WO2010014541A2 - Compositions et procédé pour le traitement de l'hypercholestérolémie - Google Patents

Compositions et procédé pour le traitement de l'hypercholestérolémie Download PDF

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Publication number
WO2010014541A2
WO2010014541A2 PCT/US2009/051837 US2009051837W WO2010014541A2 WO 2010014541 A2 WO2010014541 A2 WO 2010014541A2 US 2009051837 W US2009051837 W US 2009051837W WO 2010014541 A2 WO2010014541 A2 WO 2010014541A2
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Prior art keywords
cholesterol
pharmaceutical composition
patient
acid
inhibitor
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WO2010014541A3 (fr
Inventor
Alexey L. Margolin
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Alnara Pharmaceutical Inc
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Alnara Pharmaceutical Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/2027—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4891—Coated capsules; Multilayered drug free capsule shells
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03006—Cholesterol oxidase (1.1.3.6)

Definitions

  • the present invention generally relates to the field of biological therapeutic agents and formulations thereof.
  • the invention also relates to methods for using these formulations for the treatment of patients with hypercholesterolemia.
  • High serum cholesterol is a risk factor for cardiovascular disease (CVD).
  • CVD cardiovascular disease
  • statins HMG-CoA reductase inhibitors
  • LDL-C low density lipoprotein C
  • ezetimibe can be used as a second-line treatment. Ezetimibe reduces cholesterol absorption and thus employs a mechanism that is distinct from that of statins, which promote the clearance of LDL from the blood, principally in the liver.
  • compositions and methods for the treatment of hypercholesteremia include a pharmaceutical composition formulated for oral administration to a patient, wherein the pharmaceutical composition includes an effective amount of cholesterol oxidase and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition includes a therapeutically effective amount of cholesterol oxidase, at least one second therapeutic agent, and a pharmaceutically acceptable excipient.
  • the second therapeutic agent is selected from the group consisting of: a catalase; a peroxidase; a stomach acid reducing agent; and an enzyme that converts a triglyceride into a diglyceride, a monoglyceride, and/or glycerol and fatty acids.
  • the second therapeutic agent is a stomach acid reducing agent.
  • the stomach acid reducing agent is selected from the group consisting of: an antacid, an H 2 blocker, and a proton pump inhibitor.
  • the second therapeutic agent is an enzyme that converts a triglyceride into 1 ,3-diglyceride and/or 1 -monoglyceride.
  • the composition further comprises a second cholesterol-reducing agent.
  • the cholesterol-reducing agent is selected from the group consisting of an HMG-CoA reductase inhibitor, a bile acid sequestrant, a nicotinic acid, a fibric acid derivative, an inhibitor of cholesterol absorption, and a cholesterol esterase inhibitor.
  • the second cholesterol-reducing agent is an HMG-CoA reductase inhibitor.
  • the cholesterol oxidase is produced by a member of a bacterial genus selected from the group consisting of B revi bacterium, Cullulomonas, Nocardia, Pseudomonas, Schizophyllum, Streptomyces, Mycobacterium, Rhodococcus, and Burkholderia.
  • the pharmaceutical composition is formulated for oral administration.
  • the composition is present in an enterically-coated capsule.
  • the cholesterol oxidase is present in the composition in enterically coated granules.
  • Embodiments of the invention include a method for lowering serum cholesterol in a patient in need thereof, the method including the step of administering to the patient a pharmaceutical composition of the invention.
  • Embodiments of the invention include a method of treating a patient suffering from or susceptible to a disease or condition associated with high cholesterol levels, the method including the step of administering to the patient a pharmaceutical composition of the invention.
  • Embodiments of the invention include a method of treating a patient suffering from or susceptible to a disease or condition associated with plaque buildup in the arteries, the method including the step of administering to the patient a pharmaceutical composition of the invention.
  • Embodiments of the invention include a method for treating a patient suffering from or susceptible to obesity, the method including the step of administering to the patient a pharmaceutical composition of the invention.
  • the method for lowering serum cholesterol in a patient in need thereof, for treating a patient suffering from or susceptible to a disease or condition associated with high cholesterol levels, for treating a patient suffering from or susceptible to a disease or condition associated with plaque buildup in the arteries, or for treating a patient suffering from or susceptible to obesity includes the additional step of co-administering to the patient a second cholesterol-lowering agent.
  • the second cholesterol-lowering agent is selected from the group consisting of: an HMG-CoA reductase inhibitor, a bile acid sequestrant, a nicotinic acid, a fibric acid derivative, an inhibitor of cholesterol absorption, and a cholesterol esterase inhibitor.
  • the second cholesterol-lowering agent is an HMG-CoA reductase inhibitor.
  • the invention also provides for the use of cholesterol oxidase or cholesterol oxidase and a second therapeutic agent in the formulation of a medicament for the treatment of a patient suffering from or susceptible to a disease or condition associated with high cholesterol levels as described above.
  • the medicament further comprises a second cholesterol- reducing agent.
  • Embodiments of the invention are related, in part, to the discovery that oral administration of a cholesterol oxidase will oxidize both dietary and endogenous cholesterol in the Gl tract to produce 4-cholesten-3-one and hydrogen peroxide.
  • the conversion of cholesterol into 4-cholesten-3-one will prevent cholesterol absorption or reabsorption into the blood stream and effect a reduction of serum cholesterol to sub-normal levels.
  • the resultant hypocholesteremia caused by the use of cholesterol oxidase will also deplete arterial plaque of its cholesterol over a sufficient period of time when used alone or in combination with other cholesterol reducing therapy drugs, thus reducing or eliminating such plaque.
  • chylomicrons very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL).
  • VLDL very low density lipoprotein
  • IDL intermediate density lipoprotein
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • fats are transported in the blood via the chylomicron and then transferred to adipose tissues.
  • fats synthesized in the liver are stored in the interior of the spherical VLDL (very low density lipoprotein) particles and then transferred to adipose tissues.
  • VLDL very low density lipoprotein
  • oxidized LDL particles especially "small dense LDL” (sdLDL) particles
  • sdLDL particles are associated with atheroma formation in the walls of arteries, a condition known as atherosclerosis, which is the principal cause of coronary heart disease and other forms of cardiovascular disease.
  • HDL particles especially large HDL
  • Increased concentrations of HDL correlate with lower rates of atheroma progressions and even regression.
  • the oxidation of dietary and endogenous cholesterol in turn, will prevent the body's natural mechanism of depositing and accumulating excess cholesterol in the blood vessels, arteries and other body pools.
  • the amount of dietary cholesterol not absorbed by the body is believed to be typically up to 400 milligrams a day. Endogenous cholesterol so diverted is believed to be typically about 1200 milligrams a day.
  • conditions such as atherosclerosis may also be arrested or reversed. It is known that the elimination or lowering of cholesterol from the bloodstream will cause the body naturally to deplete or withdraw the previously deposited excess cholesterol from the various body pools. Therefore, the administration of cholesterol oxidase alone or in combination with an HMG CoA reductase inhibitor such as lovastatin or other cholesterol reducing drugs will not only lower blood cholesterol levels but further reverse or reduce the accumulation of arterial plaque in the arteries when used over an extended period of time.
  • Cholesterol oxidases which are prevalent in many bacterial species, catalyze the formation of 4-cholesten-3-one from cholesterol.
  • the oxidation of the sterol requires an FAD cofactor that is concomitantly reduced.
  • Regeneration of the oxidized cofactor is effected by the reduction of O 2 to hydrogen peroxide.
  • Embodiments of the invention relate to cholesterol oxidase as an orally delivered therapeutic for the reduction of cholesterol in the Gl tract and methods of treating patients with hypercholesteremia with such a therapeutic.
  • the value of in vivo oxidation of cholesterol as contrasted with oxidation of cholesterol in food-stuff prior to ingestion is dramatic.
  • both endogenous and dietary cholesterol is oxidized.
  • two grams per day of cholesterol is excreted in the bile acids, whereas, even with high fat diets, the amount of dietary cholesterol is less than five hundred milligrams.
  • Embodiments of the invention provide a pharmaceutically acceptable oral composition including cholesterol oxidase and a pharmaceutically acceptable carrier, thus delivering cholesterol oxidase to the Gl tract to reduce the levels of cholesterol absorbed or reabsorbed into the blood.
  • compositions of the Invention and Methods of Making Those Compositions
  • composition of the invention refers to a pharmaceutical composition comprising cholesterol oxidase.
  • the cholesterol oxidase for use in the compositions of the invention can be obtained from commercial sources, including, for example, Amano Enzyme (Amano Enzyme Inc., Japan; http://www.amano-enzyme.co.jp), SigmaAldrich (SigmaAldrich Corp., St.
  • Cholesterol oxidase may be produced by culturing a microorganism having an ability to produce cholesterol oxidase, including, for example, Cullulomonas, Nocardia, Pseudomonas, Schizophyllum, Streptomyces, Mycobacterium, Rhodococcus, Burkholderia, Brevibacterium, and the like. Methods for producing cholesterol oxidase using such microorganisms are known in the art (see, for example, Liu et al. Agric. Biol. Chem., 52: 413-418 (1988); Cheethan et al., Biochem.
  • Cholesterol oxidase produced by such organisms can be classified into two types: cholesterol oxidase I, whose cofactor is non- covalently bound to the enzyme, and cholesterol oxidase II, whose cofactor is covalently bound. Both the type I and type Il forms of cholesterol oxidase can be used in the invention.
  • useful microorganisms which produce extracellular cholesterol oxidase include Brevibacterium sterolicum, Corynebacterium cholesterolicum, Nocardia cholesterolicum, and Streptomyces violascens.
  • the cholesterol oxidase to be included in the pharmaceutical composition of the invention is obtained by purification of recombinantly expressed cholesterol oxidase.
  • the gene for cholesterol oxidase can be readily obtained by PCR amplification using primers corresponding to cholesterol oxidase gene sequences available in public databases (NCBI, www.ncbi.nlm.nih.gov; see for example, NCBI nucleotide accession number NC_007651 , Burkholderia thailandensis cholesterol oxidase, E264 chromosome I, complete sequence).
  • a cholesterol oxidase gene thus amplified can be introduced recombinantly into a prokaryotic organism commonly used for recombinant gene expression, including, for example, Escherichia coli, Salmonella typhimuirium, and the like, or into a eukaryotic cell or organism commonly used for recombinant gene expression, including, for example, Saccharomyces cerevisiae, Pichia pastoris, Candida albicans, baculovirus-infected insect cells, mammalian tissue culture cells, and the like. Methods of recombinant gene expression are well known to those of skill in the art.
  • Recombinantly expressed cholesterol oxidase is purified using methods also well established in the art (useful protocols are available in Current Protocols in Protein Science, series editor Gwen Taylor, John Wiley and Sons, Inc., 2007). Lysates of cholesterol oxidase-expressing cells, for example, are clarified by centrifugation and/or filtering and then fractionated using conventional techniques, including, for example, ammonium sulfate precipitation, chromatography using ion exchange resin, gel filtration, hydroxyapatite adsorption resin, and steroid adsorption chromatography (see U.S. Patent No. 4,374,930, incorporated by reference herein) to obtain substantially pure cholesterol oxidase. Cholesterol oxidase accumulated outside the cells may be isolated in the same manner as described above.
  • the pharmaceutical composition comprising cholesterol oxidase also comprises a second therapeutic agent.
  • the second therapeutic agent can include a catalase, peroxidase, or an enzyme capable of converting triglyceride into a diglyceride, monoglyceride, and/or glycerol and fatty acids.
  • Catalases are enzymes which convert hydrogen peroxide into water and oxygen, such as, e.g., H 2 O 2 oxidoreductase. These enzymes occur in both plant and animal cells.
  • a peroxidase is an enzyme that cleaves the oxygen-oxygen single bond of a peroxide, converting the peroxide into two alcohol molecules. Use of these enzymes is well-established in the art.
  • the second therapeutic agent of the composition can be an enzyme capable of converting a triglyceride into diglyceride, monoglyceride, and/or glycerol and fatty acids.
  • an enzyme typically belongs to the category generally called lipase (or esterase).
  • the enzyme is a lipase that converts a triglyceride into 1 ,3-diglyceride (1 ,3-DG) and/or 1 -monoglyceride.
  • nonspecific lipase capable of cleaving all the ester bonds at 1-, 2- and 3-positions in the triglyceride.
  • a lipase can be derived from microorganisms of the genera Geotrichum, Candida, and Penicillium or it can be derived from animals or plants.
  • the content of lipase with high activity in cleaving the ester bond at the 2-position in a TG is preferably increased by fractionating isozymes contained in samples of such enzymes.
  • lipases include: (1) lipase from Geotrichum candidum (for example, Lipase GC AMANO under trade name, manufactured by Amano Pharmaceutical Co., Ltd.); (2) lipase from Candida rugosa (for example, Lipase AY AMANO under trade name, manufactured by Amano Pharmaceutical Co., Ltd.); (3) lipase from Candida lipolytica (for example, Lipase L AMANO under trade name, manufactured by Amano Pharmaceutical Co., Ltd.); and (4) lipase from Pseudomonas sp. (for example, Lipase P AMANO and lipase AK AMANO under trade names, manufactured by Amano Pharmaceutical Co., Ltd.).
  • Geotrichum candidum for example, Lipase GC AMANO under trade name, manufactured by Amano Pharmaceutical Co., Ltd.
  • Candida rugosa for example, Lipase AY AMANO under trade name, manufactured by Amano Pharmaceutical Co., Ltd.
  • the enzyme that converts triglyceride into 1 ,3-DG is a 1 ,3-specific lipase.
  • 1 ,3-specific lipases include, for example, microbial lipase derived from Aspergillus or Rhizopus, and lipase having the same action, such as pancreatic lipase, derived from animals or plants.
  • a 1 ,3-specific lipase cleaves only the ester bond at the 1 -position or 3-position among the three ester bonds in a triglyceride, but by virtue of spontaneous acyl migration, the fatty acid at the 2-position can transfer to the 1 -position of the glycerol backbone.
  • lipases include: (1) lipase derived from Aspergillus niger (for example, Lipase AP AMANO TM manufactured by Amano Pharmaceutical Co., Ltd.); (2) lipase derived from Rhizopus oryzae (for example, Lipase F AMANOTM manufactured by Amano Pharmaceutical Co., Ltd.); (3) lipase derived from Rhizopus sp.
  • AMANOTM for example, Newlase AMANOTM manufactured by Amano Pharmaceutical Co., Ltd.
  • lipase derived from Mucorjavanicus for example, Lipase M AMANOTM manufactured by Amano Pharmaceutical Co., Ltd.
  • lipase derived from porcine pancreas for example, Pancreatin F AMANOTM manufactured by Amano Pharmaceutical Co., Ltd.
  • the pharmaceutical composition including cholesterol oxidase includes a stomach acid reducing agent as a second therapeutic agent.
  • Acceptable stomach acid reducing agents include, but are not limited to, antacids, histidine receptor antagonists (H 2 blockers), and proton pump inhibitors.
  • the stomach acid-reducing agent is an antacid.
  • the term "antacid” includes, but is not limited to, aluminum hydroxide, aluminum hydroxide with magnesium carbonate, calcium carbonate, magnesium hydroxide, magnesium oxide, magnesium silicate, magnesium trisillicate, magnesium metasilicate aluminate, magnesium aluminosilicate, magaldrate, dihydroxy aluminum sodium carbonate, aluminium magnesium hydroxide carbonate (hydrotalcit), exsiccated mixed aluminum hydroxide and magnesium carbonate gel, coprecipitate of aluminum hydroxide, coprecipitate of magnesium hydroxide and potassium aluminum sulfate.
  • the stomach acid-reducing agent can be used in combination with an alginate.
  • the alginate may be selected from alginic acid or sodium alginate or other pharmaceutically acceptable alginate salts, hydrates, and esters.
  • the stomach acid-reducing agent is an H 2 receptor antagonist (also known as an H 2 blocker or H 2 antagonist).
  • H 2 receptor antagonists that can be used include, but are not limited to, ranitidine, cimetidine, famotidine, and nizatidine.
  • the stomach acid-reducing agent is a proton pump inhibitor.
  • Suitable proton pump inhibitors for use in the compositions and methods of the invention include, but are not limited to, omeprazole, lansoprazole, rabeprazole, pantoprazole, esomeprazole, pantoprazole, and cogeners or racemic mixtures of the same.
  • compositions of the invention further comprise a second cholesterol-reducing agent.
  • the cholesterol-lowering agent is selected from the group consisting of: an HMG-CoA reductase inhibitor, a bile acid sequestrant, a nicotinic acid, a fibric acid derivative, an inhibitor of cholesterol absorption, and a cholesterol esterase inhibitor.
  • the at least one second therapeutic agent is an HMG-CoA reductase inhibitor.
  • compositions of the invention may be formulated with a pharmaceutically acceptable excipient for oral administration to a patient.
  • pharmaceutically acceptable such as in the recitation of a “pharmaceutically acceptable excipient,” or a “pharmaceutically acceptable carrier,” as used herein, refer to a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • carrier or “vehicle” as used herein refers to a conventional pharmaceutically acceptable carrier material suitable for drug administration, and includes any such material known in the art that is nontoxic and does not interact with other components of a pharmaceutical composition or drug delivery system in a deleterious manner.
  • compositions of the invention comprise acidifying agents, such as, e.g., acetic acid, glacial acetic acid, citric acid, fumaric acid, hydrochloric acid, diluted hydrochloric acid, malic acid, nitric acid, phosphoric acid, diluted phosphoric acid, sulfuric acid, and tartaric acid.
  • acidifying agents such as, e.g., acetic acid, glacial acetic acid, citric acid, fumaric acid, hydrochloric acid, diluted hydrochloric acid, malic acid, nitric acid, phosphoric acid, diluted phosphoric acid, sulfuric acid, and tartaric acid.
  • compositions of the invention comprise alcohol denaturants, such as, e.g., denatonium benzoate, methyl isobutyl ketone, and sucrose octacetate.
  • alcohol denaturants such as, e.g., denatonium benzoate, methyl isobutyl ketone, and sucrose octacetate.
  • compositions of the invention comprise alkalizing agents, such as, e.g., strong ammonia solution, ammonium carbonate, diethanolamine, diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, and trolamine.
  • alkalizing agents such as, e.g., strong ammonia solution, ammonium carbonate, diethanolamine, diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, and trolamine.
  • compositions of the invention comprise glidant and/or anticaking agents, such as, e.g., calcium silicate, magnesium silicate, colloidal silicon dioxide, and talc.
  • glidant and/or anticaking agents such as, e.g., calcium silicate, magnesium silicate, colloidal silicon dioxide, and talc.
  • compositions of the invention comprise antifoaming agents, such as, e.g., dimethicone and simethicone.
  • liquid compositions of the invention comprise antimicrobial preservatives, such as, e.g., benzalkonium chloride, benzalkonium chloride solution, benzelthonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol, cresol, dehydroacetic acid, ethylparaben, methylparaben, methylparaben sodium, phenol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, potassium benzoate, potassium sorbate, propylparaben, propylparaben sodium, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid
  • antimicrobial preservatives such as, e.
  • compositions of the invention comprise antioxidants, such as, e.g., ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium thiosulfate, sulfur dioxide, tocopherol, and tocopherols excipient.
  • antioxidants such as, e.g., ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium thiosulfate, sulfur dioxide, tocopherol, and tocopherols excipient.
  • compositions of the invention comprise buffering agents, such as, e.g., acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, phosphoric acid, potassium citrate, potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate, sodium lactate solution, dibasic sodium phosphate, monobasic sodium phosphate, and histidine.
  • buffering agents such as, e.g., acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, phosphoric acid, potassium citrate, potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate, sodium lactate solution, dibasic sodium phosphate, monobasic sodium phosphate, and histidine.
  • compositions of the invention comprise chelating agents, such as, e.g., edetate disodium, ethylenediaminetetraacetic acid and salts, and edetic acid.
  • compositions of the invention comprise coating agents, such as, e.g., sodium carboxymethylcellulose, cellulose acetate, cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methacrylic acid copolymer, methylcellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, carnauba wax, microcystalline wax, and zein.
  • compositions of the invention comprise colors, such as, e.g., caramel, red, yellow, black or blends, and ferric oxide.
  • compositions of the invention comprise complexing agents, such as, e.g., ethylenediaminetetraacetic acid and salts (EDTA), edetic acid, gentisic acid ethanolamide, and oxyquinoline sulfate.
  • complexing agents such as, e.g., ethylenediaminetetraacetic acid and salts (EDTA), edetic acid, gentisic acid ethanolamide, and oxyquinoline sulfate.
  • compositions of the invention comprise desiccants, such as, e.g., calcium chloride, calcium sulfate, and silicon dioxide.
  • compositions of the invention comprise emulsifying and/or solubilizing agents, such as, e.g., acacia, cholesterol, diethanolamine (adjunct), glyceryl monostearate, lanolin alcohols, lecithin, mono- and di-glycerides, monoethanolamine (adjunct), oleic acid (adjunct), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxyl 35 caster oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitan monolaurate, soritan monooleate, sorb
  • compositions of the invention comprise filtering aids, such as, e.g., powdered cellulose, purified siliceous earth, Flavors and perfumes, anethole, benzaldehyde, ethyl vanillin, menthol, methyl salicylate, monosodium glutamate, orange flower oil, peppermint, peppermint oil, peppermint spirit, rose oil, stronger rose water, thymol, tolu balsam tincture, vanilla, vanilla tincture, and vanillin.
  • filtering aids such as, e.g., powdered cellulose, purified siliceous earth, Flavors and perfumes, anethole, benzaldehyde, ethyl vanillin, menthol, methyl salicylate, monosodium glutamate, orange flower oil, peppermint, peppermint oil, peppermint spirit, rose oil, stronger rose water, thymol, tolu balsam tincture, vanilla, vanilla tinct
  • compositions of the invention comprise humectants, such as, e.g., glycerin, hexylene glycol, propylene glycol, sorbitol, Ointment bases, lanolin, anhydrous lanolin, hydrophilic ointment, white ointment, yellow ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white petrolatum, and rose water ointment.
  • humectants such as, e.g., glycerin, hexylene glycol, propylene glycol, sorbitol, Ointment bases, lanolin, anhydrous lanolin, hydrophilic ointment, white ointment, yellow ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white petrolatum, and rose water ointment.
  • compositions of the invention comprise plasticizers, such as, e.g., castor oil, lanolin, mineral oil, petrolatum, benzyl benyl formate, chlorobutanol, diethyl pthalate, sorbitol, diacetylated monoglycerides, diethyl phthalate, glycerin, glycerol, mono- and di-acetylated monoglycerides, polyethylene glycol, propylene glycol, triacetin, triethyl citrate, and ethanol.
  • plasticizers such as, e.g., castor oil, lanolin, mineral oil, petrolatum, benzyl benyl formate, chlorobutanol, diethyl pthalate, sorbitol, diacetylated monoglycerides, diethyl phthalate, glycerin, glycerol, mono- and di-acetylated monoglycerides, poly
  • compositions of the invention comprise polymer membranes, such as, e.g., cellulose acetate.
  • compositions of the invention comprise solvents, such as, e.g., acetone, alcohol, diluted alcohol, amylene hydrate, benzyl benzoate, butyl alcohol, carbon tetrachloride, chloroform, corn oil, cottonseed oil, ethyl acetate, glycerin, hexylene glycol, isopropyl alcohol, methyl alcohol, methylene chloride, methyl isobutyl ketone, mineral oil, peanut oil, polyethylene glycol, propylene carbonate, propylene glycol, sesame oil, water for injection, sterile water for injection, sterile water for irrigation, and purified water.
  • solvents such as, e.g., acetone, alcohol, diluted alcohol, amylene hydrate, benzyl benzoate, butyl alcohol, carbon tetrachloride, chloroform, corn oil, cottonseed oil, ethyl acetate, glycerin, hexylene glyco
  • compositions of the invention comprise sorbents, such as, e.g., powdered cellulose, charcoal, purified siliceous earth, bacteriostatic water for injection, and bacteriostatic sodium chloride solution for injection.
  • sorbents such as, e.g., powdered cellulose, charcoal, purified siliceous earth, bacteriostatic water for injection, and bacteriostatic sodium chloride solution for injection.
  • compositions of the invention comprise carbon dioxide sorbents, such as, e.g., barium hydroxide lime and soda lime.
  • compositions of the invention comprise stiffening agents, such as, e.g., hydrogenated castor oil, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, hard fat, paraffin, polyethylene excipient, stearyl alcohol, emulsifying wax, white wax, and yellow wax.
  • stiffening agents such as, e.g., hydrogenated castor oil, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, hard fat, paraffin, polyethylene excipient, stearyl alcohol, emulsifying wax, white wax, and yellow wax.
  • compositions of the invention comprise suppository bases, such as, e.g., cocoa butter, hard fat, and polyethylene glycol.
  • compositions of the invention comprise suspending and/or viscosity-increasing agents, such as, e.g., acacia, agar, alginic acid, aluminum monostearate, bentonite, purified bentonite, magma bentonite, carbomer 934 p, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carboxymethycellulose sodium 12, carrageenan, microcrystalline and carboxymethylcellulose sodium cellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, magnesium aluminum silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide, colloidal silicon dioxide, sodium alginate, tragacanth, and xanthan gum.
  • suspending and/or viscosity-increasing agents such as, e.g., acacia, agar
  • compositions of the invention comprise sweetening agents, such as, e.g., aspartame, dextrates, dextrose, excipient dextrose, fructose, mannitol, saccharin, calcium saccharin, sodium saccharin, sorbitol, solution sorbitol, sucrose, compressible sugar, confectioner's sugar, and syrup.
  • sweetening agents such as, e.g., aspartame, dextrates, dextrose, excipient dextrose, fructose, mannitol, saccharin, calcium saccharin, sodium saccharin, sorbitol, solution sorbitol, sucrose, compressible sugar, confectioner's sugar, and syrup.
  • compositions of the invention comprise tablet binders, such as, e.g., acacia, alginic acid, sodium carboxymethylcelluiose, microcrystalline cellulose, dextrin, ethylcellulose, gelatin, liquid glucose, guar gum, hydroxypropyl methylcellulose, methycellulose, polyethylene oxide, povidone, pregelatinized starch, and syrup.
  • tablet binders such as, e.g., acacia, alginic acid, sodium carboxymethylcelluiose, microcrystalline cellulose, dextrin, ethylcellulose, gelatin, liquid glucose, guar gum, hydroxypropyl methylcellulose, methycellulose, polyethylene oxide, povidone, pregelatinized starch, and syrup.
  • compositions of the invention comprise tablet and/or capsule diluents, such as, e.g., calcium carbonate, dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate, microcrystalline cellulose, powdered cellulose, dextrates, dextrin, dextrose excipient, fructose, kaolin, lactose, mannitol, sorbitol, starch, pregelatinized starch, sucrose, compressible sugar, and confectioner's sugar.
  • diluents such as, e.g., calcium carbonate, dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate, microcrystalline cellulose, powdered cellulose, dextrates, dextrin, dextrose excipient, fructose, kaolin, lactose, mannitol, sorbitol, starch, pregelatinized starch, sucrose, compressible sugar, and confectioner's
  • compositions of the invention comprise tablet disintegrants, such as, e.g., alginic acid, microcrystalline cellulose, croscarmellose sodium, corspovidone, polacrilin potassium, sodium starch glycolate, starch, and pregelatinized starch.
  • tablet disintegrants such as, e.g., alginic acid, microcrystalline cellulose, croscarmellose sodium, corspovidone, polacrilin potassium, sodium starch glycolate, starch, and pregelatinized starch.
  • compositions of the invention comprise tablet and/or capsule lubricants, such as, e.g., calcium stearate, glyceryl behenate, magnesium stearate, light mineral oil, polyethylene glycol, sodium stearyl fumarate, stearic acid, purified stearic acid, talc, hydrogenated vegetable oil, and zinc stearate.
  • tablet and/or capsule lubricants such as, e.g., calcium stearate, glyceryl behenate, magnesium stearate, light mineral oil, polyethylene glycol, sodium stearyl fumarate, stearic acid, purified stearic acid, talc, hydrogenated vegetable oil, and zinc stearate.
  • compositions of the invention comprise tonicity agents, such as, e.g., dextrose, glycerin, mannitol, potassium chloride, and sodium chloride.
  • compositions of the invention comprise vehicles, such as, e.g., flavored and/or sweetened, aromatic elixir, compound benzaldehyde elixir, iso-alcoholic elixir, peppermint water, sorbitol solution, syrup, tolu balsam syrup, oleaginous, almond oil, corn oil, cottonseed oil, ethyl oleate, isopropyl myristate, isopropyl palmitate, mineral oil, light mineral oil, myristyl alcohol, octyldodecanol, olive oil, peanut oil, persic oil, sesame oil, soybean oil, and squalane.
  • compositions of the invention comprise water repelling agents, such as, e.g., cyclomethicone, dimethicone, and simethicone,
  • compositions of the invention comprise wetting and/or solubilizing agents, such as, e.g., benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, docusate sodium, nonoxynol 9, nonoxynol 10, octoxynol 9, poloxamer, polyoxyl 35 castor oil, polyoxyl 40, hydrogenated castor oil, polyoxyl 50 stearate, polyoxyl 10 oleyl ether, polyoxyl 20, cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium lauryl sulfate, sorbitan monolaureate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, and tyloxapol.
  • wetting and/or solubilizing agents such as, e.g., benzalkonium chloride, benzethonium chlor
  • compositions of the invention comprise aerosol propellants, such as, e.g., butane, dichlorodifluoromethane, dichlorotetrafluoroethane, isobutane, propane, and trichloromonofluoromethane.
  • aerosol propellants such as, e.g., butane, dichlorodifluoromethane, dichlorotetrafluoroethane, isobutane, propane, and trichloromonofluoromethane.
  • compositions of the invention comprise air displacements, such as, e.g., carbon dioxide and nitrogen.
  • acceptable pharmaceutical excipients include (1) salts of amino acids, including, for example glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline, histidine, and the like; (2) carbohydrates, including, for example, monosaccharides glucose, fructose, galactose, mannose, arabinose, xylose, ribose, and the like; (3) disaccharides, including, for example, lactose, trehalose, maltose, sucrose, and the like; (4) polysaccharides, including, for example, maltodextrins, dextrans, starch, glycogen, and the like; (5) alditols, including, for example, mannitol, xylitol, lactitol
  • the excipients or ingredients can include sucrose, trehalose, lactose, sorbitol, lactitol, inositol, salts of sodium and potassium, including, for example, acetate, phosphates, citrates, borate, and the like, glycine, arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol, methoxy polyethylene glycol, gelatin, and hydroxypropyl- ⁇ -cyclodextrin.
  • Embodiments of the invention include methods for lowering serum cholesterol in a patient in need thereof, for treating a patient suffering from or susceptible to a disease or condition associated with high cholesterol levels, for treating a patient suffering from or susceptible to a disease or condition associated with plaque buildup in the arteries, or for treating a patient suffering from or susceptible to obesity, wherein the method comprises administration of a composition of the invention.
  • the method further includes the additional step of co-administering to the patient at least one second therapeutic agent, wherein the at least one second therapeutic agent is a cholesterol-lowering agent.
  • cholesterol-lowering agent and “cholesterol-lowering drug” as used herein refer to a pharmacologically active, pharmaceutically acceptable agent that, when administered to a human subject or animal which has hypercholesterolemia, has the effect of beneficially modifying serum cholesterol levels. More particularly, the cholesterol-lowering agent lowers serum low density lipoprotein (LDL) cholesterol levels, or inhibits oxidation of LDL cholesterol, whereas high density lipoprotein (HDL) serum cholesterol levels may be lowered, remain the same, or be increased.
  • LDL serum low density lipoprotein
  • HDL high density lipoprotein
  • the cholesterol- lowering agent brings the serum levels of LDL cholesterol and HDL cholesterol (and, more preferably, triglyceride levels) to normal or nearly normal levels.
  • Cholesterol-lowering agents include, but are not limited to, HMG-CoA Reductase inhibitors, bile acid sequestrants, fibric acid and derivatives thereof, nicotinic acid and derivatives thereof, cholesterol absorption inhibitors, cholesterol esterase inhibitors.
  • the second cholesterol-lowering agent is an HMG CoA reductase inhibitor.
  • HMG CoA reductase inhibitors that can be used as a second cholesterol-lowering agent include, but are not limited to, atorvastatin (Lipitor®; see U.S. Pat. No. 5,273,995), cerivastatin (also called rivastatin; Baycol®; see U.S. Pat. No. 5,177,080), fluindostatin, fluvastatin (Lescol®; see U.S. Pat. No. 5,354,772), lovastatin (Mevacor®, see U.S. Pat. No. 4,231 ,938), mevastatin (see U.S.
  • HMG CoA reductase inhibitors that may be used are, without limitation, presented in U.S. Patent No. 6,264,938 at Table 1 and U.S. Patent No. 5,622,985, columns 3 through 6.
  • HMG CoA reductase inhibitors are included in this invention.
  • Compounds that inhibit the activity of HMG CoA reductase can be readily identified by using assays well known in the art; see, as examples, the assays described or cited in U.S. Patent No. 4,231 ,938 at column 6, and in International Patent Publication WO 84/02131 at pp. 30-33.
  • the term "HMG CoA reductase inhibitor" is intended to include all pharmaceutically acceptable salt, ester, and lactone forms of compounds that have HMG CoA reductase inhibitory activity, and therefore the use of such salt, ester, and lactone forms is included within the scope of this invention.
  • the second cholesterol-lowering agent is a bile acid sequestrant.
  • Bile acids which are secreted into the intestine to aid in the digestion and absorption of lipids, are synthesized in the liver from cholesterol. Normally, approximately 97% of bile acids are reabsorbed and reused. If large amounts of bile acids are excreted, then the liver must convert more cholesterol to bile acids, lowering serum cholesterol levels, particularly LDL cholesterol levels. Although biosynthesis of cholesterol is up-regulated in this case, the net effect of increased bile acid synthesis in most individuals is to lower cholesterol, particularly LDL cholesterol, levels in the serum.
  • Bile acid sequestrants are poorly absorbed resins or other substances that bind to and sequester bile acids in the intestine. The sequestered bile acids are subsequently excreted in the feces. Any pharmaceutically acceptable bile acid sequestrant may be used in the practice of this invention. Exemplary bile acid sequestrants that may be used in this invention include, but are not limited to, cholestyramine, colesevelam, cholestipol, and poly[methyl-(3- trimethylamino-propyl)imino-trimethylene dihalide].
  • the second cholesterol-lowering agent is a fibric acid derivative.
  • Fibric acid derivatives also known as "fibrates," lower triglyceride levels, raise high density lipoprotein (HDL) levels, and have variable effects on LDL cholesterol levels in the blood.
  • fibric acid derivatives that may be used in the methods of this invention include, but are not limited to, bezafibrate (Bezalip®), beclobrate, binifibrate, ciprofibrate, clinofibrate, clofibrate, etofibrate, fenofibrate (Lipidil®.
  • the second cholesterol-lowering agent is a nicotinic acid. Nicotinic acids that may be used include, but are not limited to, niacin, nicotinic acid, and vitamin B3. In some embodiments, the second cholesterol-lowering agent is an agent that extends the release of nicotinic acid, for example niaspan.
  • the second cholesterol-lowering agent is an inhibitor of cholesterol absorption.
  • cholesterol absorption inhibitors block the uptake of micellar cholesterol, thereby reducing the incorporation of cholesteryl esters into chylomicron particles. By reducing the cholesterol content in chylomicrons and chylomicron remnants, cholesterol absorption inhibitors effectively reduce the amount of cholesterol that is delivered back to the liver.
  • Cholesterol absorption inhibitors that may be used in the methods or the compositions of the invention include, but are not limited to, ezetimibe and naturally occurring phytosterols and phytostanols.
  • the second cholesterol-lowering agent is a cholesterol esterase inhibitor.
  • Pancreatic cholesterol esterase has been proposed to have multiple functions in the intestine: 1) to control the bioavailability of cholesterol from dietary cholesterol esters; 2) to contribute to incorporation of cholesterol into mixed micelles; and 3) to aid in transport of free cholesterol to the enterocyte. It is assumed that inhibitors of cholesterol esterase limit the absorption of dietary cholesterol.
  • Cholesterol esterase inhibitors that may be used with the invention include, but are not limited to, alpha, alpha difluoro-2-naphthyl ketones (see U.S. Patent No. 5,093,371 , incorporated by reference herein), sulfated polysaccharide polymers (see U.S.
  • the oral formulations comprise enteric coatings, which protect an active ingredient from the strongly acidic environment of the stomach and enable the ingredient to be delivered to the intestinal tract.
  • Enteric coatings can be applied to tablets or capsules to prevent the release of drugs in the stomach either to maintain the stability of the drug which might otherwise be subject to degradation when exposed to the gastric environment or to reduce the risk of unpleasant side effects.
  • Most polymers that are used as enteric coating agents are polyacids that function by virtue or the fact that their solubility in aqueous medium is pH-dependent: they are insoluble in highly acidic environments but soluble in the neutral or basic environment of the duodenum.
  • Enteric coatings may be used to coat a solid or liquid dosage form of the pharmaceutical composition.
  • enteric coatings for delayed release of active ingredients in pharmaceutical compositions is well known and widely practiced in the art; see for example Hasegawa, Chem. Pharm. Bull. 33: 1615- 1619 (1985); Porter et al. J. Pharm. Pharmacol. 22: 42 (1970); Porter et al., J. Pharm. Pharmacol. 34: 5-8 (1981); Bodmeier et al., Pharmaceutical Research 11 : 882-888 (1994).
  • a wide variety of conventional enteric coatings can be employed in the present invention, including, for example cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropyl methylcellulose trimellitate (HPMCT), hydroxypropyl methyl cellulose succinate (HPMCAS), hydroxypropyl cellulose acetyl succinate; carboxymethylethyl cellulose (CMEC), polyvinyl alcohol acetate phthalate (PVAP), polyvinyl acetate phthalate;, copolymerized methacrylic acid/methacrylic acid methyl esters (such as Eudragit L 12.5, Eudragit L 100 55, and Eudragit S 100), copoylmers of methacrylic acid and ethyl acrylate, POLYQUID PA-30 (Sanyo Chemical Co), and the like.
  • CAP cellulose acetate phthalate
  • CAT cellulose acetate trimellitate
  • enteric coating polymers are used.
  • the enteric coating can further contain conventional plasticizers, pigments and/or dispersants, including, for example, polyethylene glycols, triacetin, triethyl citrate, Citroflex, dibutyl sebacate, and the like.
  • commercially obtained enterically-coated capsules i.e. Videx EC Delayed-Release Enteric-Coated Capsules are used.
  • the cholesterol oxidase is present in the pharmaceutical composition as enterically coated granules.
  • enterically coated granules Some investigators have reported that a multiple-unit type dosage form, such as granules, is superior to a single-unit type. Therefore, in some embodiments, the cholesterol oxidase is contained in an enterically coated multiple-unit dosage form.
  • the cholesterol oxidase dosage form is prepared by spray-coating granules of a cholesterol oxidase-pharmaceutical carrier-enteric coating agent solid dispersion on an inert core material. These granules can result in prolonged absorption of the drug with good bioavailability.
  • Enteric coating agents used to generate such granules include, but are not limited to, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate.
  • Various enteric coating materials may be selected on the basis of testing to achieve an enteric coated dosage form designed ab initio to have a preferable combination of dissolution time, coating thicknesses and diametral crushing strength.
  • the pharmaceutical composition is placed in enterically-coated capsules.
  • Enterically-coated capsules are commercially available, and their production is well known in the art.
  • the enteric coating may be applied in any suitable manner known in the art, such as, for example, by using a Wurster coater.
  • the enteric coating may be in the form of an aqueous dispersion in water, aqueous latex or other dispersing medium, or in the form of a solution, such as an organic solvent solution.
  • the enteric coating is applied in the form of a dispersion or solution, it is preferred that the dispersion or solution be treated with an alkali prior to applying the enteric coating to the active core in order to neutralize at least part of any free acid content.
  • the alkali may be, for example, a carbonate or a hydroxide of sodium, potassium, magnesium or calcium.
  • Coating using the enteric coating agent comprises, for example, stirring and tumbling granules or parvules of the solid dosage form while a plasticizer which is liquid at room temperature or a heat-melted liquid plasticizer is sprinkled or sprayed on them and simultaneously the enteric coating agent of 10 micrometers or less is sprinkled to coat the solid dosage form.
  • This series of operations can also be conducted by dividing it into several batches with different compositions. It is also possible to additionally sprinkle talc, aerosil (SiO 2 ), magnesium stearate or corn starch in order to prevent the granules from adhering to each other during the coating process.
  • Embodiments of the invention include methods of lowering serum cholesterol levels in patients with any disease or condition associated with or exacerbated by hypercholesterolemia.
  • Compositions of the invention can, therefore, be used to treat patients with conditions including, but not limited to, arteriosclerosis (and the most common type thereof, atherosclerosis), coronary heart disease, stroke, peripheral vascular disease, diabetes, obesity, high blood pressure, acquired lipid disorder, cushing's syndrome, familial combined hyperlipidemia, familial hypercholesterolemia, familial dysbetalipoproteinemia, retinal vascular disease (including Amaurosis fugax), Alstrom syndrome, alcoholism, kidney disease, and reduced metabolism due to thyroid problems.
  • arteriosclerosis and the most common type thereof, atherosclerosis
  • coronary heart disease stroke
  • peripheral vascular disease diabetes
  • obesity high blood pressure
  • acquired lipid disorder cushing's syndrome
  • familial combined hyperlipidemia familial hypercholesterolemia
  • familial dysbetalipoproteinemia familial dysbetalipoprotein
  • treating and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage.
  • “treating” a patient involves prevention of a particular disorder or adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual.
  • an “effective” amount or a “therapeutically effective amount” of a drug or pharmacologically active agent is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect.
  • an “effective amount” of one component of the combination is the amount of that compound that is effective to provide the desired effect when used in combination with the other components of the combination.
  • the amount that is “effective” will vary from subject to subject, depending on the age, weight, metabolism, disease severity, response to therapy, and general condition of the individual, the particular active agent or agents, local effects, pharmacodynamics, absorption, method of delivery, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • the pharmaceutical composition of the invention is orally administered in a daily dosage of at least 10 to about 1000 milligrams of cholesterol oxidase to a patient in need thereof.
  • the dosage of cholesterol oxidase administered in the compositions of the invention can be up to 3,000 mg per day.
  • the daily dosage is between 150 and 750 mg per day. Additional embodiments of the invention encompass other dosage levels that achieve beneficial results.
  • cholesterol oxidase is administered three to four times a day.
  • the daily dose of the cholesterol oxidase is approximately 10 mg to 1 g (administered from 1 to 4 times per day).
  • the HMG- CoA reductase inhibitor is administered according to accepted regimens (see, for example, treatment regimens associated with Lipitor®).
  • about 20 mg to 40 mg of the HMG-CoA reductase inhibitor is co-administered twice a day.
  • the total daily dosage is in the range of 150 mg.
  • the methods of the invention also encompass variations in dosages administered that depend on many factors which affect blood cholesterol and dietary lipid levels, including, for example, severity of cardiovascular disease (CVD), age, weight, and a host of other interactions with the catalyst activity constant, including dissipation rate and rates of hydrogenation.
  • the desired orally administered dose can be in excess of the daily dosage to ensure near complete cholesterol oxidation.
  • the enzyme may be incorporated in such suitable final dosage forms as may be satisfactorily prepared and employed by one skilled in the art.
  • the commonly employed acceptable dosage form suitable for oral administration containing the active enzyme is in sufficient concentrations to attain the desired results over an extended period of time.
  • the pharmaceutically acceptable, non-toxic carriers usually employed for such purposes may be utilized to prepare such dosage forms as tablet, capsules, lozenges, granules, powders, solutions or suspensions.
  • the method of oral administration is tablets or capsules of enteric coated granules containing the active cholesterol oxidase.
  • the active enzyme reaches the upper one-third portion of the small intestine in sufficient strength and activity to oxidize the dietary and endogenous cholesterol before it is significantly absorbed by the intestines.
  • the cholesterol oxidase is administered with meals because stomach ejections into the small intestine trigger the endogenous cholesterol contained in bile secretions, allowing both dietary and endogenous cholesterol to come into contact with cholesterol oxidase in the same place and time.
  • this is fortuitous because the concurrent rise in pH of the digested food when it enters the small intestine promotes increased activity, peaking near a pH of 7.
  • lower dosages or administrations less frequent than with every meal are prescribed.
  • Embodiments of the invention encompass lowered dosage amounts and frequencies of administration that are determined by the time frame in which the enzyme remains active and in a position in the intestine to be effective.
  • Example 1 Preparation of enterically coated capsule containing cholesterol oxidase and pharmaceutical excipients
  • Cholesterol oxidase purified using conventional methods is concentrated by centrifugal concentration and lyophilized using the following lyophilization cycle: freezing at -50 0 C, atmospheric pressure for 2 hours, annealing at -35 0 C, atmospheric pressure for 3 hours, a second freezing step at -50 0 C, atmospheric pressure for 2 hours, primary drying at -20 0 C, 100 mTorr
  • the lyophilized material is pulverized and blended with various pharmaceutical excipients.
  • Gelatin capsules
  • Blended material is encapsulated using a manual filling procedure using hard gelatin capsules. Size 9 capsules are used to generate rodent capsules and size 00 capsules are used to generate human capsules. To fill the size 00 capsules, approximately 400 mg of a formulation of pulverized lyophilized cake and excipients containing from 10-60% cholesterol oxidase, 3% sodium phosphate, 5-40% sucrose, 1-6% PEG-4000, 2% calcium silicate, 5%
  • the formulation contains 100 mg cholesterol oxidase, 500 mg sucrose, 0.5 mg polysorbate 80, 2.2 mg monobasic sodium phosphate,
  • the capsules are enterically coated with Eudragit L30 D55 (Rohm America) coat for protection of the capsule from low pH conditions (pH ⁇ 1 , gastric conditions) and disintegration and release of the contents in the higher pH conditions of the duodenum (pH > 5.5).
  • the Eudragit L30 D55 solution (111 ml) with triethyl citrate (1.62 ml; Sigma) is added to a talc (8.10 g; Luzenac) and water (49.5 ml) suspension. Capsules are sprayed with this solution with one or more
  • Capsules are stored at 4 0 C in tightly closed bottles until use.
  • Example 2 Preparation of tablets containing cholesterol oxidase and pharmaceutical excipients
  • the tablet formulations are prepared by mixing 1.5 mg mannitol as a binding agent, 0.3 mg magnesium stearate as a lubricant, 50-250 m g of cholesterol oxidase, 0-1.5 g hydroxypropylmethyl cellulose (HPMC), and 0-1.5 g polycarbophil per tablet in a blender for 2 hours. The individual mixtures are then pressed manually into a Benchtop Model Single-Punch Tablet Press (TDP) (Tablet-press.net (Athens, Ohio)) to yield 3 mm tablets.
  • TDP Benchtop Model Single-Punch Tablet Press
  • each tablet is placed in a plastic weigh boat with 20 ml of pre-warmed phosphate-buffered saline
  • PBS 37 0 C; pH 7.2
  • the buffer is removed and the size of the tablet is measured. After measurement, 20 ml of pre-warmed PBS is added and the weigh
  • Example 4 In vitro testing of cholesterol oxidase activity in orally administered capsule
  • the capsules are placed in an aqueous solution of 0.01 M HCI, Pepsin (6 mg powder [Pepsin NF powder, 1 :10,000] added for every 10 ml solution), and trypsin/chymoptrypsin (3.8 mg mixed powder for every 10 ml solution) at pH 2 at 37 0 C for 1 hour (gastric environment).
  • Pepsin 6 mg powder [Pepsin NF powder, 1 :10,000] added for every 10 ml solution
  • trypsin/chymoptrypsin 3.8 mg mixed powder for every 10 ml solution
  • Na 2 HPO 4 is first added to the solution to a concentration of 25 mM, and the pH of the solution is then raised to 6.0 by the slow addition 1 M NaOH (or 1 M HCI as necessary), maintaining the solution at 37 0 C. Aliquots of the solution are
  • 0.1 % dianisidine solution 10 mg 3,3'-Dimethoxybenzidine dihydrochloride per ml water. No pH adjustment.
  • Reagent Buffer 0.4 ml dianisidine solution and 1.4 mg peroxidase powder (Sigma Type II, horseradish peroxidase, RZ 1.0 - 1.5 No. P8250) are added to 40 ml KP buffer, mixed, and the solution diluted to 50 ml with KP buffer. The solution turns turbid when the dianisidine is added but clears when mixed. This solution is kept cold until ready to use. (Reagent buffer can be stored optionally at 4 0 C for three days, but is typically prepared fresh.) d.
  • Cholesterol solution To 10 ml Triton X-100 (S-14) heated on a hot plate, 300 mg cholesterol powder is added and mixed with stirring until solution clears. 90 ml water is added with continued stirring. Mixing of the cloudy solution is continued by swirling the flask containing the solution under a stream of cold water until the solution becomes clear. This solution is stable for one week when stored at room temperature.
  • Assay a. 1.675 ml of reagent buffer plus 0.125 ml substrate are combined in a test tube, mixed, and placed in a waterbath at 37 0 C. After five minutes, 250 ml of enzyme solution is added to give 2 ml final volume in the tube, and an initial reading at 430 nm on a Spectronic 20 spectrophotometer (Bausch and Lomb) is recorded. The tube is replaced in the waterbath. Tubes are read in the spectrophotometer every five minutes for 25 minutes. Rate of color development is determined from a plot of O. D. vs. time, followed by averaging the O. D. change throughout the linear portion of the curve, b. For continuous assay of cholesterol oxidase a Beckman recording
  • spectrophotometer is used. In this procedure, 1.675 ml of reagent buffer, 0.125 ml substrate, and water are combined in a 3 ml cuvette at 37 0 C.
  • Activity is calculated from the slope of OD 430 nm plotted vs. time. Activity is calculated using a constant previously determined for the dye system from a standard curve using known concentrations of cholesterol oxidase (enzyme preparations are diluted so that 0.005 to 0.06 units of cholesterol oxidase are used per assay tube).
  • One unit of cholesterol oxidase activity is that amount of enzyme catalyzing the production of 1 ⁇ mole H 2 O 2 per minute at 37 0 C and pH 7.0.
  • Example 5 In vitro testing of cholesterol oxidase activity in orally administered tablet
  • Example 2 The activity of cholesterol oxidase released from the enterically- coated tablets of Example 2 and the granules of Example 3 is tested in the same manner as described in Example 4. Ten ml of test solution are used per tablet or dose of granules in the test sample.
  • Example 6 Antihypercholesterolemic effect of cholesterol oxidase in mice.
  • mice deficient in both the LDL receptor and APOBEC-1 are housed in a full-barrier facility and fed a high-fat diet. The mice are then divided into two groups, and one group is fed 200 mg lyophilized cholesterol oxidase in their meals.
  • the plasma level of cholesterol and triglycerides in each animal is measured with enzymatic kits (Wako; Sigma).
  • the distribution of lipids within the plasma lipoproteins is assessed by size fractionation of pooled sera by fast performance liquid chromatography (FPLC).
  • Plasma lipoprotein electrophoresis is performed on 1% agarose gels. Dried gels are stained for neutral lipids with Fat Red 7b (Sigma).
  • the plasma lipoproteins are isolated by ultracentrifugation at 100,000 rpm for 3 hours and the protein content of the lipoproteins is assessed by 4-20% gradient SDS-PAGE gels stained with Coomassie blue. Quantitation of mouse apoBIOO in plasma samples is done with a sandwich radioimmunoassay using two different mouse antibodies.
  • mice are anesthetized and exsanguinated, and perfused through the left ventricle with PBS followed by 10% formalin. Aortas are removed and incised from the aortic arch to just beyond the iliac bifurcation, pinned onto black wax, postfixed overnight with formalin, and stained with Sudan IV. Images are captured and the total area of the aorta and the atherosclertoic plaque area are measured using the histogram function of Adobe Photoshop. Histopathological examination is done on 3 ⁇ m sections stained with hematoxylin and eosin.

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Abstract

La présente invention concerne des compositions et procédés destinés au traitement de patients souffrant d'hypercholestérolémie. En l'occurrence, les modes de réalisation de l'invention concerne une composition pharmaceutique formulée pour administration au patient par voie orale. Cette composition pharmaceutique comprend une quantité suffisante d'oxydase de cholestérol, éventuellement au moins un second agent thérapeutique ou un hypocholestérolémiant, et un excipient pharmaceutiquement admis pour l'administration par voie orale.
PCT/US2009/051837 2008-07-28 2009-07-27 Compositions et procédé pour le traitement de l'hypercholestérolémie Ceased WO2010014541A2 (fr)

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Cited By (1)

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WO2015025336A3 (fr) * 2013-08-22 2015-07-02 Council Of Scientific & Industrial Research Composition probiotique comprenant la nouvelle souche bactérienne isolée de brevibacterium casei ap9

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RU1826911C (ru) * 1990-07-10 1993-07-07 Научно-Исследовательский Институт Экспериментальной Медицины Амн Ссср Способ профилактики и лечени атеросклероза в эксперименте
US6083497A (en) * 1997-11-05 2000-07-04 Geltex Pharmaceuticals, Inc. Method for treating hypercholesterolemia with unsubstituted polydiallylamine polymers
US7407668B2 (en) * 2002-01-24 2008-08-05 Boston Scimed, Inc. Medical articles having enzymatic surfaces for localized therapy

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015025336A3 (fr) * 2013-08-22 2015-07-02 Council Of Scientific & Industrial Research Composition probiotique comprenant la nouvelle souche bactérienne isolée de brevibacterium casei ap9
US10058575B2 (en) 2013-08-22 2018-08-28 Council Of Scientific & Industrial Research Probiotic composition comprising the novel isolated bacterial strain of brevibacterium casei AP9

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