Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C7/00—Other dairy technology
  • A23C7/04—Removing unwanted substances other than lactose or milk proteins from milk
  • A23C7/043—Removing unwanted substances other than lactose or milk proteins from milk using chemicals in liquid or solid state, e.g. flocculating, adsorbing or extracting agents
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
  • A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
  • A23L5/273—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption using adsorption or absorption agents, resins, synthetic polymers, or ion exchangers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/716—Glucans
  • A61K31/724—Cyclodextrins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/74—Synthetic polymeric materials
  • A61K31/765—Polymers containing oxygen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/74—Synthetic polymeric materials
  • A61K31/765—Polymers containing oxygen
  • A61K31/78—Polymers containing oxygen of acrylic acid or derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/74—Synthetic polymeric materials
  • A61K31/785—Polymers containing nitrogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
  • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F8/00—Chemical modification by after-treatment
  • C08F8/14—Esterification
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F8/00—Chemical modification by after-treatment
  • C08F8/26—Removing halogen atoms or halogen-containing groups from the molecule

Definitions

• 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.
• sterols particularly cholesterol
• Background of the Invention It is widely accepted that serious health risks attach to high plasma cholesterol levels.
• 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.
• cholestyramine 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**
• Bile acid Cholestyramine Sequesters bile acids sequestrant ( uestran, 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
• Butylphenol Probuco1 (Lorelco) After being incorporated into LDL, increases the latter's removal by liver, through mechanism other than LDL receptor.
• 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 has not been tested as they have only recently been released.
• ⁇ - cyclodextrin is a cyclic oligosaccharide of seven glucose 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.
• 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.
• the amphiphilic cholesterol molecules would be available for complexing with cyclodextrin in an aqueous phase.
• 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.
• 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.
• 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.
• the cholesterol complexation ability of the absorbent polymer is at least 10%, and preferably at least 25%, of that of free cyclodextrin.
• the cyclodextrin is ⁇ - cyclodextrin, however, ⁇ - cyclodextrin or / - cyclodextrin can also be used.
• 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 ⁇ , ⁇ or ⁇ cyclodextrin and CDX is modified a, ⁇ or ⁇ cyclodextrin. It can been seen that the polymer falls into four general categories:-
• Poly (A) in which each or some of the monomer units (A) are covalently linked to ⁇ -cyclodextrin (BCD-A) as shown in figure 1A.
• the active polymer is a copolymer of (BCD-A) and (B), or (BCD-B), as shown in Figure IB and 1C.
• the polymers are essentially linear chains, however, there may be occasional cross-links between chains.
• 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.
• 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.
• the essentially linear polymer should be non-toxic and resistant to digestion by the enzymes of the gastrointestinal tract.
• 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.
• 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.
• cholestyramine that cyclodextrins are more specific and would consequently have fewer side effects.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the food is a dairy product.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the polymers PCM and PCG were obtained from ⁇ - cyclodextrinmethacrylate and ⁇ - cyclodextringlycidyl- methacrylate respectively as described under preparation No. 4.
• Example 6 Reaction of Polyacrylic Acid with BCD NH 2 fPC ⁇
• 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) .
• 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
• 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) .
• Plasma treated with BCD contained 0.6% residual BCD but no BCD was detected in PCA treated plasma.
• 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.
• Cholesterol emulsion (4 ml) was mixed with BCD or PCA (60 mg) and incubated at 40°C for 15 min. then
• 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.
• 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.

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