AU2555692A - Composition and method for reducing cholesterol concentration - Google Patents

Description

Composition and Method for Reducing Cholesterol Concentration

Field of the invention

The present invention relates to an absorbent polymer and method using this absorbent polymer for reducing the level of or removing sterols, particularly cholesterol, from blood, plasma, food materials, fatty substances and oils or fats of biological origin. Background of the Invention It is widely accepted that serious health risks attach to high plasma cholesterol levels. In Australia, coronary heart disease is responsible for more than 50,000 deaths every year, and death from coronary heart disease is twice as frequent as death from cancer. Drugs for reducing cholesterol and triglycerides are listed in Table 1. Cholestyramine is the only drug listed in Table 1 which is not absorbed and which does not enter the metabolic pathways of the body. It has been considered safe, even for children with blood cholesterol and lipid disorders. By sequestering bile acids, cholestyramine inhibits their intestinal absorption, thus interrupting the enterohepatic bile acid cycle. The conversion of liver cholesterol into bile acid is increased and the liver then removes cholesterol from the blood by increasing the number of LDL receptors on liver cells.

The daily recommended intake of cholestyramine ranges from 20 to 30g daily. Only 10% of the ion-exchange capacity is utilised for binding bile acids, the remaining capacity is capable of binding other anions in the digesta. Thus the drug is not without side effects; the most common being constipation which occurs in 66% of patients. It can also give a feeling of satiety and cause heartburn. Some patients dislike the gritty, sandy mouthfeel and full compliance with this drug has often been questioned. TABLE 1: Drugs for treatment of hyperlipidaemia**

Type of Drug Names of Drug Mechanism of Action

Bile acid Cholestyramine Sequesters bile acids sequestrant ( uestran, the intestinal lumen

Cholybar) interrupts

Colestepol enthrohepatic bile

(Colestid) acid cycle. Not absorbed from the intestinal lumen.

Niacin Nicotinic acid Primary action, is on (Vitamin B3) the liver, where it reduces VLDL production. Reduces blood triglycerides and cholesterol.

HMG CoA reductase Lovastatin Reduces cholesterol inhibitor Simvasta in (Zocor) synthesis in the

Pravastatin liver and increases

(Pravachol) cholesterol uptake

Fluvastatin from the blood into

(LoChol) the liver. Fibric acid Gemfibrozil (Lopid) Primarily used to

Clofibrate reduce blood

(Atromid-S) triglycerides

Fenofibrate through activation of

(Lipidil)* lipoprotein lipase. Increases cholesterol output into the bile.

Butylphenol Probuco1 (Lorelco) After being incorporated into LDL, increases the latter's removal by liver, through mechanism other than LDL receptor.

* Not yet approved by the Food and Drug Administration for prescription use. ** Modified from. P.O. Kwiterovich Jr. (1989) "Beyond

Cholesterol" The Johns Hopkins University Press,

Baltimore USA. By reducing the effective concentration of bile acid in the upper small intestine, cholestyramine interferes with the absorption of fat-soluble vitamins. The drug also interferes with the absorption of a variety of medications, such as thiazines, anticoagulants, digitalis and coumarin derivatives.

The remaining drugs in Table 1 act by modifying metabolic processes in the body; they lack specificity and are also not without side effects. The long term safety of drugs that inhibit cholesterol synethesis (lovastatin and others listed) has not been tested as they have only recently been released.

Thus most if not all the cholesterol-lowering drugs currently available have disadvantages. In particular there has been no alternative to cholestyramine to bind bile acids (and preferably cholesterol also) within the intestine and inhibit their absorption.

One method of controlling cholesterol levels involves dietary modification. Dairy products, and eggs in particular, are perceived as contributing significantly to dietary cholesterol - butter fat and egg yolk, for example, contain approximately 3mg and 15mg per gram respectively - consequently there is widespread interest in reducing the cholesterol level of such products. European Patent Application No 8400175.9 and Australian Patent Application No 55112/90 describe methods for using β - cyclodextrin to extract cholesterol from fats and oils of animal origin, β - cyclodextrin is a cyclic oligosaccharide of seven glucose units. As a consequence of the Cl conformation of the glucopyranose units, the secondary OH-groups protrude outwardly from the wide edge of the torus-like cyclodextrin molecule while the primary OH-groups are located on the narrower edge. The central cavity is therefore hydrophobic, giving the molecule an affinity for non-polar molecules such as cholesterol; additionally the radius of the cavity is such as to accommodate a cholesterol molecule almost exactly and this confers a highly specific ability to form an inclusion complex with cholesterol and remove it from food emulsions or fats or oils.

While the aforementioned methods allow removal of as much as 90% cholesterol, they are not without disadvantages. For instance, there can be significant costs associated with a loss of cyclodextrin during separation and regeneration steps, also unavoidable residues of cyclodextrin in the treated product may not be acceptable in certain countries.

To avoid the disadvantages which are associated with the use of free cyclodextrin, treatment with solid phase adsorbants has been proposed (see, for example Australian Patent Application No 54768/90 and 85601/90), however the specific adsorbants disclosed do not meet all the criteria for commercial viability. Essentially a solid phase adsorbant should satisfy the following requirements:

be non- oxic and leave no residues in the food.

be specific for cholesterol and not remove other materials from the food.

remain stable when brought into contact with food.

have a high adsorptive capacity ie a high proportion of accessible binding sites per unit weight.

have a high affinity constant for cholesterol.

be capable of performing at low temperatures in order to reduce the risk of microbiological spoilage or deterioration of food quality.

be of a physical form which facilitates intimate contact between the adsorbant and the fatty particles of the food, and at the same time allows easy separation from the food after the adsorption treatment.

be capable of being easily and cheaply regenerated (ie freed of complexed cholesterol).

Of particular importance is the need for a high proportion of accessible binding sites. The fat in milk is emulsified and, being amphiphilic, the cholesterol molecules are concentrated at the surface of the fat globules where they would be accessible to cyclodextrin in an aqueous phase. In egg, most of the fat and cholesterol is carried in the yolk lipoproteins. Again, the amphiphilic cholesterol molecules would be available for complexing with cyclodextrin in an aqueous phase. For efficient cholesterol adsorption, however, it is necessary that each fat-associated cholesterol molecule has the opportunity to make intimate contact with a cyclodextrin molecule. It is probably in this respect that the prior art solid phase adsorbants fall short of meeting the above criteria. In Australian Patent Application No 85601/90 the examples reveal only modest cholesterol reduction, ^' thus 30% from milk and 52% from cream, whereas had the same amounts of unbound cyclodextrin been used, chc. )sterol reduction would have been of the order of 90% The adsorbants were in fact beads of cyclodextrin cross-linked with epichlorohydrin; the cross-linking results in a network structure where a considerable proportion of the cyclodextrin molecules are located away from the surface of the beads and hence are unavailable for complexing with cholesterol. Another problem is that the beads swell taking up water and water soluble compounds from the food being treated, and this complicates the food processing and hinders adsorbant regeneration.

The present inventors have developed absorbent polymers which are not only very effective in inhibiting the adsorption of cholesterol and bile acid from the intestine but which is also useful for removing cholesterol and other sterols from food materials and from fatty substances of biological origin more efficiently than has hitherto been possible.

These polymers are based on cyclodextrin. Summary of the Present Invention Accordingly, in a first aspect the present invention consists in an absorbent polymer for use in reducing cholesterol levels, the absorbent polymer comprising an essentially linear polymer in which all or a substantial proportion of the monomer units carry a covalently linked cyclodextrin or modified cyclodextrin molecule.

In a preferred embodiment of the present invention the cyclodextrin or modified cyclodextrin molecules are covalently linked to the monomer units in a manner such that at least 5% and, preferably at least 25%, of the cyclodextrin or modified cyclodextrin molecules are readily accessible for cholesterol complexations.

In a further preferred embodiment of the present invention the cholesterol complexation ability of the absorbent polymer is at least 10%, and preferably at least 25%, of that of free cyclodextrin.

In a preferred embodiment of the present invention the cyclodextrin is β - cyclodextrin, however, α - cyclodextrin or _/ - cyclodextrin can also be used.

In a further preferred embodiment of the present invention at least 50% of the monomer units carry a covalently linked cyclodextrin or modified cyclodextrin molecule.

Figure 1 shows schematic representations of the absorbent polymer of the present invention in which CD is α, β or γ cyclodextrin and CDX is modified a, β or γ cyclodextrin. It can been seen that the polymer falls into four general categories:-

1. An essentially linear polymer. Poly (A), in which each or some of the monomer units (A) are covalently linked to β -cyclodextrin (BCD-A) as shown in figure 1A.

2. A more generalised form in which the monomer units (BCD-A) are interspersed with another monomer (B) . Thus the active polymer is a copolymer of (BCD-A) and (B), or (BCD-B), as shown in Figure IB and 1C.

3. A form in which the cyclodextrin is itself chemically modified in addition to being bonded to the monomer unit A or B as shown in Figure ID.

4. Essentially linear copolymers produced by combining any two or more of types 1, 2 and 3 in different proportions.

The polymers are essentially linear chains, however, there may be occasional cross-links between chains.

In a preferred embodiment of the present invention the absorbent polymer is selected from the group consisting of poly(3 - cyclodextrinacrylate) , poly(/3 - cyclodextringlacturonate) , poly(_/3 - cyclodextrinmethacrylate) , poly(3 - cyclodextringlycidyl ethacrylate) , poly(3 - cyclodextrinvinylchloride), poly(N-/3 - cyclodextrinacrylamide) and copolymers thereof.

As used herein the phrase "readily available for cholesterol co plexation" means that the cyclodexation molecule is covalently bound to the monomer unit in such a manner that when the polymer is bought into contact with cholesterol containing material that the cholesterol forms a complex with the cyclodextrin molecules. This is to be contrasted with the situation where, due to the nature of the binding of the cyclodextrin molecule, the cyclodextrin molecule is not able to form a complex with cholesterol.

Where the absorbent polymer of the present invention is to be used as a pharmaceutical, or as the active ingredient for pharmaceutical composition, the essentially linear polymer should be non-toxic and resistant to digestion by the enzymes of the gastrointestinal tract. In such a form the polymers of the present invention could be administered in the treatment of hypercholesterolaemia to adsorb cholesterol or bile acids in the intestine and inhibit their absorption.

Cyclodextrins also form complexes with bile acids and inhibit their absorption from the intestine. Thus modified cyclodextrins or cyclodextrins attached to essentially linear polymers" provide another potentially safe means of lowering plasma cholesterol by - like cholestyramine - interrupting the enterohepatic bile acid cycle. Again, there is the great advantage over cholestyramine that cyclodextrins are more specific and would consequently have fewer side effects. Accordingly, in a second aspect the present invention consists in a method of treating hypercholesterolaemia in a subject comprising administering to the subject an effective amount of the absorbent polymer of the first aspect of the present invention. Another adaptation of this invention would be for the treatment of hypercholesterolaemia by the extraction of cholesterol directly from the blood plasma. Blood could be withdrawn from the patient, treated with a suitable form of the absorbent polymer and then returned to the body. Various techniques have been developed for extracorporeal adsorption and removal of harmful substances from blood plasma (Parker and Studebaker, Methods in Enzymology, 137, 466-478, 1988). Typically these techniques use immobilised enzymes or immunoadsorption systems on plasma after separation of the red blood cells. LDL particles rich in cholesterol can be removed using specially prepared immunoadsorption columns which can be regenerated with buffers for reuse. In another method, plasma is subjected to a column treatment to remove LDL particles rich in cholesterol (Yokohama, S., Yamamoto, A., Hayashi, R. et al., Jap. Circ. J. 5_1, 1116-1122, 1987). The treated plasma is then recombined with red blood cells and returned to the patient's blood circulation. Both these techniques suffer from serious disadvantages. Valuable proteins, phospholipids and vitamins are lost from the blood plasma in the course of treatment. Also immunoadsorption columns cannot be heat sterilised and are likely to induce immune reactions to the antibodies which are derived from animal sources to prepare them.

Accordingly, in a third aspect the present invention consists in a method of reducing blood cholesterol levels in a subject comprising withdrawing blood from the subject, contacting the blood with the absorbent polymer of the first aspect of the present invention and returning the treated blood to the subject.

In a preferred embodiment of this aspect of the present invention the blood cells are removed from the blood and the plasma is contacted with the absorbent polymer of the first aspect of the present invention. Subsequently, the blood cells and the treated plasma are recombined and returned to the subject.

An inert polymer, free from protein, which selectively removes cholesterol would therefore be highly advantageous compared with existing techniques. The cyclodextrin polymers of this invention are capable of meeting the stringent health and safety requirements which would be required for a cholesterol adsorbant for use with blood plasma. Another important advantage is that the polymers selectively remove the LDL cholesterol which is associated with heart disease risk. The protective HDL cholesterol is left behind.

A further form of this invention could be used to selectively and specifically extract cholesterol or bile acids from appropriate source materials such as bile (an abattoir waste) . Cholesterol and bile acids are useful substances in their own right, and more so as precursors for synthesis of steroid-based drugs. A suitable form of the adsorbant would extract these materials and they could be recovered selectively by washing with different solvents or solvent mixtures.

As alluded to above the absorbent polymer of the present invention may also be used in the removal or reduction of cholesterol and other sterol levels from food materials. Accordingly, in a fourth aspect the present invention consists in a method of removing or reducing the level of cholesterol in the sample comprising contacting the food material comprising contacting the food material with the absorbent polymer of the first aspect of the present invention.

In preferred forms of this aspect of the present invention the food is a dairy product.

Where the absorbent polymer of the present invention is to be used in removal or reduction of cholesterol from food materials and the like in the same way as treatments using free cyclodextrin, the adsorption of cholesterol using the bound cyclodextrin polymer as described in this application is carried out in an aqueous medium. The adsorption takes place at the oil-water interface, so the fat or oil is preferably exposed to the adsorbant in the form of an oil-in-water emulsion, as this maximises the available interfacial area. Since the fat in milk, cream or egg is already in this form such products can be simply exposed to the adsorbant, however some minimal pretreatment, such as homogenisation or dilution may facilitate rapid interaction.

The product is exposed to the active polymer by mixing with efficient stirring, allowing time for reaction (10 seconds to 2 hours and preferably 15 minutes) and then separating the polymer by filtration or centrifugation. Alternatively, the product can be passed through a bed of the polymer in particulate form. The temperature can range from 0 C to 60 C and is preferably in the "chilled" range, below 15°C.

The adsorption process can be carried out at any temperature at which water is in the liquid phase. For more delicate food products, such as milk, cream or egg yolk, the temperature would be kept close to 0 to avoid microbiological spoilage and deterioration of flavour or functional properties. Low temperature would also make the removal of cholesterol by the absorbent polymer more specific and would preclude complex formation with other constituents such as fatty acids, phospholipids and proteins. Other products, such as lard or tallow, might more conveniently be processed at high temperature where the fat is liquid. There is, however, no necessity for the dispersed fat to be above its bulk melting temperature. The adsorptive power of the absorbent polymer of the present invention can be restored by washing through with acetic acid, or other organic acids, alkali solutions or other suitable solvent, which will remove the cholesterol without degrading the adsorbant. The present inventors have also developed novel method of producing the absorbent polymers of the present invention. Accordingly, in a fifth aspect the present invention consists in a method of producing an absorbent polymer for use in reducing cholesterol levels, the absorbent polymer comprising an essentially polymer in which all or a substantial portion of the monomer units carry a covalent linked cyclodextrin or modified cyclodextrin molecule, the method comprising reacting the cyclodextrin or modified cyclodextrin molecules with monomer units such that the cyclodextrin or modified cyclodextrin molecules are covalently bound to the monomer units and subsequently preliminising the monomer units. In a sixth aspect the present invention consists in method of producing an absorbent polymer for use in reducing cholesterol levels. The absorbent polymer comprising an essentially linear polymer in which all or a substantial proportion of the monomer units carry a covalently linked cyclodextrin or modified cyclodextrin molecule, the method comprising preliminising the monomer units and subsequently reacting the polymer with cyclodextrin or modified cyclodextrin molecules such that the cyclodextrin or modified cyclodextrin molecules such that the cyclodextrin or modified cyclodextrin molecules are covalently bound to the monomer units.

In a preferred embodiment of these aspects of the present invention the reaction of the cyclodextrin or ^' modified cyclodextrin molecules with the monomer units or polymer is carried out in the presence of dicyclohexylcarbodiimide or one, three-diisopropylcarbodiimide.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples. Examples Example 1

Preparation of Poly( β - c clodext in acrylate) (PCA la and lb) Acrylic acid (AA) was distilled under vacuum. Dimethylformamide (DMF) was dried over CaH₂ and distilled under vacuum, β - cyclodextrin (BCD) was dried at 100° under vacuum. BCD (56.8g) was dissolved in DMF (710 ml) and to this solution AA (3.6 ml) was added, followed by dicyclohexylcarbodiimide (DCC)

(11.33 g) . The solution was left to stand in a tightly stoppered flask at room temperature overnight. Most of the DMF was removed by vacuum distillation (64°C, 34 mmHg) and the remainder by rotary evaporation. The dicyclohexylurea (DCU) formed during the reaction was removed by Soxhlet extraction with methylene dichloride. The resulting polymer was washed free of unreacted BCD by water and dried. Example 2 Preparation Of ?Qlγ{β - cyclodextrin galacturonate) (PCG) Polygalacturonic acid (PGA) and BCD were dried under vacuum. Dimethylsulphoxide (DMSO) was vacuum distilled. BCD (11.35g) was dissolved in DMSO. PGA (1.94g) was dissolved in DMSO (50 ml) and added to the BCD solution and heated with stirring at 90°C for 70 minutes. DCC (2.9g) was added and the solution left overnight at room temperature with stirring. Most of the DMSO was removed by rotary evaporation. Then 10-times its volume of ethyl acetate was added to the remaining solution. The white precipitate that formed was collected and the DCU removed by Soxhlet extraction with methylene dichloride. The polymer so obtained was repeatedly washed with water and dried at 60 C under vacuum. Example 3

Preparation of Polymer of Polyacrylic Acid with BCD fPCA-2^

Polyacrylic acid (0.72g) was dissolved in dry DMF (3 ml) and 1,3-Diisopropylcarbodiimide (DIC) (1.26g) was added to it with cooling. The reaction mixture was stirred for additional 1 hr. Dry BCD (11.33 g) was dissolved separately in 50ml DMF and added to the stirred reaction mixture. After 30 min. dimethylaminopyrindine (DMAP) (100 mg) was added and the stirring continued overnight. The solvent was rotary evaporated and the residue triturated with acetone and then ethanol. It was dialysed to remove unreacted BCD and dried in vacuo at 60°C for 24 hr. Example 4 Preparation of Poly(β -cyclodextrinacrylate) from monomer (PCA-3)

Acrylic acid (1.44 g) was taken in dry DMF (10 ml). DIC (1.3 g) was added to it with cooling. The reaction mixture was stirred for 1 hr. BCD (11.33 g) was dissolved in dry DMF (30 ml) and added all at once to the above reaction mixture. DMAP (100 mg) was added and the reaction mixture stirred overnight. The solvent was rotary evaporated and the residue triturated with acetone followed by ethanol. It was dried in vacuo at 60 C for 24 hrs. Exam l 5

Preparation Of β - cycloxdextrinmethacrylate and β - cyclodextringlycidylmethacrylate (PCM-1 and PCG-1)

BCD (11.33 g) was dissolved in dry DMF (30 ml). Methacrylic anhydride (4.2g) was added to it in one portion followed by the DMAP (100 mg) . The reaction mixture was heated with stirring at 90 C for 3 hrs. After the usual work up β - cyclodextrinmethacrylate was obtained in ) 92% yield. Similarly, β - cyclodextringlycidylmethacrylate was prepared from BCD and glycidylmethacrylate.

The polymers PCM and PCG were obtained from β - cyclodextrinmethacrylate and β - cyclodextringlycidyl- methacrylate respectively as described under preparation No. 4. Example 6 Reaction of Polyacrylic Acid with BCD NH₂ fPC ^

Polyacrylic acid (0.210 g) was dissolved in dry DMF (5 ml) and DIC (4 g) was added to it with cooling and stirring. The mixture was stirred for 1 hr. BCD NH„ (3.3 g) in dry DMF (4 ml) was added to the reaction mixture and stirred overnight. After removal of the solvent, the residue was triturated with acetone and then ethanol. It was dialysed to remove unreacted BCD NH-. The residue was dried at 60°C in vacuo for 24 hrs. Example 7 Reaction of BCD with Polyvinylchloride

Dry BCD (20 g) was dissolved in DMF (250 ml) and NaH (0.4 g) added to it at room temperature and the mixture stirred overnight. PVC (3.3 g) was added to it and stirring continued for 24 hrs. Half of the DMF was removed by rotary vaporator and the mixture thrown into an excess of ethyl acetate. It was filtered and the orange solid obtained was dialysed at room temperature for 24 hrs, filtered and dried in vacuo at 50°C overnight. E ample 8 Preparation of Poly (β - cyclodextrinmethacrylate) (PCM-2) Polymethacrylic acid (0.860 g) was taken in dry DMF. DIC (1.3 g) was added to it with cooling. The reaction mixture was stirred for 1 hr. BCD (11.33 g) was dissolved in dry DMF (30 ml) and added all at once to the above reaction mixture. DMAP (1000 mg) was added and the reaction mixture stirred overnight. The solvent was. rotary evaporated and the residue triturated with acetone followed by ethanol. The residue thus obtained was dialysed for 72 hrs to remove unreacted BCD. After filtration the solid was dried in vacuo at 60 for 2 hrs. Measurement of Cholesterol Adsorption

3 A standard emulsion containing H-labelled cholesterol was used for routine screening of adsorbants.

The emulsion was prepared in isotonic phosphate buffer and contained 14C-labelled Cr-EDTA added to provide a means of monitoring any uptake of water by the adsorbant. It contained cholesterol (0.25 mM) , oleic acid (1.2 mM) , monoolein (6.0 mM) and sodium taurocholate (10.0 mM) (the same cholesterol concentration as in milk) .

Example 9 Absorption of Cholesterol from Loops of Small Intestine in vivo in the Rat

3 A standard emulsion containing H-labelled cholesterol was used for routine screening of adsorbants.

The emulsion was prepared in isotonic phosphate buffer and contained 14C-labelled Cr-EDTA added to provide a means of monitoring any uptake or intake of water. It contained cholesterol (O.lmM), oleic acid (1.2mM), onolein (6.0mM) and sodium taurocholate (lO.OmM).

Male Wistar rats (300-40Og body weight) were used for studying cholesterol absorption in vivo. After anaesthetising the animals with intraperitoneal sodium pentobarbitol (60mg/kg body weight), the abdomen was ^' opened by midline incision and two segments (each about 20cm in length) were isolated, one from the upper jejunum and one from the lower ileum. Inflow cannulae were fitted, distal openings were established and the digesta from the lumen of each segment was washed out with isotonic saline at 37 C . The distal openings were closed by ligatures and cholesterol emulsion (4 ml) was injected. The segment was then closed with another ligature and returned to the abdominal cavity. The abdominal wall was closed with surgical clips. After 30 min the cavity was opened, the segments were removed, washed in isotonic saline to remove blood and drained of water on filter paper. The length of each segment was measured and the contents washed into a 25 ml volumetric flask. The concentration of cholesterol remaining was

3 determined from the H radioactivity. Chromium EDTA labelled with 14C was used as a non-absorbable marker to check for water influx or efflux. Cyclodextrin or its polymers were added to the emulsion for studying their effect on cholesterol absorption.

Table 2: Adsorption of cholesterol (nmol/cm/min.) from rat intestinal segments in vivo (means of results obtained from three animals in each treatment) .

Treatment Upper Segment Lower Segment

Control 0.403(-)* 0.187(-)

BCD 0.490 (+21) 0.478(+156)

PCA 0.040 (-90) 0.013 (-93)

PCG 0.088 (-78) 0.037 (-80)

NB The results achieved using PCA and PCG show that the polymers have an enhanced ability to bind cholestrol compared with that of unmodified BCD.

* Values in parentheses indicate percentage difference from the control value.

Example 10

Removal of Cholesterol from Blood Plasma

To a sample of human blood plasma (5 ml) was added 3.5%

(by weight) of the absorbent polymer and mixed at 18°C for 10 min. It was then cooled in ice and centrifuged at below 4°C. The concentration of cholesterol in the supernatant was determined by standard colorimetric and enzymatic procedures (Rudel and Morris J. Lipid Res. H, 364, 1973; and Boehringer Mannheim Monotest (R) kits for total and HDL cholesterol) .

Table 3 - Removal of cholesterol from blood plasma

Treatment Cholesterol Percentage

Concentration Change

Control 211

BCD 53.9 -74 PCA 59.8 -72

PCG 126 -40

Example 11

Differential Removal of Cholesterol from LDL Particles from Human Blood

A sample of human blood plasma was mixed with solid β - cyclodextrin (3.5% w/w) at 18°C. It was then cooled in ice for 20 minutes and centrifuged at 3,000 G for 15 minutes, the results are shown in Table 4. The supernatant was analysed for tital and HDL cholesterol. Table 4: Removal of Cholesterol from LDL Particles

Concentration mg/lOOml plasma

Untreated Treated Change (%)

Total Cholesterol 222.5 42.6 -81

HDL Cholesterol 50.3 23.2 -54

HDL/Total

Cholesterol 0.23 0.54 +235 The total cholesterol concentration was reduced by 81%. The high density lipoprotein fraction (HDL) was also reduced, but not to the same extent (54%). Thus the bulk of the cholesterol reduction was in the LDL fraction and the ratio of HDL to total cholesterol was greatly increased by the extraction procedure. The cholesterol depleted HDL particles would pick up cholesterol readily when returned to blood circulation through the action of lipoprotein lipase from LDL and cholymicrons, thus reducing the concentration of potentially harmful cholesterol. Example 12 Residue of Cyclodextrin in Treated Plasma

2ml of plasma treated with BCD or its polymer (PCA) was diluted with 8.8 ml of physiological saline (9g NaCl/L) and 5 ml of this solution dialysed against physiological saline (15ml) using a cellulose membrane tubing, β - cyclodextrin in the dialysate was determined by complexing with phenolphthalein as described by Makela et al. (J. Agric. Chem. 2£._r 83-88, 1988).

Plasma treated with BCD contained 0.6% residual BCD but no BCD was detected in PCA treated plasma. Example 13

Regeneration of PCA Packed Columns Column used to remove cholesterol from the emulsion was washed with saline (9g NaCl/L) followed by a solution of isopropanol and acetic acid (3:1). By this procedure it was possible to remove 98% of the cholesterol adsorbed onto the column. Example 14

A sample of emulsion (2.5 ml) was shaken with adsorbant (as specified in the appropriate Table) for

15 min. at 18 C. It was then cooled in ice for 30 min.

3 an centrifuged at 3,000 g for 15 min. The H-activity of cholesterol remaining in the supernatant was determined to give the percentage of cholesterol removed. TABLE 5 Removal of Cholesterol from a Standard Emulsion by BCD. PCA and PCG

* This is calculated on the equivalent number of cavities present per unit mass of BCD or polymer Our estimates suggest that only 70% of carboxylic groups on acrylate polymer (PCA) were occupied by cyclodextrins and on this basis the polymer contained to the extent of 67% by weight of BCD monomers as compared to free cyclodextrin.

Similarly PCG contained only 57.02% of cyclodextrin monomers by weight. If it is assumed further that the cavities of all the cyclodextrins attached to PCA and PCG are readily accessible for cholesterol complexation it can be shown that per cyclodextrin monomer the PCA la, PCA-2, PCM 2 and PCG were decidedly more efficient than free cyclodextrin. Example 15

Cholesterol emulsion (4 ml) was mixed with BCD or PCA (60 mg) and incubated at 40°C for 15 min. then

3 centrifuged without cooling. The H-activity of cholesterol remaining in the supernatant was determined to give the percentage of cholesterol removed (see Table 6) .

Removal of Cholesterol from a Standard Emulsion by Pol (Cyclodextrin Acrylate) ( CA^ at 40°C

Treat Concentration Cholesterol Cholesterol ♦Efficiency -ment of Absorbent in Supernatant Removed of Cholesterol polymer after Treatment (2) removed %

(Z w/w) (³H DPM)

None 19,193

BCD 1.5 14,990 21.9 100

PCA-Ia 1.5 8,661 58.0 395 Example 1

10ml samples of homogenised whole milk were mixed with 200 mg of BCD or its polymers at 15 C and shaken for 30 min using a rotary shaker. The samples were cooled in ice to below 4°C, allowed to stand for 30 min and centrifuged at 4000 g for 15 min using a refrigerated centrifuge. The supernatant containing cream and liquid milk was poured off and lipids extracted into 10 ml of hexane using the method described by Low and Dunkley (1971), J.Dairy Sci. , 1699.

Aliquot volume (5 ml) of hexane solution was evaporated to determine total lipid content of milk and saponified. The non-saponifiable material was extracted into hexane and cholesterol determined by gas chromatography and colorimetric methods. For gas chromatographic determinations β - cholestane was used as an internal standard. Colorimetric method was based on the method described by Rudel and Morris (J.Lipid Res. 15, 364, 1973). Results on cholesterol reduction are given in Table 7.

Table 7

Cholesterol Removal from Milk

Cholesterol Cholesterol Efficiency o mg/g fat Reduction % Cholesterol

Removal

Treatment Colori GC Colori GC Average metric metric

# This is calculated on equivalent number of cavities available for complexation per unit mass of BCD or polymers.

Represents two batches synthesized separately. Example 17

Cholesterol Removal from Egg Yolk

Egg yolk was separated from white and diluted by adding 3 or 4 volumes of 0.4 M NaCl. Pure cyclodextrin or PCA polymer was added (4% w/v), mixed at 15 C for 20 min, cooled in iced water to below 4 C and centrifuged after holding for 30 min. The cholesterol content of the supernatant was determined by using colorimetric method. The results are given in Table 8.

Table 8

* This is calculated on the equivalent number of cavities present per unit mass of BCD or polymer.

Example 18

Use of Packed Columns for Cholesterol Removal

A glass column with 1.0 g PCA-I and 5 ml cholesterol

3 emulsion containing 0.25 mM [ H]-labelled cholesterol was percolated through the column. Reduction in cholesterol is shown in Table .9.

Table 9 Cholesterol Reduction by Column Percolation

3 [ H]-cholesterol disintegrations per min

Amount Initially Adsorbed Adsorbed (%)

Placed on the

Column

946500 677000 71.5

Example 19 Regeneration of the Column 3 [ H]-cholesterol adsorbed by the column was eluted by passing 8 ml of isopropanol:acetic acid (3:1 v/v). The elution was carried out at a temperature of 50 C. Over

98% of the radioactivity adsorbed on the column, was recovered by this procedure. Example 20

Residue of Cyclodextrin in Treated Emulsions/Food Materials Egg yolk diluted with 0.4 m saline (1+3) was treated with β - cyclodextrin or its linear polymer (PCA) for cholesterol removal. It was then dialysed against water using a cellulose membrane tubing, β - Cyclodextrin in the dialysate was determined by complexing with phenophthalein as described by Makela et al. (J. Agric. Chem. 83-88, 1988). Egg yolk treated with β - cyclodextrin contained 0.6% BCD whereas no β - cyclodextrin could be detected in PCA treated egg yolk. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (26)

CLAIMS :

1. An absorbant polymer for use in reducing cholesterol levels, the absorbant polymer comprising an essentially linear polymer in which all or a substantial proportion of the monomer units carry a covalently linked cyclodextrin or modified cyclodextrin molecule.

2. An absorbent polymer as claimed in claim 1 in which the cyclodextrin or modified cyclodextrin molecules are covalently linked to the monomer units in a manner such that at least 5% of the cyclodextrin or modified cyclodextrin molecules are readily accessible for cholesterol complexation.

3. An absorbent polymer as claimed in claim 1 or claim 2 in which the cyclodextrin or modified cyclodextrin molecules are covalently linked to the monomer units in a manner such that at least 25% of the cyclodextrin or modified cyclodextrin molecules are readily accessible for cholesterol complexation.

4. An absorbant polymer as claimed in any one of claims 1 to 3 in which the cyclodextrin is β - cyclodextrin.

5. An absorbant polymer as claimed in any one claims 1 to 4 or claim 2 in which at least 50% of the monomer units carry a covalently linked cyclodextrin or modified cyclodextrin molecule.

6. An absorbant polymer as claimed in any one of claims 1 to 5 in which at least 50% of the cyclodextrin or modified cyclodextrin molecules are readily accessible for ' cholesterol complexation.

7. An absorbent polymer as claimed in any one of claims 1-6 in which the cholesterol complexation ability of the absorbent polymer is at least 10% of that of free cyclodextrin.

8. An absorbant polymer as claimed in any one of claims 1 to 7 in which the cholesterol complexation ability of the absorbant polymer is at least 25% of that of free cyclodextrin.

9. An absorbant polymer as claimed in any one of claims 1 to 8 in which the absorbant polymer is selected from the group consisting of poly(β - cyclodextrinacrylate), poly(β - cyclodextringlacturonate) , poly(β - cyclodextrinmethacrylate), poly(β - eyelodextringlycidylmethacrylate) , poly(β - cyclodextrinvinylchloride) , poly(N-β - cyclodextrinacrylamide) and copolymers thereof.

10. Poly (b - cyclodextrinacrylate).

11. Poly (b - cyclodextringlacturonate).

12. Poly (b - cyclodextrinacrylate).

13. Poly (b - cyclodextringlycidylmethacrylate) .

14. Poly (b - cyclodextrinvinylchloride).

15. Poly(N-β - cyclodextrinacrylamide)

16. A method of treating hypercholesterolaemia in a subject comprising administering to the subject an effective amount of the absorbant polymer as claimed in any one of claims 1 to 15

17. A method of reducing blood cholesterol levels in a subject comprising withdrawing blood from the subject, contacting the blood with the absorbant polymer as claimed in any one of claims 1 to 15 and returning the treated blood to the subject.

18. A method as claimed in claim 17 in which the blood cells are removed from the blood and the plasma is

^' contacted with the absorbant polymer and subsequently, the blood cells and the treated plasma are recombined and returned to the subject.

19. A method of removing or reducing the level of cholesterol in food material comprising contacting the food material comprising contacting the food material with the absorbant polymer as claimed in any one of claims 1 to 15.

20. A method as claimed in claim 19 in which the food is a dairy product.

21. A method as claimed in claim 19 or 20 in which the food material is contacted with the absorbant polymer by mixing with stirring, allowing time for reaction and then separating the absorbant polymer by filtration or centrifugation.

22. A method as claimed in claim 19 or 20 in which the food product is passed through a bed of the absorbant polymer in particulate form.

23. A method as claimed in any one of claims 19 to 22 in which the contacting of the food product with the polymer is conducted at a temperature in the range of 0°C to 60°C, and preferably below 15°C.

24. A method of producing an absorbent polymer for use in reducing cholesterol levels, the absorbent polymer comprising an essential linear polymer in which all or a substantial proportion of the monomer units carry a covalently linked cyclodextrin or modified cyclodextrin molecule, the method comprising reacting the cyclodextrin or modified cyclodextrin molecules with the monomer units such that the cyclodextrin or modified cyclodextrin molecules are covalently bound to the monomer units and subsequently polymerising the monomer units.

25. A method of producing an absorbent polymer for use in reducing cholesterol levels, the absorbent polymer comprising an essentially linear polymer in which all or a substantial proportion of the monomer units carry a covalently linked cyclodextrin or modified cyclodextrin molecule, the method comprising polymerising the monomer units and reacting the polymer which cyclodextrin or cyclodextrin molecules such that the cyclodextrin or modified cyclodextrin molecules are covalently bound to the monomer unit.

26. A method as claimed in claim 24 or claim 25 in which the monomer units or polymer is reacted with the cyclodextrin or modified cyclodextrin molecules in the presence of dicyclohexylcarbodiimide or one, three-diisopropylcarbodiimide.