly suitable for preparing products ultimately for human consumption. In overview, this preferred method, adapted for use within the present invention, comprises heating the phytosterol (s) with a vegetable oil fatty acid ester (preferably a methyl ester) at a temperature from 90-120°C and subsequently adding a suitable catalyst such as sodium ethylate. The catalyst is then removed/destroyed by any one of the techniques known in the art e.g. adding water and/or filtration/centrifugation.

Another method which may be used in accordance with the present invention is described in US Patent Serial No. 4,588,717, which is also incorporated herein by reference. A preferred method is to mix the phytosterol and the fatty acid together bringing the mixture to a temperature of from about 15°C to about 45°C at about atmospheric pressure for approximately one to three hours.

Tocotrienols

Within the scope of the present invention, any tocotrienol (tocotrienol, alpha-tocotrienol, beta-tocotrienol, delta-tocotrienol and gamma-tocotrienol) alone or together or any derivative thereof may be combined with the phytosterol/phytostanol component to form the desired composition or condensed with the phytosterol/phytostanol component to form the desired ester analogue.

The term "tocotrienols" as used herein encompasses all natural and synthetically-derived tocotrienols, including isomers, racemic mixtures, analogs and derivatives thereof. The richest sources of tocotrienols are cereals such as barley, oats, rice, wheat and rye and vegetable oils such as palm oil, rice bran oil, and olive oil. Within one embodiment of the present invention, it is preferred that tocotrienols isolated from natural sources be used as the active ingredient in the compositions/analogues and method of the present invention. A natural source of tocotrienols is described in the literature, Sodano et al. (22) and is incorporated herein by reference. Similarly, the procedures as described in detail in 22a, 22b, and 22c, (all of which are incorporated herein by reference) may be used to obtain the tocotrienols for use within the scope of the present invention.

Suitable methods of preparing synthetic tocotrienols are also known to those in the art inclduing Kato et al. (23), which is suitable for the synthesis of gamma-tocotrienol and US Patent Serial No. 5,217,992, to Wright et al., which describes the synthesis of alpha, beta and delta-tocotrienols and which is incorporated herein by reference. Similarly, US Patent Serial No. 5,393,776 to Pearce, incorporated herein by reference, discloses the preparation of tocotrienol analogs which may be suitably used in the composition/analogue of the present invention.

In one embodiment of the present invention, the actual vegetable oil comprising tocotrienols may be mixed directly with the phytosterol and/or phytostanol component to form the composition. The most preferred of these vegetable oils is rice bran oil which is rich in tocotrienols (23a). In another embodiment, the tocotrienols may be extracted from the natural source prior to mixing with the phytosterol and/or phytostanol component. In yet another embodiment, the tocotrienols are synthesized using the techniques described above prior to mixing with the phytosterol and/or phytostanol component.

There are a number of tocotrienol-based nutritional supplements available on the market which may be used within the scope of the present invention in combination with the phytosterol and/or stanol component. For example, NuTriene™, developed by the Eastman Chemical Company which comprises naturally occurring tocotrienols and tocopherols and Tocotrienol Complex Genesis 6:21™, developed by Covenant Products may suitably be used.

Novel Analogues

The analogues of the present invention are represented by the following formula:

R2

R 3- -0 R

wherein R is a phytosterol or phytostanol moiety; R2 is oxygen or hydrogen (H2) and R3 is an alpha, beta, delta or gamma tocotrienol or derivative thereof.

The general formula of the present invention noted above defines phytosterol/phytostanol-based esters or ethers.

Ether Formation

To form the ether derivative, one preferred method is to convert the phenolic OH on the phytosterol/phytostanol to halogen by reacting the sterol/stanol with PX3, wherein X is selected from Br, Cl or I; reacting the tocotrienol with a strong base; then condensing the two components under suitable reaction conditions.

Another method involves the formation of the alkoxide form of the phytosterol/phytostanol by reaction of the latter with a strong base such a sodium hydride, sodium amide, sodium alkoxide, lithium diisopropyl amide, in an anhydrous inert solvent such as diethyl ether, tetrahydrofuran or benzene, toluene or similar aromatic solvent. The resulting anionic phytosterol/phytostanol derivative is then condensed with a suitable derivative of the tocotrienol. In general, the latter derivative is formed by reduction of the tocotrienol to the corresponding primary alcohol and the latter is transformed to the halide by reaction of the alcohol with thionyl chloride, phosphorus trihalide, phosphorus pentahalide or by treatment with mineral acid. Condensation of these two units in an inert anhydrous solvent such as diethyl ether, tetrahydrofuran or benzene, toluene or similar aromatic solvent generally at room or elevated temperature, results in the desired ether formation.

Delivery Systems

Although it is fully contemplated within the scope of the present invention that the compositions and/or analogues may be administered directly and without any further modification, it is preferred and greater efficacy is achieved when the compositions are treated to ensure even distribution throughout the food, beverage, pharmaceutical, nutraceutical and the like to which they are added. Such enhancement may be achieved by a number of suitable means such as, for example, solubilizing or dispersing the compositions to form emulsions, solutions and dispersions or self-emulsifying systems; reducing the particle size by mechanical grinding (milling, micronisation etc.), lyophilizing, spray drying, controlled precipitating, or a combination thereof; forming solid dispersions, suspensions, hydrated lipid systems; forming inclusion complexations with cyclodextrins; and using hydrotopes and formulations with bile acids and their derivatives.

Prior to the solubiiity/dispersability enhancement techniques of the present invention, it is preferred that the phytosterol and/or phytostanol component be isolated from the source and formed into a solid powder through precipitation, filtration and drying, spray drying, lyophilization or by other conventional work-up techniques. This powder form may be added to the tocotrienol component and then physically modified as described below to enhance the solubility and dispersability of the phytosterol(s) and/or phytostanol(s) in the chosen delivery medium.

Each of the techniques which may be used in accordance with the present invention are described below. It is to be understood that the term "composition" can be used interchangeably with the term "anaolgue". Emulsions

Emulsions are finely divided or colloidal dispersions comprising two immiscible phases, e.g. oil and water, one of which (the internal or discontinuous phase) is dispersed as droplets within the other (external or discontinuous phase). Thus an oil-in-water emulsion consists of oil as the internal phase, and water as the discontinuous or external phase, the water-in-oil emulsion being the opposite. A wide variety of emulsified systems may be formed which comprise phytosterols or phytostanols or mixtures thereof and tocotrienols or derivatives thereof including standard emulsions, microemulsions and those systems which are self-emulsifying (emulsify on exposure to agitated aqueous fluids such as gastric or intestinal fluids).

Generally, emulsions may include oil and water phases, emulsifiers, emulsion stabilizers and optionally preservatives, flavouring agents, pH adjusters and buffers, chelating agents, antifoam agents, tonicity adjusters and anti-oxidants. Suitable emulsifiers (wherein bracketed numerals refer to the preferred HLB values) include: anionic surfactants such as alcohol ether sulfates, alkyl sulfates (30-40), soaps (12-20) and sulfosuccinates; cationic surfactants such as quaternary ammonium compounds; zwitterionic surfactants such as alkyl betaine derivatives; amphoteric surfactants such as fatty amine sulfates, difatty alkyl triethanolamine derivatives (16-17); and nonionic surfactants such as the polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, saturated fatty acids and alkyphenols, water-soluble polyethyleneoxy adducts onto polypropylene glycol and alkyl polypropylene glycol, nonylphenol polyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxy- polyethoxyethanol, polyethylene glycol, octylphenoxy-poiyethoxyethanol, lanolin alcohols, polyoxyethyiated (POE) alkyl phenols(12-13), POE fatty amides, POE fatty alcohol ethers, POE fatty amines, POE fatty esters, poloxamers (7-19), POE glycol monoethers (13-16), polysorbates (17-19) and sorbitan esters (2-9). This list is not intended to be exhaustive as other emulsifiers are equally suitable.

Appropriate emulsion stabilizers include, but are not limited to, lyophilic colloids such as polysaccharides, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, tragacanth, xanthan gum; amphoterics (e.g. gelatin) and synthetic or semi-synthetic polymers (e.g. carbomer resins, cellulose ethers and esters, carboxymethyl chitin, polyethylene glycol-n (ethylene oxide polymer H(OCH2CH2)nOH); finely divided solids including clays (e.g. attapulgite, bentonite, hectorite, kaolin, magnesium aluminum silicate and montmorillonite), microcrystalline cellulose oxides and hydroxides (e.g. aluminum hydroxide, magnesium hydroxide and silica); and cybotactic promoters/gel lants (including amino acids, peptides, proteins lecithin and other phospholipids and poloxamers).

Suitable anti-oxidants for use in the formation of emulsions include: chelating agents such as citric acid, EDTA, phenylalanine, phosphoric acid, tartaric acid and tryptophane; preferentially oxidized compounds such as ascorbic acid, sodium bisulfite and sodium sulfite; water soluble chain terminators such as thiols and lipid soluble chain terminators such as alkyl gallates, ascorbyl palmitate, t-butyl hydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, hydroquinone, nordihydroguaiaretic acid and alpha-tocopherol. Suitable preservatives, pH adjustment agents, and buffers, chelating agents, osmotic agents, colours and flavouring agents are discussed hereinbelow under "Supensions", but are equally applicable with respect to the formation of emulsions.

The general preparation of emulsions is as follows: the two phases (oil and water) are separately heated to an appropriate temperature, the same in both cases, generally 5- 10°C above the melting point of the highest melting ingredients in the case of a solid or semi-solid oil, or where the oil phase is liquid, a suitable temperature as determined by routine experimentation). Water-soluble components are dissolved in the aqueous (water) phase and oil-soluble components such as tocotrienols or derivatives thereof, are dissolved in the oil phase. To create an oil-in water emulsion, the oil phase is vigorously mixed into the aqueous phase to create a suitable dispersion and the product is allowed to cool at a controlled rate with stirring. A water-in-oil emulsion is formed in the opposite fashion i.e. the water phase is added to the oil phase. When hydrophilic colloids are a part of the system as emulsion stabilizers, a phase inversion technique may be employed whereby the colloid is mixed into the oil phase rather than the aqueous phase, prior to addition to the aqueous phase. In using any phytosterol or phytostanols or mixtures thereof, it is preferred to add these to the oil phase prior to heating.

Microemulsions, characterized by a particle size at least an order of magnitude smaller (10-100 nm) than standard emulsions and defined as "a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid" (26), may also be formed comprising phytosterols or phytostanols or mixtures thereof and tocotrienols or derivatives thereof. In a preferred form, the microemulsion comprises a surfactant or surfactant mixture, a co-surfactant (usually a short chain alcohol) the chosen phytosterols or phytostanols or mixtures thereof, tocotrienols or derivatives thereof, water and optionally other additives.

This system has several advantages as a delivery system for the phytosterols or phytostanols or mixtures thereof having relatively high lipophilicity. Firstly, microemulsions tend to be created spontaneously, that is, without the degree of vigorous mixing required to form standard emulsions. From a commercial perspective, this simplifies the manufacturing process. Secondly, microemulsions may be sterilized using microfiltration techniques without breaking the microstructure due to the small diameter of the microdroplets. Thirdly, microemulsions are highly thermodynamically stable. Fourthly, microemulsions possess high solubilizing power which is particularly important as they allow for an increased solubilization of the poorly hydrosoluble phytostanols and phytosterols.

Surfactant or surfactant mixtures which are suitable for use in the formation of microemulsions can be anionic, cationic, amphoteric or non-ionic and possess HLB (hydrophile-lipophile balance) values within the range of 1-20, more preferably in the ranges 2-6 and 8-17. Especially preferred agents are non-ionic surfactants, selected from the group consisting of polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, saturated fatty acids and alkyphenols, water-soluble polyethyleneoxy adducts onto polypropylene glycol and alkyl polypropylene glycol, nonylphenol poiyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxy-polyethoxyethanol, polyethylene glycol, octylphenoxy- polyethoxyethanol, lanolin alcohols, polyoxyethylated (POE) alkyl phenols (12-13), POE fatty amides, POE fatty alcohol ethers, POE fatty amines, POE fatty esters, poloxamers (7-19), POE glycol monoethers (13-16), polysorbates (10-17) and sorbitan esters (2-9).

There are a number of methods known and used by those skilled in the art for making microemulsions. In a preferred method of forming microemulsions of the present invention, a surfactant, a co-surfactant, and phytosterols or phytostanols or mixtures thereof (pre-dissolved in a suitable proportion of an appropriate oil along with the tocotrienaol component) are mixed and then titrated with water until a system of desired transparency is obtained.

In a further preferred embodiment, the formation of microemulsions may be achieved by mixing the phytosterols or phytostanols or mixtures thereof and tocotrienols or derivatives thereof with hydrotropic agents and food-grade surfactants (refer to 27).

Solutions and Dispersions

Phytosterols or phytostanols or mixtures thereof along with tocotrienols or derivatives thereof may be dissolved or dispersed in a suitable oil vehicle, with or without additional excipients, and used in this form, for example, in general food usage, in basting meats and fish, and for incorporation into animal feeds.

Suitable solubilizing agents include all food grade oils such as plant oils, marine oils (such as fish oil) and vegetable oils, monogiycerides, diglycerides, triglycerides, tocopherols and the like and mixtures thereof.

Self-Emulsifying Systems

Phytosterols or phytostanols or mixtures thereof along with tocotrienols or derivatives thereof may be mixed with appropriate excipients, for example, surfactants, emulsion stabilizers (described above) and the like, heated (if necessary) and cooled to form a semi-solid product capable of forming a spontaneous emulsion on contact with aqueous media. This semi-solid product may be used in numerous other forms such as filler material in two-piece hard or soft gelatin capsules, or may be adapted for use in other delivery systems.

Reducing Particle Size

Many techniques of particle size reduction are suitable for use within the present invention including, inter alia, dry and wet milling, micropulverization, dry or wet fluid energy size reduction, controlled precipitation, lyophilisation and spray-drying. Each of these techniques is well known in the art and will not be discussed in any detail other than to provide reference to 28 and 29, the former showing preferred processes of spray- drying and the latter summarizing the other techniques listed above.

It has been found that reducing the particle size to under 500um and most preferably under 20um allows suitable dispersability/solubility of the phytosterol or phytostanol or mixture thereof along with the tocotrienol component in the carriers and dosage forms described further below.

Solid Dispersions

An alternative means of increasing the solubility/dispersability of phytosterols or phytostanols or mixtures thereof along with tocotrienols or derivatives thereof involves the use of solid dispersion systems. These dispersions may include molecular solutions (eutectics), physical dispersions or a combination of both.

For example, solid dispersions may typically be prepared by utilizing water-soluble polymers as carriers. Without limitation, these carriers may include, either alone or in combination: solid grade polyethylene glycois (PEG's), with or without the addition of liquid grade PEG's; polyvinylpyrrolidones or their co-polymers with vinyl acetate and cellulose ethers and esters. Other excipients, such as additional members of the glycol family e.g. propylene glycol, polyols, e.g. glycerol etc.. may also be included in the dispersions.

Solid dispersions may be prepared by a number of ways which are familiar to those in the art. These include, without limitation, the following methods:

(a) fusing the ingredients, followed by controlled cooling to allow solidification and subsequent mechanical grinding to produce a suitable powder. Alternatively, the molten (fused) dispersion may be sprayed into a stream of cooled air in a spray drier to form solid particles (prilling) or passed through an extruder and spheroniser to form solid masses of a controlled particle size. In a further alternative, the molten dispersion is filled directly into two-piece hard gelatin capsules; (b) dissolving the ingredients in a suitable solvent system (organic, mixed organic, organic-aqueous) and then removing the solvents e.g. by evaporating at atmospheric pressure or in vacuo, spray drying, lyophilizing and the like; or, in a variation of the foregoing, and

(c) dissolving the ingredients in a suitable solvent system, subsequently precipitating them from solution by the use of an immiscible solvent in which the ingredients have little or no solubility, filtration, removing the solvent, drying and optionally grinding to provide a suitable powder form.

Other commercially available agents for enhancing solubility of the phytosterols or phytostanols or mixtures thereof along with tocotrienols or derivatives thereof through the formation of solid dispersions are considered to fall within the purview of this application. For example, the commercial excipient marketed under the trade-mark Gelucire™ by Gattefosse comprising saturated polyglycolised glycerides may readily be used herein.

Suspensions

Suspensions, which may be used to enhance the solubility and/or dispersability of phytosterols or phytostanols or mixtures thereof along with tocotrienols or derivatives thereof , comprise a solid, perhaps finely divided, internal phase dispersed in an oily or aqueous external phase (the vehicle). In addition, the solid internal phase may be added to an emulsion as described above during its' formation to produce a delivery system having properties common to both suspensions and emulsions.

As tocotrienols are not soluble in an aqueous phase, additional steps must be taken in order to convert the oily tocotrienol component into a solid powder form suitable for incorporation into an aqueous phase. For example, the tocotrienol component may be spray dried along with a suitable carrier to produce a solid powder which can then be mixed with the phytosterol/stanol component in an aqueous suspension. Alternatively, the tocotrienol component may be absorbed onto a suitable base, optionally spray dried and then mixed with the phytosterol/stanol component in an aqueous suspension. Examples of bases include hydrophobic or hydrophilic colloidal silicone dioxides. These and other appropriate bases absorb the oily tocotrienol and allow dispersion of the tocotrienol in an aqueous supension.

In the case of an oily external phase, the tocotrienol component may be dissolved first, followed by the phytosterol/stanol component, without the need for additional steps.

Numerous excipients, which are commonly used in the art, may be suitable for producing a suspension within the scope of the present invention. Typically, a suspension comprises an oily or aqueous vehicle, the dispersed (suspended) internal phase, dispersing and/or wetting agents (surfactants), pH adjustment agents/buffers, chelating agents, antioxidants, agents to adjust ionic strength (osmotic agents) colours, flavours, substances to stabilize the suspension and increase viscosity (suspending agents) and preservatives.

Appropriate vehicles include, but are not limited to: water, oils, alcohols, polyols, other edible or food grade compounds in which the phytosterol composition is partially or not soluble and mixtures thereof. Appropriate dispersing agents include, but are not limited to: lecithin; phospholipids; nonionic surfactants such as polysorbate 65, octoxynol-9, nonoxynol-10, polysorbate 60, polysorbate 80, polysorbate 40, poloxamer 235, polysorbate 20 and poloxamer 188; anionic surfactants such as sodium lauryl sulfate and docusate sodium; fatty acids, salts of fatty acids, other fatty acid esters, and mixtures thereof.

Agents/buffers for pH adjustment include citric acid and its salts, tartaric acid and its salts, phosphoric acid and its salts, acetic acid and its salts, hydrochloric acid, sodium hydroxide and sodium bicarbonate. Suitable chelating agents include edetates (disodium, calcium disodium and the like), citric acid and tartaric acid. Suitable antioxidants include ascorbic acid and its salts, ascorbyl palmitate, tocopherols (especially alpha-tocopherol), butylated hydroxytoluene, butylated hydroxyanisole, sodium bisulfite and metabisulfite. Suitable osmotic agents include monovalent, divalent and trivalent electrolytes, monosaccharides and disaccharides. Suitable preservatives include parabens (Me, Et, Pr, Bu and mixtures thereof), sorbic acid, thimerosal, quaternary ammonium salts, benzyl alcohol, benzoic acid, chlorhexidine gluconate and phenylethanol. Colours and flavours may be added as desired and may be selected from all natural, nature-identical and synthetic varieties.

Hydrated Lipid Systems

In a further embodiment of the present invention, the solubility/dispersability of the compositions of the present invention may be enhanced by the formation 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 lipids 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 lipids and the phytosterols or phytostanols or mixtures thereof and the tocotrienol component 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 lipids, 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 δf 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 30 and 31 , 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.

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 composition 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-cyicodextrin. 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 composition 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 phytotsterol, phytostanol or mixture thereof 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 enhance the solubility and dissolution rate and increase the stability of the phytosterols or phytostanols or mixtures thereof. For a review of cyclodextrin complexation, please refer to 32.

Complexation with Bile Salts

Bile acids, their salts and conjugated derivatives, suitably formulated, may be used to solubilize the compositions of the present invention, thereby improving the solubility and dispersion characteristics of these compositions. Examples of suitable bile acids include: cholic acid, chenodeoxycholic acid, deoxycholic acid, dehydrocholic acid, and lithocholic acid. Examples of suitable bile salts include: sodium cholate, sodium deoxycholate and their other salt forms. Examples of suitable conjugated bile acids include: glycochenodeoxycholic acid, glycholic acid, taurochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid and their salts. A suitable system for solubilizing the composition of the present inventionconslsts of the sterol and/or stanol component, the tocotrienol component plus one or more bile acids, salts or conjugated bile acids. Further materials may be added to produce formulations having additional solubilization capacity. These materials include, but are not limited to: phospholipids, glycolipids and monoglycerides. These ingredients may be formulated either in the solid phase or by the use of suitable solvents or carrier vehicles, with appropriate isolation and, optionally, particle size reduction using techniques described hereinabove.

Since bile acids and their derivatives have an unpleasant taste and may be irritating to the mucous membranes of the stomach and upper regions of the gastro-intestinal tract, a suitable enteric coating may be applied to the solid formulation particulates, using techniques known to those skilled in the art. Typical enteric coatings include, inter alia: cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinylacetate phthalate, acrylate polymers and their derivatives (e.g. appropriate members of the Eudragit™ series), ethylcellulose or combinations thereof. Additional excipients may be added to the coating formulation to modify membrane functionality or to aid in the coating process (e.g. surfactants, plasticisers, channeling agents, permeability modifiers and the like). Coating formulation vehicles may comprise aqueous or organic systems, or mixtures of both.

Hydrotropic Complexation

Compounds which are capable of opening up the water structure associated with hydrophobic (lipophilic) and other molecules are referred to as hydrotropes. These compounds may be used to enhance the aqueous solubility of poorly water-soluble substances such as phytosterols, phytostanols and their esters. Examples of hydrotopes include, inter alia, sodium benzoate, sodium hydroxybenzoates, sodium salicylate, nicotinamide, sodium nicotinate, sodium gentisate, gentisic acid ethanolamide, sodium toluates, sodium aminobenzoates, sodium anthranilate, sodium butylmonoglycolsulfate, resorcinol and the like.

Complex formation, which is non-covalent in nature, may be achieved by mixing appropriate ratios of the phytosterols or phytostanols or mixtures thereof, the tocotrienols or derivatives thereof and the hydrotope or mixtures thereof in a suitable liquid^" vehicle, which may be aqueous, organic or a combination of both. Additional excipients such as surfactants, polyols, disaccharides etc.. may be added to facilitate complexation or to aid in dispersability. The resultant complex is isolated as a dry powder by any process known in the art (co-precipitation and drying, evaporation of the liquid vehicle, spray drying, lyophilization etc.). Particle size may be reduced by any standard technique such as those described previously herein, if desired. The resultant hydrotope complex may be used without further modification or may be compounded into a variety of other formulations or vehicles as required.

Methods of Use

The composition of the present invention may be administered to animals, in particular humans, directly and without further modification or may be treated to enhance the solubility and/or dispersability of the composition as described in detail above. Alternatively, and optionally in conjunction with any one of these solubility and/or dispersability enhancement methods, the composition may be incorporated into various vehicles as described further below in order to treat and/or prevent CVD, its underlying conditions such as hypercholesterolemia, hyperlipidemia, arteriosclerosis, hypertension, thrombosis, related diseases such as Type II diabetes, as well as other diseases that include oxidative damage as part of the underlying disease process such as dementia, aging, and cancer. In populations, which are considered "high-risk" for CVD or any of the oxidation related disorders, it is contemplated that the composition of the present invention be used in primary, secondary and tertiary treatment programs.

Without limiting the generality of the foregoing, the composition 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 composition, the examples below are provided. The ratios and concentrations of the tocotrienol and phytosterol and/or stanol components of the composition will vary depending upon, among other factors, the mode of delivery, the patient size and condition, the result to be achieved, as well as other factors known to those skilled in the art of food additives and medicinal agents. Generally, however, it is preferred that the composition of the present invention be administered to humans in a form comprising up to 6 grams of phytosterols and/or phytostanols and up to 4 grams of the tocotrienol component per day.

In a most preferred form, the composition, for daily administration, comprises from 0.1 to 3.0 grams of the phytosterols and/or stanol component, from 0.05 to 1.0 grams of the tocotrienol component.

1 ) Pharmaceutical Dosage Forms:

It is contemplated within the scope of the present invention that the composition 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 composition 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. Although the precise mechanism of action is unclear, the composition of the present invention, administered intra-venously, lowers serum cholesterol. It is believed that certain phytosterol-based compositions may have, in addition to the role as an inhibitors of cholesterol absorption in the intestine, a systemic effect on cholesterol homeostasis through bile acid synthesis, enterocycte and biliary cholesterol excretion, bile acid excretion and changes in enzyme kinetics and cholesterol transport between various compartments within the body (PCT/CA97/00474 which was published on January 15, 1998). See also paper to Peter Jones (under publication). 2 ) Foods/Be veraαes/N utraceutical s :

In another form of the present invention, the composition 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 composition 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 composition 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

Example 1 Softgel Capsule Dosage Form.

Phytosterols finely divided (micronized), were mixed with a dispersing/emulsifying agent, lecithin, in combination with a medium chain mono/di/triglyceride, an edible oil carrier and the tocotrienol component. Depending on the purpose for which the combination product is used, the necessary dosage was supplied in one or two capsules taken with each meal.

Example 2 Oral Microemulsion.

Phytosterols, which were finely divided (micronized) and the tocotrienol component were mixed with appropriate excipients, to form a self-emulsifying drug delivery system which presented itself as a microemulsion in the gastrointestinal fluids. Suitable excipients comprised a blend of medium chain mono- and diglycerides having HLB values within the range 2-7, e.g. the CAPMUL (trademark) series; a medium chain triglyceride, e.g. a member of the CAPTEX (trademark) series; a high HLB emulsifier (HLB value 10-16), e.g. polysorbate 20 ; an edible oil and water. Appropriate flavouring agents, preservatives and anti-oxidants were also incorporated. Depending on the purpose for which the combination product is used, the necessary dosage would be supplied in 5-10 mL of preparation taken with each meal.

Example 3 Hard Gelatin Capsule and Tablet Dosage Form.

Phytosterols, which were finely divided (micronized) and the tocotrienol component were dissolved/dispersed in a suitable organic solvent, e.g. a mixture of chloroform or dichloromethane and an alcohol, e.g. methanol or ethanol. An appropriate dispersing/emulsifying agent, e.g. lecithin, and colloidal silicon dioxide were blended into the mixture. A suitable anti-oxidant, selected from ascorbyl palmitate, butylated hydroxytoluene, and alpha tocopherol, was also added. The resultant formulation was spray dried, to remove the solvent vehicle, and blended with suitable disintegrants, e.g. croscarmellose sodium; lubricants, e.g. magnesium stearate, talc; glidants, e.g. colloidal silicon dioxide and diluents, e.g. microcrystalline cellulose, lactose. The blend could then suitably be filled into two-piece hard gelatin capsules or compressed into tablets, as desired. Depending on the purpose for which the combination product is used, the necessary dosage would be supplied in one or two units taken with each meal.

Example 4 Dietary Food Bar

Grain-based bars with various food ingredients, flavourings with or without preservatives are prepared and designed to be consumed as either snacks or additions to regular meals. One to three such bars would supply the total dose of phytosterols and tocotrienols needed for efficacy/or prevention of disease. In the same bar, some additional benefit might be obtained by inclusion of omega-3 fatty acids

Example 5 Dietary Chocolates

These are be targeted specifically for Type II diabetics which besides having elevated cholesterol are very susceptible to cardiovascular disease. However, the same dosage forms could be taken by non-diabetic hypercholesterolemics. Chocolate dosage forms would be approximately 10 g weight containing cocoa butter, vegetable and dairy fat, an artificial sweetener such as maltilol, flavourings & fillers, phytosterols and tocotrienols. One to three such chocolates would supply the total daily dose needed for efficacy/or prevention of disease.

Compositions comprising one or more phytosterols, phytostanols or mixtures of both and one or more alpha, beta, delta, or gamma tocotrienols or derivatives thereof and use of the compositions in treating or preventing cardiovascular disease and other disorders

Example 6 Formulations

In order to evaluate the applicability of various formulation approaches for phytosterols ( hereinafter may be referred to as "FCP-3P1") and tocotrienols, examples of potential formulae were investigated. Unless otherwise stated, FCP-3P1 Batch FM-PH-42 (composition: campesterol 14.35%, campestanol 3.07%, β-sitosterol 54.67%, and sitostanol 15.76%) was used in the formulation work. Content uniformity data was referenced to the total phytosterol content of the batch, ie 87.85%.

The tocotrienol oil used in this study (Tocomin™ 50%) was provided by H. Reisman Corporation, Orange, New Jersey (Lot No. B888-2-300399). Literature accompanying the product stated that it contained, among other components, alpha-tocopherol 11.8%, alpha-tocotrienol 12.2%, gamma-tocotrienol 21.0%, and delta-tocotrienol 5.9%.

Macroemulsion formulation

A 10% w/w solution of FCP-3P1 was prepared by adding 1.39227 g of FCP-3P1 to 386.91 mg of Tocomin™ 50% and 13.20822 g soybean oil and heating to 63° C to give a clear solution. The surfactant, Span 60 [polyoxyethylene-(20)-sorbitan monostearate], was added (0.82811 g). This constituted the oil phase. This surfactant has a Hydrophile- Lipophile Balance (HLB) value of 4.7 ± 1.0.

The aqueous phase consisted of a 15 mL solution of 0.84233 g Tween 40 [polyoxyethylene-(20)-sorbitan monopalmitate] and 22.33 mg EDTA (ethylene diamine tetraacetic acid) in distilled de-ionized water, 31.53 mg methyl paraben and 33.24 mg propyl paraben. Tween 40 has an HLB value of 15.6 ± 1.0.

Both oil and aqueous phases were individually heated to 70° C, combined and vigorously mixed using a Polytron Model PCV li mixer, on the high speed setting, for 1 minute. The product was left to cool to ambient temperature. This gave an oil in water emulsion, with an oil (dispersed) phase of 40% in an aqueous continuous phase, containing a dual surfactant system having an overall HLB of 10.0 ± 1.0 and an active loading of ca 4% w/v.

Drug was in the oil phase.

Phase Separation Assessment

15 mL of emulsion was poured into a graduated centrifuge tube, which was subsequently sealed. Daily visual inspection over 4 days indicated no phase separation.

PH

The measured pH of the system was 5.35. Oil Phase Droplet Size

This parameter was evaluated using an optical microscope equipped with a calibrated eyepiece under polarizing conditions. Sample preparation involved diluting 1 part of emulsion with 4 parts water and examining on a microscope slide, under a cover slip, at 400x magnification. The dispersed oil phase consisted of droplets ranging from ca 2.5 - 20 microns and no evidence of FCP-3P1 crystallization was observed. Small droplets (< 2.5 microns diameter) outnumbered larger droplets ( 2.5 - 20 microns) by roughly a 300:1 ratio.

FCP-3P1 Content Uniformity Determination

This was assessed on 6 samples, removed from the bulk according to a pre-determined sample plan. Each sample (0.5 mL) was extracted by vortexing for 10 minutes with dichloromethane (DCM, 5 mL), followed by centrifugation at 4000 rpm for 2 min to separate the two phases. The analytical sample was withdrawn from the DCM layer and assayed by GC-FID, using a cholestane internal standard. Results are reported in Table

1.

Table 1: Content uniformity of FCP-3P1 in macroemulsion formulation

* Reflects total of major phytosterols only (campestanol + campesterol + β-sitosterol + sitostanol = 87.85% of sample weight)

Content uniformity is acceptable (36.03 ± 2.44 mg/mL) and indicates satisfactory emulsion homogeneity. This dosage delivery system has successfully incorporated both FCP-3P1 Tocomin 50% in a single formulation.

Self emulsifying drug delivery system formulation

The self emulsifying drug delivery system (SEDDS) was designed such that dilution of the oily formulation with aqueous solvent would produce an emulsion with only moderate agitation. An oily formulation was made up consisting of 3% FCP-3P1 by combining 309.20 mg of FCP-3P1 with 167.20 mg Tocomin™ 50%, 5.2042 g Capmul MCM, and 4.5608 g Tween 80. These were mixed together in a 125 mL Erlenmeyer flask, and heated at 55° C in a sonicatting water bath for 20 minutes to obtain a clear mixture. A magnetic stir bar was used to agitate the SEDDS while normal saline was added to the formulation in quantities of between 2 - 5 mL per addition. After each addition the formulation was observed for evidence of emulsion formation. After 15 mL and after 30 mL of aqueous phase (oil:aqueous 2:3 and 1 :4 respectively) had been added, a small sample was removed for microscopic examination.

Phase separation assessment

Initial additions of aqueous solution (up to 20 mL) produced a 2 phase liquid which could be described as yellow, opaque and viscous. Further addition of aqueous solution resulted in a reduction in the viscosity of the formulation (assessed by observing the ease with which the stir bar could move) and the formulation becoming progressively more translucent.

Following the additon of 30 mL of aqueous solution (25% oil in water) the formulation was allowed to stand at room temperature while being agitated with the magnetic stir bar. Over 20 minutes a clear liquid was obtained. On standing overnight without agitation, the formulation became translucent.

Microscopic observation

Samples were viewed under the microscope when the oil: aqueous ratios were 2:3 and 1 :4. Samples were prepared by placing a drop of the formulation on the microscope slide and covering it with a cover slip prior to viewing using a 400X lens. No droplets could be seen, and no evidence of separate phases (neither oil nor crystals) were observed:

This dosage formulation has successfully incorporated FCP-3P1 and Tocomin™ 50% in a single dosage form, which, when exposed to an aqueous environment with only mild agitation at room temperature, spontaneously results in a single phase solution.

Microemulsion formulation

The formation of a clear, one phase system in the SEDDS formulation indicated that a microemulsion, albeit an unstable one, had been formed. Adjustments to the formulation could be made to increase the stability of the system as a microemulsion.

Example 7 Solutions and dispersions (oil-based)

An oil-based soft gelatin capsule formulation was obtained by taking the oil phase from the macroemulsion, with or without modification, and filling the solution into a soft gelatin capsule. Potential modifications could include increasing the content of FCP-3P1, by forming a dispersion or paste; altering the ratio of FCP-3P1 to tocotrienols, with appropriate adjustment of the soybean oil diluent; inclusion of Tween 40 or other suitable surfactant at an appropriate level. In the event that a dispersion or paste is required, the particle size of the FCP-3P1 may be modified by milling in the dry state, or dispersed in some or ail of the oil components.

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Claims

We Claim:

1. A composition for use in preventing and treating cardiovascular disease, its underlying conditions and other disorders which comprises one or more phytosterols, phytostanols or mixtures of both, and one or more tocotrienols or derivatives thereof.

2. The composition of claim 1 wherein the phytosterols, phytostanols or mixtures of both are esterified.

3. The composition of claim 1 wherein the phytosterols are selected from the group consisting of sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol and all natural or synthesized, isomeric forms and derivatives thereof.

4. The composition of claim 1 wherein the phytostanols are selected from the group consisting of sitostanol, campestanol, stigmastanol, brassicastanol, desmostanol, chalinostanol, poriferastanol, clionastanol and all natural or synthesized, isomeric forms and derivatives thereof.

5. The composition of claim 1 wherein the tocotrienol is selected from the group consisiting of alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta- tocotrienol.

6. The composition of claim 1 wherein the tocotrienol is provided in the form of one or more vegetable oils.

7. The composition of claim 1 wherein the underlying conditions are selected from the group comprising atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension and thrombosis.

8. The composition of claim 1 wherein the other disorders are selected from the group consisitng of Diabetes Type II and those diseases which include oxidative damage as part of the disease pathology.

9. The composition of claim 1 incorporated into a vehicle selected from the group consisting of a food, a beverage, a pharmaceutical and a nutraceutical.

10. The composition of claim 1 additionally comprising a pharmaceutically acceptable carrier material.

11. The composition of claim 1 formed into one of an emulsion or microemulsion.

12. The composition of claim 1 modified to form a delivery vehicle selected from the group consisiting of solid dispersions, suspensions, hydrated lipid systems and inclusion complexations with cyclodextrins, hydrotopes and bile salts.

.

13. A method of treating and preventing cardiovascular disease, its underlying conditions and other disorders in an animal which comprises administering to the animal a composition comprising one or more phytosterols, phytostanols or mixtures of both, and one or more tocotrienols or derivatives thereof.

14. The method of claim 13 wherein the underlying conditions are selected from the group consisting of atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, and thrombosis.

15. The method of claim 13 wherein the other disorders are selected from the group consisting of Diabetes Type II and those diseases having oxidative damage as part of the disease pathology.

16. The use of a composition comprising one or more phytosterols, phytostanols or mixtures of both and one or more tocotrienols or derivatives thereof to treat and/or prevent cardiovascular disease, its underlying conditions and other disorders.

17. A structural analogue for use in preventing and treating cardiovascular disease, its underlying conditions and other disorders having the following formula:

R2

R 3 C O R

wherein R is a phytosterol or phytostanol moiety; R2 is oxygen or hydrogen (H2) and R3 is an alpha, beta, delta or gamma tocotrienol or derivative thereof.

18. The analogue of claim 17 wherein the phytosterols are selected from the group consisting of sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol and all natural or synthesized, isomeric forms and derivatives thereof.

19. The analogue of claim 17 wherein the phytostanols are selected from the group consisting of sitostanol, campestanol, stigmastanol, brassicastanol, desmostanol, chalinostanol, poriferastanol, clionastanol and all natural or synthesized, isomeric forms and derivatives thereof.

20. The analogue of claim 17 wherein the tocotrienol is selected from the group consisiting of alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta- tocotrienol.

21. The analogue of claim 17 wherein R2 is hydrogen (H2).