Attorney Docket No. AKBM -14409/US-11/CON
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`BIOEFFECTIVE KRILL OIL COMPOSITIONS
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`CROSS-REFERENCE TO RELATED APPLICATIONS
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`This application is a continuation of pending U.S. Patent Application No. 12/057,775,
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`filed March 28, 2008, which claims the benefit of expired U.S. Provisional Patent Application
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`No. 60/920,483, filed March 28, 2007, expired U.S. Provisional Patent Application No.
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`60/975,058, filed September 25, 2007, expired U.S. Provisional Patent Application No.
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`60/983,446, filed October 29, 2007, and expired U.S. Provisional Patent Application No.
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`61/024,072, filed January 28, 2008, all of which are incorporated by reference herein in their
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`entirety.
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`FIELD OF THE INVENTION
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`This invention relates to extracts from Antarctic krill that comprise bioactive fatty acids.
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`15 BACKGROUND OF THE INVENTION
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`In the Southern Ocean, off the coast of Antarctica, Antarctic krill (Euphausia superba)
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`can be found in large quantities, ranging from 300-500 million metric tons ofbiomass. It feeds on
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`phytoplankton during the short Antarctic summer. During winter, however, its food supply is
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`limited to ice algae, bacteria, marine detritus as well as depleting body protein for energy.
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`In order to isolate the krill oil from the krill, solvent extraction methods have been used.
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`See, e.g., WO 00/23546. Krill lipids have been extracted by placing the material in a ketone
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`solvent (e.g. acetone) in order to extract the lipid soluble fraction. This method involves
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`separating the liquid and solid contents and recovering a lipid rich fraction from the liquid
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`fraction by evaporation. Further processing steps include extracting and recovering by
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`evaporation the remaining soluble lipid fraction from the solid contents by using a solvent such as
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`ethanol. See, e.g., WO 00/23546. The compositions produced by these methods are characterized
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`by containing at least 75 !lglg astaxanthin, preferably 90 !lglg astaxanthin. Another krill lipid
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`extract disclosed contained at least 250 !lglg canastaxanthin, preferably 270 !lglg canastaxanthin.
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`Krill oil compositions have been described as being effective for decreasing cholesterol,
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`inhibiting platelet adhesion, inhibiting artery plaque formation, preventing hypertension,
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`controlling arthritis symptoms, preventing skin cancer, enhancing transdermal transport, reducing
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`the symptoms of premenstrual symptoms or controlling blood glucose levels in a patient. See,
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`e.g., WO 02/102394. In yet another application, a krill oil composition has been disclosed
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`comprising a phospholipid and/or a flavonoid. The phospholipid content in the krill lipid extract
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`could be as high as 60% w/w and the EP A/DHA content as high as 35% (w/w). See, e.g., WO
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`03/011873.
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`Furthermore, nutraceuticals, pharmaceuticals and cosmetics comprising the phospholipid
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`extract were disclosed. Previously, it was also shown that supercritical fluid extraction using neat
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`C02 could be used to prevent the extraction of phospholipids in order to extract the neutral lipid
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`fraction from krill, which comprised of esterified and free astaxanthin. See, e.g., Yamaguchi et
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`al., J Agric. Food Chem. (1986), 34(5), 904-7. Supercritical fluid extraction with solvent
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`modifier has previously been used to extract marine phospholipids from salmon roe, but has not
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`been previously used to extract phospholipids from krill meal. See, e.g., Tanaka et al., J. Oleo
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`Sci. (2004), 53(9), 417-424.
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`The methods described above rely on the processing of frozen krill that are transported
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`from the Southern Ocean to the processing site. This transportation is both expensive and can
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`result in degradation of the krill starting material. Data in the literature showing a rapid
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`decomposition of the oil in krill explains why some krill oil currently offered as an omega-3
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`supplement in the marketplace contains very high amounts of partly decomposed
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`phosphatidylcholine and also partly decomposed glycerides. Saether et al., Comp. Biochem
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`Phys. B 83B(l): 51-55 (1986). The products offered also contain high levels of free fatty acids.
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`What is needed in the art are methods for processing krill that do not require transport of
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`frozen krill material over long distances and the products produced by those methods.
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`SUMMARY OF THE INVENTION
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`In a first aspect of the invention is a composition characterized by comprising at least 65%
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`(w/w) phospholipids.
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`In another aspect of the invention is a composition obtained from aquatic or marine
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`sources, characterized by comprising 65% (w/w) phospholipids.
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`In yet another aspect of the invention is a composition obtained from krill, characterized
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`by comprising at least 65% (w/w) phospholipids.
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`In another aspect of the invention is a composition obtained from krill, characterized by
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`comprising at least 65% (w/w) phospholipids and at least 39% omega-3 fatty acids (w/w).
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`In yet another aspect of the invention is a composition obtained from krill, characterized
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`by comprising at least 65% (w/w) phospholipids, at least 39% omega-3 fatty acids (w/w) and at
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`least 580 mg/kg astaxanthin esters.
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`In another aspect of the invention is a composition obtained from krill, characterized by
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`comprising at least 39% omega-3 fatty acids (w/w) and at least 580 mg/kg astaxanthin esters.
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`In yet another aspect of the invention is a composition obtained from krill, characterized
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`by comprising at least 65% (w/w) phospholipids and at least 580mg/kg astaxanthin esters.
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`In yet another aspect, the present invention provides a krill oil effective for reducing
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`insulin resistance, improving blood lipid profile, reducing inflammation or reducing oxidative
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`stress.
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`In some embodiments, the present invention provides compositions comprising: from
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`about 3% to 10% ether phospholipids on a w/w basis; from about 35% to 50% non-ether
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`phospholipids on w/w basis, so that the total amount of ether phospholipids and non-ether
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`phospholipids in the composition is from about 48% to 60% on a w/w basis;
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`from about 20% to 45% triglycerides on a w/w basis; and from about 400 to about 2500 mg/kg
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`astaxanthin. In some embodiments, the ether phospholipids are selected from the group
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`consisting of alkylacylphosphatidylcholine, lyso-alkylacylphosphatidylcholine,
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`alkylacylphosphatidylethanolamine, and combinations thereof. In some embodiments, the ether
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`lipids are greater than 90% alkylacylphosphatidylcholine. In some embodiments, the non-ether
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`phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylserine,
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`phosphatidylethanolamine and combinations thereof. In some embodiments, krill oil composition
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`comprises a blend of lipid fractions obtained from krill. In some preferred embodiments, krill is
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`Euphausia superba, although other krill species also find use in the present invention. Other krill
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`species include, but are not limited to E. pacifica, E. frigida, E. longirostris, E. triacantha, E.
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`vallentini, Meganyctiphanes norvegica, Thysanoessa raschii and Thysanoessa inermis. In some
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`embodiments, the compositions comprise from about 25% to 30% omega-3 fatty acids as a
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`percentage of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids
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`are attached to said phospholipids. In some embodiments, the present invention provides a
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`capsule containing the foregoing compositions.
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`In further embodiments, the present inventions provide compositions comprising: from
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`about 3% to 10% ether phospholipids on a w/w basis; and from about 400 to about 2500 mg/kg
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`astaxanthin. In some embodiments, the compositions further comprise from about 35% to 50%
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`non-ether phospholipids on w/w basis, so that the total amount of ether phospholipids and non-
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`ether phospholipids in the composition is from about 38% to 60% on a w/w basis. In some
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`embodiments, the compositions further comprise from about 20% to 45% triglycerides on a w/w
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`basis. In some embodiments, the ether phospholipids are selected from the group consisting of
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`alkylacylphosphatidylcholine, lyso-alkylacylphosphatidylcholine,
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`alkylacylphosphatidylethanolamine, and combinations thereof. In some embodiments, the ether
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`lipids are greater than 90% alkylacylphosphatidylcholine. In some embodiments, the non-ether
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`phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylserine,
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`phosphatidylethanolamine and combinations thereof. In some embodiments, krill oil composition
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`comprises a blend of lipid fractions obtained from krill. In some preferred embodiments, krill is
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`Euphausia superba, although other krill species also find use in the present invention. Other krill
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`species include, but are not limited to E. pacifica, E. frigida, E. longirostris, E. triacantha, E.
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`vallentini, Meganyctiphanes norvegica, Thysanoessa raschii and Thysanoessa inermis. In some
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`embodiments, the compositions comprise about 25% to 30% omega-3 fatty acids as a percentage
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`of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids are attached
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`to said phospholipids. In some embodiments, the present invention provides a capsule containing
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`the foregoing compositions.
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`In some embodiments, the present invention provides a composition comprising at least
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`65% (w/w) of phospholipids, said phospholipids characterized in containing at least 35% omega-
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`3 fatty acid residues. In some preferred embodiments, the composition is derived from a marine
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`or aquatic biomass. In some further preferred embodiments, the composition is derived from
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`krill. In some embodiments, the composition comprises less than 2% free fatty acids. In some
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`embodiments, composition comprises less than 10% triglycerides. In some preferred
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`embodiments, the phospholipids comprise greater than 50% phosphatidylcholine. In some
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`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters. In some
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`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters and at least 36%
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`(w/w) omega-3 fatty acids. In some embodiments, the composition comprises less than about
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`0.5g/100g total cholesterol. In some embodiments, the composition comprises less than about
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`0.45% arachidonic acid (w/w).
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`In some embodiments, the present invention provides a krill lipid extract comprising at
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`least 500, 100, 1500, 2000, 2100, or 2200 mg/kg astaxanthin esters and at least 36% (w/w)
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`omega-3 fatty acids. In further embodiments, the present invention provides a krill lipid extract
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`comprising at least 100 mg/kg astaxanthin esters, at least 20% (w/w) omega-3 fatty acids, and
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`less than about 0.45% arachidonic acid (w/w).
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`In some embodiments, the present invention provides methods comprising administering
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`the foregoing compositions to a subject in an amount effective for reducing insulin resistance,
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`reducing inflammation, improving blood lipid profile and reducing oxidative stress.
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`In some embodiments, the present invention provides a krill lipid extract comprising
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`greater than about 80% triglycerides and greater than about 90, 100, 500, 1000, 1500, 200, 2100
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`or 2200 mg/kg astaxanthin esters. In some embodiments, the krill lipid extract is characterized in
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`containing from about 5% to about 15% omega-3 fatty acid residues. In some embodiments, the
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`krill lipid extract is characterized in containing less than about 5% phospholipids. In some
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`embodiments, the krill lipid extract is characterized in comprising from about 5% to about 10%
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`cholesterol.
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`In some embodiments, the present invention provides a krill meal composition comprising
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`less than about 50g/kg total fat. In some embodiments, the krill meal composition comprises
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`from about 5 to about 20 mg/kg astaxanthin esters. In some embodiments, the krill meal
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`composition comprises greater than about 65% protein. In some embodiments, the krill meal
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`composition of comprises greater than about 70% protein. In some further embodiments, the
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`present invention provides an animal feed comprising the krill meal composition.
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`In some embodiments, the present invention provides methods of increasing flesh
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`coloration in an aquatic species comprising feeding said aquatic species a composition
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`comprising the krill meal described above. In some embodiments, the present invention provides
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`methods of increasing growth and overall survival rate of aquatic species by feeding the krill
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`meal described above.
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`In some embodiments, the present invention provides methods of producing krill oil
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`comprising: a) providing krill meal; and b) extracting oil from said krill meal. In some
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`embodiments, the krill meal is produced by heat-treating krill. In some embodiments, the krill
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`meal is stored prior to the extraction step. In some embodiments, the extracting step comprises
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`extraction by supercritical fluid extraction. In some embodiments, the supercritical fluid
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`extraction is a two step process comprising a first extraction step with carbon dioxide and a low
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`concentration of a co-solvent (e.g., from about 1-10% co-solvent) and a second extraction step
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`5 with carbon dioxide and a high concentration of a co-solvent (e.g., from about 10-30% co(cid:173)
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`solvent). In preferred embodiments, the co-solvent is a C1-C3 monohydric alcohol, preferably
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`ethanol. In some embodiments, the present invention provides oil produced by the foregoing
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`method.
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`In some embodiments, the present invention provides methods of production of krill oil
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`comprising: a) providing fresh krill; b) treating said fresh krill to denature lipases and
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`phospholipases in said fresh krill to provide a denatured krill product; and c) extracting oil from
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`said denatured krill product. In some embodiments, the denaturation step comprises heating of
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`said fresh krill. In some embodiments, the denaturation step comprises heating said fresh krill
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`after grinding. In some embodiments, the methods further comprise storing said denatured krill
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`product at room temperature or below between the denaturation step and the extraction step. In
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`some embodiments, the enzyme denaturation step is achieved by application of heat. In some
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`embodiments, the extraction step comprises use of supercritical carbon dioxide, with or without
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`use of a polar modifier. In some embodiments, the extraction step comprises use of ethanol. In
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`some embodiments, the extraction step is comprises ethanol extraction followed by acetone to
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`precipitation of phospholipids. In some embodiments, the denatured krill product is a meal. In
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`some embodiments, the present invention provides oil produced by the foregoing method.
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`In some embodiments, the present invention provides a composition comprising oil
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`extracted from krill having a phosphatidylcholine content of greater then about 50% (w/w). In
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`some embodiments, the oil has a phosphatidylcholine content of greater then about 70% (w/w).
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`In some embodiments, the oil has a phosphatidylcholine content of greater then about 80% (w/w).
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`In some embodiments, the composition comprises less than 2% free fatty acids. In some
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`embodiments, the composition comprises less than 10% triglycerides. In some embodiments, the
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`composition comprises at least 500 mg/kg astaxanthin esters. In some embodiments, the
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`composition comprises less than about 0.45% arachidonic acid (w/w).
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`In some embodiments, the present invention provides composition comprising odorless
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`krill oil. In some embodiments, the odorless krill oil comprises less than about 10 mg/kg (w/w)
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`trimethylamine. In some further embodiments, the present invention provides an odorless krill
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`oil produced by the method comprising: extracting a neutral krill oil from a krill oil containing
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`material by supercritical fluid extraction to provide a deodorized krill material, wherein said
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`neutral krill oil contains odor causing compounds and extracting a polar krill oil from said
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`deodorized krill material by supercritical fluid extraction with a polar entrainer to provide an
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`essentially odorless krill oil.
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`In some embodiments, the present invention provides a composition comprising krill oil
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`containing less than about 70 micrograms/kilogram (w/w) astaxanthin esters. In some
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`embodiments, the compositions comprise less than about 50 micrograms/kilogram (w/w)
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`astaxanthin esters. In some embodiments, the compositions comprise less than about 20
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`micrograms/kilogram (w/w) astaxanthin esters. In some embodiments, the compositions
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`comprise less than about 5 micrograms/kilogram (w/w) astaxanthin esters.
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`In some embodiments, the present invention provides a krill oil produced by the process
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`comprising: pumping fresh krill from a trawl onto a ship, heating the krill to provide a krill
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`15 material, and extracting oil from the krill material.
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`In further embodiments, the present invention provides a blended krill oil composition
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`comprising: from about 45% to 55% w/w phospholipids; from about 20% to 45% w/w
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`triglycerides; and from about 400 to about 2500 mg/kg astaxanthin. In some embodiments, the
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`blended krill oil product comprises a blend oflipid fractions obtained from Euphausia superba.
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`In some embodiments, the composition comprises from about 25% to 30% omega-3 fatty acids as
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`a percentage of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids
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`are attached to said phospholipids.
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`In still other embodiments, the present invention provides a Euphausia superba krill oil
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`composition comprising: from about 30% to 60% w/w phospholipids; from about 20% to 50%
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`triglycerides; from about 400 to about 2500 mg/kg astaxanthin; and from about 20% to 35%
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`omega-3 fatty acids as a percentage of total fatty acids in said composition, wherein from about
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`70% to 95% of said omega-3 fatty acids are attached to said phospholipids.
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`In still further embodiments, the present invention provides a dietary supplement
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`comprising encapsulated Euphausia superba krill oil comprising from about 30% to 60% w/w
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`phospholipids; from about 20% to 50% triglycerides; from about 400 to about 2500 mg/kg
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`astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage of total fatty acids
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`in said composition, wherein from about 70% to 95% of said omega-3 fatty acids are attached to
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`said phospholipids.
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`In some embodiments, the present invention provides methods of making a Euphausia
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`superba krill oil composition comprising: contacting Euphausia superba with a polar solvent to
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`provide a polar extract comprising phospholipids; contacting Euphausia superba with a neutral
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`solvent to provide a neutral extract comprising triglycerides and astaxanthin; combining said
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`polar extract and said neutral extract to provide Euphausia superba krill oil comprising from
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`about 30% to 60% w/w phospholipids; from about 20% to 50% triglycerides; from about 400 to
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`about 2500 mg/kg astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage
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`of total fatty acids in said composition, wherein from about 70% to 95% of said omega-3 fatty
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`acids are attached to said phospholipids. In some embodiments, the methods further comprise the
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`step of encapsulating the Euphausia superba krill oil. In some embodiments, the present
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`invention provides a Euphausia superba krill oil produced by the methods described above.
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`In some embodiments, the present invention provides methods of producing a dietary
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`supplement comprising; contacting Euphausia superba with a polar solvent to provide an polar
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`extract comprising phospholipids; contacting Euphausia superba with a neutral solvent to provide
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`a neutral extract comprising triglycerides and astaxanthin; combining said polar extract and said
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`neutral extract to provide Euphausia superba krill oil comprising from about 30% to 60% w/w
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`phospholipids; from about 20% to 50% triglycerides; from about 400 to about 2500 mg/kg
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`astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage of total fatty acids
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`in said composition, wherein from about 70% to 95% of said omega-3 fatty acids are attached to
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`said phospholipids; and encapsulating said Euphausia superba krill oil.
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`In some embodiments, the present invention provides methods of reducing diet-induced
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`hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy, serum adiponectin reduction or
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`hepatic steatosis comprising in a subject exposed to a high fat diet: administering to said subject
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`exposed to a high fat diet an effective amount of a krill oil composition under conditions such that
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`a condition selected from the group consisting of diet-induced hyperinsulinemia, insulin
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`insensitivity, muscle mass hypertrophy, serum adiponectin reduction and hepatic steatosis is
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`reduced. The present invention is not limited to any particular krill oil composition. In some
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`embodiments, the krill oil composition is a Euphausia superba krill oil composition. The present
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`invention is not limited to any particular formulation of krill oil. In some embodiments, the krill
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`oil composition is encapsulated. In some preferred embodiments, the effective amount of a krill
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`oil composition is from 0.2 grams to 10 grams of said krill oil composition. In some
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`embodiments, the krill oil composition comprises: from about 45% to 55% w/w phospholipids;
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`from about 20% to 45% w/w triglycerides; and from about 400 to about 2500 mg/kg astaxanthin.
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`In some embodiments, the krill oil composition comprises a blend of lipid fractions obtained
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`from Euphausia superba. In some embodiments, the krill oil composition comprises from about
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`25% to 30% omega-3 fatty acids as a percentage of total fatty acids and wherein from about 80%
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`to 90% of said omega-3 fatty acids are attached to said phospholipids. In some embodiments, the
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`krill oil composition comprises from about 30% to 60% w/w phospholipids; from about 20% to
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`10
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`50% triglycerides; from about 400 to about 2500 mg/kg astaxanthin; and from about 20% to 35%
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`omega-3 fatty acids as a percentage of total fatty acids in said composition, and wherein from
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`about 70% to 95% of said omega-3 fatty acids are attached to said phospholipids.
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`In some embodiments, the present invention provides methods of reducing diet-induced
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`hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy, serum adiponectin reduction or
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`hepatic steatosis comprising in a subject consuming a high fat diet or a normal fat diet:
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`administering to said subject consuming a high fat diet or a normal fat diet an effective amount
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`of a krill oil composition under conditions such that a condition selected from the group
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`consisting of diet-induced hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy,
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`serum adiponectin reduction and hepatic steatosis is reduced. The present invention is not limited
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`to any particular krill oil composition. In some embodiments, the krill oil composition is a
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`Euphausia superba krill oil composition. The present invention is not limited to any particular
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`formulation of krill oil. In some embodiments, the krill oil composition is encapsulated. In some
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`preferred embodiments, the effective amount of a krill oil composition is from 0.2 grams to 10
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`grams of said krill oil composition. In some embodiments, the krill oil composition comprises:
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`25
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`from about 45% to 55% w/w phospholipids; from about 20% to 45% w/w triglycerides; and from
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`about 400 to about 2500 mg/kg astaxanthin. In some embodiments, the krill oil composition
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`comprises a blend oflipid fractions obtained from Euphausia superba. In some embodiments,
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`the krill oil composition comprises from about 25% to 30% omega-3 fatty acids as a percentage
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`of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids are attached
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`30
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`to said phospholipids. In some embodiments, the krill oil composition comprises from about
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`30% to 60% w/w phospholipids; from about 20% to 50% triglycerides; from about 400 to about
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`2500 mg/kg astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage of total
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`fatty acids in said composition, and wherein from about 70% to 95% of said omega-3 fatty acids
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`are attached to said phospholipids.
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`In some embodiments, the present invention provides methods of inducing diuresis in a
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`subject comprising: administering to said subject an effective amount of a krill oil composition
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`under conditions such that diuresis is induced. In some embodiments, the present invention
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`provides methods of increasing muscle mass in a subject, comprising:
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`administering to said subject an effective amount of a krill oil composition under conditions such
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`that muscle mass is increased. In some embodiments, the present invention provides methods of
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`decreasing protein catabolism in a subject, comprising: administering to said subject an effective
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`amount of a krill oil composition under conditions such that protein catabolism is decreased. In
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`some embodiments, the present invention provides methods of decreasing lipid content in the
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`heart of a subject, comprising: administering to said subject an effective amount of a krill oil
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`composition under conditions such that lipid content in the heart of the subject is decreased. In
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`some embodiments, the present invention provides methods of decreasing lipid content in the
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`liver of a subject, comprising: administering to said subject an effective amount of a krill oil
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`composition under conditions such that lipid content in the liver of the subject is decreased.
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`DESCRIPTION OF THE FIGURES
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`Figure 1. 31P NMR analysis of polar lipids in krill oil.
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`Figure 2. Blood lipid profiles in Zucker rats fed different forms of omega-3 fatty acids
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`(TAG= FO, PLI = NKO and PL2 = Superba).
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`Figure 3. Plasma glucose concentration in Zucker rats fed different forms of omega-3
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`fatty acids.
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`Figure 4. Plasma insulin concentration in Zucker rats fed different forms of omega-3 fatty
`
`acids.
`
`acids.
`
`Figure 5. Estimated HOMA-IR values in Zucker rats fed different forms of omega-3 fatty
`
`Figure 6. The effect of dietary omega-3 fatty acids on TNF production by peritoneal
`
`30 macrophages.
`
`Figure 7. The effect of dietary omega-3 fatty acids on lipid accumulation in the liver.
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`Attorney Docket No. AKBM -14409/US-11/CON
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`Figure 8. The effect of dietary omega-3 fatty acids on lipid accumulation in the muscle.
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`Figure 9. The effect of dietary omega-3 fatty acids on lipid accumulation in the heart.
`
`Figure 10. Relative concentrations ofDHA in the brain in Zucker rats supplemented with
`
`omega-3 fatty acids.
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`5
`
`Figure 11. Mean group body weights (g) in the collagen-induced male DBA/I arthritic
`mice. B- PL2 is the krill oil group. * p<0.05, significantly different from Group A (Positive
`Control - Fish Oil) and Group C (Control).
`
`Figure 12. Body weight for the various treatment groups.
`
`Figure 13. Muscle weight for the various treatment groups.
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`Figure 14. Muscle to body weight ratio for the various treatment groups.
`
`Figure 15. Serum adiopnectin levels (ng/ml) for the various treatment groups.
`
`Figure 16. Serum insulin levels for the various treatment groups.
`
`Figure 17. Blood glucose (mmol/1) levels in the various treatment groups.
`
`Figure 18. HOMA-IR values for the various treatment groups.
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`Figure 19. Liver triglyceride levels ().!mol/g) for the various treatment groups.
`
`DEFINITIONS
`
`As used herein, "phospholipid" refers to an orgamc compound having the following
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`20
`
`general structure:
`
`0
`II
`1
`O -C -R
`
`0
`II
`2
`- -O -C -R
`
`0
`II
`3
`O - r - _ O-R
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`Attorney Docket No. AKBM -14409/US-11/CON
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`wherein R1 is a fatty acid residue, R2 is a fatty acid residue or -OH, and R3 is a -H or nitrogen
`
`containing compound choline (HOCH2CH2N+(CH3) 30K), ethanolamine (HOCH2CH2NH2),
`
`inositol or serine. R1 and R2 cannot simultaneously be OH. When R3 is an -OH, the compound
`
`is a diacylglycerophosphate, while when R3 is a nitrogen-containing compound, the compound is
`
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`
`a phosphatide such as lecithin, cephalin, phosphatidyl serine or plasmalogen.
`
`An "ether phospholipid" as used herein refers to a phospholipid having an ether bond at
`
`position 1 the glycerol backbone. Examples of ether phospholipids include, but are not limited
`
`to, alkylacylphosphatidylcholine (AAPC), lyso-alkylacylphosphatidylcholine (LAAPC), and
`
`alkylacylphosphatidylethanolamine (AAPE). A "non-ether phospholipid" is a phospholipid that
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`
`does not have an ether bond at position 1 of the glycerol backbone.
`
`As used herein, the term omega-3 fatty acid refers to polyunsaturated fatty acids that have
`
`the final double bond in the hydrocarbon chain between the third and fourth carbon atoms from
`
`the methyl end of the molecule. Non-limiting examples of omega-3 fatty acids include,
`
`5,8,11,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexanoic acid (DHA) and
`
`15
`
`7,10,13,16,19-docosapentanoic acid (DPA).
`
`As used herein, astaxanthin refers to the following chemical structure:
`
`OH
`
`HO
`
`0
`
`As used herein, astaxanthin esters refer to the fatty acids esterified to OH group in the
`
`astaxanthin molecule.
`
`20
`
`As used herein, the term w/w (weight/weight) refers to the amount of a given substance in
`
`a composition on weight basis. For example, a composition comprising 50% w/w phospholipids
`
`means that the mass of the phospholipids is 50% of the total mass of the composition (i.e., 50
`
`grams of phospholipids in 100 grams of the composition, such as an oil).
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`Attorney Docket No. AKBM -14409/US-11/CON
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`DETAILED DESCRIPTION OF THE INVENTION
`
`This invention discloses novel krill oil compositions characterized by containing high
`
`levels of astaxanthin, phospholipids, included an enriched quantities of ether phospholipids, and
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`5
`
`omega-3 fatty acids. The krill oils compositions are extracted from krill meal using supercritical
`
`fluid extraction (SFE) with a co-solvent modifier. The krill meal has been processed on board a
`
`ship in Antarctica using live krill as starting material in order to ensure the highest possible
`
`quality of the krill meal. The krill oils are extracted from the krill meal in two stages, in step 1 the
`
`neutral fraction is extracted using neat supercritical C02 or in combination with 5% ethanol. The
`
`1 0
`
`neutral fraction consisted mostly of triglycerides and cholesterol. In stage 2, the polar lipids
`
`(phospholipids) are extracted by adding at least 20% ethanol to the supercritical C02 extraction
`
`medium.
`
`The present invention provides methods to avoid decomposition of glycerides and
`
`phospholipids in krill oil and compositions produced by those methods. The product obtained by
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`
`these new methods is virtually free of enzymatically decomposed oil constituents. The solution to
`
`the problem is to incorporate a protein denaturation step on fresh krill prior to use of any
`
`extraction technology. Denaturation can be achieved by thermal stress or by other means. After
`
`denaturation, the oil can be extracted by an optional selection of nonpolar and polar solvents
`
`including use of supercritical carbon dioxide. Krill is adapted to a very efficient nutrient digestion
`
`20
`
`at very low temperatures. Therefore the enzymes are sensitive to heat and the step of applying
`
`thermal denaturation of lipases and phospholipases does not imply use of very high temperatures.
`
`Surprisingly, it has been found that the use of mild denaturation conditions can greatly enhance
`
`the quality of krill oil.
`
`Additionally, a major obstacle of several processes of extraction is the cost o