AU2007200897B2 - Sterol compositions from pulping soap - Google Patents

Description

474467DIV2 DIA:JO P/00/011 Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant: THE UNIVERSITY OF BRITISH COLUMBIA Actual Inventor(s): EGON NOVAK, JAMES P KUTNEY, PETER J JONES Address for Service: COLLISON & CO.,117 King William Street, Adelaide, S.A. 5000 Invention Title: STEROL COMPOSITIONS FROM PULPING SOAP Details of Associated Provisional Application: Australian Patent Application Nos. 35597/95 dated 29 September 1995, 59393/99 dated 15 November 1999 and 2002300278 dated 24 July 2002 The following statement is a full description of this invention, including the beat method of performing it known to us: -lA STEROL COMPOSITIONS FROM PULPING SOAP FIELD OF THE INVENTION This invention relates to sterol compositions and to the use of these compositions and derivatives thereof as agents to present or treat primary and 5 secondarydyslipidemias. BACKGROUND OF THE INVENTION The direct cause of heart attack and angina is a degenerated process known as atherosclerosis. Atherosclerosis results from a number of integrated 10 inherited (genetic) and environmental factors. The interplay of these faclors, of which diet in our civilization appears to be the most important, leads to the development of atherosclerosis. Growth of cholesterol filled atherosclerotic plaques ultimatelycuts of blood supply to the heart mus cle, or alternatelyto brain or legs, depending on the location of the plaque in the arterial tree. 15 One of the major risk favors for atherosclerosis that is potentially modifiable is the level of blood cholesterol. A number of well documented studies have shown thatthe blood cholesterol level is indeed an important predictor for the risk of heart attack and also for strokes. The relationship between blood concentration of cholesterol and risk of these disorders is coninuous (spans 20 across all levels of cholesterol) graded (the higher the level, the more likely the disease) with no apparent threshold (even by lowering so-called low levels, one can further decrease riskof the disease). For example, in people over 40 years of age, a blood cholesterol level of 7.0 mmo/L presents a risk of coronary artery disease three to four times that associated with levels below 5.0 mmoVL. The 25 relations hip becomes especiallysteep when the levels are above 5.2 mmoVL. For instance, the death rate among men with cholesterol of levels 8.Onmol/L was almost sixtimes that among men with levels of 4.0'mmol/L. These more recent findings are consistentwith earlier studies. Other large clinical trials have shown clearly that by lowering high 30 cholesterol levels, one can reduce the risk of fatal and non-fatal myocardial -2 infarctions, angina, changes in electrocardiograms and in coronary artery bypass surgery. The best known and thefirst of these trials was at Lipid Research Clinics at which Coronary Primary Prexention Trials showed thatwith every 1% reduction in total blood cholesterol level, there was a 2% reduction in the risk of coronary artery 5 disease. For any long term prewntative therapy of hypercholesterolemia to be successful, it has to be commenced at a relatively early age and continue indefinitely. While a low-fatdietis the corner stone of such long term therapy, up to 60% of patients become non-compliant after six months. The difficulty in 10 non-compliance is marked in many Wesern countries by a general diet which is high in fat. The poor cholesterol profile of many patients is exacerbated by the premelence of additional riskfacbrs for cardiovascular disease such as high blood pressure, diabetes, obesity and smoking. Dietary modification as a therapy for atherosclerosis and other 15 cardiovascular diseases has been refined significantly overthe past 10 to 15 years. In particular, it has been recognizd by researchers thatplant steinls (phytosterols) are effective in lowering plasma cholesterol levels: Lees et al. Atherosclerosis, 28 (1977) 325-338; Kudehodkar et al., Atherosclerosis, 23 (1076) 239; Day. Artety, 18(3):125-132 (1991). 20 Phybsterols are sterol-like compounds synthesised in plants with no nutritional value to humans. In plants theyare required forcell function in a manner similar to the way in which cholesterol is required in humans. The average Western diet contains up to 360 mg of phybsterols per day. Recently, these dietary plant sterols have received a great deal of attention because of their possible 25 anti-cancer properties and their ability to decrease cholesterol levels when fed to a number of mammalian species, including humans. Chemically, phybsterols closely resemble cholesterol in structure. The major phybsterols are beta-sitosterol, campesterol and stignasterol. Others include stigmastanol (beta-sitostanol), sitostanol, desmosterol, chalinasterol, 30 poriferasterol, clionasterol and brassicasterol. The chemical structures of beta- -3 s itosteroi, cam pesterol and stigmasterol are as follows: beta-sitosirol 5 10 campesterol 15 20 stigmasterol -4 The mechanism by which phybsterols lower blood cholesterol in animals is unclear, butit appears to involve the inhibition of cholesterol absorbtion from the proximum jejunum by competing with cholesterol at specific uptake sites. 5 Research data has also suggested thatsome phybsterols are not absorbed at the proximal jejunum at all (sibstanol) and, when there is absorbtion (beb-sitosterol), it is in verylimited quantities. Based on these research findings, the use of phybsterols as a diebry supplement to reduce cholesterol absorbtion has been investigated. Lees 10 et al., supra; Pollak, Pharmac. Ther., 31 (1985) 177-208; Raicht et al., Biochimica et Biophysica Aca, 388 (1975) 374-384. In Lees et al., supra, a corn parison was made between the effects of siterosterol preparations from two sources, soy sterols and tall oil sterols, on plasma cholesterol. Plant sterol preparations were found to be effective in treating 15 patients with hypercholesterolern ia. Pollak, supra, is a survey paper of phybsterols and their effect on serum lipids. Raicht, supra, describes furmher the effect of beta sitosterolon sterol balance and rate-limiting enzynes of sterol metabolism. It is generally accepted that phybsterols offer a unique combination of long-term safety, efficacy, and versatility in human treatment. The ongoing 20 chalenges with respect to phybsterols is in their isolation and purification from plant sources, in determining additional sources which are cost-effecive and manageable on a large-scale and in finding combination of phybsterols which best exhibit hypocholestbrem ic effects. Traditionally, phybsterols have been isolated from sources such as 25 corn oil, wheat germ oil, soya bean pitch and com oil pitch, Similarly, tall oil pitch, which is obtained during the process of preparing paper from wood, particularly pine wood, has been used as a phybsterol source. Generally, in this- process, wood chips are digested with caustic soda to produce a pulp or "soap". The soap is then disilled to remove the volatile materials leasing a "pibh" as the residue. It is 30 from this pitch that researchers have isolated phybsterols.

-5 There are some marked disadvantages to these traditional sources of phylosterols. The tall oil pitch is an extremely complex material comprising resins, faty acids, oxidation products, esterified materials and phybsterols. AJthough the pitch is inepensive in that it is the tailing left from various 5 manufacturing processes, it is verydificultto recover high molecular weight sterols from it in good yields and at the high purities required fbr pharmaceutical uses, United Stabs Patent No.'3,840,570 to Julian proddes a process for preparing sterols from tall oil pitch by extraction in a water - alcohol - hydrocarbon mixture followed by saponificalon and subsequent purification. The starting 10 material in this process is tall oil pitch from which are extracted phybsterols and various impurities. It is recognized that, in any tall oil pitch purification process, the long-chain alcohol and acid impurities are parlicularly difficult to separate from the sterols (which are, themselves, high molecular weight alcohols). Other researchers have addressed the issue of sterol purification 15 from tall oil pitch: Unied Stabs Patent No.2,835,682 to Steiner and Fritz; United Stabs Patent No. 2,715,638 to Abrecht and Herrlinger; Unied Stats Patent No. 2,573,891 to Christenson. It is important to note that in each of these known purification processes, the starting material was tall oilpitch which has the recovery problems discussed abow. 20 It is an object of the present intention to obviate or mitigate these and other disadvantages.

-6 SUL4RYOF THE INVENTION The present invention provides unique compositions which are effective in preventing or treating dyslipidemias and which comprise beta-sitosterol, 5 campesterol and stigmastano or derivatives thereof. The phybsterol compositions provided herein are significantly different from those found in plants, foods and oils. In paricular, the provision of stigmastanol, in conjunction with the other components, appears to enhance the efficacy. These compositions may additionally comprise various co-occurring compounds, which may or may not be 10 phybsterols. In paricular,these co-occurring compounds mayinclude trirpenes, long chain alcohols and other alcohol-soluble organic compounds. The present invention further provides the use of the compositions described herein to prevent or treat primary and secondary dyslipidemias and atherosclerosis including coronary heart disease, peripheral vas cular disease and 15 strokes in humans and animals. The present invention further proddes a process of preparing a phybsterol composition which comprises blending a mixtre of both hydrogenated and non-hydrogenated phybsterols. The unique compositions of the present invention have exhibited 20 excellent results in lowering total (TC) and low density lipoprotein (LDL) blood cholesterol. In addition, and quite surprisingly, the compositions of the present invention were found, in diflirent animal species, to maintain or elevete plasma levels of high-density lipoprotein (HDL) blood cholesterol. This feature of the present invention is critically important given the fact that research has shown that 25 irrespective of TC levels, as the plasma HDL level decreases, the risk of atherosclerosis incmases. Phybsterols isolated from tall oil pith soybean and other sources have not to the knowledge of the present inventors, exhbited this unique HDL effect. Athough it is known to produce some types of phylosterols from the 30 pitch disilled from the soap of wood chip treatments, phylosterol compositions -7 have not heretofore been produced from the pulping soap component of the wood chip treatment press. The tall oil pitd1 is significantly different in composition from the pulping soap. It is believed thatthe surprising effect of the compositions of the present invention is due, at least partially, to the use of the pulping soap as the 5 starting material and to the unique separation process. BRIEF REFERENCE TO THE DRAWINGS: Various aspects of the invention will be illustrated by the following non-limiting drawings wherein: 10 Figure 1 is a gas-chromabgraphy profile for one composition (hereinafter Forbes-2) within the scope of the present invention; Figure 2 is a representation of the profile in Figure 1 from 35 to 45 minutes retention time; Figure 3 is a representation of the profile in Figure 1 from 22 to 27 15 minutes retention time; Figure 4 is an indexof thegas-chromabgraphypmille of Figure'1; Figure"5 is a gas-chromabgraphy profile for another composition (hereinafter Forbes-3) within the scope of the present invention; Figure 6 is a representation of the proile in Figure 5 from 32 to 48 20 minutes retention time; Figure 7 is an indexof the gas-chromabgraphyproile of Figure"5; Figure 8 represents a bar graph illustrating the effects of Forbes-1 and Forbes-2 on TC concentrations in rats; Figure 9 represents a bar graph illustrating the effects of Forbes-1 25 and Forbes-2 on LDL-cholesterol concentrations in rats; Figure 10 represents a bar graph illustrating the effects of Forbes-1 and Forbes-2 on HDL-cholesterol concentrations in rats; Figure 11 represents a bar graph illustrating the effects of Forbes-3 on serun TC in hamsters; 30 Figure 12 represents a bar graph illustrating the effects of Forbes-3 -8 on serum LDL-cholesterol in hamsters; Figure 13 represents a bar graph illustrating the effects of Forbes-3 on serum HDL-cholesterolin hamsters; Figure 14 represents a bar graph illustrating the effects of various 5 dietary treatments on cholesterol levels in male and female hamsters; Figure 15 represents a bar graph illustrating the effects of various dietary treatments on plasma cholesterol levels in male hamsters; Figure 16 represents a bar graph illustrating the effects of various diearytreatments on plasma cholesterol levels in female hamsters; 10 Figure 17 represents a bar graph illustrating the effects of various dietarytreatments on plasma triglyceride levels in male and female hamsters; Figure 18 represents a bar graph illustrating the effects of various dietary treatments on HDL/APO-B ratios in male and female hamsters; and Figure 19 represents a bar graph illustrating the effects of dietary 15 treatments on total cholesterol in hamster 45 daystudy Figure 20 represents a bar graph illustrating the effects of dietary treatments on cholesterol correlation with sitostanol; Figure 21 represents a bar graph illustrating the effects of dietary treatments on HDL levels in hamsters over45 days; 20 Figure 22 represents a bar graph illustrating the effects of dietary treatments on non-apo Napo A ratios in hamsters over45 days; Figure 23 represents a bar graph illustrating the effects of dietary treatments on non-apo Asterols in hamsters over45 days; Figure 24 is a graph representing the plasma lipid concentrations to 25 total cholesterol, HDL cholesterol, triglycerides, and LDL cholesterol in healthy male and female subjects consuming either the compositions of present invention or other diets; Figure 25 is a graph representing the plasma lipid concentrations of total cholesterol, HDL cholesterol, triglycerides, and LDL cholesterol in healthy 30 male subjects consuming either the compositions of present invention or other -9 diets; Figure 26 is a graph representing the decrease in plasma lipid concentrations of total cholesterol, HDL cholesterol, triglycerides, and LDL cholesterol in healthy female subjects consuming either the compositions of 5 present invention or other dies; Figure 27 is a graph representing the decrease in plasma lipid concentrations of total cholesterol, HDL cholesterol, triglycerides, and LDL cholesterol in healthy female and male subjects consuming either the compositions of present invention or other diets; 10 Figure 28 is a graph representing the decrease in plasma lipid concentrations of total cholesterol, HDL cholesterol, triglycerides, and LDL cholesterol in healthy male subjects consuming either the com positions of present invention or other diets; Figure 29 is a graph representing the decrease in plasma lipid 15 conontrations of total cholesterol, HDL cholesterol, triglycerides, and LDL cholesterol in healthy female subjects consuming either the compositions of present invention or other dies; Figure 30 is a graph showing phybsterol concntrations in in blood plasma in various treatment groups including the group administered the 20 composition of the present invention andshoawing the relative concentrations of the constituents; Figure 31 is a graph showing the level of LCAT enzyne activity as between various treatment groups; Figure 32 is a graph representing the FER values of each of the 25 treatment groups; Figure 33 is a graph representing the IVER proile of each of the treatment groups; Figure 34 is a bar graph representing the effects of phybsterol administration on plasma lipid levels; 30 Figure 35 is a graph representing the effects of phytsterol -10 administration on plasma HDL cholesterol levels; Figure 36 is a graph representing the effects of phybsteral administration on plasma triglyceride levels; Figure 37 is a graph representing the effects of phybsterol 5 administration on plasma total cholesterol levels; Figure 38 is a graph representing the effects of phybsterol adminis tration on plasma LDL cholesterol levels; Figure 39 is a bargraph showing plasma phybsterol levels relative to plasma cholesterol levels; 10 Figure 40 is a graph representing the effects of phybsterol administration on free cholesterol fractional synthetic rates; Figure 41 is a bargraph showing the percentage hemolysis in control and treated groups; Figure 42 is a phoiomicrograph of the nuchal skin; 15 Figure 43 is a representative phobmicrograph of the kidneys from one control and one treated mouse; Figure 44 is a representative phobmicrograph of the livers from one contol and one treated mouse; Figure 45 is a representative photmicrograph of the tests from one 20 control and one treated mouse; Figure 46 is a graph showing the effect of a phybsterol-enriched diet on total cholesterol; Figure 47 is a graph s howing the effect of a phybsterol-enriched diet on lowering total cholesterol; 25 Figure 48 is a graph showing the effect of a phybsterol-enriched diet on LDL cholesterol Figure 49 is a graph showing the effect of a phybsterol-enriched diet on LDL cholesterol; Figure 50a is a graph showing an extrapolation of cholesterol 30 lowering effects of a phybsterol-enriched diet -11 Figue 50b is a graph showing an extrapolation of LDL cholesterol lowering effects of a phybsterol-enridched diet Figure 51 is a graph showing the effect of a phylosterol-enriched diet on HDL cholesterol; 5 Figure 52 is a graph showing the effect of a phybsterol-enriched diet on triglycerides; and Figure 53 is a graph showing the effect of a phybsterol-enriched diet on total cholesteroVlHDL cholesterol. 10 PREFERRED EMBODIMENTS OF THE INVENTION The present invention includes unique phytsterol compositions which comprise beta-sitosierol, campesterol and stigmastanol andbr derivatives thereof. As described furher hereinbelow, these unique compositions may partially comprise tall-oil pulping soap derived phybsterols and/br phybstanols 15 obtained by any suitable extraction process or synthesis, including the extraction process described hereinbelow. In a further embodiment, these compositions may partially comprise phybsterols and/br phybstanols derived from other vegetable sources . The process of blending both hydrogenated and non hydrogenated phybsterols from various sources to achieve optional theraputic 20 ratios of the constituents is also proUded herein. It is to be understood at the outset thattheApplicant does notwish to be bound byanyone method of extracting the phyosterols from their natural sources. The keyfbcus of the present invention is the compositions perse. 25 ExtractionProcess One suitable method of extracting phybisterols from a tall-oil derived pulping soap is described in US Patent No.5,770,749 which issued on June 23, 1998 to the present Applicant and which comprises the steps of (A) obtaining or preparing the starting material, a plant-derived pulping 30 soap; -12 (B) extracting from the soap a creamy precipitate using an appropriate soent; and (C) purifying from the creamy predpitate a phylosterol composition. There are numerous possible sources of the plant-derived pulping 5 soap. Generally, in a known process (the "Kralt" process) wood chips are treated with caustic soda to produce a soap. The wood chips may be derived from any hard wood or softwood variety of tree including, but not limited to, fir, cedar, pine, spruce, oak, hemlock and poplar. Most preferably, the chips are derived from any Pacific NorthwestAmerican or European forestvarietyof woods. 10 In the extraction phase, the soap is mixed with a ketone and water solution. A hydrocarbon sohent is used to extract the sterols. This step can be performed at temperatures generally from about 25oC to about 1500C, but most preferablyfrom about 5OoC to about 100-C. Most preferably, this extraction phase is continued over 15b 24'hours. It is important to note that the use of alcohol is 15 required nor suggested during the extraction phase. The extraction process of the present invention is conducted using a Ketone-water-hydiocarbon solvent. The ketone is selected from the group having the general structure RCOR where R and R' are alkyl groups. Preferably the alki groups are C, - Ce groups. Most preferably, the ketone is 2-propanone (acetone). The hydrocarbon 20 may be selected from the group comprising all Cs - C1o hydocarbons. Most preferably, the hydrocarbon is hexane. As depicted in Figure'1, the product of the extraction phase is a creamy predpitate or residue from which is purified the phybsterol composition. This purification phase may be conducted by crystallization, chromatographic 25 separation or by any other suitable procedures. Most preferably, the creamy predpitate is dissolved in alcohol, cooled slowly, then filtered and washed with cold alcohol. The residue is dried, and the resultant product is a phybsterol composition. In a preferred form, the alcohol used in the purification phase is 30 selected from the group having the general structures R-CHOHR, R-CI-bOH, and -13 RCOH where R is a CI - C 4 alk group. Most prefrably, the alcohol is methanol. The cooling phase maybe affected at temperatures from 10 Celsius to 0o Celsius, most preferablyat 3 to 4oCelsius for 24'hours. 5 The resultant composition of this extraction process is a mixkire of phybosterols comprising beta-sitosirol, campesterol and stigmastanol. Phytosterol Compositions The phylsterol compositions of the present invention comprise beta 10 sitosterol, campesterol, stigmastanol (beta-sitostanol) and all natural or synthesized forms and derivatives thereof, including isomers and optionally includes campestanol and all natural or syntihesized forms and derivatives thereof, including isomers. This compositions per se, as well as the preferred concentration ratios described further hereinbelow are distinct from the sitosterol 15 based compositions which have been previously investigated by researchers. Generally, in the most preferred compositions of the present invention, the relative beta-sitosarol concentration is lower than in the compositions advocated in the references. Conversely, the relative concentration of stigmastanol is higher in the compositions of the present invention. it is believed that the extr-ordinary effect of 20 the compositions of the present invention in preenting and treating atherosclerosis, and its underlying disorders, is the result kwfly of the unique paricular combination of three phybsterols in concert and secondly, in the preferred form, of the unique ratios of these three phytsterols in concert. The compositions of the present invention exhibit a marked ability to lower total serum 25 cholesterol levels and LDL cholesterol levels while concomittantly increasing both the beneficial HDL serum cholesterol level and HDLLDL ratio. Nowhere in the art is there an appreciation of these features and advantages. The phyosterols for use in the corn positions of the present invention 30 maybe procured from a variety of natural sources, including extraction from tall oil -14 soap in accordance with the method described in detail herein (and in US Patent No. 5,770,749, referred to above) and extraction from vegetable sources, For example, phybsterols maybe obtained from the processing of plant oils (induding aquatic plants) such as com oil and other vegetable oils, wheat germ oil, soy 5 extract, rice extract, rice bran, rapeseed oil, sesame oil and fish oils. These latlr extraction methods are known in the art. The compositions of the present invention may be administered to animals, including humans, directly or may be incorporated into various other 10 vehicles for delivery such as pharmaceuticals, foods, beverages, nutraceuticals and the like including dietary supplements and vitamin formulations for the treatment andkbr prevention of atherosclerosis and its consequences, strokes, heart attacks and peripheral vascular disease. 15 Within one embodiment of the present invention, the phybsterol compositions described herein may be provided in the form of medications with suitable adjuvants or carriers. Furthermore, the compositions maybe combined or prescribed with other known compounds which inhibit cholesterol syntiesis, such as stains, or other selected lipid-lowering agents, to decrease the necessary 20 dosage, and hence the toxicity, of these later compounds, Additionally. the compositions of the present invention may be formulated into foods, beverages and nutracueticals. The phybsterol compositions of the present invention have exhibited a marked ability to modify lipoproteins, even at lower phylosterol concentrations 25 than in known formulations. More surprisingly, however, has been the effect of these compositions on increasing plasma levels of high density lipoproteins (HDL), an effect heretofore not associated with any other tall oil-derived phybsterol composition. It is believed that this unique effect may be due to the prodsion of stirnastanol as keyelement of the composition. 30 In one embodiment of the invention, the composition comprises at 15 least 10% campesterol, no more than 75% beta-sitosLrol and stigmastanol, Further, in another preferred form, the composition comprises from 10-25% campesterol, 10-45% stigmastanol and from 45-75% beta-sitosierol. Optionally, the composition comprises from 1-10% campestanol, most prefrably2-6%. It is 5 to be understood, however, that other additional phybsterols maybe added to the composition in order to enhance the theraputic effects. In order to achieve compositions having the preferred phybsteroVphytostanol ratios defined above, one may 10 1.syithesim or otherwise extract each of the components de novo and combine in the required ratios; 2.follow the tall oil pulping soap eAraction probcol outlined above and covered in 1S US Patent No.5,770,749 which issued to the Applicants; or 3. blend various sources of phybsterols/phybstanols such as hydrgenated and non-hydrogenated vegetable phybsterols, optionally with tall oil derived 20 phybsterols and/br stanols as described in more detail below. Practically and from a commercial perspective, options 2 and/br 3 are preferred although the Applicant does not wish the compositions to be limited to anyone mode of preparation. 25 Preferred Ratios of Phytosterols/Phytostanos As described abow, the preferred ratios of the three primary components of the composition are: at least 10% campesterol, no more than 75% beta-sitosbrol and stigmastanol. More prebrably, the ratios are as follows: 10 30 25% campesterol, 45-75% beta-sitosierol and 10-45% stigmastanol. Additionally, -16 campestanol maybe present. Furlher more preferred ratios include: beta-siterosterol (1); campesterol (0-2-0.4) and stigmastanol (0.2 - 0.5). In a pretred form of the composition, campesterol 5 and stigmastanol together represent at least 50% of the total concentration of beta sitosterol. In another preferred form, the compositions of the present invention comprise the following ratio of phylosterols Ratio of Known Phytosterols Approxdmate B-Sitosterol Campesterol Stigmastanol Purity(%) Forbes-1 91.0 1 0.354 0A14 Forbes-2 77.0 1 0.330 0.203 Forbes-3 90.0 1 0.268 0.299 10 The composition and purity of two other extracts within the scope of the present invention are as follows: Composition (%) Approximate B-Sitosterol Campesterol Stigmastanol Purity (%) Forbes-4 99.0 62.6 16.6 23.2 Forbes-5 98.3 64.7 16A 17.2 Other preferred compositions are defined as follows: 15 Phybsterol(%) -17 sitosterol sitostanol campesterol Forbes-6 39 32 14 Forbes-7 32 29 14 Forbes-B 25.5 42.5 7 5 Another preferred form of the composition within the scope of the present invention corn prises cam pestanol, most preferably from 1-10%. Co-occurring Compounds 10 In every composition prepared by the extraction process from tall oil pulping soap described herein, there maybe additional compounds present which may or may not be phytsterols. In order to determine the nature of these co occurring compounds, gas liquid chromatography analysis has been conducted on each of the mostpreftrred compositions of the present invention. 15 Gas chromatography running conditions for the phybsterols were: initial temperature 80cC which was held for 1'minute; ramp to 120cC at 20cC per minute, which was held for7minutes; ramp to 24C at 20OC per minute which was held for 15"minutes; and ramp to 269cC at 20cC per minute which was held for 25'minutes. At the end of each run, the temperature was ramped to 320cC and 20 held for a minimum of minutes. The injection temperature was 300CC and the detector temperature was 320cC. The column flow rate was I'mi per minute and 1420A6\02404S4.WP -18 the split vent flow rate was 4'ml minute. The purge vent flow-rate was 4.5'ml minute. The carrier gas was helium. The results of the gas liquid chromatography analysis for two of the most preferred com positions of the present invention are depicted in Figures'1-7. 5 With respect to the Forbes-2 composition, the known sterols appear in the 35'-'45'minute region in Figures 1 and 2. Beta-sitostrol is indicated at peak 87; campesterol is indicated at peak'81 and stigmastanol is indicted at peak'84. Peaks'65, 66 and 77 in Figure'2 are co-occurring compounds which may exhibit hypocholestrolemic effects. It is possible, however, that these co-occurring 10 compounds mayhave a synergistic effect on the actions of the known phybsterols in the compositions. Simiarly, in Figures 5 and 6, campesterol, stigmastano and beta-sitoslerol are represented at peaks 6, 7 and 8 respectively. Blending Phytosterols/Phytostanol 15 The compositions of the present invention are unique in that the combination of the three core phybsterol components has not heretofore been recognized for treating or preventing primary and secondary dislipidemias and atherosclerosis including coronary artery disease, peripheral vacsular disease and stroke. 20 One preferred means to achieve a composition with the desired 142086\0240454.WP -19 ratios is to blend phybsterols andbr phytostanols from varying sources. This way, one may take advantage of particularly high levels of one component from one source (i.e. sitosterol), while another source may be richer in another required component (i.e. sitstanol). 5 Generally, phybsterols and their hydrogenated or saturated counterparts phytostanols are present in manynatural sources as outlined above. While each of these sources may be used within the scope of the present invention to prepare the core compositions and the blended compositions as 10 defined herein, it is most preferred that one or both of tall oil derived and vegetable derived phybsterols and phybstanols be used. In a most preferred form, the blending process comprises hydrogenating vegetable derived phybsterols yielding a product of approximately 15 80% beta-sitostanol and 20% campestanol. This intermediary product is then blended with tall oil derived phybsterolsfphybstanols to yield the desired composition. Alternatively, both non-hydrogenated and hydrogenated vegetable derived phytsterols may be blended with a tall oil derived mixture in order to yield the desired composition. What is important to note is that the end result of the 20 blending should be a phybstero/stano composition having the ratios as defined herein foroptirnal efficacy. 142086\0240454.WP -20 For example, and in one preferred form, hydrogenated soy derived phylosteros are blended in a 1:1 ratio with tall oil derived phylosterols, prepared using the promss of extraction such as thatdescribed hereinabove. This particular 5 blended composition which comprises 25.5% beta-sitostrol, 42.5% sitostanol, 7% campesterol, and 17.5% campestanol, has been found to be highly effective in treating the conditions underlying atherosclerosis. In another preferred form, the blended composition of the present invention comprises hydrogenated soy phylbsterols, non-hydrogenated soyphybsterols and tall oil denied phybsterols in 10 a 1:1:1 ratio yielding the following constituents: 32% beta-sitostrol, 29% sitostanol, 14% capesterol 12% cam pestanol, and 6% stigmasterol. Blending of the constituent phybsterol/stand constituents of the composition from various sources may be achieved by a number of different 15 methods. In a most preferred form, the constituent phybsterolstanols are dissolved into a suitble solent mixture and recrystallized to ensure unitrmity in the composition. Other blending methods are also appropriate. 142086\02t454.WP -21 Hydrogenation of the phylosterol in accordance with the present invention maybe achieved by a number of methods known and applied in the art including the method described in Augustine and Reardon. The Palladium S catalyzed hydrogenation of cholesterol. Org Prep. And Proceed. 1969;1:107-109, the contents of which are incorporated herein by reference. This reference is based on the use of Pd/C catalyst in organic solvents. Other suitable catalysts include plalnum and Raney nickel. 10 EXAIVPLE 1 - Extraction and Purification A batch of 3 kg of pulping soap was obtained from B.C. Chemicals Inc. Amixkure of 3 L of acetone and 1.5 L of water was prepared to which the soap was added. The mixtre was extracted coninuously with 4.5 L of hexane at 50oC for 24 hours using an 18 L evaporator. The resultant extraction product was then 15 dried over sodium sulphate and allowed to evaporate. This produced 460 g of residue or creamy precipitate. The creamy predpitate was warmed and stirred using a magnetic bar and 460ml of methanol was slowly added. The mixbre was reiluxed under stirring for15 min. and cooled slowly for 3-5 hours. The mixiure was refrigerated at 3-4*C 20 ovemight and then filtered and washed (twice) with 150 mi cold methanol. Finally, the mixtre was maintained in a vacuum for 2 days yielding 100 g of mixhre with a 142086\0240454.WP -22 purity of 82% (i.e. 82 g of phybsterols). EXANPLE 2 - Evaluation of the Effects of Phybsterol Compositions in Rats Ninety male Wisar rats (80-100 g) were divided into 3 experimental modules: Forbes-1 composition; Forbes-2 composition and soybean. The thirty 5 rats within each mode were further divided into 5 dietary regimes as indicated in Table 2. The rats were kept on reverse lighting cycle, and fed for 10 days with a basal semi-purified diet (Table'1) supplemented with different amounts of cholesterol and phystosterol (Table'2). Within each. of the.5 dietary groups, 2 rats were administered the Forbes-1 composition, 2 rats were administered the 10 Forbes-2 composition and 2 rats were administered soybean-derived phybsterol (Sigma). Table 1. Composition ofexperimental diet Ingredients % Cas ein 20 Cornstarch 21.5 Sucrose 35 Fixed-oil* 18 Dl-methionine 0.5 Mineral miAure 4.00 Vitamin miAure 1.00 'Safflower and lard mbed in a 1:3 ratio. 142O86\0243454 .WP1 -23 Table 2. Dietaryregimens Groups Sterols added to the basal diet(%) Cholesterol Phybsterol 1 0 0 2 1 0 3 1 0.2 4 1 0.5 5 1 1 At the end of the feeding period, the rats were intrapedtoneally injected with deutrium odde (0.4 ml) and deprived of food and water for at least 2 5 hours. The rats were then anaesthetimd with halothane. Blood samples were withdrawn from the heart. Samples of liver, small intestine and musde were quickly removed, weighed, put in liquid nitrogen and stored at 80"C until determination of cholesterol synthesis. Total cholesterol, LDL and HDL cholesterol were determined with a commercial kit (Biopacific Diagnostic Inc). 10 The results of the effects of the phylosterol compositions on total cholesterol, LDL and HDL are represented in Figures 8,9 and 10 respectively. The efficacy of the Forbes-1 and Forbes-2 is evident from the reduction in LDL cholesterol shown in Figure"9 and in the increase in HDL-cholestrol shown in Figure 10, particularly by Forbes-1. In Figure"8. the addition of cholesterol (dietary 15 group"2) to the base diet(group'l) rested in an increase in circulating cholesterol concentrations. Progressive addition of increasing levels of phybsterols 142086\024454.WP -24 (groups"3-5) rested in a normalization of cholesterol levels in groups fed Forbes-2 and Forbes-1, but not soybean phybsterols, as determined by regression analysis. Figure9 shows that Forbes-2 and Forbes-I phybsterols possess better cholesterol - lowering efficacy than the soybean phylosterols for 5 LDL. Figure'10 demonstrates the greater HDL-raising ability of the preferred compositions of the present invention, particularly Forbes-i, compared to the soybean phybsterols. EXAIVPLE 3 - Evaluation of Effects of Phybsterol Compositions in Hamsters 10 The present study was to examine the effect of dietary phybsterol compositions of the present invention on the dietary cholesterol-induced election of serum cholesterol concentrations in hamsters. A total of 40 male hamsters (80-100g), housed individually in stainless mesh cages werefed rodent chow and acclimated forthree days in an air 15 conditions room (20-220C, lights on 1700-0500). Hamsters were then divided into five groups of 8 animals each group, and fed for 34 days, a basal semi-purified diet (Table 3) supplemented with different amounts of cholesterol and one of the phylosterol compositions of the present invention (Forbes 3) (Table 4). 142096\02C454.WP -25 Table 3. Composition ofexperinental diet Ingredients % byweight Casein 20 Cornstarch 28 Sucose 36.3 Corn oil 5.0 Cellulose 5.0 Dl-methionine 0.5 Mneral miAure 4.00 Vitamin miAure 1.00 Choline bibrtrate 0.2 Cholesterol 0.025, 0.25 Table 4. Dietaryregim ens Groups Cholesterol added to Phybsterol added to control'diet contol*diet % %0 1 0.025 None 2 0.25 None 3 0.25 0.25 4 0.25 0.5 5 0.25 1.0 At the end of the feeding period the animals were intraperitoneally 5 injected with deutrium oxide (0.4 ml), and deprived of food and water for at least 2 hours. The hamsters were then anaesthetizd with halothane. Blood samples were withdrawn from the heart. Other tissue samples including liver, small intestine and musde were quickly removed, weighed, put in liquid nitrogen and stored at-800C unti determination of cholesterol synthesis. Total cholesterol, HDL 142086\0240454.WP -26 and LDL cholesterol were determined using a commercial kit The results were statistically evaluated with ONEWAY analysis of variance procedure (SYSTAT). Hamsters fed the high cholesterol diet had significantly higher serum total cholesterol and LDL cholesterol than did those fed the normal cholesterol 5 (0.025%) diet The supplementation of phybsterol at levels of 0.5% and 1% remarkably abolished these increases induced by high cholesterol consumption (Figures 11 and 12). The LDL cholesterol concentration in group 5 was lower compared to the levels in hamsters fed normal cholesterol-containing diets (Figure 12). Furthermore, there was negative regression association of total cholesterol 10 and LDL cholesterol to the level of phybsterol-added in diet (Figure 13). Supplementation of phybsterol caused a slight increase in HDL, but without yielding a significantdifference (Figure 13). EXAMPLE 4 - Evaluation of Phytosterol Composition Effects in Hamsters 90 day 15 trial Six groups of 20 hamsters (10 males, 10 females) were fed semi-purified diets containing 30% fat (polyunsaturated/saturated fat ratio = 0.3) for 90 days. Diet I was cholesterol free. Diets 2-6 contained 0.25% (wt/wt) dietary cholesterol. 20 Diets 3 and 4 contained Forbes phytosterols (greater than 90% purity) at 0.5 and I %. respectively. Diets were made from primary ingredients every week. Fat, phytosterol and cholesterol levels were determined by gas liquid chromatography. All animals had free access to water and diets throughout the experimental period. Animals were weighed weekly. Food intake was also 142086\024454-IP -27 determined every day and averaged per week by weighing food cups before and after each 24h feeding period. After 90 days feeding, animals were sacrificed using halothane and blood was collected for lipoprotein profile analyses. Circulating total, apoB containing particles and HDL cholesterol and triglyceride 5 levels were determined. Also, just prior to sacrifice, cholesterol absorption and synthesis rates were determined using 14C-cholesterol disappearance from the gut and deuterium incorporation into tissue cholesterol methods, respectively. Uptake of phytosterols into intestine and other tissues was also examined. In addition, samples of Intestine and 10 liver were stored to be provided to Bio-Research Laboratories of Senneville, Quebec, for histopathological, carcinogenicity and enzyme function analyses. Results: Results are shown in Figures 14-18. Groups in these Figures are 15 often referred to by number, which correspond to those described in Experimental Design. Letters above bars in bar-graphs identify significant differences between groups. Where letters are given, bars sharing the same letter are not significantly different, while those with different letters are different at a statistical level of p < 0. 05. 20 Shown in Figure 14-16 are the circulating cholesterol data for male and female hamsters consuming the test diets over 90 days. Significant effects of sex were observed in total circulating cholesterol levels for animals consuming diet 2, the base diet with added cholesterol only, and diet 5 containing cholesterol + 25 soyabean phytosterols. Females, although no different from males in cholesterol levels on the basal diet (group 1), exhibited a higher response to added cholesterol alone compared with males. Adding phytosterols negated this difference, with the exception of group 5. 142056\0240454.WP -28 Cholesterol level data broken down into sexes are shown in Figures 15 and 16. For males (Figure 15), addition of cholesterol alone to the basal diet resulted in a significant increase in total circulatory cholesterol level. Addition of Forbes 5 phytosterols at 0.5 percent resulted in a trend towards a decrease in cholesterol level, however, addition of Forbes phytosterols at 1 % elicited a statistically significant reduction in cholesterol, to approximately the same levels of the control group without added cholesterol. When soyabean phytosterols were added to the diet at 0.5 or 1 %, there was no significant decrement in circulating 10 total cholesterol level in males. For HDL, a differential effect of Forbes versus soyabean was observed, where Forbes feeding produced no change in the HDL levels of the group given cholesterol alone (group 2), however, feeding soyabean resulted in a significant decline in HDL values at both levels tested (groups 5 and 6). There were no significant differences across apoB containing cholesterol 15 particles, however, there was a trend towards lower levels with the feeding of cholesterol Forbes at 1 %, compared with other groups. Data for females are shown in Figure 16. For total and HDL cholesterol, there was a pronounced influence of adding dietary cholesterol alone. Addition of 20 either type of phytosterol source at 1 % resulted in significant and similar declines in total and HDL cholesterol concentrations. ApoB containing particle levels were not influenced by diet. Circulating triglyceride levels in hamsters consuming the test diets for 90 days are shown in Figure 17. There was an increase in circulating triglyceride levels in female animals given the basal diet 25 with cholesterol and Forbes 0. 5 % compared to the basal diet alone, however, no other inter-group differences were observed in either sex. In males, there was no effect or trend of diet on triglyceride concentrations. 142086\0240454.WP -29 In summary form, the ranking of male results on the test diets are as follows: Table 5: 5 Total Cholesterol LDL HDL Forbes 0. 5 % 4 2 1 Forbes 1.0% 1 1 2 10 Soya 0.5% 2 3-4 4 Soya 1.0% 3 4-3 3 15 The advantage of the Forbes compositions (those of the present invention) can be clearly seen. In addition, similar rankings were found for the HDL:ApoB ratio in males (Figure 18): Forbes 0. 5 % 2 20 Forbes 0. 1 % 1 Soya 0. 5 % 4 Soya 1.0% 3 It was also found that the HDL:LDL ratio for the Forbes compositions were 25 almost double those of B-sitosterol alone. EXAMPLE 5 - Effects of Phytosterol Composition on Rabbits 142086\024454.W -30 .in this study, two rabbits were assessed over 43 days with respect to the effects of one of the compositions of the present invention (Forbes 1% in diet) on their total cholesterol profiles. The results are as follows: 5 Table 6: Rabbit Total Cholesterol Profiles (mg/di) Date Rabbit A Rabbit B 14/06/95 95 215 18/06/95 129 162 10 26/06/95 - starting feeding with Forbes 1 % 07107195 75 106 09/07/95 - starting feeding with Forbes 1 % 18/07195 90 112 20 25/07195 82 118 15 01/08/95 95 119 08/08195 76 114 A decrease in total cholesterol can be seen in both Rabbits A and B during the two weeks of phytosterol composition (Forbes 1 %) administration. This effect 20 continued even after the discontinuation of Forbes 1 %. The effects of the compositions of the present invention on total cholesterol lowering linger past the initial administration phase. EXAMPLE 6 - Effects of Phytosterol Composition on Apo-E Deficient Mice 25 Animals: Nineteen 5-week old male Apo-E deficient mice were purchased from Jackson Laboratory, USA. Animals were randomly divided into 2 groups, 9 animals in control group and 10 mice in experimental Forbes group. After 5 days as an adaptation period mice were bled from tale into capillary tubes and plasma 30 was separated by centrifugation of blood. Mouse plasma lipids were estimated. Diet: Low-fat, low-cholesterol mouse chow was purchased from Jamieson's Pet Food Distributors Ltd., Vancouver, B.C. Tall-oil derived phytosterols were 142086\O24454.WP -31 extracted from tall-oil soap, using the process described in the present invention. The purity and percentage of each individual phytosterol in the mixture of final product were assessed by gas liquid chromatography. The fmal produce showed up to 95 % in purity and contained 69% sitosterol, 15% campesterol and 5 16% stigmastanol. Mouse chow was ground to a fine powder state. To this powder 0.15% (w/w) cholesterol (Sigma) was added and mixed well. A portion of this cholesterol supplemented diet was repelleted, dried and used for feeding control group of mice, and another portion of that was supplemented with 2% (w/w) tall-oil extracted phytosterols, repelleted, dried and, used for feeding of 10 experimental group of mice. Biochemical assays: Plasma total cholesterol and triglyceride were measured using enzymatic kit (Boehringer Mannheim) and HDLcholesterol was estimated by previously published precipitation method using polyethylene glucol 6000. 15 Body weight and food consumption: Mouse body weight and food consumption were measured weekly. Table 7 Mouse mean body weight (g) (only monthly measurements are reported) 20 Date Forbes group Control group 20/06/95 21.42 21.02 25 24/07/95 29.82 28.00 22/08/95 32.12 29.73 12/09/95 34.16* 30.78 *p < 0.05 30 Table 8: Mouse mean weekly food consumption (g) (only monthly measurements are reported) 142086\024454.hP -32 Date Forbes group Control group 26/06-03/07/95 19.61 21.21 01/08-08/08/95 32.62* 29.20 S 15/08-22/08/95 29.32 27.10 05109-12/09/95 35.56 30.25 p<0.05 10 Other findings: No side effects were observed with regard to new diets. All animals from both groups look normal, with normal habits including bowel habit. One mouse in the control group was found dead on 11/07/95. Since the mouse body was not kept, the autopsy was not performed. Another mouse from Forbes group was found dehydrated with body weight lost, The animal was sacrificed 15 and the reason for its sickness was found to be due to malocclusion (teeth overgrowth). Statistical analysis: Results were analyzed using t-test two-samples assuming equal variances. 20 Results: Results up to the present time are summarized in the following Tables. Table 9: Mouse mean plasma total cholesterol level (mg/dl). 25 Date Forbes group Control group 20/06/95 606.28 599.61 30 18/07/95 1027.96* 1622.56 06/09/95 1168.57* 1508.63 P<0.0001 Table 10: Mouse mean plasma triglyceride level (mg/di). 35 Date Forbes group Control group 142086\024454.WP -33 20/06/95 110.97 120.31 18/07/95 210.17 143.71 06/09/95 , 224.48 152.25 5 Table 11: Mouse mean HDL-cholesterol level (mg/dl). Date Forbes group (9) Control group (7) 08/07/95 42.00* 18.29 P<0.001 10 It can be seen from the results that the Forbes composition group showed a significant (33 %) decrease in total cholesterol, an insignificant increase in triglycerides, and a significant increase in HDL cholesterol (> 100%). 15 EXAMPLE 7 - Effect of Phytosterol Composition on Hamsters - 45 day Trial Fifty GS hamsters were accommodated for two weeks in an animal care facility before feeding them a semi-purified diet for 45 days. They were divided into five groups fed 0.25% cholesterol along with one of four mixtures of plant sterols: soy 20 bean, tall oil, pure sitostanol, and artificial mixture representing tall oil phytosterols. The control group received 0.25% cholesterol only. Their food Intake was monitored during the study period every three days. Their body weight was measured every week, and at the time of tissue collection. Three days before sacrificing the GS hamsters, they were lightly anaesthetized with diethyl 25 ether and injected intravenously through the jugular vein with 0. 4 ml intralipid containing 0. 18 mg. 13-C-cholesterol. Directly after the injection, the animals were fed by gavage, 0.6 ml of lipid mixture (coconut, olive, and safflower oils) containing 0.44 mg 18-cholesterol. Then, the GS hamsters were kept in their wired cages for 72 hours, provided water and food ad libitum. 30 On the day of sacrificing, each GS hamster was injected i.p., with 1 ml deuterated 1420S6\0240454.WP -34 water, and left for one hour before the killing. The animals were anaesthetized with diethyl ether, and blood samples were collected by cardio-puncture. Liver, gall bladder, small intestine, large intestine, and heart were collected, frozen in liquid nitrogen, and stored in the freezer at -80 degrees C. Their carcasses and 5 faces were stored at -20 degrees C for further total lipid and sterol analysis. Food Intake, Body and Liver Weight Measurements: GS hamsters food intake was measured every three days. The statistical 10 analysis shows no significant difference among the five groups in their food consumption. The average daily intake in the five groups varied from 8.84 to 9.34g per day, p-value = 0.4. The animals showed a significant increase in their body weight of about 25 to 40g during the study period, p-value<0.05 (paired t test). The final measurement of their body weight ranged from 112.5 to 154.3g. 15 No statistical significance was noticed among the different treatment groups, p value = 0.43. Liver weights varied significantly among the different divisions. The sitostanol treated group showed the lowest liver weights as compared to the control and 20 other groups fed 0.25% cholesterol and different phytosterols respectively. In addition, the soy bean treated group presented similar significant difference to the sitostanol treated one when the data are statistically analyzed using Newman-Keuls test. In general, all groups fed plant sterols showed lower liver weight as compared to control fed 0.25 % cholesterol, p-value = 0.01. The tall oil 25 sterols and the sitostanol fed GS hamsters had an average liver weight of 15% and 20% less than that of the control, respectively. The natural tall oil and the artificially prepared tall oil demonstrated similar values of liver weights suggesting that the missing compound in the soy bean plant sterol, sitostanol, 142006\0240454.WP -35 play a major role in decreasing the sterol content in the liver. Lipid Analysis: 1. Total cholesterol: 5 Total cholesterol values were determined by using a commercial enzymatic reagent kits on a VP autoanalyzer. Blood samples were measured twice, and the average of the two values was used in the final statistical analysis. One way ANOVA, with Newman-Keuls, and Bonferroni methods were used for the different lipid analysis. Sitostanol decreased significantly total cholesterol level in GS 10 hamsters plasma by 34% as compared to the control. The mean value for control group was 226.9 mgfdl, and the one for sitostanol treated group was 151.2 mg/dI, p-value = 0.007. Tall oil phytosterols, and the artificial tall oil mixtures showed similar decrease in plasma cholesterol (17.5%), 118.4 mg/dl and 186.1 mg/dl respectively. However, tall oil phytosterols (Forbes) showed 15 significant decrease In total cholesterol value (175.2 mg/dl) when one off-scale sample value was excluded from the analysis, p-value <0.02 in the tall oil group as compared with the control (0,25 % cholesterol only) group. The correlation between the presence of sitostanol and lower plasma level was significant, p value <0.0001, and r=0.46. 20 [I. HDL cholesterol: The apoA portion of the lipoproteins present in the HDL cholesterol did not show any significant changes in their values among the five different groups, p-value = 0. 18. A non significant decrease of 15 % in the mean HDL value in the 25 sitostanol treated group was observed. Nevertheless, this element did not affect the significant decrease in the total cholesterol which was of 34%. The ratio of the non apoA lipoproteins to the apoA (HDL) lipoproteins did not vary significantly among the groups. The confounding effect of the non-significant lower values of 142016\024454 WP -36 HDL in the sitostanol and tall oil groups contributed in creating a non significant result in the non apoA/apoA (HDL) values, In general, the different types of phytosterols did not vary the HDL cholesterol level in GS hamsters plasma. 5 Il1. LDL and/or non apoA sterols: Sitostanol was efficient in decreasing the non apoA sterols in the plasma. A 55 % decrease in the non apoA lipoproteins was shown, p-value - 0.02. Similarly to their effect on total cholesterol, tall oil and artificial mixture tall oil decreased the non apoA sterols by 21 % respectively, again suggesting of a strong correlation 10 existing between sitostanol content in the phytosterols and their beneficial effects in decreasing cholesterol levels in the GS hamsters plasma. However, this decrease was not statistically significant due to the variability in the triglyceride values. 15 IV. Triglyceride: When applying the one way ANOVA method on the TG values, they did not pass the normality test. GS hamsters were sacrificed in a non fasting condition (important status for the future cholesterol synthesis, kinetics, and absorption 20 analysis). Because of such situation, different values were off scale. With ANOVA on Ranks, the TG levels showed no statistical difference among the groups. Plant sterols did not affect the plasma TG levels in GS hamsters. Table 12: Potency Rank 25 Total Cholesterol HDL LDL TG Control c c c c 30 Soybean B-sitostanol 4 1 3-4 1-4 142086\0243454.WP -37 Tall oil (Forbes) 2-3 2-2 2-3 1-4 Sigmastanol 1 4 1 1-4 5 Artificial tall oil 3-2 3-2 4-3-2 1-4 In conclusion, the tall oil soap-derived composition of the present invention exhibited the most favourable profile due to the increase in HDL cholesterol and 10 decrease in total cholesterol. This HDL effect was not seen in the artificial tall oil composition. Similarly, the overall effect of sitostanol Is not favourable due to the significant decrease in HDL. Although it is not entirely clear, it does appear that, with respect to plant sterols, 15 the relatively hydrophobic sterols inhibit more total cholesterol absorption while the relatively hydrophilic sterols have more influence on the level of HDL The phytosterol compositions of the present Invention are unique in that both of these effects are preserved. 20 EXAMPLE 8: Human Feeding Experiment - General The aim of this study was to examine how sitostanol-enriched plant sterols added to the diet influence body's level and rate of production of circulating cholesterol. Before study, 10 health normo-lipidemic subjects aged 18-45 were 25 examined by a physician to ensure that they were in good health. Five males and five females were selected. A blood sample (20mL) were taken for laboratory to confirm the absence of health abnormalities and to measure blood lipid levels, Subjects then consumed a diet provided by the Metabolic Kitchen within the M2086\02454.WP -38 Clinical Research Laboratory for 9 days. This diet contained normal foods and was fed as three meals per day over the 9 day period. To the diet was added at a level of 1.5 g/day of one of: olive oil alone (Olive); olive oil in combination with a composition in accordance with the present invention and comprising sitostanol 5 (Forbes) ; olive oil in combination with a soybean plant sterol composition (Nulife); or corn oil alone (Corn). These materials were added directly to the diet mixed with a small amount of the dietary oil. This study enabled comparison between data obtained form a separate Heart and Stroke funded research project where the question was to examine whether the higher levels of plan 10 sterols in corn oil were responsible for the cholesterol synthesis-raising action that we previously observed when we fed subjects these oils. In this regard, three diet phases'have been studied already; corn (high in phytosterols), olive (very low in phytosterols) and olive with 1.5*g added soybean phytosterols. It is the present objective, in order to use these previous phases as controls, to use 15 olive oil as the dietary oil with added 1.5*g Forbes phytosterols (one of the compositions of the present invention). Diets were prepared in our metabolic testing facilities, with all meals consumed under supervision by the research staff. Diets were comprised of a two-day 20 rotating menu with food given as three meals per day. The level of food consumed by each subject was formulated to maintain that individual at weight 142086\024454.WP -39 balance, using predictive equations based on the subjects' weight, height, age and sex. Any weight changes were monitored and the amount of food given adjusted accordingly. During the study duration, subjects were given the phone number of a physician that could be contacted should subjects feel any 5 discomfort with the diet. Over day 9 of the diet period, subjects were requested to drink 25mL water labelled with a "stable isotope" tag, deuterium oxide. On each of the mornings of days 9 and 10 subjects provided a blood sample (20mL) for assessment of 10 cholesterol levels and cholesterol synthesis rate. These analyses were conducted by standard procedures. In brief, blood plasma taken at each timepoint over the study was separated and analyzed for total, low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol and total triglyceride concentrations. The circulatory levels of plants sterols were also 15 measured as documented. Levels of sitostanol, sitosterol and campesterol were assessed. In addition, cholesterol synthesis was assessed using the rate of uptake of the tracer deuterium from body water into circulating free cholesterol. Analytical methodologies have been described in the literature for these procedures. At the end of the diet period subjects were again examined by a 20 physician to ensure that they were in good health. A portion of the blood sample taken on day 10 was used again to confirm the absence of health abnormalities. 142086\0240454.W -40 Subjects were tested in two groups of 5. Each group was studied over 10 days. A 4-10 day interval separated the two trials. All procedures were conducted at the Metabolic Testing Facility at the Macdonald Campus of McGill University under 5 the direction of Dr. Peter Jones, Director, Dietetics and Human Nutrition. Phytosterol Analysis: Plasma phytosterols were extracted and quantitated by gas liquid 10 chromatography (GLC). 5-alpha-cholestane was used as an internal standard. The standard was added to 1.0 ml of plasma and saponified with 50% KOH and methanol (6:94 v/v) for 2h at 100-C. Plasma was then extracted three times with petroleum ether. Sterols were injected into the GLC (HP 5890 Series II) equipped with flame ionization detection. Separation was achieved on an RTx-1, 15 30'm capillary column, 0.25mm ID, 0.25um film thickness (Restek Corp. Bellefont, PA). Samples were injected at 80aC. The oven remained for 20 min. The oven temperature then increased to 320C (20cC/min) for at least 5 min before subsequent analyses. The injector and detector were set at 1.2 mI/min with the inlet splitter set at 10.:1. Phytosterol (campesterol, sitosterol, and 20 sitostanol) peak identification was confirmed using authentic standards. 142086\024454.WP -41 Fractional Synthetic Rate Deterrnination: Deuterium (D) enrichment was measured in red blood cell (RBC) free cholesterol and plasma water. RBC lipid extraction was performed in duplicate. 5 Methanol, hexane/chloroform (4:1 v/v), and doubly distilled water was added to the plasma. The mixtures were shaken mechanically, centrifuged at 1500 rev./min and supernatants were collected. The extraction procedure was repeated and solvent layers were combined. The supernatant was dried under nitrogen and the residue was then dissolved in chloroform and 10 chromatagraphed on silica plates. Plates were developed in hexane/diethyl ether/acetic acid (70:30:1) and the cholesterol band were identified according to a co-chromatagraphed free cholesterol standard. The cholesterol based was scraped from plates and extracted three times by shaking the silica in chloroform for 15 min followed by centrifugation. 15 Dried cholesterol samples were transferred to 18-cm combustion tubes. Cupric oxide (0.5g) and a 2-cm length of silver wire were added and tubes were sealed under vacuum of less than 20 mtorr pressure. The cholesterol samples were combusted for 4 hours at 520oC and the water generated was then vacuum 20 distilled into 10 cm combustion tubes containing 60 mg zinc reagent. These samples were reduced to zinc oxide and hydrogen gas at 520cC for 30 min. 142086\0240454.WP -42 Plasma samples were diluted 20 fold with water to reduce D enrichment to within the normal analytical range. Baseline samples were not diluted. Duplicate samples were vacuum-distilled into zinc containing combustion tubes. These 5 plasma water samples were also reduced to zinc oxide and hydrogen gas at 52OoC for 30 min. The deuterium enrichments of cholesterol and plasma samples were measured by differential isotope ratio mass spectometry using a triple inlet system with 10 electrical H3 compensation. RBC fractional synthetic rate (FSR) values were calculated as cholesterol deuterium enrichment relative to that of the precursor body water pool adjusted for the fraction of hydrogens of cholesterol derived from labelled substrate. The hepatic FSR values were derived using the equation: 15 FSR(per day) = chol. enrichment (1OOo/oo) *24h/interval period (h) plasma water enrichment (1000/oo '0.478 The cholesterol enrichment value covers the period of the time between first 20 consumption of deuterated water on the morning of day 9 and when the blood is drawn on the morning of day 10. The multiplication factor of 0.478 accounts for 142086\020454.WP -43 the fraction of deuterium atoms obtained from body water during cholesterogenesis. FIGURE LEGENDS 5 Fig. 24 Plasma lipid concentrations of total cholesterol (TOT-C), high density lipoprotein cholesterol (HDL-C), triglycerides (TG) and low density lipoprotein cholesteml (LDL-C) in healthy male and female subjects (n=11) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife plant 10 sterols and an oliver oil based diet (Nulife), an olive oil diet alone (Olive) or a corn oil diet alone (Corn) for 9 days. Results are expressed as mean - S.E.M. Diet treatment group means within each parameter having different subscripts differ significantly (p(0.05 using Tukeys post hoc comparison). 15 Fig. 25 Plasma lipid concentrations of total cholesterol (TOT-C), high density lipoprotein cholesterol (HDL-C), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) in healthy male subjects (n=6) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife plant sterols and an olive oil based diet (Nulife), an olive oil diet along (Olive) or a corn diet alone (Corn) for 20 9 days. Results are expressed as mean - S.E.M. Diet treatment group means within each parameter having different subscripts differ significantly (p(O.05 using 142Ol6\024454.WP -44 Tukeys post hoc comparison). Fig. 26 Plasma lipid concentrations of total cholesterol (TOT-C), high density lipoprotein cholesterol (HDL-C), triglycerides (TG) and low density lipoprotein 5 cholesterol (LDL-C) in healthy female subjects (n=5) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife plant sterols and an olive oil based diet (Nulife), an olive oil diet along (Olive) or a corn diet alone (Corn) for 9 days. Results are expressed as mean - S.E.M. Diet treatment group means within each parameter having different subscripts differ significantly (p(O.05 using 10 Tukeys post hoc comparison). Fig. 27 Decrease in plasma lipid concentrations of total cholesterol (TOT-C). high density lipoprotein cholesterol (HDL-C), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) in healthy male and female subjects (n-11) 15 consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife plant sterols and an olive oil based diet (Nulife), an olive oil diet alone (Olive) or a corn oil diet alone (Corn) for 9 days compared to plasma lipid concentrations prior to diet treatment. Results are expressed as mean - S.E.M. Diet treatment group means within each parameter having different subscripts differ 20 significantly (p(O.05 using Tukeys post hoc comparison). 142086\02c454.WP -45 Fig. 28 Decrease in plasma lipid concentrations of total cholesterol (TOT-C), high density lipoprotein cholesterol (HDL-C), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) in healthy male subjects (n=6) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife plant sterols and 5 an olive oil based diet (Nulife), an olive oil diet alone (Olive) or a corn oil diet alone (Corn) for 9 days compared to plasma lipid concentrations prior to diet treatment. Results are expressed as mean - S.E.M. Diet treatment group means within each parameter having different subscripts differ significantly (p(O.05 using Tukeys post hoc comparison). 10 Figure 29 Decrease in plasma lipid concentrations of total cholesterol (TOT-C). high density lipoprotein cholesterol (HDL-C), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) in healthy female subjects (n=5) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife plant 15 sterols and an Oliver based diet (Nulife), an olive oil diet along (Olive) or a corn oil diet alone (Corn) for 9 days compared to plasma lipid concentrations prior to diet treatment. Results are expressed as mean - S.E.M. Diet treatment group means within each parameter having different subscripts differ significantly (p(0.05 using Tukeys post hoc comparison). 20 Fig. 30 shows the concentration of individual phytosterols in plasma of subjects 142086\0240454.WP -46 consuming either a Forbes (present invention) and olive oil based diet, Nulife phytosterols and olive oil based diet, olive oil diet alone (Olive) and corn oil diet along (corn). 5 RESULTS The results of these human clinical trials ware outlined in the following Tables 13-32 and further compiled into Figures 24-30, Mean plasma total cholesterol concentration in the Forbes phytosterol 10 supplemented group was significantly lower than that for the olive oil group. It was also lower, though non-significantly, than the Nulife Phytosterol supplemented group (Table'13, Figure'24). Mean HDL cholesterol concentration was highest in the Forbes phytosterol 15 group. However, this difference was non-significant relative to the other groups due to much variation among subjects within groups (Table"14, Figure*24). Mean triglyceride levels were lower in the group fed corn oil than that consuming the Nulife sterols (Table"15, Figure 24). 20 Mean LDL cholesterol concentration in the Forbes phytosterol supplemented 14200\024454.WP -47 group was significantly lower than the mean concentrations for both the Nulife and oil groups (Table16, Figure 24). Within the male subset of subjects, total cholesterol levels were significantly 5 different between the Forbes and olive oil groups and between corn oil and Nulife groups. LDL cholesterol levels were significantly different between the Forbes and Nulife groups, the Forbes and olive oil groups, and olive oil and corn oil/groups. There was no statistical significance between treatment groups for the female subjects (Figures 25, 26). 10 FSR values were highest in the Forbes phytosteroLgroup. This difference is significant with respect to the olive oil group. Nulife means FSR is substantially lower than the Forbes mean FSR. However, the difference is non-significant due to much variation within the Nulife group (Figure 27). 15 The mean values in the corn oil group for each of the preceding parameters were in all cases minimally or non-significantly different from the Forbes mean values. Results of the GLC analysis indicate absorption of phytosterols (campesterol 20 and sitosterol) from the intestine into the bloodstream is lowest in the Forbes phytosterol supplemented group- The corn group demonstrated high 142086\0240454.WP -48 concentrations of phytosterols in plasma, and thus the greatest absorption of phytosterols into the blood stream. Olive and Nulife groups demonstrated similar intermediate plasma phytosterol concentrations (Figure 28). Plasma campesterol concentrations following the Forbes treatment diet were significantly 5 different from those following the Nulife and corn treatment diets. Sitosterol concentrations in the Forbes group were significantly different from those in all other treatment groups. Correlational analyses were performed to examine for relationships between 10 circulating phytosterol levels and indices of lipid level and synthesis (Table"21 25). Significant associations were observed between campesterol and HDL cholesterol concentration in the FCP group, between sitosterol and LDL cholesterol and between campesterol and total and LDL cholesterol level in the olive oil group. In corn oil fed individuals, sitostanol was correlated to HDL 15 cholesterol levels also. Individual data points for all subjects and males versus females for circulating campesterol, sitosterol, campesterol/sitosterol ratio, total cholesterol, HDL cholesterol and LDL cholesterol, as well as FSR are provided for subjects on 20 each dietary trial in Tables 21-32. IA2056\024454.WP -49 Table 13 Plasma Cholesterol Concentration (mg/dl) following Diet Treatment Subject Sex Diet Treatment Forbes Nulife Olive Corn I M 143.6 154.2 153.6 126.3 2 M 112.6 115.1 126.2 110.4 3 M 116.4 119.4 116.7 107.3 4 M 136.5 138.8 142.0 136.0 5 M 104.9 124.7 116.8 115.7 6 M 108.4 110.0 122.7 103.1 Subtotal mean 120.4 127.0"a 1 29

.

7 b 116.5" -SEM -6.5 -6.8 -6,1 -5.1 7 F 144.9 163.9 114.8 119.8 8 F 94.6 117.9 177.2 106.9 9 F 146.1 154.4 137.4 135.3 10 F 146.0 146.9 133.4 136.3 11 F 120.4 126.9 140.7 116.1 -13.1 Subtotal mean 130.4 142.0 13 4 .1b 122.9 -SEM -10.2 -8.6 -6.2 -5.7 mean 124.9" 133.8 119.42 -SEM -5.7 -5.6 -3.7 5 Plasma total cholesterol concentration in healthy male and female subjects (n=1 1) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife phytosterols and an olive oil based diet (Nulife), an olive oil diet alone (Olive) or a corn oil diet alone (Corn) for 9 days, Diet treatment group 10 means within each parameter having different subscripts differ significantly (p<0.05 using Tukeys post hoc comparison). 142086\024454.WP -50 Table 14 Plasma High Density Lipoprotein Cholesterol Concentration (mg/dl) following Diet Treatment Subject Sex Diet Treatment Forbes Nulife Olive Com 1 M 49.1 41.4 32.7 28.6 2 M 35.0 37.5 33.7 32.5 3 M 40.5 29.1 34.8 33.6 4 M 33.0 36.5 40.7 35.0 5 M 40.0 41.1 39.2 42.1 6 M 50.5 46.0 47.0 46.0 Subtotal mean 41.3 38.6 38.0 36.3 -SEM -2.9 -2.3 -2.2 -2.6 7 F 49.3 50.1 36.7 8 F 36.3 44.1 44.5 44.8 9 F 39.1 39.1 42.7 34.4 10 F 65.2 58.5 50.9 57.6 11 F 49.1 48.3 47.0 49.7 Subtotal mean 47.8 48.0 48.3 44.6 -SEM -5.1 -3.2 -1.8 -4.3 TOTAL mean 44.28 42.8 41.3 40.1 b -SEM -2.8 -2.4 -2.6 -2.6 5 Plasma high density lipoprotein cholesterol concentration in healthy male and female subjects (n=1 1) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife phytosterols and an olive oil based diet (Nulife), an olive oil diet alone (Olive) or a corn oil diet alone (Corn) for 9 days. Diet treatment 10 group means within each parameter having different subscripts differ significantly (p<0.05 using Tukeys post hoc comparison). 14206\02454.WP -51 Table 15 Plasma Triglyceride Concentration (mg/di) following Diet Treatment Subject Sex Diet Treatment Forbes Nulife Olive Corn I M 48-0 59.7 69.2 60.4 2 M 55.3 56.4 43.7 46.2 3 M 67.5 95.4 58.7 81.8 4 M 91.5 70.5 60.1 76.4 5 M 77.5 59.6 62.6 41.7 6 M 74.7 82.7 97.4 86.0 Subtotal mean 69.1 70.7 65.3 65.4 -SEM -6.4 -6.3 -7.3 -7.7 7 F 62.5 59.8 44.7 8 F 99.6 130.1 85.5 83.1 9 F 57.8 105.3 135.9 84.9 10 F 65.3 60.9 62.7 50.6 11 F 55.2 78.0 83.8 52.8 Subtotal mean 68.1 86.8 92.0 63.2 -SEM -8.1 -13.6 -15.5 -8.6 TOTAL mean 68.6 78.02 76.0 6 4 .4b -SEM -4.8 -7.1 -8.3 -5.4 142086\024B454.WP -52 Plasma triglyceride concentration in healthy male and female subjects (n=1 1) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife phytosterols and an olive oil based diet (Nulife), an olive oil diet alone (Olive) or a 5 corn oil diet alone (Corn) for 9 days. Diet treatment group means within each parameter having different subscripts differ significantly (p<0.05 using Tukeys post hoc comparison). Table 16 Plasma Low Density Lipoprotein Cholesterol Concentration (mg/dl) 10 following Diet Treatment Subject Sex Diet Treatment Forbes Nulife Olive Corn 1 M 84.8 100.8 107.1 85.5 2 M 66,5 66.3 83.7 68.7 3 M 62.4 71.2 70.2 57.4 4 M 85.2 88.2 89.3 85.8 5 M 49.8 71.8 65.1 65.3 6 M 42.9 47.4 56.3 39.8 Subtotal mean 65.33 74.3 * 78.6" 67.1 be -SEM -7.1 -7.5 -7.5 -7.2 7 F 83.1 101.9 74.1 8 F 38.4 47.8 53.2 45.5 9 F 95.4 94.3 107.3 84.0 10 F 66.3 75.8 74.0 97.4 11 F 60.3 63.0 69.6 55.8 Subtot mean 68.7 76.5 76.0 71.3 -SEM -9.8 -9.9 -11.3 -9.4 TOTAL mean 68.88 75.3b 7 7 .6b 69.0** --SEM -5.6 -5.8 -6.0 -5.5 14208M\024M54.WAP -53 Plasma low density lipoprotein cholesterol concentration in healthy male and female subjects (n=1 1) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife phytosterols and an olive oil based diet (Nulife), an 5 olive oil diet alone (Olive) or a corn oil diet alone (Corn) for 9 days. Diet treatment group means within each parameter having different subscripts differ significantly (p<0.05 using Tukeys post hoc comparison). Table 17 Decrease in Plasma Cholesterol Concentration (mg/dl) following Diet 10 Treatment Compared to Baseline Subject Sex Diet Treatment Forbes Nulife Olive Com I M 18.4 7.8 8.4 35.7 2 M 27.7 25.2 14.1 29.9 3 M 18.0 15.0 17.6 27.0 4 M 22.5 20.3 17.1 23.1 5 M 9.6 -10.2 -2.3 -1.2 6 M 19.6 17.9 5.2 24.9 Subtotal mean 19.3c 1 2

.

7 "a 10.0b 23.2 -SEM -2.4 -5.2 -3.2 -5.2 7 F 0.1 -18.9 25.3 8 F 53.9 30.6 33.8 41.7 9 F 32.0 23.7 1.0 42.8 10 F 4.6 3.7 13.2 14.4 11 F 49.8 43.3 36.7 54.0 Subtotal mean 28.1 16.5 a 21.2 35.6 -SEM -11.1 -10.9 -8.5 -7.0 TOTAL mean 23.3* 1 4

.

4 b 1 4

.

4 b 28.8 -SEM -5.1 -5.4 -4.0 -4.5 142055\024454.WP -54 Decrease in plasma cholesterol concentration in healthy male and female subjects (n=1 1) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife phytosterols and an olive oil based diet (Nulife), an olive oil 5 diet alone (Olive) or a corn oil diet alone (Corn) for 9 days compared to plasma lipid concentrations prior to diet treatment. Diet treatment group means within each parameter having different subscripts differ significantly (p<0.05 using Tukeys post hoc comparison). 10 Table 18 Decrease in Plasma High Density Lipoprotein Cholesterol Concentration (mg/di) following Diet Treatment Compared to Baseline Subject Sex Diet Treatment Forbes Nulife Olive Corn 1 M -13.5 -5.8 2.94 7.0 2 M 16.9 14.4 18.1 19.4 3 M 26.5 37.9 32.2 33.4 4 M 15.5 12.0 7.9 13,5 5 M 13.1 11.7 13.5 10.6 6 M 0.4 4.9 4.0 4.9 Subtotal mean 9.8 12.5 13.1 14.8 -SEM -5.8 -5.9 -4.5 -4.3 7 F 16.6 15.8 29,3 8 F 45.8 38.0 37.6 37.3 9 F 13.5 13.5 9.9 18.2 10 F 5.11 1.6 9.2 2.5 11 F 6.6 7.3 8.6 5.9 Subtotal mean 15.5 15.3 16.3 18.6 -SEM -8.4 -6.2 -7.1 -6.7 TOTAL mean 12.48 13.8 14.4 16.5 -SEM -4.8 -4.1 -3.7 -3.7 142056\O241454.WP -55 Decrease in plasma high density lipoprotein cholesterol concentration in healthy male and female subjects (n=1 1) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife phytosterols and an olive oil based diet 5 (Nulife), an olive oil diet alone (Olive) or a corn oil diet alone (Corn) for 9 days compared to plasma lipid concentrations prior to diet treatment. Diet treatment group means within each parameter having different subscripts differ significantly (p<0.05 using Tukeys post hoc comparison). 10 Table 19 Decease in Plasma Triglyceride Concentration (mg/dl) following Diet Treatment Subject Sex Diet Treatment Forbes Nulife Olive Com 1 M -9.9 -21.5 -31.0 -22.3 2 M -26.5 -27.6 -14.9 -17.4 3 M 5.5 -22.4 14.3 -8.8 4 M -26.6 -5.5 4.8 -11.4 5 M -30.3 -12.4 -15.5 5.5 6 M -13.0 -21.1 -35.7 -24.4 Subtotal s mean -16.8 -18.4 -13.0 -13.1 -SEM -5.6 -3.3 -8.0 -4.5 7 F 0.7 3.4 18.5 8 F -6.3 -36.8 7.8 10.2 9 F 42.8 -4.6 -35.3 15.7 10 F 3.0 7.4 5.5 17.7 11 F 25.7 2.9 -2.9 28.1 Subtotai mean 13.2 -5.6 -6.2 18.0 -SEM -9.1 -8.0 -10.0 -2.9 TOTAL mean -3.173 -12.6" -10.3 1.0b -SEM -6.8 -4.3 -6.0 -5.6 142085\024S4.P -56 Decrease in plasma triglyceride concentration in healthy male and female subjects (n=1 1) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife phytosterols and an olive oil based diet (Nulife), an olive oil 5 diet alone (Olive) or a corn oil diet alone (Corn) for 9 days compared to plasma lipid concentrations prior to diet treatment. Diet treatment group means within each parameter having different subscripts differ significantly (p<0.05 using Tukeys post hoc comparison). 10 Table 20 Decrease in Plasma Low Density Lipoprotein Cholesterol Concentration (mg/dl) following Diet Treatment Compared to Baseline Subject Sex Diet Treatment Forbes Nulife Olive Corn 1 M 33.9 17.9 11.6 33.2 2 M 16.1 16.3 -1.1 13.9 3 M -9.7 -18.5 -17.5 -4.6 4 M 12.4 9.3 8.3 11.8 5 M 2.5 -19.4 -12.7 -12.9 6 M 21.7 17.2 8.4 24.8 Subtotal mean 12.8" 3.8 * -0.5 11.0it -SEM -6.2 -7.3 -5.0 -7.1 7 F -16.6 -35.4 -7.7 8 F 9.4 0.0 -5.4 2.3 9 F 10.0 11.1 -1.9 21.5 10 F 10.6 1.1 2.9 -20.5 11 F 38.0 35.3 28.7 42.5 Subtotal mean 10.3 2.4 6.1 7.6 -SEM -8.6 -11.4 -7.2 -11.1 TOTAL mean 11.7 3.2 b. 9.5 -SEM -4.9 -6.2 -4.2 -6.0 142065\024M54.V -57 Decrease in plasma low density lipoprotein cholesterol concentration in healthy male and female subjects (n=11) consuming either Forbes phytosterols and an olive oil based diet (Forbes), Nulife phytosterols and an olive oil based diet 5 (Nulife), an olive oil diet alone (Olive) or a corn oil diet alone (Corn) for 9 days compared to plasma lipid concentrations prior to diet treatment. Diet treatment group means within each parameter having different subscripts differ significantly (p<0,05 using Tukeys post hoc comparison). 10 Table 21 FORBES PHYTOSTEROLS SAMPLES Campes sitoste FSR LDL HDL Tot. camp/ Name t mg/dl pools/ mg/dl mg/di chol sit mg/dl d mgdl Alan___ ___ 0.30 5,130 66. 35.0 112.60 Z 1 M Dave_ 1.11 0.68 6.3 42.9 5i050 106.40 1.5 Elizabeth 1.16 0.59 4.00 -90-& 49.1 120A0 1.94 _Gran~t____3.4_6- 1.63 26 48 491 421 Johan__ __1.35_ 1.33 . a 8. .0 105 . John 0.28 2 5. 2 49.8 .. AQAM0 10.90 1.22 Ma n . 0.37 0.13 4.0 8. 493,1.0 2. Mar, 5.90 66.5 65.0 146.00 _4 Paula _____3.20 .......iA 39.10 146.10 trice 5 .30 42.4 40.50 118.40 1.54 Stephanie 052-0 6._1_ 7.60 38.4 36-30 94.0k 4.46 E 1.07 a Q .60 66.83 49.1 124.65 2.21 STD ____2 15.33 171 .00 18.12 1.0 1405.40314M14P -58 Table 22 OLIVE___ SAMPLES Campest sitoste FSR LDL HDL Tot. camp/ Name mgd mg/dl pools! mg/dl mg/dl chol sit d ____ mgldl Dave 6.2Q .

56 47.00 122.70 0.79 Elizabeth 8.30 9.40 69.6 47 133.40 0.88 .han 3.60 7.60 89. 40.70 .A4Z±QQ 0.47 John6.90 15 - 6. 39.20 116.80 0.44 4.30 9.80 50.90 747. 0.4 Paula _107.3 42.70 114.80 Patrice 1 4.80 70.2 34.80 1 . 7 0 St~a~e 1.0 14.10l 53.2 44.502 114.80 1.02 Grant 31 3.-- 1.30 i._QZAO Q-32.70, 153.60 .~ MEA .N5 7690 -413J 12 0.74 STD 3.87 0 18.92 13.33 142086\024D454.WP -59 Table 23 PLANT STEROL TABLEI1 ___ __ SAMPLES Campest sitoste FSR LDL HOL Tot camp/ Name mg/dl mg/dI pools/ mgldl mg/u chol sit di mgldl Dave 7, 47. 46.00 11000 O.88 Eiaeh5.70 5.90 63 48.30 -126.90 0.97 Johan 4~A.7 100 88.2 36.50 138.80 0.46 John 12.60 7i.8 ~ 41.1 124.7 0.82 M 11.00 13.20 75.8 5-3 0 i0-90 8 Paula 12.00 14.50 4.3. 39.10 135.30 .83 Patric 4.20 - 4 ion0i 29.10 119.40 0.88 Stephanie M1 m n i4.4 _ _-47.- 44.10 17.90 0.90 Ala 1.60 - 66 3 .3750 11110____ Grant 2.40 4.54 0.60 =Q=8 41.40 154.20 . EAN 7.76 9.74 1.10 STD 1 .04 7 142066\24D4S4.WP -60 Table 24 CORN SAMPLES Campest sitoste FSR LDL HDL Tot. camp/ Name mg/dI mg/dl pools/ mg/dl mg/dl chol sit d mg/dI Dave 9.70 10.50 39.8 46.00 103.10 0.92 EH 2;beth 9.30 11.50 55,8 49.70 116.10 0.81 Johan 13.90 i OL 85.8 35,00 136.00 1.99 John 12.30 13.90 65.3 42.10 116.70 0.88 Mary 18.40 21.50 97.4 57.6U 136.30 0.86 Paula 6.70 7.00 84 34.40 135.30 0.96 Patrice - 5,$ 5.60 - - 57.4 33.60 10Q7.30 0.98 Stephanie 4.40 10.00 45.5 44.0 106.90 0.44 Alan 7.10 68.70 32.50 110AQ.40 Grt_ 10.96 9.91 2.10 85i5i 28.60 126.30 .1J.1 MEAN 10.13 1 0.77 4.60 6852_ 40.43 119.34 1.00 ST__ 4.15 .4,0 2.50 18.20 65 12.39 0.411 142086\024D454-WP -61 Table 25 FORBES.PHYTOSTE: MALES SAMPLES Campest sitoste FSR LDL HDL Tot. camp/ Name mg/dl mg/dl pools/ mg/dl mgfdl chol sit d mg/dl Alan 0,30 0.13 5.30 66.5 35.00 112.6Q 2.34 Dave 1.11 0.68 6.3 42.9 50.50 108.40 1.64 Grant 3 1.63 2.60 84,8 49.10 143,$0 2.12 Johan 1.35 1.33 5.30 852 33.00 136.50 1.02 John 0.28 0.23 5.20 49.8 40.00 104.90 1.22 Patrie 5.00 .2, 40.50 116.40 MEAN 1.30 0.80 4.951 6 1351 120.4 1.67 STD 1.16 0.59 1.13 15.96 6.54 14.48 0.51 Table 26 FORBES PHYTOSTE: FEMALES SAMPLES Campest sitoste FSR LDL HDL Tot. camp/si Name mgldl mg/dl pools/ mg/dl mg/dl chol t d mgldl Elizabeth 1.16 0.59 60.3 49.10 120.40 1. Manon 0.13 4.70 __ 49.30 144.90 2.92 MQ 66.3 65.20 146.00 Paula __21.0 14. 10 Stephanie 0.52 0.12 7. .384 36.30 94.60 4.46 MEAN 08 0.28 5.08 68.70 ... !5 10.40 3.11 STD 0.341 0.221 1.541 19.57 10. ... A 1.04 142006\02404S4-WP -62 Table 27 OLIVE; MALES SAMPLES Campest sitoste FSR LDL HDL Tot. camp/ Name mg/dl mg/dl pools/ mg/di mgldl chol sit d mgldl_ Dave 6.20 7.0 56.3 47.00 122.70 0.79 Johan 3.60 7.60 89.3 40.70 142.00 0.47 John 6.90 15.60 55.1 39.20 116.60 0.44 patriw,. . 4.80 5.40 70 2 34.80 116.70 0.89 Alex 3,43 1.21 2.60 91.90 42.30 148.15 2.82 Grant 3.13 3.23 1.30 107.10 32.70 153.Q 0.97 Jean 3.02 -1.1 1.30 96.80 40.80 14L.5 2.53 MEAN 4.44 6.00 1.3 82.39 39.64 135.49 1.27 STD 1.45 466 0 17.26 4.40149 0.91 5 Table 28 OUVE: FEMALES SAMPLES Campest sitoste FSR LDL HDL Tot. camp/ Name mg/dl mg/dl pools/ mg/dl mg/dl chol sit d mg/dl Elizabeth 8.30 ._90 69.5 47.00 .iL4... 0.88 Mary Paula 107.3 42.70 114.8 3kphanit..... __1.4 .4.1. _.3.,2 _44.50. _114..8_ ___L2. Lucv 0.81 0.60 3.70 89.40 68.70 174.Q 1.35 MEN 7.84 8.03 .3J0 79.88 50.73.1.34,2. 1.08 STD 5. 56. 5601 0.00 20.37 10,49 24.17Q0.2 142086\02C454.WP -63 Table 29 PLANT: MALES SAMPLES (Nulife) Campest sitosto FSR LDL HDL Tot camp/ Name mgldl mgldl pools/ mg/dI mg/dl chol sit d mg/dl Dave 6.60 7.50. 47.4 -00 110.0 0.88 John 4.70 .20 8.8_2 36.50 138.8 0.46 _12p I Q.30 12.60 71.8 41.10 124.7 0.82 Patrice 4.20 119.4 0.88 Alan 1.60 66.30 5 115.1 Grant 4.54 -L0fi0100.80 41.40 L 0.55 0.50 8 41.90 139.2 _ MEAN 5.66 -0.90 76a 39.07 128.7 0,2 2.67 0.50 16.29 4.99 14.84 18 142006\02 454.WP -64 Table 30 PLANT: FEMALES SAMPLES (Nullfe) Campest sitoste FSR LDL HDL Tot camp/ Name mg/dl mg/dl pools/ mg/dI mg/dI chol sit d mg/dl Elizabeth 5.70 5.90 63 _48,3 126.9 0.97 Mary 11Q. 13.20 753 6850 146.9 __0.83 Py__a 12.00 14.50 94.3 39.10 135.3 0. Stephanie .1290 14.40 47.6 44.10 117.9 0.90 -L ______ ___5.60 91.80 42,80 1472 MEN 10.40 12.00 5.60 74.54 46.56 134J 0.8__08 STD 2.80 3.56 0.00 1L54 6.6 0.08 142086\02 4454.WP -65 Table 31 CORN; MALES_ _ _ SAMPLES Campest sitoste FSR LDL HDL Tot camp/ Name mgldI mg/dl pools/ mg/dl mg/dI choll sit di mg/i___ 9.70 10.50 39. 103.1 0.92 Johan- 13.90 --. P.Q 85.8 n.00 -in-±f 1-99 Jh~ 12.30 13.90 fi5.3 42.10 115.7 1 .8 Patrice 5.50__5.6Q 57 33.60 0 0.98 Alan 6.32 2.33 7.10 68.7 2 110. 2.71 rant 196 9.91 2.10___85. 28.60 128.3 1.11 Je4n 11.21 1-0 4,40 82,40 40.20 152.8 1.64 Simon 6.10 9..2 45.30 155.5 14 72.A\ 2 91 123.6 \2.4 STD ~ ~ .9 8.=§ ~59 __ i42O86\024D454.WP -66 Table 32 CORN: FEMALES SAMPLES Campest sitoste FSR LDL HDL Tot. camp/ Name mg/dl mg/dl Pools/ mg/dl mg/dl chol sit d mg/dl Elizabeth 9.30 11.50 55.8 49.70 ; 11. J1 Marvy_ 1S-40 2.50 ,9$7.4 57,60 136.3 0.88 Pu la 6.70 7.00 4 34.40 135.3 0.96 Steghanie 4.40 10.00 45.5 44.80 J1DL9_0.44 Manon .50 741 0 36.70 119.8 MEAN 9 70 12.50 8,50 71.36 44.6 122.8 0.77 5.1 54 0.0 8.50 1.36 0.2 STDp 5.31 5.44 .0__8.74 ___.0_[_3 DISCUSSION 5 In humans, two common patterns of lipoprotein cholesterol and triglycerides are known. The female pattern spans across reproductive years and consists of relatively low TC* and LDL-Ch ("bad" cholesterol") and high HDL-Ch"* ("good" cholesterol) with high TG**". The typical male pattern has relatively higher TC and LDL-Ch and lower HDL-Ch and TG. Not only did the Forbes' composition 10 reverse the male pattern and enhance female cholesterol patterns (TC, LDL-Ch, HDL-Ch) but also decreased female TG excess considered to be a risk factor for atherosclerosis and modified undesirable olive oil dietary effect on blood cholesterol. These are very significant and unexpected results. 15 The trial results are summarized in Table 32a 142086\0243454.WP -67 Table 32a Normocholesterolemic Subjects On Average Western Diet With Added Olive Oil, Olive Oil and Forbes FCP-3PI Composition, Olive Oil and Soybean Plant Sterol Composition (Nulife) Male Female Relative changes in % with olive oil diet as a baseline (100.0%) Diet TC LDL- HDL-C TG TC LDL - TG Olive Oil* 100 100 100% 100% 100% 100 100 100 Olive Oil + 92.8 83.0 108.3% 105.8% 92.6% 90.9 103.2 74% Forbes %___ % A % Olive Oil + 97.9 94.5 101.5% 108-2% 100.9 100.6 103.6 94.3 Nulife % % % % % 5 (Expressed as an average percent deviation from olive oil diet baseline) TC* - Total Cholesterol LDL-Ch** - Low Density Lipoprotein Cholesterol HDL-Ch**" - High Density Lipoprotein Cholesterol 10 TG**** - Triglycerides DISCUSSION The results of this study indicate that consumption of Forbes phytosterol mixture 15 significantly reduces plasma total and LDL cholesterol levels with respect to olive oil. LDL cholesterol levels were also significantly lowered in the Forbes group with respect to the Nulife phytosterol group. A compensatory increase in fractional cholesterol synthetic rate was observed in the Forbes phytosterol group. This increased bisynthesis of cholesterol likely followed the reduction in 20 plasma cholesterol levels through intestinal cholesterol losses. 142085\024454.WP -68 There is significantly less absorption of phytosterols into the bloodstream in the Forbes phytosterol group versus the other treatment groups. Moreover, sitostanol was nondetectable in subjects' plasma, regardless of the phytosterol 5 mixture consumed. It is believed that sitostanol inhibits the absorption of phytosterols as well as cholesterol into the bloodstream, The olive treatment group served as a control for the Forbes and Nulife treatment groups - in that all variables were constant 10 except for the additional of the respective phytosterol mixtures. The fact that total and LDL cholesterol values were greatly reduced in the Forbes group as opposed to the Nulife group, without a corresponding increase in plasma sitosterol concentrations, suggests that the sitostanol inhibits both cholesterol and its own absorption more than any of the Nulife component phytosterols. 15 There remains the possibility that it is an interactive effect of different phytosterols mixed together in different proportions rather than the effect of an individual component which is influencing cholesterol metabolism. However, this possibility seems unlikely given that the effects of pure sitostanol versus Forbes phytosterol mixture was tested in hamsters and total and LDL cholesterol levels 20 were lower in the former group (unpublished results). Curiously, the HDL cholesterol levels in the sitostanol group were lower than those for the Forbes 142056\02C454.WP -69 group. Possibly, sitostanol is the more "active" ingredient in terms of cholesterol metabolism modification, but its action is enhanced by the presence of other phytosterols in the Forbes mixture. 5 It is interesting to note that although B-sitosterol is a main constituent of the Forbes phytosterol mixture, it is campesterol that is more concentrated in the plasma. It is possible that the presence of sitostanol selectively inhibits the absorption or facilitates the elimination of sitosterol relative to campesterol. It is speculated that this action may act as a secondary effect concurrent to the 10 inhibition of cholesterol absorption. While the results indicate that sitostanol is more effective than campesterol and sitosterol in terms of modifying cholesterol absorption into the bloodstream, this increase coincides with an increase in fractional synthetic rate. The not 15 hypocholesterolemic effect of phytosterols is likely the result of an inhibition in cholesterol absorption or altered cholesterol synthesis and excretion. Phytosterols may thus displace cholesterol from mixed micelles during fat absorption, resulting in a reduction in the absorption rate of intestinal cholesterol. It is yet unknown whether phytosterols exert their influence in the gut lumen by 20 trapping cholesterol or in the intestinal mucosa by interfering with the assembly and secretion of chylomicrons into the blood. 142C86\02C454-WP -70 A favourable response to the administration of the Forbes composition has been demonstrated across virtually the entire lipid and lipoprotein profile both in male and female. 5 The low plasma HDL-Ch is regarded both in male and female as a cardiovascular risk factor. The individuals with high HDL-Ch have lower risk of coronary heart disease than individuals with low HDL-Ch even in the presence of other risk factors such as high LDL-Ch. Thus, a high HDL-Ch:LDL-Ch ratio can 10 be considered a "negative" cardiovascular risk factor. 1420W\1Zs454.WP -71 Table 32b HDL I LDL Ratio* Diet Olive Oil Olive Oil + Forbes Olive Oil + Nulife Male 0.483 0.632 0.519 Female 0.609 0.695 0.627 * the higher the ratio the lesser the risk of atherosclerosis 5 10 EXAMPLE 9 The following example examines the effect of plant sterols derived from soybean, the composition of the present invention (Forbes) or pure sitostanol on plasma lecithin-cholesterol acetyl transferase ("LCAT") activity and on free cholesterol 15 esterification rate in New Zealand White (NZW) male rabbits fed 0.5% cholesterol for 65 days U2086\024454.P -72 Methods Twenty four NZW rabbits weighing 1.6 to 1.8 Kg were divided into four groups of n=6. They were accommodated for two weeks in the animal care facility at McGill 5 University prior to a 65 days feeding period of semi-purified diet. All groups received 0.5% cholesterol in their diet in addition to one of three mixture of plant sterols for the non control groups: soybean (65% §-sitosterol, 20% campesterol and 15% dihydrobrassicasterol), Forbes as per one of the embodiments of the present invention (65% §-sitosterol, 16% campesterol and 17% sitostanol) and 10 sitostanol (89% purity with dy weight every one week during the study period. LCAT activity and plasma cholesterol esterification rate were accomplished in Ste Paul's hospital at the ASL facility in Vancouver (Dr. Frholich's lab). For detailed procedures refer to Dr. Pritchard, ASL director. Briefly, 30 pl of labeled H 3 15 cholesterol in ethanolosomes with 10 pl of apoA-1 and 85 pl of assay buffer were mixed together, incubated for 30 min at 3700, then added to 15 pl plasma sample in a glass culter tube to which 60 pi of BSA/§-mercaptoethanol was added. The sample-mixture was incubated for 30 min at 37 0 C. The enzymatic reaction was stopped with 1 ml of 99% ethanol. Assay samples were incubated 20 for 1 h at 60 0 C, centrifuged for 10 min at 3000 rpm (1710 x g), then transferred to clean glass tubes and dried under nitrogen at 60 0 C. An internal standard of free 142096\0240454.WP -73 cholesterol and ester-cholesterol was added to each sample. The lipid extracts were resuspended in 50 pi chloroform and streaked on pre-coated TLC plastic sheets silica gels 60 F 25 4 (E. Merck). The plates were developed in a glass tank containing and saturating with petroleum ether, diethyl ether and acetic acid 5 (105:18:1.5, V:V:V) for 8-10 min. Unesterified cholesterol and cholesterol ester bands were visualized with iodine, transferred to 5 ml scintillation vials. Toluene containing omnifluor (4g/l) was added to each vial and then left for 1 h before reading. Cholesterol esterification rate (FER) was measured after equilibration and labelling of internal free cholesterol for 24 h with 3 H-cholesterol. The 10 procedures applied in measuring esterified cholesterol are similar to the previous ones. Results 15 Food intake among different groups did not show significant variations. Rabbit's daily food intake ranged between 54.6 to 87.8 g. The rabbits' daily chow consumption during the accommodation period was around 100 g. LCAT activity and cholesterol esterification rate 20 The results obtained showed a decrease in LCAT activity by 31.1% in the Forbes 142086\024454.bP -74 fed group as compared to the control one; mean values are 11.58 - 2.06 and 16.81 - 5.34 nmol/h/ml respectively. However, the mean of the values were not significant, P = 0.16 (Figure'8). LCAT activity from human control showed a value of 28.86 nmol/h/ml which a rabbit LCAT activity gave a value of 49.44 nmol/h/mI. 5 Both values are considered with in the expected normal range. FER values were the lowest in the soybean and tall oil groups; the means were 0.496 - 0.2 and 0.82 - 0.19 %/h respectively. The mean activity was significantly different as compared to the sitostanol group, P = 0.014 (Figure 33) Mean plasma cholesterol esterification rate was the highest in the sitostanol treated group with 10 soybean being the lowest; both groups were significantly different, P<0.05 (Figure 10). The ratio of unesterified esterified cholesterol ranged between 24 to 42%. No difference in the mean values was observed among the different groups. 15 LCAT activity was the lowest in the Forbes treated group. LCAT activity in normal rabbits (no cholesterol or phytosterol fed rabbits) was >168% higher than any of the four cholesterol groups. The first explanation could be that the enzyme content was a limiting factor in the assay. That is, with extremely high lipidimic rabbits (200 to 577 mg/d unesterified cholesterol (UC)), LCAT would esterify a 20 certain concentration of endogenous and exogenous UC (30 wg) but not all the UC available. LCAT will be depleted before esterifying all the endogenous and 142086\0240454.WP -75 exogenous UC. Another possible explanation is that a high plasma cholesterol levels, lipoproteins are aggregated in bigger particles which become less accessible to LCAT enzyme. This factor will explain the low activity observed, but it will not explain why the Forbes group had lower activity than the other three 5 groups. Sitostanol treated group showed similar LCAT activity as the control group, while that of soybean was lower and Forbes the lowest, Sitostanol has the lowest absorption rate as compared to the other two plant sterol groups which suggests that sitostanol did not exhibit any internal effect on the LCAT enzyme while the more absorbed phytosterols showed a certain effect on LOAT 10 activity. FER values were the highest in the sitostanol group which showed the lowest cholesterol concentration. It correlates with the high LCAT activity in the same group suggesting that at this total cholesterol concentration (635 mg/dl). LCAT was not down regulated with cholesterol. 15 In summary, blood cholesterol esterification leads to an increase in cholesterol atherogenicity. In this rabbit study, one of the compositions of the present invention resulted in a 31.1% LCAT activity decrease. The total serum phytosterol-cholesterol ratio was 2:1 indicating an "intrinsic" effect and confirming the dual phytosterol effect described herein 20 EXAMPLE 10 DAY ORAL ADMINISTRATION OF SITOSTANOL-ENRICHED FCP3PI PHYTOSTEROLS VERSUS SITOSTANOL-FREE SOYBEAN L420O6\024454.WP -76 PHYTOSTEROLS IN MALE AND FEMALE NORMOLIPIDEMIC HUMANS: EFFECTS ON STEROL METABOLISM The present study was designed to examine the effects of phytosterols in varying S compositions on cholesterol metabolism in humans. The FCP-3PI phytosterol mixture was added as a fourth arm to a previous study funded by the Heart and Stroke Foundation of Canada involving three 10 day diets administered in a random crossover design. The four diets consisted of the same base diet with the only difference being the replacement of 213rd's of the total fat with: 10 1, CORN OIL 2. OLIVE OIL 3. OLIVE OIL plus a soybean based mixture of phytostorols (sitostanol-free NuLife brand containing 62% b-sitosterol, 14% stigmasterol, and 24% 15 campesterol) administered at a dose of 0.55 g phytosterol per 1000 kcal 4. OLIVE OIL plus FCP-3PI mixture of phytosterols (containing 62% bsitosterol. 16% campesterol, and 21% sitostanol) administered at a dose of 1.5 g phytosterol per 70 kg body weight 20 For all treatment groups, the following parameters of endogenous cholesterol metabolism were monitored: *Plasma total. LDL- and HDL-cholesterol concentrations as well as triglyceride concentrations 25 *plasma phytosterol concentrations (b-sitosterol, campesterol, and sitostanol) as an index of absorption of cholesterol and phytosterols 142086\024D454.WP -77 *in vivo cholesterol synthesis rates via deuterium incorporation OBJECTIVES 5 1. To determine the effects of the addition of sitostanol-enriched FOP-3PI and sitostanol-free soybean based phytosterols to an olive oil based diet on plasma total, LDL, and HDL cholesterol, and triglyceride concentrations in normolipidemic men and women. 10 II To compare in vivo cholesterol synthesis rates in normolipidemic humans receiving corn oil, olive oil, and olive oil plus phytosterol mixtures containing sitostanol-enriched FCP-3PI or sitostanol-free soybean based phytosterols. METHODS 15 Subjects Eleven healthy volunteers with plasma total cholesterol <5.1 mmol/l, LDLcholesterol <2.5 mmol/l, plasma triglyceride (TG) <2.8 mmol/l, and BMI<27 were recruited by poster and Internet advertisements. After verbal screening 20 which provided medical history, there was no indication of any chronic illness, they did not smoke, and were not taking any medication known to affect lipid metabolism (lipid-lowering drugs, beta-blockers, diuretics, or hormones). All subjects gave informed consent on the understanding they could withdraw at any time from the study which was approved by the Ethical Committee of McGill 25 University. Experimental Protocol 142086\02 4 4 4.WP -78 A randomized, cross-over design was used for three of the four 10 day diet cycles with a minimum 2 week wash out period between each phase where the subjects consumed their habitual ad libitum diets. An addition fourth 10 day diet cycle of olive oil plus the FCP-3PI phytosterol mixture (21% sitostanol) was 5 added a minimum of 4 weeks following the third diet cycle. For each 10 day diet cycle, subjects consumed a solid food diet designed to containing 55% of energy as carbohydrate, 35% fat and 15% protein where all non-oil constituents of each diet were identical. The only factor that was manipulated in the diet was the type of oil that the diet is based on. The four study diets contained 2/3 fat 10 (approximately 21% of energy) as either (i) corn oil, (ii) olive oil, (iii) olive oil plus soy-based phytosterols (62% b-sitosterol) fed at a level of 0.55 g per 1000 kcal, or (iv) olive oil plus phytosterols (addition of 21% sitostanol) administered at 1.5 g per 70 kg body weight. The diets were designed by a registered dietician so as to meet normal nutrient needs, as indicated by Recommended Nutrient Intakes 15 for Canadians (Health and Welfare Canada 1990). The diets were administered as three isocaloric meals per day prepared using fresh, canned, and frozen ingredients purchased at a local supermarket; wherever possible, treatment oils were used in all baking and cooking. All 20 ingredients were weighed to the nearest 0.5 g. Meals were prepared and consumed on site under supervision at the Metabolic Research Unit, Macdonald Campus, McGill University. In rare cases when subjects could not be present for meals at the unit, pre-prepared meals were packaged for take-out. Subjects were continually monitored for compliance, instructed to eat all foods provided, 25 and requested not to eat or drink any additional food or drink, except water, during each 10 day trial. For each diet cycle, subjects received identical caloric loads individually determined by the Mifflin predictive equation and multiplied by an additional activity factor of 1.7 to yield total daily energy requirements for younger, 142016\0240454.bP -79 active individuals. Body weights were monitored daily during each diet cycle to ensure compliance and correct caloric intake. Any adjustments needed to caloric intake were made only within the first three days of the first cycle. This caloric load then remained consistent over all three diet phases. 5 On day 9 and 10 of each dietary phase, duplicate fasting blood samples were collected just before and 24 hour after dosing with deuterium oxide (D20) for the determination of plasma lipid levels, sterol levels, and deuterium (D) incorporation into cholesterol. An oral bolus dose of 0.7 g of D20 per kilogram 10 body water (estimated as 60% of body weight) was administered pre-breakfast on Day 9 at approximately 8:00 am to each subject following the initial blood sample. Any water consumed during the following 24 hours included trace amounts of D20 (1.4 g per kg water consumed). 15 Macronutrient Analysis of Diets Homogenized mixtures of each meal (2 day rotation) for each diet phase were chemically analysed for macronutrient content (see Table 33). Moisture, ash, crude fat, and protein content of the homogenized food mixtures were analysed in 20 accordance with AOAC guidelines (1987). Proximate compositions were reported as grams of macronutrient per 100 grams wet weight. Carbohydrate contents of food samples were calculated by subtracting the sum of moisture, protein, crude fat, and ash values from 100 g (therefore, overestimation of carbohydrate content was from I % to 5% due to inclusion of crude fibre content). 25 Final values are presented as percent of total energy. Fatty Acid and Phytosterol Analysis 142086\240454.WP -80 Following lipid extraction and boron trifluoride methylation of homogenized meals, fatty acid methyl esters were analysed on a Hewlett-Packard 5890 Series I gas-liquid chromatograph (GLC) equipped with a flame ionization detector and fitted with a 30 in x 0.25 mm ID fused-silica capillary column (SP2330). The 5 carrier gas was helium at, 1.0 ml/min with the inlet splitter set at 50:1. Runs were temperature programmed in the range of 200-250degreesC at a heating rate of 4degreesC/min. Fatty acid methyl esters were identified by matching retention times with 99% pure commercial standards (Nu-Chek Prep) (Table 33). 10 Phytosterol analysis was carried out following lipid extraction and saponification of the dietary oils, homogenized meals, and plasma. The nonsaponifiable lipid contents were analysed on a Hewlett-Packard 5890 Series 11 gas liquid chromatograph (GLC) equipped with a flame ionization detector and fitted with a SAC-5 (30 m x 0.25mm ID) capillary column. The carrier gas was helium at 1.0 15 ml/min with the inlet splitter set at 50:1. Isothermal runs were made at 275degreesC with the injector and detector heaters at 300C. Beta-sitosterol, campesterol, and other sterol peaks were identified by comparison of retention times with those of authentic standards and quantitated with the use of a 5-a cholestane internal standard (Sigma Chemical Co., St. Louis, MO). Absolute 20 values of plasma phytosterol levels have been provided (mmol/l), However, to eliminate the effects of varying concentrations of plasma lipoproteins transporting plant sterols, plasma phytosterol concentrations have also been expressed in terms of mmol/mol of plasma total cholesterol. 25 Plasma Lipids Duplicate fasting blood samples obtained on the morning of day 9 and 10 of each diet cycle were collected in vacutainer tubes containing EDTA (0.1%). Blood 142096\02*454.UP -81 samples were centriftiged at 1500 rpm at 4degreesC for 15 min within 2 hours of the blood draw, separated, and stored at -80&C until further analysis. The only exception to this routine was with the initial lipid screening values which were analysed immediately after centrifuging. With the use of an autoanalyzer (Abbott 5 VG Supersystem), total plasma cholesterol and total plasma triglyceride (TG) concentrations were assayed enzymatically using 75-100 ul aliquots of sample. HDL levels was measured similarly after precipitation of apo B-containing lipoproteins. LDL levels were calculated by subtraction of triglyceride and HDL concentrations from total cholesterol using the Friedewald formula. 10 Cholesterol Synthesis Determinations Briefly, erythrocyte total lipids were extracted in duplicate from 3 g erythrocyte samples and separated by TLC. Separtation by TLC was achieved with the use 15 of hexane-diethyl ether-acetic acid 105:45:1.5 (vlv/v). Cholesterol bands were identified, scraped from the TLC plates, and then eluted from the silica gel with the use of hexane:chooroform:diethyl ether 5:2:1 (v/v/v). After drying the sample down under nitrogen, the resultant cholesterol was transferred into combustion tubes containing 0.5 g cupric oxide and silver wire (2 - 2.5 cm). The combustion 20 tubes were then sealed under vacuum and heated in an oven for 4 hours at 520oC. The water that ensued was then vacuum distilled into reduction tubes containing 0.06 g zinc reagent. The resultant gas was analysed for deuterium enrichment. For measurement of body water deuterium enrichment, plasma samples were diluted with known volumes of a water standard to bring 25 enrichments within the range of the working standards. Samples were reduced over zinc at 520*C before analysis of deuterium enrichment. Deuterium enrichment was determined using isotope ratio mass spectrometry 142086\024054.WP -82 (VG Isomass). The mass spectrometer was calibrated daily against water standards of known isotopic abundance. All samples for each subject phase were analysed in replicate using a single standard set. 5 Calculations Calculation of fractional synthetic rate (FSR) of cholesterol in RBC is based on the methods using tritiated water by Dietschy and Spady (1984) as adapted by Jones et al. (1988;1993). In brief, synthesis is calculated using the difference between deuterium abundance of erythrocyte cholesterol at t=O and t=24 h 10 relative to the enrichment of the body water pool. As the enrichment plateau during constant ingestion of D20 for erythrocyte cholesterol requires months to attain, the initial rate of uptake, independent of circadian rhythm, is highly linear. The initial rate provides a direct index of synthesis, independent of the total production rate. The equation for the FSR of cholesterol is as follows: 15 FSR (pools/day= d (0/00)cholesterol d (0/oo)plasma water X 0.478 where dcholesterol and dplasmawater refer to deuterium enrichments above 20 baseline level over 24 hours expressed as parts per thousand (0/00.) relative to the Standard Mean Ocean Water (SMOW) calibrated reference standard. The fraction "0.478" represents the proportion of protons in newly synthesized cholesterol which originate from water or water-based sources. 25 Statistics Descriptive data are expressed as MEANS + SD, whereas Inferential data pertaining to the objectives are expressed as MEAN + SEM. The inferential data determined for each treatment group were analysed for within and between 1A209s\U244S.WP -83 differences using a two way analysis of variance (ANOVA) with diet and subject as dependent variables. Due to the use of male and female subjects, gender effects were tested. If any significant within group difference was detected, the data would be stratified accordingly. Where the two way ANOVA attained a 5 significance of P < 0.05, specific group differences were evaluated using Tukey's HSD post-hoc analysis. The significant difference between group differences are expressed as mean differences along with their corresponding 95% confidence intervals (ie: mean difference (95% Cl)). 10 RESULTS Six men and five women participated in this study (see Table 34). The mean age of the subjects was 23+/- 2 years and the average body mass index was 23.21- 2.4 (mean .LSD). All subjects were normolipidemic with mean screening 15 plasma total and LDL cholesterol levels of 3.9+1-0.5 mmol/I and 2.0+1-0.6 mmol/l, respectively. The body weights of the volunteers were similar at the start of each study and were stable throughout each of the study periods (70.01+1-0.15 kg). The compliance was excellent with all subjects completing every dietary treatment. There was good agreement between the estimate macronutrient 20 content and the chemical analysis of the homogenized meals (see Table 33) The fatty acid and sterol profiles of the test oils are listed in Table 34. The fatty acid profile of each of the diets reflects the test oil the diet was based on. Total plant sterol and b-sitosterol contents differed across the test oils, with corn oil containing approximately 3 1/2 times more total plant sterols than olive oil. Sterol 25 analysis of homogenized meals reflected this sterol compositional difference (see Table 33). . The phytosterols administered in diets 3 and 4 differed in composition (see 14206\0240454.WP -84 Table 36). Both mixtures contained equal amounts of b-sitosterol, the difference was seen in the addition of sitostanol and elimination of stigmasterol in the FCP 3P1 mixture. The averaged daily dose given to subjects was 160 g (range 1.23 to 1.88 g) of the Nu-Life mixture and 1.51 g (range 1.22 to 1.93 g) of the sitostanol 5 enriched FCP-3PI mixture. T'he small difference between the two groups in absolute phytosterol administered was mainly due to limited availability of the sitostanol-enriched FCP-3PI mixture, therefore, slightly less was used. Table 36 displays the dose of b-sitosterol and sitostanol, as well as the ratio of each phytosterol to cholesterol. Despite the small absolute daily dose of b-sitosterol 10 used during each of the phytosterol trials (0.99 g in NuLife, 0.93 g in sitostanol enriched FCP-3PI), the ratios of this phytosterol to cholesterol, although low, have been shown to elicit a cholesterol lowering effect by other research groups (2.64:1 for sitostanol-free NuLife, 2.50:1 for sitostanol-enriched FCP-3P). With the addition of the phytosterols found in the meals, the mean daily phytosterol 15 intake in the Nu-Life and sitostanol-enriched FCP-3PI groups was 1.80 g and 1.71 g, respectively (see Table 36). Both phytosterols were tolerated well by all subjects, with no side -effects reported. Serum Lipids 20 Gender effects were tested for all plasma lipid measurements and there was no significant difference between male and female subjects, therefore, the results were analysed as one group. Figure 33 summarizes the plasma lipid levels of subjects consuming the four test diets. Total and LDL-cholesterol were 25 the only two lipid fractions to demonstrate a significant change in plasma levels (p<0.05). Plasma HDL cholesterol and triglyceride levels did not significantly change across the four diet groups (see Table 37 and Figure 35 for HDL-c and Table 38 and Figure 36 for .triglycerides) 142086\0240454.WP -85 As displayed in Figure 36 and Table 39, total cholesterol significantly increased with the exchange of olive oil for corn oil with a mean increase of 0.382 mmol/l (95% confidence interval: 0.138, 0.627). With the addition of the soybean based 5 phytosterol, there was also a significant increase in total cholesterol when compared to the corn oil based diet (0.378 mmol/l (0.137, 0.614)); however, this difference was not significantly different from the rise seen with the diet of olive oil alone (-0.004 mmoI (-0.251, 0.238)). Therefore, it can be concluded that the soybean based phytosterol mixture had no effect on plasma total cholesterol 10 levels. The addition of the sitostanol-enriched FCP-3PI phytosterol to the olive oil based diet significantly decreased plasma total cholesterol levels (-0.238 mmol/I (-0.482, -0.007)), where the FCP-3PI mixtures addition to the olive oil based diet decreased the overall plasma total cholesterol to a level that was no longer significantly different from the corn oil based diet (0.145 mmol/l (-0.094, 0.383)). 15 Thus, the sitostanol-enriched FCP-3PI mixture appeared to be more effective than the sitostanol-free NuLife soybean based mixture at decreasing plasma total cholesterol levels. The addition of the sitostanol-enriched FCP-3PI mixture to the olive oil based diet 20 was the only dietary factor that significantly decreased plasma LDL-cholesterol levels (-0.279 mmol/ (-0.506, -0.053)) (see Figure 38 and Table 40. No other significant difference was detected among the other diet treatments. However, it should be reported that with the substitution of olive oil for corn oil to the base diet, there was a substantial increase in the plasma LDL-C concentration, even 25 though this difference did not prove to be statistically significant (0,223 mmol/I ( 0.005, 0.450)). It is also worth noting the difference observed between the sitostan-l-free NuLife and the sitostanol enriched FCP-3PI mixtures (0.218 mmol/ (-0.004, 0A40)), where the sitostanol-enriched FCP-3PI mixture appeared 142086\02C454.P -86 to be more effective at lowering plasma LDL-C concentrations. This decrease in plasma LDL-cholesterol levels was reflected in the significant increase observed in the plasma HDL/LDL cholesterol ratio (0.145 (0.047, 0.243)) when the sitostanol-enriched FCP-3P tali-oil based phytosterol mixture 5 was added to the olive oil diet (see Table 41). There was also a significant difference detected in the plasma HDL/LDL cholesterol ratio when the two phytosterol mixtures where compared, where the sitostanol-enriched FCP-3PI phytosterol mixture was more effective at increasing the HDL/LDL ratio than the sitostanol-free NuLife phytosterols mixture (0-107 (0.011, 0.202)). 10 Plasma Phytosterol Levels Plasma phytosterols were extremely variable among individuals, therefore no significant differences were detected among any of the groups, however trends 15 were detected between the means of the treatment groups. Overall, plasma campesterol levels were higher than plasma sitosterol levels (see Tables 42-45. This was evident in the ratio of campesterol to sitosterol (see Table 46). When the plasma phytosterol levels relative to cholesterol were compared 20 between the treatment groups, campesterol levels were highest in the corn oil (24.60+/-5.84 mmol/mol) and sitostanol-free NuLife supplemented (28.911-5.72 mmol/mol) groups (see Table 43). The olive oil group (19.57+1-3.67 mmol/mol) had a slightly lower plasma campesterol level, however, the sitostanol-enriched FCP-3PI supplemented group had the lowest campesterol level (13.43+1-4.03 25 mmol/mol). Plasma sitosterol levels relative to cholesterol were considerably lower than those for campesterol, with the olive oil (1.58+1-0.30 mmol/mol) and sitostanol free NuLife supplemented (1.65+/-0.24 mmol/mol) groups having the highest 1420S\O24D4s4.P -87 concentration, and the corn oil (1.24+/-0.35 mmol/mol) group having a slightly lower concentration of sitosterol in the plasma (see Table 45). The sitostanol enriched FCP-3P supplemented (3.04+1-1.12 mmol/mol) group appeared to have the highest concentration of sitosterol relative to cholesterol in the plasma, S although, these values were not statistically significant due to the intersubject variability. Campesterol to sitosterol (C:S) ratios varied from 1.05 to 51.44 (see Table 46). However, the C:S ratio did appear to follow the trend found with the plasma 10 campesterol levels relative to cholesterol, with the corn oil (22.46+/-5.79) and the sitostanol-free NuLife supplemented (18.65+/-3.97) having the highest ratios, the olive oil (16.43+/-4.82) ratio being slightly lower, and the sitostanol-enriched FCP-3PI supplemented (11,69+1-6.64) group having the lowest overall ratio of C:S. 15 Free Cholesterol Synthesis Rates Deuterium incorporation rates are shown in Figure 40and Table 47 for subjects consuming each diet. Rates are expressed as calculated fractional synthetic 20 rates (FSR, pools per day). Tlie only significant difference in FSR was detected between the corn oil and olive oil treatment groups, where the FSR was significantly higher in the corn oil (0.0701+/-0.0120 pools/day) compared to the olive oil group (0.0295+/-0.0049) with a mean difference of -0.0406 pools/day (95% Cl: -0.0697, -0.0114). However, a trend was detected where the FSR 25 increased and became increasing similar to the corn oil FSR with the addition of either phytosteral mixture to the olive oil based diet; nevertheless, the difference from the olive oil phase did not reach statistical significance. There appeared to be more of an effect on increasing FSR with the addition of the sitostanol 142086\02t454.WP -88 enriched FCP-3PI mixture to the olive oil diet (mean difference from olive oil diet of 0.0210 pools/day (95% CI:-0.0082, 0.0501) compared to the addition of the sitostanol-free NuLife mixture (mean difference from olive oil diet of 0.0139 pools/day (-0.0152, 0.0431)) , although the difference between the sitostanol S enriched FCP-3Pl and the sitostanol-free NuLife mixtures (0.0070 pools[day) was not statistically significant (95%CI;-0.0221, 0.0362). DISCUSSION 10 T'he present study demonstrates that with the addition of sitostanol-enriched FCP3PI phytosterol mixture to an olive oil based diet there is a significant decrease in plasma total and LDL cholesterol concentrations, along with a corresponding increase in the HDL/LDL ratios in male and female subjects with normal plasma lipid levels. The study also demonstrates that the sitostanol 15 enriched FCP-3PI mixture was more effective than the sitostanol-free NuLife soybean based phytosterol mixture at increasing the HDLILDL ratio in normolipidemic humans. The addition of sitostanol-free NuLife soybean based phytosterols to an olive oil based diet had no additional effect on plasma lipid levels. These results suggest a more effective plasma lipid lowering phytosterol 20 mixture is created with the addition of sitostanol to a phytosterol mixture. This is best exemplified with the sitostanol-enriched FCP-3PI mixture being statistically more effective at increasing the HDL/LDL ratio than the sitostanol-free mixture. The overall conclusion from these reports is that sitostanol supplementation 25 effectively decreases both total and LDL plasma cholesterol levels with a correponding increase in the HDLJLDL ratio. The key difference between this current project and previous research is the dose of phytosterol administered. Previous studies have used 2 to 3 grams of pure sitostanol. whereas, the 142086\024454.WP -89 present study only used an average of 0,32 grams (range of 0.26-0.41 g), yet a similar response was induced.. When plasma phytosterol levels were compared, no significant differences were detected. Nevertheless, despite this lack of significance, there were distinct trends seen that are consistent with what 5 is currently known about phytosterol absorption. Recent research has determined that a longer side chain at the C-24 position of the cholesterol molecule is negatively correlated with the absorption of that molecule (Heinemann et al 1993). Thus, campesterol, with an extra methanol at C-24 of the cholesterol molecule, is absorbed at a higher percentage than b-sitosterol, 10 with an extra ethanol at the C-24 position. It has also been demonstrated that the Sa-saturated derivative of b-sitosterol, better known as sitostanol, is almost completely unabsorbed (Heinemann et al 1991). Reports have indicated sitostanol may act not only to impede cholesterol absorption more effectively but also to impede the absorption of other phytosterols. This is demonstrated by 15. comparing the level of plasma campesterol relative to cholesterol concentrations across the treatment groups. With the addition of sitostanol to the phytosterol mixture, there was a decrease in the campesterol to cholesterol ratio (see Table 43. This is further exemplified when this effect is examined in the plasma campesterol to sitosterol ratio (see Table 46 ), with the sitostanol-enriched FCP 20 3PI mixture supplemented diet displaying the lowest plasma C:S ratio. However, this effect may have arisen due to the lower level of campesterol available for absorption in the sitostanol-enriched FCP-3PI mixture diet (see Table 36). Consequently, further studies would be required before it can be elucidated that sitostanol blocks campesterol absorption and thus induces a 25 lower plasma campesterol level. The consensus is that the primary mechanism by which phytosterols achieve a change in lipid profile is through altering the absorption of cholesterol. The 1A2086\0240454WP -90 absorption of cholesterol can be monitored in vivo through measuring the compensatory change in cholesterol synthesis. This study monitored cholesterol synthesis through measuring deuterium incorporation rates. The results indicate that there is no significant increase in in vivo cholesterol 5 synthesis rates with the addition of either phytosterol mixture to an olive oil based diet. Nevertheless, there was a graded increase with the addition of the phytosterols mixtures (see Figure.39). There was an initial increase in the FSR with the addition of the sitostanol-free NuLife mixture and a further increase in the FSR with the addition of the sitostanol-enriched FCP-3PI mixture to a level that 10 was no longer significantly different from the corn oil based diet. Hence, this response indicates that the addition of sitostanol to a phytosterol mixture may be more effective in inhibiting cholesterol absorption. At larger doses of the FCP 3P1 mixture, this effect may become more pronounced. 15 In summary, the present experiment builds on existing knowledge to strongly suggest that at relatively low doses, the sitostanol-enriched FCP-3P phytosterol mixture effectively impedes cholesterol absorption, thus improving the plasma plasma lipid profile through decreasing total and LDL-cholesterol levels as well as increasing the HDLJLDL ratio. Hence, it can be concluded that the addition of 20 sitostanol to a phytosterol mixture creates a more effective plasma cholesterol lowering agent in normolipidemic humans. Table 33 25 Composition of Experimental Diets CORN OIL DIET OLIVE OIL DIET (% of total energy) Carbohydrate* 52.7+1- 3.7 52.4+/- 3.9 Protein 15.0+/- 1.5 15.0+/- 1.4 30 Fat 32.3+/- 2.8 32.7+1- 2.8 142086\0240454.W -91 Fatty Acids (% of total fat) Saturated: 6.7 7.4 C8:0 0.1 0.1 CIO:0 0.4 0.2 5 C12:0 0.7 0.5 C14:0 0.6 0.6 C16:0 3.8 4.6 C18:0 1.2 1.5 MUFA: 8.6 20.9 10 C16:In7 0.3 0.5 C18:1n9 8.3 20.4 PUFA- 17.0 4.3 C 18:2n6 16.7 3.9 C18:3n3 0.3 0.4 15 Cholesterol 128.9 128.9 (mg/1 000kcal) *Calculated by subtracting wet weights of moisture, protein, crude fat, and ash 20 from 100g, therefore, overestimated by 1-5% due to crude fibre content. Abbreviations: MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. 25 Table 34 Characteristics of the Study Subjects Characteristic Women Men Mean n=5 n=6 n=11 30 Age, y 22+/-1 24 +/- 2 23+/-2 Body weight, kg 59+/-5 77+/-9 68+1- 11 Height, cm 163+1-9 178+/-5 171+/- 10 Body mass index, 35 kg/m2 22.3 +/- 2.4 24.3 +/- 2.1 23.3+/- 2.4 Total cholesterol, mmol/l 4.1+/- 0.4 3.6 +/- 0.5 3.9 +/- 0.5 40 LDL-C, mmol/ 2.0+/- 0.6 1.9 +/- 0.6 2.0 +/- 0.6 HDL-C, mmol/l 1.7+/-0.3 1.4+1-0.2 1.5 +/-0.3 142086\024454.W -92 Triglycerides,08/0, mmoili 0.9+/- 0.2 0.7 +/- 0.3 0.8 +/- 0.3 5 Values expressed as mean +/- SD. Abbreviations: LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol. 10 Table 35 15 Composition of Dietary Oils CORN OIL OLIVE OIL Phytosterol content 20 PHYTOSTEROLS (mg/100g) (% of total) (mg/100g) Campesterol 210 25.3 0 Stigmasterol 65 7.8 0 B-Sitosterol 555 66.9 225 Sitostanol ND ND trace 25 Total 830 100 225 FATTY ACIDS: Fatty acid composition (% total fat) 30 Saturated: 13.9 17.8 C14:0 0.0 0.0 C16:0 12.3 16.5 C18:0 1.6 1.3 35 MUFX 27.8 72.9 C16:1n7 0.0 0.9 C18:1n9 27.8 72.0 PUFA 58.3 9.3 40 C 18:2n6 55.5 8.2 C18:3n3 2.8 1.1 142086\024454.W -93 Abbrev.iations: ND, none detected; trace, trace amount detected, MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids 5 Table 36 Characteristics of Phytosterols Administered Phytosterol Mixtures 10 Phytosterol Nulife FCP Composition (% of total) Campesterol 23.7 16.1 Stigmasterol 14.4 0.0 B-Sitosterol 61.9 62.0 15 Sitostanol 0.0 21.1 Average dose (range) /g Total. 1.60 (1.23,1.88) 1.51 (1.22,1.93) 20 B-Sitosterol 0.99 (0.76,1.16) 0.93 (0.76,1m2O) Sitostanol 0.00 0.32 (0.26,0.41) Ratio to dietary cholesterol 25 Total. 4.27 4.03 B-Sitosterol 2.64 2.50 Sitostanol 0.00 0.85 30 Table 37 The effects of dietary treatment on human plasma HDL-cholesterol levels (mmol/1) 142086\024454.WP -94 Plasma HDL-c (mmol/I) Subject Gender Corn Olive Nulife FCP 5 1 m -0.91 1.06 0.95 0.86 2 m 0.87 0.91 0.76 1.05 3 m 1.20 1.22 1.20 1.31 4 m 1.10 1.02 1.07 1.03 5 m 0.74 0.85 1.08 1.28 10 6 m 0.84 0.88 0.98 0.91 7 f 1.16 1,16 1.15 0.94 8 f 1.50 1.32 1.52 1.69 9 f 1.29 1.22 1.22 1.28 10 f 0.89 1.11 1.02 1.02 15 11 f 0.95 - 1.30 1.28 MEAN: 1.04 1.07 1.12 1.15 +SEM: 0.07 0.05 0.06 0.07 20 Results are expressed as mean +/- SEM. Treatment group means having different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: HDL-C, high density lipoprotein cholesterol-, m= rnale; f= fermal 25 Table 38 The effects of dietary treatment on human plasma triglyceride levels (mmov/l) Plasma Triglycerides (mmol/) Subject Gender Corn Olive Nulife FCP 30 1 m 0.84 0.66 0.78 1.01 2 m 0.90 0.65 1.05 0.74 3 m 0.95 1.07 0.91 0.82 4 m 0.46 0.69 0.66 0.85 35 5 m 0.66 0.76 0.66 0.53 6 m 0.51 0.48 0.62 0.61 7 f 0.91 0.94 1.43 1.10 8 f 0.56 0.69 0.67 0.72 9 f 0.58 0.92 0.86 0.61 40 10 f 0.93 1.50 1.16 0.64 11 f 0.49 0.66 0.68 142086\02'454.WP -95 MEAN: 0.71 0.84 0.86 0.75 +SEM: 0.06 0.09 0.08 0.05 Results are expressed as mean +/- SEM. Treatment group means having 5 different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: m rnale; f, female. Table 39 10 The effects of dietary treatment on human plasma total cholesterol levels (mmol/l) Plasma Total Cholesterol (mmol/) 15 Subject Gender Corn Olive Nulife FCP 1 m 3.54 3.69 3.61 3.55 2 m 2.79 3.03 3.10 3.03 3 m 2.68 3.19 2.86 2.82 4 m 3.01 3.04 3.24 2.73 20 5 m 3.28 3.99 4-01 3.73 6 m 2.87 3.28 2.99 2.93 7 f 2.79 2.98 3.07 2.46 8 f 3.54 3.57 3.82 3.80 9 f 3.02 3.47 3.30 3.13 25 10 f 3.52 4.61 4.01 3.80 11 f 3.11 4.26 3.77 MEAN: 3.10 a 3.49 b 3.48 b 3.25 a +SEM: 0.10 0.16 0.15 0.15 30 Results are expressed as mean +/- SEM. Treatment group means having different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: m male; f, female. 142086\0240454.W -96 Table 40 The effects of dietary treatment on human plasma LDL-cholesterol levels (mmol/l) Plasma LDL-c (mmol/) Subject Gender Corn Olive Nulife FCP 10 1 m 2.23 2.32 2.29 2.22 2 m 1.49 1.82 1.85 1.62 3 m 1.04 1.46 1.23 1.12 4 m 1.70 1.69 1.87 1.30 5 m 2.22 2.78 2.62 2.21 15 6 m 1.79 2.18 1.72 1.73 7 f 1.18 1.38 1.24 1.00 8 f 2.53 1.92 1.97 1.72 9 f 1.45 1.81 1.64 1.57 10 f 2.18 2.79 2.45 2.48 20 11 f 1.93 --- 2.65 2.16 MEAN: 1-79 2.02 a 1.96 1.74 b +/-SEM: 0.16 0.16 0.15 0.16 Results are expressed as mean +/- SEM. Treatment group means having 25 different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: LDL-C, low density lipoprotein cholesterol; m male; f, female. 142086\0244S4.WP -97 Table 41 The effects of dietary treatment on human plasma HDL/LDL cholesterol ratio HDL/LDL Cholesterol Ratio 5 Subject Gender Corn Olive Nulife FCP 1 m 0.407 0. 455 0.413 0.388 2 m 0.586 0.496 0.409 0.649 3 m 1.159 0,835 0.970 1.176 10 4 m 0.646 0.603 0.572 0.795 5 m 0.335 0.305 0.410 0.579 6 m 0.473 0.403 0.566 0.526 7 f 0.984 0.836 0.923 0.944 8 f 0.592 0.687 0.771 0.983 15 9 f 0.891 0.675 0.745 0.814 10 f 0.409 0.398 0.414 0.410 11 f 0.495 - 0.492 0.594 MEAN: 0.634 ab 0.569 a 0.608 a 0.714 b +/-SEM: 0.080 0.059 0,064 0.075 20 Results are expressed as mean +/- SEM. Treatment group means having different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: HDL. high density lipoprotein ; LDL, low density lipoprotein; m male; f, female. NuLife mixture is sitostanol-free, FCP mixture contains 21% sitostanol. 25 Table 42 The effects of dietary treatment on human plasma campesterol levels (103 X mmol/I) 30 Plasma Campesterol Levels (103 X mmol/l) Subject Gender Corn Olive Nulife FCP 1 m 63.3 27.0 88.8 33.8 35 2 m 27.0 39.4 92.8 36.0 3 m 83.3 55.1 108.0 27.8 4 m 184.9 85.2 - 7.0 7 f 51.4 18.8 56.9 13.0 8 f 60.6 75.0 44.8 146.1 40 9 f 84.9 105.0 -- 29.0 10 f 45.8 153.9 204.2 78.1 142086\024D454.WP -98 MEAN: 75.14 69.92 99.26 46.35 +SEM: 17.06 15.90 23.09 16.11 Results are expressed as mean +/- SEM. Treatment group means having 5 different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: m male; f, female. NuLife mixture is sitostanol-free. FCP mixture contains 21% sitostanol. 10 Table 43 The effects of dietary treatment on human plasma campesterol levels relative to total cholesterol (mmol/mol of cholesterol) 15 CampesteroL:Cholesterol Ratio (mmol/mol) Subject Gender Corn Olive Nulife FCP 1 m 17.90 7.31 24.60 9.52 2 m 9.67 12.98 29.90 11.91 20 3 m 31.07 17.27 37.77 9.86 4 m 61.47 28.05 - 2.57 7 f 18.51 6.30 18.57 5.29 8 f 17.09 20.99 11.74 38.50 9 f 28.10 30.27 - 9.27 25 10 f 13.01 33.41 50.87 20.55 MEAN: 24.60 19.57 28.91 13.43 +SEM: 5.84 3.67 5.72 4.03 30 Results are expressed as mean +/- SEM. Treatment group means having different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: m male; f, female. NuLife mixture is sitostanol-free, FCP mixture contains 21% sitostanol. 142086\024)454.WP -99 Table 44 The effects of dietary treatment on human plasma sitosterol levels (103 X mmol/l) Plasma Sitosterol Levels (103 X mmol/I) Subject Gender Corn Olive Nulife FCP I m 0.00 3.39 3.89 32.15 10 2 m 3.74 6.09 3.89 0.00 3 m 2.93 3.80 7.72 16.44 4 m 9.69 2.56 - 5,56 7 f 3.74 5.54 6.11 2.90 8 f 6.69 1.80 5.23 3.14 15 9 f 1.65 8.09 --- 14.26 10 f 1.62 13.60 6.12 3.14 MEAN: 3.76 5.61 5.49 9.70 +SEM: 1.10 1.35 0.60 3.81 20 Results are expressed as mean +/- SEM. Treatment group means having different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: m male; f, female. NuLife mixture is sitostanol-free, FCP mixture contains 21% sitostanol. 25 Table 45 The effects of dietary treatment on human plasma sitosterol levels relative to total cholesterol (mmol/mol of cholesterol) 30 Sitosterol:Cholesterol Ratio (mmol/mol) Subject Gender Corn Olive Nulife FCP I m 0.00 0.92 1.08 9.06 2 m 1.34 2.01 1.25 0.00 3 m 1.09 1.19 2.70 5.84 35 4 m 3.22 0.84 - 2.04 7 f 1.35 1.86 1.99 1.18 8 f 1.89 0.50 1.37 0.83 9 f 0.55 2.33 -- 4.56 10 f 0.46 2.95 1.52 0.83 40 MEAN: 1.24 1.58 1.65 3.04 +SEM: 0.35 0.30 0.24 1.12 142086\02C454.WP -100 Restdts are expressed as mean +/- SEM. Treatment group meam having different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: m male; f, female. 5 NuLife mixture is sitostanol-free, FCP mixt= contains 21% sitostanol. Table 46 10 The effects of dietary treatment on human plasma campesterol:sitosterol ratio Campesterol:Sitosterol Ratio Subject Gender Corn Olive Nulife FCP 1 m no sito 7.95 22.83 1.05 15 2 m 7.22 6.46 23.83 no sito 3 m 28.47 14.51 13.99 1.69 4 m 19.09 33.27 -- 1.26 7 f 13.74 3.39 9.32 4.49 8 f 9.05 41.58 8.57 46.50 20 9 f 51.44 12.98 - 2.03 10 f 28.19 11.32 33.39 24.84 MEAN: 22.46 16.43 1 8.65 11.69 +SEM: 5.79 4.82 3.97 6.64 25 Results are expressed as mean +/- SEM. Treatment group means having different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: no sito, no sitosterol detected; m male; f, female. NuLife mixture is sitostanol free, FCP mixture contains 21% sitostanol. 30 142086\02CD454.Ad -101 Table 47 The effects of dietary treatment on human in vivo cholesterol fractional synthetic rate (pools per day) 5 FSR (pools per day) Subject Gender Corn Olive Nulife FCP 1 m 0.0753 0.0230 0.0380 0.0524 2 m 0.0929 0.0201 0.0329 0.0497 10 3 m 0.0717 0,0294 0.0948 0,0693 4 m 0.0091 0.0355 0.0350 0.0521 5 m 0.0210 0.0130 0.0060 0.0260 6 m 0.0710 0.0660 0.0160 0.0530 7 f 0.1596 0.0188 0.0440 0.0762 15 8 f 0.0668 0.0381 0.0967 0.0585 9 f 0.0416 0.0069 0.0493 0.0397 10 f 0.0770 0.0402 0.0602 0.0316 11 f 0.0850 0.0340 0.0050 0.0470 20 MEAN: 0.0701 a 0.0295 b 0.0435 ab 0.0505 ab +SEM: 0.0120 0.0049 0.0094 0.0045 Results are expressed as mean +/- SEM. Treatment group means having different subscripts differ significantly (p<0.05 Tukey's HSD test). Abbreviations: FSR, fractional synthetic rate; m males; f, females. 25 30 EXAMPLE 11 35 Tolerability of a phytosterol mixture (FCP-3P1= one of compositions of the present Invention) at an anti-atherogenic dose over a period of 18 weeks has been evaluated in male apo E-deficient mice. The mice were divided into treated (n=9) and control (n=6) groups which were matched for plasma lipid levels and 40 body weight, Control group was fed with a diet containing 9% (w/w) fat and 0.15% 14ZD86\020454.W -102 (w/w) cholesterol and treated group with the same diet supplemented with 2% (w/w) FCP-3P1. At the end of the study the treated group had a significantly lower plasma total cholesterol level (30.4-2.5 vs 39.2-2.1 mmol/L, p<0,05) and higher body weight (34.2 +1- 2.0 vs 30.8 - 1.9 g; p<0.05), while free cholesterol, 5 triglyceride and glucose levels were comparable between the two groups, All urinalysis and hematological data were comparable between the two groups except for significantly (p.<0,04) lower platelet counts in the treated animals as compared to those in controls (681.6-118.9 vs 857.1-185.4, X 10 9/L). Gross inspection showed skin lesions in 2 mice (33%) in the control _group, but none 10 in the treated group. Histological examination revealed non-specific, ORO negative vacuolation in the iver and kidney of the control animals, but not in the treated mice. Thus, FCP-3P1 treatment prevented skin lesion formation and appearance of non-specific vacuolation in the kidneys and livers. Treated animals showed a mild testicular atrophy on H&E stained sections. We 15 conclude that a dose (2% w/w) of FCP-3PI which lowers plasma cholesterol levels and reduces atherosclerotic lesion formation is safe for medium to long term use in apo E-deficient mice. Both pharmacological and non-pharmacological agents have been extensively 20 used in an attempt to decrease the mortality and morbidity of hypercholesterolemia-related disorders. A major limitation of using conventional lipid-lowering agents, particularly for long-term administration, is their side effects; myopathy and liver dysfunction miay occur in patients treated with wildly used lipid-lowering agents such as fibrates or statins (HMG-CoA reductase 25 inhibitors). Thus, the aim of the present study was to test tolerability of orally administered FCP-3Pi at a rate of 2% (w/w) to male apo E-deficient mice by assessing several 1A2096\024D454.WP -103 histological, hematological and biochemical parameters. Materials and Methods 5 Animals Fifteen male four-week old apo E dficient mice were purchased from Jackson Laboratory. Animals were divided into two groups matched for body weight and plasma lipid levels. One group (n=6) was fed with a Western diet containing 9% (wlw) fat and 0.15% (w/w) cholesterol and served as the control group; the other 10 group (n=9, treated) was fed with the same diet supplemented with 2% (w/w) FCP3PI. The mice were housed individually and had access to food and water ad libitum for 18 weeks of the experiment. Body weight and food consumption were monitored weekly. There were no changes in animals' activity or behavior during the experimental course. 15 Diet and phytosterol mixture PicoLab Mouse diet 20 (9% fat w/w) was purchased from Jamieson's Pet Food Distributor Ltd., Delta, BC. Its chemical composition is summarized in Table 48. Extraction and purification of FCP-3P1 as well as diet preparation were carried 20 out as previously described. Blood sampling Blood samples for hematological and biochemical tests were taken from tail veins or from the right ventricle (at the end of experiment from pentobarbital 25 anaesthetized) into the heparinized or EDTA ElectroCoated tubes (Fisher Scientific,. Pittsburgh, PA and Becton Dickinson Company, respectively) as previously described in the literature. 142086\024054.WP -104 Plasma Lipid Anaylses Plasma levels of total cholesterol (TC), free cholesterol (FC) and triglyceride (TG) were measured at the baseline and at the end of experiment. Analyses were performed using enzymatic assays [Boehringer Mannheim GmbH, Mannheim, 5 Germany (TC, TG); Wako Chemicals GmbH, Nissanster, Germany (FC) as previously described in the literature. Hematology Aliquots of the whole blood in EDTA-coated tubes was sent to Hematology 10 Laboratory at Children's Hospital, Vancouver, BC, for blood film and other hematological assessments in a blinded fashion by using standard diagnostic procedures. Erythrocyte Fragility Test 15 Erythrocyte fragility test was performed as previously described in the art. Briefly, the erythrocytes were obtained from the blood by centrifugation at 13,000 rpm for 3 minutes using an ICMCentra centrifuge. Cells were washed twice with isotonic saline and once with 0.15 M NaCI-15 mM Tris pH 7.0 and then resuspended in 0.15 M NaCI-15 mM Tris. Aliquots of red cell suspension were added into the 20 tubes containing 0.05 M NaCl solutions. After 15 minutes incubation at room temperature samples were centrifuged and supernatant absorbance at 540 nm read using a Perkin-Elmer Lambda 3B UVNIS spectrophotometer. Percentage of total hemolysis for each sample was calculated. 25 Urinalysis Urine was collected directly from the bladder of each anesthetized mouse (at the end of the study) in a tuberculin syringe. Urinalysis was performed in a blinded fashion by dripping the urine sample on the reagent strips for urinalysis (Multistix. 1420t6\0243454.P 105 Milles Canada Inc., Etobicoke, Ont.). Plasma glucose level Aliquots of plasma obtained at the end of the experiment were used for 5 determination of glucose level as previously described. Organ pathology All organs located in the thoracic and abdominal cavities were carefully inspected. Specimens from the heart, lung, brain, kidney, skeletal muscle, skin, 10 esophagus, stomach, small and large intestines, liver, adrenal gland, spleen, pancreas, bladder and testis were fixed in 10% buffered-formalin (Fisher Scientific, Fair Lawn, New Jersey). Four micrometer sections were cut from paraffin-embedded specimens of all the above organs and stained with hematoxylline-eosine (H&E). Oil red 0 (ORO) staining was performed on the 10 15 um frozen sections cut from OCT-embedded specimens as needed. Statistical analyses A two-tailed Student t test assuming equal variances was used to evaluate the significance of differences between results in the two groups, Data are 20 expressed as mean - SD. Results Effects of FCP-3P1 on body weight and plasma glucose level 25 Both control and treated groups of mice had similar body weight at the baseline of the study (21.4 - 1.1 vs 21.0 - 0.8 g). However, by the end of the experiment, the treated group showed a significantly higher body weight than the control group (34.2 - 2.0 vs 30.8 - 1.9 g; p<0.05). 142086\024454.WP -106 Treatment with phytosterols did not alter plasma glucose level (14.6+1-2-3 vs 13.9-1.9 mmolIL). 5 Effects of FCP-3PI on plasma lipid levels Table 49 demonstrates the level of plasma lipids of the. mice during the study. Both groups of mice had similar plasma lipid levels at the initiation of the experiment. As previously shown, after 18 weeks of consumption of the experimental diet, the treated group showed a significantly lower plasma total 10 cholesterol level compared to controls (30.4-2.5 vs 39.2-2.1 mmol/L, p<0.05). The levels of plasma FC and TG were comparable between the two groups. Effects of FCP-3P1 on hematological parameters Table 50 summarizes the hematological data obtained in the treated and control. 15 animals. Hemoglobin concentration, red cell counts and hematocrit were comparable between the two groups, but a statistically significant difference was noted in platelet counts [681.6 - 118.9 (in treated) vs 857.1 - 185.4 (in controls), x 10 9/L, p<0.04]. Leukocyte counts showed a large but not significant variation between the two groups. This may be related to stress which results in the 20 release of dormant leukocytes from the vascular wall during the blood sampling. Blood film examinations revealed no abnormalities and no differences between the two groups. Effects of FCP-3P1 on hemolysis 25 Figure 41 shows the percentage of hemolysis in both groups when exposed to 0.05 M NaCl for 15 minutes at room temperature. Erythrocytes from treated animals were significantly less fragile at the given concentration of NaCl compared to controls (83.3- 6,7 vs 95.5 - 2.3; per cent; p<0.001), 142086\02404.WP -107 Effects of FCP-3P1 on urinalysis No significant changes were observed in the urine parameters including specific gravity, glucose, erythrocytes, hemoglobin, leukocytes, nitrates, protein, bilirubin, 5 urobilinogen, pH and ketone levels of the treated mice as compared to those in controls. Gross and microscopic tissue examination Macroscopic examination of the organs from both groups of animals revealed no 10 abnormality except for skin lesions (thickened, red, alopecia) in two control mice (Fig.42, Panel A). Histological examination of the affected skin revealed numerous cholesterol crystals, cholesterol granulomas along with cellular reaction with eosinophils and histiocyte (Fig. 42, Panels B & C). Histological examination was also performed on a number of other tissues from both groups 15 of animals. Routine histochemical staining (H & E) revealed no histological abnormalities in the tissues examined except for slight histological changes in certain kidneys, livers and testes. Sections from a number of kidneys of both treated and control animals showed a little focal and non-specific interstitial inflammation: in some cases it was extended to the pelvic area of the affected 20 kidneys. Sections taken from the kidneys of five controls and one treated animal showed vacuolation appearance when stained with H&E (Fig. 43). Similar vacuolation was also observed on the sections taken from the livers of control but not from treated animals as examined by H&E staining (Fig. 44). ORO staining of the frozen sections obtained from the affected kidneys and livers failed to 25 demonstrate lipid deposits as the cause of vacuolation. Testicular sections from almost all treated animals showed a mild atrophy in seminiferous tubules. In some tubules It was more significant and profound in such a way that there was no evidence of active spermatagenesis in more severely affected tubules (Fig. Z42o86\02D454.wP -108 45). In spite of similarity between the chemical structures of phytosterols and cholesterol, intestinal absorption of phytosterols is very low compared to that of 5 cholesterol. The rate of intestinal absorption varies among individual phytosterols. Heinemann et al. compared the rate of intestinal absorption of cholesterol to that of several plant sterols in 10 healthy men who underwent intestinal perfusion over a 50cm segment of the upper jejunum. They found the highest absorption rate for cholesterol (as much as 33,0%) followed by 10 campestanol, campesterol, stigmasterol. beta-sitosterol and sitostanol at 12.6%, 9.6%, 4.8%, 4.2% and 0.0%, respectively, Components of FCP-3PI have been shown to be absorbed and reduce atherosclerosis and plasma cholesterol levels in a number of experimental animal species. In the present study we have tested the tolerability of FCP-3P1 in young male apo- E deficient mice over 18 15 weeks of the experiment. Although certain dietary plant sterols are to some extent absorbed; the absorbable plant sterols may have systemic activities including unwanted effects. Consumption of beta-sitosterol for four years by humans resulted in no 20 adverse effects as determined by kidney and liver function tests, hematology, urinalysis, electrocardiogram records and gall bladder visualization. In addition, long-term (22 months) oral administration of beta-sitosterol to rats, rabbits and dogs was found to be safe. Chronic administration of beta-sitosterol subcutaneously to both male and female rats was well tolerated without any 25 evidence of tissue damage in either sex. The concentration of plant sterols in the plasma may play a significant role in physiological functions of the tissues. Sitosterolemic patients develop 142086\0240454.WP -109 atherosclerosis and have more rigid/fragile red cells. In contrast to this, the present applicants showed that dietary phytosterols at the level of 2% (w/w) are anti-atherogenic and that this effect of plant sterols was accompanied by a significantly less fragile red cells, unlike in sitosterolemia. Absorbed 5 components of FCP-3P1 used may have been incorporated into the cell membranes of the red cells and made them more resistant to hemolysis. These findings may suggest a rationale for the amount and route of administration of plant sterols. Moreover, lipid analysis showed a significant reduction in plasma total cholesterol level in the treated group as compared to that in the control 10 group which was in accordance with previous reports, Hematological tests revealed a significant decrease in the number of platelets in FCP-3P1 treated animals compared to controls. This is unlikely due to the effects of the phytosterols/cholesterol on the hematopoietic tissues because a) 15 other hematological measurements were comparable between the two groups; b) blood film examination revealed no abnormalities in cellular components of the blood from either group; and c) control animals had comparable platelet counts with that in C57BL/6 mice which are the wild type counterparts for apo E deficient mice used in the present study. In agreement with this observation, 20 Clayton et al. reported thrombocytopenia in five children with parenteral nutrition induced phytosterolemia. This condition was improved by reducing intake of lipid emulsion containing phytosterols. Although the reason for this finding is unclear, we observed a significant decrease in the plasma level of fibrinogen in male apo F-deficient mice fed with the same amount (2% w/w) of FCP-3P1. Moreover, 25 other studies reported a stimulation effect of beta-sitosterol for releasing tissue plasminogen activator from cultured bovine carotid artery endothelial cells, but not from human umbilical vein endothelial cells. Altogether, the applicant s and other reasearcher s observations may suggest an anticoagulant activity for the 142086\0240454.WP -110 plant sterols. Thus beta-sitosterol has been proposed as an agent for prevention and treatment of thrombosis. Gross inspection of organs failed to detect any damage attributable to phytosterol 5 treatment. Histological studies revealed no abnormalities in the treated animals except for a slight atrophy in testicular tissues. The latter observation is in accordance with previous findings in which the administration of beta-sitosterol resulted in a reduction in testicular weight and impaired spermatogenesis in albino rats. These observations may be explained by the estrogenic effects of 10 beta-sitosteroi. Similar effects of plant sterols have been also shown in female animals. Observed cellular vacuolation in the kidneys of the control animals probably does not have a functional consequence since urinalysis failed to demonstrate any 15 abnormalities. Although we did not perform liver function tests, similar conclusions may apply to observed hepatic vacuolation. The rnild but significant increase in the body weight of the treated mice might be due to periodical increased food intake as we showed before and/or to 20 decreased plasma cholesterol levels as we observed in our other studies. The increased body weight was accompanied by a non-significant increase in the plasma glucose level of the treated animals as compared to that in control group (14.6 - 2-3 vs 139 - 1.9 mmol/L). It is of interest that there is a marked difference between plasma glucose level in apo-E deficient mice and that in their 25 wild type counterparts C57BU6J (13.9 - 1.9 vs 6.5 - 0.1 mmol/L). Although the "volume effects" (due to high lipid levels) might contribute to this difference, high fat diet (58% fat on energy basis) significantly increased the levels of insulin, glucose and total cholesterol in the plasma of male C57BL/6J. Therefore, our 1420O5\2D454.Wp

AI

observation in regard to plasma glucose level suggests that deletion of apo-E gene which leads to a very high level of plasma cholesterol may alter the metabolism of glucose which in turn could attribute to accelerated atherosclerosis. Investigation of the relationship between lipids, apo-E and 5 glucose metabolism seem to be necessary for better understanding of accelerated atherosclerosis in apo-E deficient mice. In summary, FOP-3P1, one form of composition in accordance with the present invention, has been found significantly to alter the cholesterol metabolism 10 (reduction of levels), to delay atherosclerotic processes, to decrease peripheral platlete counts and to reduce red blood cell susceptability to hemolysis in 0.05 M NaCl. These findings support extensive systemic effects of the present composition. The dose of FCP-3P1 adminstered was relatively well tolerated in the mice with no apparent toxic effects as assessed by biochemical, histological 15 and hematological tests. In accordance with the previous findings phytosterol treatment caused a slight histological atrophy in the testes of the mice. Findings of the present study support the safety of medium to long-term use of FCP-3P1. Figure legends 20 Figure 41- Percentage of hemolysis at 0.05 mM NaCl concentrations in the control and treated groups, *p<0.001. Figure 42 -- Skin lesion on the shoulder area of a control mice (A). 25 Photomicrograph of the nuchal skin illustrating marked infiltration by lipids and presence of cholesterol granulomas and cholesterol clefting. Neutral lipids are highlighted in Panel B on ORO staining. (ORO stain, B, x5O; H & E stain, C, x 125). 142086\024454.WP -112 Figure 4 3 -Representative photomicrographs of kidneys from one control (Panel A) and one treated (Panel B) mouse. S evident non-specific vacuolation is senn in the kidney of the control but not the treated mouse (H & E stain, A & B, x 16). 5 Figure 44 --Representative photomicrographs of livers from one control (Panels A & C) and one treated (Panels B & D) mouse. A evident non-specific vacuolation is seen only in the liver of the control but not the treated mouse. (H & E stain, A & B x 16; B & D, x25). 10 Figure 4 5~-Representative photomicrographs of a testis from one treated mouse. Atrophy of the seminiferous tubules is illustrated. (H & E stain, A, x50; B, x2O). Table 48 Chemical composition of PicoLab mouse diet 20. Composition percentage 15 protein 20.5 fat 9.0 cholesterol 285.0 ppm carbohydrate 53.0 20 ash and vitamins 4.8 fiber 2.7 moisture 10.0 Table 49 Plasma lipid levels (mmol/L) at the begining and at the end of the 25 experiment (mean-SD). 1 4 2 086\024454.w -113 Groups Baseline Final Control Treated Control Treated 5 TC 15.6- 1.8 15.7-2,1 39.2-2.1 30.4-2.5* TG 1.32-0.7 1.21-0.7 1.67-0.9 2.46-1.8 10 FC 10.4-1.3 9.6-1.9 TC= total cholesterol; TG= triglyceride; FC= free cholesterol, ap<0.05 Table 50 Effects of phytosterol mixture on the hematological parameters 15 (mean-So)) Parameter Control (6) Treated (9) leukocyte (xO 9/L) 14.5 - 5.2 12.7 -4.3 erythrocyte (x 10 12/L) 10.6- 0.7 10.1 - 0.7 platelet (x 10 9/L) 857.1 - 185.4 681.6 - 118.9* 20 hemoglobin (g/L) 153.0 - 10.3 140.0 - 13.7 hematocrete 0.53 - 0.03 0.49 - 0.04 p<0.04 25 EXAMPLE 12 Our objective was to examine whether tall oil plant sterols provided at 1.5 g/day (per 70 kg body weight) would alter lipoprotein cholesterol levels in hyperlipidemics 30 when provided as a food supplement suspended in margarine. The null hypothesis tested was that when sitostanol containing phytosterols are provided across 3 meals of a fixed foods prudent North American diet to hyperlipidemic men for 30 days. lipoprotein cholesterol profiles will be no different that when the diet is provided alone. 35 Methodology 1420I6\024D454.Wp -114 Subjects Thirty three males (ages 25-60 yrs) with primary familial hypercholesteroemia were selected. Subjects were screened for total cholesterol and triglyceride 5 levels prior to acceptance into the study. Criteria for acceptance were total circulating cholesterol levels between 6.5 and 10 mmol/ and total circulating triglyceride levels of less than 5 mmol/L. Individuals with diabetes, heart disease and hypothyriodism or who had not been off medication for hypercholesteroemia for at least 4 weeks were excluded. Subjects were systematically randomized 10 into the two treatment groups to equalize initial total circulating cholesterol levels. Protocol and Diet The study was a randomized double-blind clinical trial. All subjects were provided with a North American solid foods diet considered to be healthy in terms 15 of macronutrient and fat content for 30 days. One group (n=16) received diet alone, the second group (n=16) received diet with sitostanol containing phytosterols suspended in the margarine component of the diet. Subjects were systematically randomized into the two groups on the basis of screening plasma total cholesterol level. The diet was formulated to meet Canadian 20 Recommended Nutrient Intakes and provide fat, fiber and carbohydrate sub components consistent with Health Canada Recommendations. Dietary protein, carbohydrate and fat made up 15, 40 and 35% of ingested energy, respectively, The dietary fat was made up of 11, 14 and 10% as energy of saturated, monounsaturated and polyunsaturated fats, respectively, using a blend of butter, 25 olive oil and margarine. Diets, fed at levels determined to maintain body weight throughout the 30 day trial were fed under supervision as three meals of equal energy proportions per day. Diets were comprised of a three day rotating cycle, each cycle possessing very similar macro- and micronutrient contents. Meals 14 2086\02454.WP -115 were prepared within the Clinical Nutrition Research Unit for consumption on site or in certain cases for take out, as previously described. During meal preparation, foods weighed out to the nearest 0.5 . Subjects were instructed not to consume any foods or beverages other than provided by the diet. Alcoholic 5 and caffienated beverage consumption was prohibited over the course of the trial. Subjects were provided with decaffienated energy free carbonated beverages to drink between meals. Phytosterols were added to the diets at a level of 21.4 mg/kg body weight 10 (1.5g/70 kg individual). Phytosterol composition of the material utilized in the study is provided in Table 52. Sitostanol comprised about 21% of the mixture by weight. Administration was performed by suspending the phytosterols in 30 9 of pre-warmed margarine each day, providing a ratio of approximately 20:1 (wt/wt) margarine: phylosterol mixture. The 30 g was divided equally across the 3 meals 15 and mixed directly with food ingredients during preparation before cooking. Subjects underwent routine physical examinations and detailed blood chemistry analyses before and on days 10, 20 and 30 of study to evaluate the safety of the phytosterol materials. A physician was on continual call throughout the trial for subjects to contact in case they experienced any discomfort. 20 Analyses Plasma total, low density and high density lipoprotein cholesterol and total triglyceride levels were determined at days 0, 10, 20. 29 and 30 of the trial. In addition, levels were determined in subjects after cessation of the diet on days 25 40 and 50 to track the die-away of any cholesterol-modulating effect. Lipid levels were analyzed with enzymatic kits and standardized reagents and standards 142086\02C454.WP -116 using a VPAutoanalyser (Abbott Laboratories, North Chicago, IL, USA). For LDL cholesterol determinations, the Friedewald equation was utilized. Statistical and Modeling Methods: 5 Plasma lipid level data were evaluated using repeated measure analysis of variance procedures with tests for time and diet effects. In addition, tests for significant differences from the day 0 point were conducted within each treatment. Linear regression models were computed for time series data using the SAS system. In addition, Linear and quadratic modeling procedures were 10 also employed to construct prediction curves for the plasma total and LDL cholesterol level data over a 60 day period. These prediction curves took into account the assumption that a plateau would be attained after some finite time interval. 15 Results; Thirty three subjects commenced the feeding trial with 32 subjects completing the total study. All individuals tolerated the experimental diet without any reported adverse effects. In addition, blood and urine chemistry parameters were normal at the onset of the period and throughout the duration of the trial. Screening 20 check-ups conducted at each 10 day time point of the trial revealed no suggestion of any clinical irregularity. Overall, subjects were found to maintain excellent health through the duration of the experiment, with the exception of one subject in the phytosterol group who reported considerable diarrhea associated with a bout of influenza over the final 4 study days. 25 The sitostanol-containing phytosterol mixture was highly inert. Subjects reported no particular abnormal or atypical smell, taste or mouthfeel effects of meals on either diets. Subjects were not able to identify which treatment diet they were 14206\z40254.wp -117 consuming. There were no reported abnormalities with stool consistency or color, with the exception noted above. No after taste was reported at any time by any subjects. 5 Individual and group mean weight changes across the 30 day feeding period were minor, suggesting that study subjects were receiving energy at levels consistent with their actual individual requirements. Mean weights of subjects at commencement and end of the study are shown in Table 53. Seven study subjects had their level of caloric intake altered by 10-20 percent over the first 10 week of each trial. Three subjects either lost weight or reported lack of satiation, thus had their energy level increased. Four subjects reported feelings of excessive intake, thus had their energy intake levels reduced. In instances where energy intakes were adjusted, relatively steady weight was achieved over the remainder of the period. 15 Total cholesterol levels measured across the 30 day feeding period showed substantial variation in pattern across subjects. For the control diet, the tertile (n=5) of individuals showing greatest response to diet showed on average a 32% decline, while the tertile (n=5) with the least response showed an average 20 increase in total cholesterol levels by 3% (data not shown). On the phytosterol enriched diet the variability was similar. Here, the tertile of individuals showing greatest response to diet had on average a 31% decline, while the tertile with the least response showed an average drop in total cholesterol levels of only 6% (data not shown). Variance In response was not associated with change In body 25 weight or number of meals consumed away from the Clinical Nutrition Research Unit. Mean group changes in total circulatory cholesterol levels over the 30 day feeding 14206\024M54.wP -118 period and the subsequent 20 day wash-out are shown in Figure 46. As subjects of the two treatment groups were matched for screening total cholesterol levels, values at day 0 were very similar across groups. A significant influence of time was observed in the data overall. Versus day 0, total cholesterol 5 levels declined significantly commencing at day 10, with these differences persisting until day 40 of study for control and day 50 for phytosterol enriched diets. A distinct effect of diet treatment in general is evident from the nadir of cholesterol levels at days 29 and 30, with a trend towards resumption of pre-diet levels over days 40 and 50, regardless of treatment. The rate of return to 10 baseline appeared to be more rapid for the phytosterol-containing diet. These data reveal that both diets caused a progressive decline in total circulatory cholesterol levels over time on treatment, however, the decline was more rapid with the phytosterol-enriched diet. 15 Figure 46 also reveals that at days 29 (p<0.038) and 30 (p<0.035) of the experiment a diet effect was observed relative to time 0. Thus, at these time points the control diet, despite reducing cholesterol levels itself, was shown to be significantly less effective in cholesterol-reduction ability, compared with the phytosterol-enriched 20 diet. The phytosterol-enriched diet also produced a pattern of cholesterol reduction which appeared to differ from that of the control diet. While the latter produced an effect over the 30 day feeding trial which showed a tendency towards attainment of plateau, the slope of the phytosterol-enriched decline was nearly linear. Comparison of curves from the two dietary treatments suggests 25 divergence between them, with the full cholesterol reducing potential of the phytosterol-enriched diet not fully realized. Figure 47 shows the not decline in total cholesterol levels expressed as a 142086\0240454.WP -119 difference between the mean of days 29 and 30 versus the level on day 0. For the control diet, this value was 10.4%. For the phytosterol-enriched diet, the level of decline was 18.7%. When linear regression models were applied across the 0 to 30 day time-set data for each subject, then pooled and compared, the mean 5 decline in total cholesterol levels over 30 days was 10.5 and 17.4% for control and phytosterol enriched diets, respectively. For low density lipoprotein cholesterol levels measured across the 30 day feeding period, substantial variation in pattern across subjects was also 10 observed, For the control diet, the tertile (n=5) of individuals showing greatest response to diet had on average a 24% decline, while the tertile (n=5) with the least response showed an average drop in LDL cholesterol levels by 4% (data not shown), On the phytosterol enriched diet the variability was similar. Here, the tertile of individuals showing greatest response to diet had on average a 37% 15 decline, while the tertile with the least response showed an average drop in LDL cholesterol levels of only 8% (data not shown). Patterns of response for the groups as a whole for LDL cholesterol levels over the 30 day feeding period, and subsequent 20 day wash-out period, are shown in 20 Figure 48. A significant influence of time was observed across both diets. Versus day 0, LDL cholesterol levels declined significantly commencing at day 10, with these differences persisting across the entire 50 day study for the phytosterol-enriched diet. A similar was observed for the control diet, but differences were not significant for days 10 and 50. A distinct effect of diet 25 treatment in general also is evident from the nadir of LDL cholesterol levels at days 29 (p<0.06) and 30 (p<0.032) in contrast with day 0, with a trend towards resumption of pre-diet levels over days 40 and 50, regardless of treatment. These data reveal that both diets caused a progressive decline in circulatory LDL 142085\02C454.WP -120 cholesterol levels over time on treatment. Figure 48 also shows the diet effects observed relative to tirne 0 for LDL levels. At this time point the control diet, despite reducing cholesterol levels itself, was 5 shown to be less effective in its cholesterol-reduction ability, compared with the phytosterol-enriched diet. The phytosterol-enriched diet also produced a pattern of LDL cholesterol reduction which appeared to differ from that of the control diet. As with the situation for total cholesterol levels, while the control diet produced an effect over the 30 day feeding trial which showed a tendency towards 10 attainment of plateau, the slope of the phytosterol-enriched decline was steeper and nearly linear. Comparison of curves from the two dietary treatments suggests divergence between them, with the full LDL cholesterol reducing potential of the phytosterol-enriched diet not yet fully achieved. 15 The net decline in LDL cholesterol revels expressed as a difference between the mean of days 29 and 30 versus the level on day 0 is shown in Figure 49. -For the control diet, this value is 10.5%. For the phytosterol-enriched diet, the level of decline is 21.5%. When linear regression models were applied across the 0 to 30 day time-set data for each subject, then pooled and compared, as described 20 for total cholesterol levels above, the mean declines in LDL cholesterol levels over 30 d were 9.8 and 21.6% for control and phytosterol-enriched diets, respectively. The SAS 60 day projected extrapolation curves for total and LDL cholesterol 25 levels using incremental data to 30 days are shown in Figure 50. For total cholesterol levels, the projected cholesterol-lowering effects at 60 days were 13.4% and 24.9% for diet and diet with phytosterols, respectively (Figure 50a), For LDL cholesterol, the 142056\0241454.WP -121 projected cholesterol-lowering effects at 60 days were 14.2% and 28.3% for diet and diet with phytosterols, respectively (Figure 50b) 5 Data for HDL levels over time as a function of diet are shown in Figure 51. The group mean starting HDL level of individuals consuming the control diet was 15% lower over those consuming the phytosterol-enriched diet, however, this difference was not statistically significant. Neither time nor diet exhibited significant effect on 10 HDL cholesterol levels in subjects over the duration of the trial. Slight declines in HDL levels were observed for both control (2.8%) and phytosterol-enriched (6.6%) groups over the 30 days of the trial. Circulating triglyceride concentrations on each treatment are depicted on Figure 15 52. Subjects consuming the control diet had triglyceride levels at day 0 which were 22% lower compared to those on the phytosterol-enriched diet. These differences persisted over the course of the trial and were significant for days 10, 40 and 50. Also, there was no effect of time on circulating triglyceride levels over the 50 day study period. Changes in triglyceride levels from day 0 to day 30 were 20 - 13.3 and 6,3% for control and phytosterol-enriched diet groups, respectively. Changes in total cholesterol/LDL ratio across dietary treatments are shown In Figure 53. For the control diet alone there was no effect of time on the ratio relative to day 0. However, in the diet plus phytosterol group, effects of time 25 relative to day 0 were evident at days 20, 29 and 30. Discussion: The efficacy of action of plant sterols in lowering circulating lipid levels has been 142086\024045.Wp -122 previously demonstrated for both unsaturated and saturated phytosterols. These materials work through inhibition of cholesterol absorption across the intestinal wall. However, use of both pure unsaturated and saturated plant sterols as cholesterol lowering agents possess substantial drawbacks. Unsaturated 5 phytosterols, although effective in considerably reducing total and LDL cholesterol levels, must be utilized in such high dosages to preclude application as a practical food adjunct. Mixtures of unsaturated sitosterol, campesterol and other phytosterols of over 10 9/day must be provided to see good efficacy of action in most cases, although, Pelletier et al reported efficacy with much lower 10 doses of unsaturated phytosterols. Similarly, saturated sitostanol, although more effective than sitostero in cholesterol-lowering ability, possesses a major drawback in that the material must be prepared chemically through hydrogenation from sitosterol. Further 15 modification is required to produce the sitostanol ester utilized in certain of these studies. These procedures add substantially to the cost, time and effort of production. Thus, supply and price disadvantages limit the utility of saturated phytosterols as effective broad-use dietary adjuncts. 20 In contrast, the present study has demonstrated cholesterol-lowering efficacy of a mixture of plant sterols in accordance with the present invention which possesses advantages over pure unsaturated and saturated species used previously. At least two advantages of use of the present phytosterol blend combining 25 unsaturated and saturated plant sterols as cholesterol-lowering adjuncts are apparent. First, the present phytosterol blend lowers total and LDL cholesterol levels in mildly hypercholesterolemic subjects by about 10 %, over and above the action of diet alone, after 30 days of use. This level of reduction, within the range 142086\024454

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-123 of that achieved with fully saturated stanol esters, was achieved in subjects given a modest dose of 1.5 g/70 kg body weight per day blended phytosterols. When used in conjunction with a prudent North American diet, the extent of cholesterol lowering is approximately twice this level; LDL declined 22% in these 5 hyperlipidemic subjects when the North American diet and phytosterols were used in combination. These levels of cholesterol lowering, achieved over the longer term, are sufficient to produce substantial declines in cardiovascular disease risk in the general population. 10 Second, the mixture of phytosterols used presently was obtained from tall oil without hydrogenation or chemical manipulation after extraction. Avoiding these stages of production increases ease and cost of preparation, which are important considerations if the material is to be used in the larger scale commercial context within the food industry. Moreover, a major advantage exists 15 in that the starting material tall oil is present on a world-wide scale in abundant quantity. Thus, with appropriately upscaled extraction facilities, tail oil can be expected to provide the amount of material necessary for full scale international commercialization and application. 20 Independent of the present observation that this phytosterol mixture, when provided as a suspension in margarine, reduces total and LDL cholesterol values in hyperlipidemic males is the finding, that the full effect of this material is likely attained after more prolonged usage. For both total and LDL cholesterol data, a distinct divergence in the slopes of the lipid level curves was observed 25 across days 0 through 30 (Figurel 50). Whereas the control diet data demonstrated a degree of plateauing, indicating that the greatest action of the diet occurred during the initial 10 to 20 day period, for both total and LDL cholesterol levels the trajectory of decline during consumption of the phytosterol 142086\O240454.WP -124 diet was steep and linear. Thus, it is suggested from these data that phytosterol's action is occurring in a manner that is mechanistically separate from that of diet alone and requires a more prolonged time window to be fully manifested. The final plateau was obtained at about 6 months. Thus, the 5 present data indicate that phytosterol possibly elicits a more substantial effect than observed at 30 days when consumed for more prolonged periods. Our results contrast those of Denke who provided 3 g of sitostanol per day to hyperlipidemic subjects and found no significant effect on circulating lipid levels 10 In summary, the present study results demonstrate pronounced efficacy of a highly obtainable, safe plant sterol mixture in lowering total and LDL cholesterol levels in hyperlipidemic meals, particularly when provided in conjunction with a prudent diet. It is concluded that the compositions of the present invention possess good potential for lowering of heart disease risk in susceptible IS populations. Table 52 Composition of phytosterols 20 Plant Sterol Mixture Corn Starch Placebo Campesterol (%) 16.1 0 Campestanol (%) 0.9 0 Beta-sitosterol (%) 62.0 0 25 Sitostanol (%) 21.1 0 Table 53 Mean, standard deviation and decline % of subjects' body weights during 30 day trial for control and phytosterol-enriched diets (n = 16) 30 Diet Day 0 Day 30 Body Weight change (%) 142086\0243454 .WP -125 Control Mean 79.8 78.9 _1 SD 9.6 6.5 1A Phytosterol Mean 87.8 86.7 -1.2 SD 15.2 15.1 1.4 10 Figure Captions: Figure46 Influence of phytosterol-enriched diet over time on total cholesterol of hypercholesterolemic subjects. The *and show significance differences 15 (5%, 1 % and 0, 1% levels, respectively) for time effect and the + shows the significance difference (5% level) for diet effect in contrast with day 0, Bars are 25% SD and ns stands for non-significant effect. 20 Figure 47 Influence of phytosterol-enriched diet on lowering total cholesterol of hypercholesterolemnic subjects. Figure 48 Influence of phytosterol-enriched diet over time on LDL-cholesteroi 25 of hyper-cholesterolemic subjects. The *, ** and *** show significance differences (5%, 1 % and 0. 1% levels, respectively) for time effect and the + shows the significance difference (5% level) for diet effect in contrast with day 0. Bars are 25% SD and ns stands for non-significant effect. 30 Figure 49 Influence of phytosterol-enriched diet on lowering LDL-cholesterol of hypercholesterolemic subjects. Figure 50a Extrapolation of cholesterol-lowering effects of control and 35 phytosterol-enriched diets on total cholesterol of hypercholesterolemic subjects. Figure 50b Extrapolation of LDL-cholesterol-lowering effects of control and phytosterol-enriched diets on LDL-cholesterol of hypercholesterolemic subjects. 40 Figure 51 Influence of phytosterol-enriched diet over time on HDL-cholesterol of hypercholesterolemic subjects. Bars are 25% SD. 142006\024M54.WP -126 Figure 52 Influence of phytosterol-enriched diet over time on triglyceride of hypercholesterotemicsubjectsBars subjects. Bars are 25% SD and different letters show significance effect (5% level 5 Figure 53 Influence of phytosterol-enriched diet over time on total cholesterol/HDL-cholesterol of hypercholesterolemic subjects. The * and show significance differences (at 5% and 71% levels, respectively) for time effect in contrast with day 0, Bars are 25% SD and ns stands for non-significant effect. 14206\024454.WP

Claims (7)

1. Use of cholesterol-lowering composition comprising beta-sitosterol, campesterol and further comprising stigmastanol, in which campesterol and stigmastanol together comprise at least 50% of the concentration of beta sitosterol and wherein the ratio of beta-sitosterol is 1.0, campesterol is between 0.2 and 0.4 and stigmastanol is between 0.2 and 0.5 in the manufacture of a medication for use in a method of lowering the plasma concentration of low density lipoprotein-oholesterot.

2. Use according to claim 1 wherein the ratio of beta-sitosterol is 1.0, and either a) campesterol is 0.354 and stigmastanol is 0.414 or b) campesterol is 0.330 and stigmastanol is 0.203, or c) campesterol is 0.268 and stigmastanol is 0.299.

3. Use according to claim 1 or claim 2 in which the method is for prevention or treatment of primary and secondary dislipidemias and atherosclerosis.

4. Use according to any of claims 1 to 3 in which the composition comprises phytosterols and co-occurring compounds selected from triterpenes, long chain alcohols and other alcohol-soluble organic compounds.

5. A medication comprising beta-sitosterol, campesterol and further comprising stigmastanol, in which campesterol and stigmastanol together comprise at least 50% of the concentration of beta-sitosterol and wherein the ratio of beta-sitosterol is 1.0, campesterol is between 0.2 and 0.4 and stigmastanol is between 0.2 and 0.5, and a pharmaceutically effective carrier therefor.

6. A medication according to claim 5 wherein the ratio of beta-sitosterol is 1.0, and either a) campesterol is 0.354 and stigmastanol is 0.414 or b) campesterol is 0.330 and stigmastanol is 0.203, or c) campesterol is 0.268 and stigmastanol is 0.299.

7. A composition comprising beta-sitosterol, campesterol and further comprising stigmastanol, in which campesterol and stigmastanol together comprise at least 50% of the concentration of beta-sitosterol and wherein the ratio of beta-sitosterol is 1.0, and either a) campesterol is 0.354 and stigmastanol is 0.414 or b) campesterol is 0.330 and stigmastanol is 0.203, or c) campesterol is 0.268 and stigmastanol is 0.299.