`(10) Patent No.:
`US 9,072,752 B1
`
`Bruheim et a].
`(45) Date of Patent:
`*Jul. 7, 2015
`
`USOO9072752B1
`
`(54) BIOEFFECTIVE KRILL OIL COMPOSITIONS
`
`-
`.
`(71) Appl1cant.
`
`(72)
`
`Inventors:
`
`:ISGESR BIOlélARgNE ANTARCTIC
`,
`tamsun (N )
`
`Inge Bruheim, Volde (NO); Snorre
`Tilseth3 Bergen (NO); Daniele
`Mancinelli Orsta (NO)
`’
`
`.
`.
`(73) Ass1gnee. AKER BIOMARINE ANTARCTIC
`AS, Stamsund (NO)
`
`( * ) Notice:
`
`'
`'
`Subject to any disclaimer, the term of this
`31:13 1155:);Slbtil) (Eyegldluswd under 35
`’
`’
`’
`’
`~
`~
`~
`~
`~
`_
`Tlhrs patent 1s subject to a terminal d1s
`C almer-
`
`(21) Appl. No.: 14/620,784
`
`(22)
`
`Filed:
`
`Feb. 12, 2015
`
`Related US. Application Data
`(63) Continuation of application No. 12/057,775, filed on
`Mar. 28, 2008.
`
`AU
`Ei
`
`5,006,281 A
`5,266,564 A
`5,434,183 A
`6,214,396 B1
`6,346,276 B1
`6,537,787 B1
`6,800,299 B1
`7,488,503 B1
`7,666,447 B2
`8,030,348 B2
`8,278,351 B2
`8,383,675 B2
`8,697,138 B2
`2002/0076468 A1
`3883;89411431212; :1
`2004/0241249 A1
`2006/0078625 A1
`2006/0193962 A1
`2008/0166419 A1
`2008/0166420 A1
`2010/0143571 A1
`2010/0160659 A1
`2011/0130458 A1
`
`4/1991 Rubin et a1.
`11/1993 Modolell
`7/1995 Larsson-Backstrom
`4/2001 Barrier
`2/2002 Tanouchi et 31.
`3/2003 Breton
`10/2004 Beaudoin et a1.
`2/2009 Porzio et a1.
`2/2010 Rockway
`10/2011 Sampalis
`10/2012 Sampalis
`2/2013 Sampalis
`4/2014 Bruheim et a1.
`6/2002 Saxby
`0s 1t0m1 eta .
`33883 $381211? et €11~
`1
`12/2004 Sampalis
`4/2006 Rockway
`8/2006 Kamiya et a1.
`7/2008 Sones
`7/2008 Sones
`6/2010 Breivik
`6/2010 Catchpole
`6/2011 Breivik
`
`............
`
`426/643
`
`................ 426/615
`
`FOREIGN PATENT DOCUMENTS
`
`2002322233
`figgéggg
`
`2/2003
`431/1981
`,
`(Continued)
`
`(60) Provisional application No. 60/920,483, filed on Mar.
`28, 2007, provisional application No. 60/975,058,
`filed on Sep. 25’ 2007’ prov1s1onal applicat1on NO'
`60/983,446,
`filed on Oct. 29, 2007, prov1s1onal
`1‘
`tion No 61/024 072 filed on Jan 28 2008
`app lea
`’
`’
`’
`’
`’
`’
`
`(51)
`
`(2015.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`Int- Cl-
`A61K 35/612
`A61K 31/122
`A61K 31/20
`A61K 9/48
`A61K 31/235
`(52) U'S' Cl'
`CPC """"""" A61K 35/612 (201301); A61K 31/122
`(2013.01); A61K 31/20 (2013.01); A61K 9/48
`.
`(2013.01); A61K31/235 (2013.01)
`.
`(58) Fleld of Class1ficat10n Search
`CPC ............ A61K 2300/00; A61K 31/122; A61K
`31/685; A61K 35/612; A61K 31/683; A61K
`31/23; A61K 31/202; A61K 45/06; A61K
`9/4858; A61K 31/133; A61K 31/198; A61K
`31/201; A61K 31/232; A61K 31/575; A61K
`31/661; A61K 38/1767; A61K 9/2009;
`A61K 9/2054
`See application file for complete search history.
`
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`
`,
`.
`.
`.
`Primary hammer * Debl’le K Ware:
`(74) Attorney, Agent, or Flrm 7 Cas1m1r Jones S.C.
`
`ABSTRACT
`(57)
`This invention discloses new krill oil compositions charac-
`terized by having high amounts ofphospholipids, astaxanthin
`esters and/or omega-3 contents. The krill oils are obtained
`from krill meal using supercritical fluid extraction in a two
`stage process. Stage 1 removes the neutral lipid by extracting
`with neat supercritical CO2 or CO2 plus approximately 5% of
`a co-solvent. Stage 2 extracts the actual krill oils by using
`supercritical CO2 in combination with approximately 20%
`ethanol. The krill oil materials obtained are compared with
`commercially available krill oil and found to be more bioef—
`fective in a number of areas such as anti-inflammation, anti-
`oxidant effects, improving insulin resistances and improving
`blood lipid Profile
`
`20 Claims, 19 Drawing Sheets
`
`RIMFROST EXHIBIT 1118 Page 0001
`RIMFROST EXHIBIT 1118 Page 0001
`
`
`
`US 9,072,752 B1
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`WO 2001/082928 filed herewith.
`English Abstract; JP 2004-525180; See abstract from corresponding
`WO 2002/083122 filed herewith.
`English Abstract; JP 2006-528233; See abstract from corresponding
`WC 2004/ 100943 filed herewith.
`English Abstract; JP 2007-502805; See abstract from corresponding
`WO 2005/018632 filed herewith.
`English Abstract; JP 2007-509131; See abstract from corresponding
`WO 2005/037848 filed herewith.
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`WO 2005/070411 filed herewith.
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`WO 2002/09254 filed herewith.
`English Abstract; JP 2006-502196; See abstract from corresponding
`WO 2004/028529 filed herewith.
`
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`Fig. 2
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`Fig. 4
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`.....................................................................................................................................................................................
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`Sheet 9 of 19
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`Sheet 12 of 19
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`US 9,072,752 B1
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`Fig. 12
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`RIMFROST EXHIBIT 1118 Page 0017
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`US 9,072,752 B1
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`Fig. 14
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`Sheet 15 of 19
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`US 9,072,752 B1
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`US 9,072,752 B1
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`Fig. 17
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`1
`BIOEFFECTIVE KRILL OIL COMPOSITIONS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation of pending US. patent
`application Ser. No. 12/057,775, filed Mar. 28, 2008, which
`claims the benefit of US. Provisional Patent Application No.
`60/920,483, filed Mar. 28, 2007, US. Provisional Patent
`Application No. 60/975,058, filed Sep. 25, 2007, US. Provi-
`sional PatentApplication No. 60/983,446, filed Oct. 29, 2007,
`and US. Provisional PatentApplication No. 61/024,072, filed
`Jan. 28, 2008, all of which are incorporated by reference
`herein in their entirety.
`
`FIELD OF THE INVENTION
`
`This invention relates to extracts from Antarctic krill that
`
`comprise bioactive fatty acids.
`
`BACKGROUND OF THE INVENTION
`
`In the Southern Ocean, off the coast ofAntarctica, Antarc-
`tic krill (Euphausia superba) can be found in large quantities,
`ranging from 300-500 million metric tons ofbiomass. It feeds
`on phytoplankton during the short Antarctic summer. During
`winter, however, its food supply is limited to ice algae, bac-
`teria, marine detritus as well as depleting body protein for
`energy.
`In order to isolate the krill oil from the krill, solvent extrac-
`tion methods have been used. See, e.g., WO 00/23546. Krill
`lipids have been extracted by placing the material in a ketone
`solvent (e.g. acetone) in order to extract the lipid soluble
`fraction. This method involves separating the liquid and solid
`contents and recovering a lipid rich fraction from the liquid
`fraction by evaporation. Further processing steps include
`extracting and recovering by evaporation the remaining
`soluble lipid fraction from the solid contents by using a sol-
`vent such as ethanol. See, e.g., WO 00/23546. The composi-
`tions produced by these methods are characterized by con-
`taining at
`least 75 ug/g astaxanthin, preferably 90 ug/g
`astaxanthin. Another krill lipid extract disclosed contained at
`least 250 ug/g canastaxanthin, preferably 270 ug/g canastax-
`anthin.
`
`Krill oil compositions have been described as being effec-
`tive for decreasing cholesterol, inhibiting platelet adhesion,
`inhibiting artery plaque formation, preventing hypertension,
`controlling arthritis
`symptoms, preventing skin cancer,
`enhancing transderrnal transport, reducing the symptoms of
`premenstrual symptoms or controlling blood glucose levels
`in a patient. See, e.g., WO 02/102394. In yet another appli-
`cation, a krill oil composition has been disclosed comprising
`a phospholipid and/or a flavonoid. The phospholipid content
`in the krill lipid extract could be as high as 60% w/w and the
`EPA/DHA content as high as 35% (w/w). See, e.g., WO
`03/01 1 873.
`
`Furthermore, nutraceuticals, pharmaceuticals and cosmet-
`ics comprising the phospholipid extract were disclosed. Pre-
`viously, it was also shown that supercritical fluid extraction
`using neat CO2 could be used to prevent the extraction of
`phospholipids in order to extract the neutral lipid fraction
`from krill, which comprised ofesterified and free astaxanthin.
`See, e.g., Yamaguchi et al., J. Agric. Food Chem. (1986),
`34(5), 904-7. Supercritical fluid extraction with solvent
`modifier has previously been used to extract marine pho spho-
`lipids from salmon roe, but has not been previously used to
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`
`extract phospholipids from krill meal. See, e.g., Tanaka et al.,
`J. Oleo Sci. (2004), 53(9), 417-424.
`The methods described above rely on the processing of
`frozen krill that are transported from the Southern Ocean to
`the processing site. This transportation is both expensive and
`can result in degradation of the krill starting material. Data in
`the literature showing a rapid decomposition of the oil in krill
`explains why some krill oil currently offered as an omega-3
`supplement in the marketplace contains very high amounts of
`partly decomposed phosphatidylcholine and also partly
`decomposed glycerides. Saether et al., Comp. Biochem Phys.
`B 83B(1): 51-55 (1986). The products offered also contain
`high levels of free fatty acids.
`What is needed in the art are methods for processing krill
`that do not require transport of frozen krill material over long
`distances and the products produced by those methods.
`
`SUMMARY OF THE INVENTION
`
`In a first aspect of the invention is a composition charac-
`terized by comprising at least 65% (w/w) phospholipids.
`In another aspect ofthe invention is a composition obtained
`from aquatic or marine sources, characterized by comprising
`65% (w/w) phospholipids.
`In yet another aspect of the invention is a composition
`obtained from krill, characterized by comprising at least 65%
`(w/w) phospholipids.
`In another aspect ofthe invention is a composition obtained
`from krill, characterized by comprising at least 65% (w/w)
`phospholipids and at least 39% omega-3 fatty acids (w/w).
`In yet another aspect of the invention is a composition
`obtained from krill, characterized by comprising at least 65%
`(w/w) phospholipids, at least 39% omega-3 fatty acids (w/w)
`and at least 580 mg/kg astaxanthin esters.
`In another aspect ofthe invention is a composition obtained
`from krill, characterized by comprising at least 39% omega-3
`fatty acids (w/w) and at least 580 mg/kg astaxanthin esters.
`In yet another aspect of the invention is a composition
`obtained from krill, characterized by comprising at least 65%
`(w/w) phospholipids and at least 580 mg/kg astaxanthin
`esters.
`
`In yet another aspect, the present invention provides a krill
`oil effective for reducing insulin resistance, improving blood
`lipid profile, reducing inflammation or reducing oxidative
`stress.
`
`In some embodiments, the present invention provides com-
`positions comprising: from about 3%