111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US008278351B2
`
`c12) United States Patent
`Sampalis
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,278,351 B2
`*Oct. 2, 2012
`
`(54) NATURAL MARINE SOURCE
`PHOSPHOLIPIDS COMPRISING
`POLYUNSATURATED FATTY ACIDS AND
`THEIR APPLICATIONS
`
`(75)
`
`Inventor: Fontini Sampalis, Laval (CA)
`
`(73) Assignee: Neptune Technologies &
`Bioressources, Inc., Laval QC (CA)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 13/189,714
`
`(22) Filed:
`
`Jul. 25, 2011
`
`(65)
`
`Prior Publication Data
`US 2012/0028922 Al
`Feb. 2, 2012
`
`Related U.S. Application Data
`(63) Continuation of application No. 10/485,094, filed as
`application No. PCT/CA02/01185 on Jul. 29, 2002,
`now Pat. No. 8,030,348.
`(60) Provisional application No. 60/307,842, filed on Jul.
`27,2001.
`
`(51)
`
`Int. Cl.
`(2006.01)
`A61K 311215
`(2006.01)
`A61K 311661
`(52) U.S. Cl. .......................................... 514/506; 514/75
`(58) Field of Classification Search .................. 514/506,
`514/75
`See application file for complete search history.
`
`(56)
`
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`
`(Continued)
`Primary Examiner- James D Anderson
`Assistant Examiner- Gregg Polansky
`(74) Attorney, Agent, or Firm- Cooley LLP
`ABSTRACT
`(57)
`A phospholipid extract from a marine or aquatic biomass
`possesses therapeutic properties. The phospholipid extract
`comprises a variety of phospholipids, fatty acid, metals and a
`novel flavonoid.
`94 Claims, 4 Drawing Sheets
`
`000001
`
`

`

`US 8,278,351 B2
`Page 2
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`Psychopharmacology 132:217-226 ( 1997).
`Wiegand and Anderson, "Phospholipid Molecular Species of Frog
`Rod Outer Segment Membranes," Exp. Eye Res. 37(2):159-173
`(1983).
`Yamaguchi eta!., "Supercritical Carbon Dioxide Extraction of Oils
`from Antarctic Krill," J. Agric. Food Chern. 34:904-907 (1986).
`Yarochkin et a!., "Technochemical Characteristics of the Canned
`Food 'Natural Antarctic Krill Meat' and Its Food Value," Voprosy
`pitaniia Mar.-Apr.(2):69-72 (1985).
`Yongmanitchai and Ward, "Positional distribution of fatty acids, and
`molecular species of polar lipids, in the diatom Phaeodactylum
`tricornutum," J. Gen. Microbiol. 139:465-472 (1993).
`Youdim et a!., "Essential fatty acids and the brain: possible health
`implications," Int. J. Devl. Neuroscience 18(4-5):383-399 (2000).
`Fricke et al., "Lipid, Sterol and Fatty Acid Composition of Antarctic
`Krill," Lipids, 19( 11 ): 821-827 ( 1984).
`Gordeev et al., "Fatty Acid Composition of the Main Phospholipids
`of the Antarctic Krill Euphausia superba," translated from Khimiya
`Prirodnykh Soedinenii, No.2, 181-187 (1990).
`Kuroda eta!., "Comparison of Hypocholesterolemic Effect among
`Three Phospholipids Containing Different Fatty Acid and the Related
`Oils in Rats," Jpn. J. Nutr., 48(5):213-220 (1990).
`Makuta eta!., "Effects of EPA and Use in Health Foods," Japan Food
`Science 25(1):29-35 (1986).
`Aker Biomarine's Corrected Request for Reexamination of Patent
`No. 8,030,348 (U.S. Appl. No. 95/001,774).
`Declaration of Bjorn Ole Haugsgjerd, submitted inAker Biomarine's
`Corrected Request for Reexamination of Patent No. 8,030,348 (U.S.
`Appl. No. 95/001,774).
`Declaration of Thomas Gundersen, submitted in Aker Biomarine's
`Corrected Request for Reexamination of Patent No. 8,030,348 (U.S.
`Appl. No. 95/001,774).
`Non Final Office Action issued in the Reexamination of Patent No.
`8,030,348 (U.S. Appl. No. 95/001,774).
`Aker Biomarine's Corrected Request for Reexamination of Patent
`No. 8,057,825 (U.S. Appl. No. 95/00 1,819).
`Declaration ofNils Hoem, submitted in Aker Biomarine's Corrected
`Request for Reexamination of Patent No. 8,057,825 (U.S. Appl. No.
`95/00 1,819).
`
`Complaint filed by Neptune in Neptune Technologies v. Aker
`BiomarineASA, eta/. -Case 1: 11-cv-00894-GMS.
`Answer filed by Aker/Schiff in Neptune Technologies v. Aker
`BiomarineASA, eta/. -Case 1: 11-cv-00894-GMS.
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`Limited, eta/. -Case 1:11-cv-00895-GMS.
`Answer filed by Enzymotec in Neptune Technologies v. Enzymotec
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`Decision of Board in Opposition ofEP 1417211 (Dec. 30, 2009).
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`2002322233 (Apr. 22, 2009).
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`Analysis by Professor Andersen (Feb. 29, 2008) in Opposition to EP
`1417211.
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`www.dilesta.com/Tina.htrn.
`Sampalis eta!., "Evaluation of the Effects of Neptune Krill Oil™ on
`the Management of Premenstrual Syndrome and Dysmenorrhea,"
`Alternative Medicine Review 8(2), 171-178 (2003).
`Extract from online KEGG database for Lucenin-2.
`Levy et al., "The novel Flavonoid Chemistry and Phylogenetic Origin
`of Phloxjloridana," Evolution 29:487-499 (Sep. 1975).
`Bandyukov eta!., "Natural Flavonoid C-Glycosides," Chemistry of
`Natural Compounds, vol.l7, No.1 Jan.-Feb.l981-Translatedfrom
`Khimiya Prirodnykh Soedinenii, No. 1, pp. 5-24 (Jan.-Feb. 1981).
`Voirin et al., "Separation of Flavone C-Glycosides and Qualitative
`Analysis of Passiflora
`incarnata L. by Capillary Zone
`Electrophoresis," Phytochem. Anal. 11, 90-98 (2000).
`Iwashina, "The Structure and Distribution of the Flavonoids in
`Plants," J. Plant Res. 113:287-299 (2000).
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`Since 1986, Ed. J.B. Harborne, Chapter 3 ( 1994), ISBN 0 412 480700
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`loaded Jan. 23, 2008.
`Definition of "aglycon," IUPAC Compendium of Chemical Termi-
`nology, 2nd Edition, ISBN 0865426848 (1997).
`Enzymotec's Submission of a Letter from Igal Gozlan of the Tami-
`IMI Institute of Research and Development to Enzymotec Ltd. (Jan.
`14, 2008) in Opposition to EP 1417211.
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`tions from Spanish to English) (Sep. 1997 and Oct. 1999).
`* cited by examiner
`
`000004
`
`

`

`U.S. Patent
`
`Oct. 2, 2012
`
`Sheet 1 of 4
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`US 8,278,351 B2
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`U.S. Patent
`
`Oct. 2, 2012
`
`Sheet 2 of 4
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`US 8,278,351 B2
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`

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`U.S. Patent
`
`Oct. 2, 2012
`
`Sheet 3 of 4
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`US 8,278,351 B2
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`4H-1-Benzopyran-4-one, 2-(3,4-dihydroxyphenyl)-6,8-
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`Name (s)
`di-beta-D-glucopyranosyl-5, 7-dihyroxy-
`2) Lucenin 2
`3) Luteolin 6, 8-C-diglucoside
`4) 6, 8-Di-C-glucosylluteolin
`5) Flavone, 6, 8-diglucopyranosyl-3' ,4' ,5,7-tetrahydroxy-
`6) 6, 8-Diglucopyranosyl- 3', 4', 5, 7 -tetrahydroxyflavone
`7) 6, 8-Di-C-beta-glucosylluteolin
`Formula:
`C27H30016
`MW
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`CAS#
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`NIST#
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`ID#
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`Database
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`

`

`US 8,278,351 B2
`
`1
`NATURAL MARINE SOURCE
`PHOSPHOLIPIDS COMPRISING
`POLYUNSATURATED FATTY ACIDS AND
`THEIR APPLICATIONS
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`
`This application is a continuation of U.S. application Ser.
`No. 10/485,094, filed Jul. 13,2004, now U.S. Pat. No. 8,030, 10
`348, which is a §371national phase application ofPCT Appli-
`cation Serial No. PCT/CA2002/001185, filed Jul. 29, 2002,
`now expired, which claims the benefit of U.S. Provisional
`Patent Application Ser. No. 60/307,842, filed Jul. 27, 2001,
`which is incorporated herein by reference in its entirety.
`
`15
`
`FIELD OF THE INVENTION
`
`The present invention is directed to nutraceutical, pharma-
`ceutical or cosmetic compositions, particularly to phospho-
`lipid compositions derived from natural marine or aquatic
`sources.
`
`BACKGROUND OF THE INVENTION
`
`30
`
`WO 92/21335 published on Dec. 10, 1992 and correspond-
`ing U.S. Pat. No. 5,434,183 issued on Jul. 18, 1995 describes
`a phospholipid emulsion derived from marine and/or syn-
`thetic origin comprising polyunsaturated fatty acids and hav-
`ing anti-inflammatory and immunosuppressive effects and
`which promotes-normal brain or retinal development and
`function. U.S. Pat. No. 5,434,183 does not disclose the pres-
`ence of flavonoids or nervonic acid (a mono-unsaturated fatty
`acid) in the composition.
`JP 2215351, published on Aug. 28, 1990, discloses a 35
`method for extracting and purifYing phospholipids from fresh
`hill. Krill is lyophilized and then extracted with ethanol to
`produce an extract which is fractionated by absorption col-
`unm chromatography to produce high purity phosphatidyl
`choline and phosphatidyl ethanolamine. There is no disclo- 40
`sure of a composition comprising a flavonoid or nervonic
`acid.
`WO 00/23546, published onApr. 27, 2000, discloses meth-
`ods for extracting lipid fractions from marine and aquatic
`animal material by acetone extractions. The resulting non- 45
`soluble and particulate fraction is further solvent extracted
`with ethanol or ethylacetate to achieve further lipid extrac-
`tions.
`Hosokawa et a!. (35), published in 2000, discloses the
`conversion of docosahexanoic acid containing phosphatidyl-
`
`2
`cholines (DHA-PC) from squid skin lecithin to docosahex-
`anoic acid containing phosphadylserines (DHA-PS) via
`transphosphatidylation with phospholipase D
`(PLD).
`According to Table 2 of this reference, the fatty acid compo-
`sition of the phospholipid includes important portions of
`eicosapentanoic acid. There is no disclosure concerning any
`pharmaceutical, nutraceutical, or cosmetic use of a composi-
`tion comprising a flavonoid.
`Henderson eta!. (36), published in 1994, discloses lipid
`compositions of the pineal organ from rainbow trout compris-
`ing phospholipids. According to Table 4 of this reference, said
`phospholipids contain fatty acids corresponding to eicosap-
`entanoic and docosahexanoic acid. Similarly, Bell eta!. (37),
`published in 1991, discloses phospholipid compositions
`derived from different organs of cod. Moreover, Wiegand et
`a!. (38), published in 1983, discloses polyene derivatives of
`20 phosphatidylcholine as phospholipid molecular species of
`frog receptor membranes. However, there is no disclosure in
`any of these references concerning any pharmaceutical,
`nutraceutical, or cosmetic use of a composition comprising a
`25 flavonoid.
`WO 97/39759, published on Oct. 30, 1997, discloses w-3
`fatty acids and w-3 phosphatidylcholine in the treatment of
`bipolar disorder. The preferred w-3 phosphatidylcholine
`derivatives comprise eicosapentanoic and/or docosahexanoic
`acid. However, there is no disclosure concerning any phar-
`maceutical, nutraceutical, or cosmetic use of phospholipids
`beyond the treatment of bipolar disorder or the use of a
`composition comprising a flavonoid.
`EP 0609078 A1, published on Mar. 8, 1994, discloses a
`phospholipid comprising two different unsaturated fatty
`acids, wherein a preferred phospholipid contains both eicosa-
`pentanoic and docosahexanoic acid. Furthermore, the phos-
`pholipid can be used in the preparation of foods, skin care
`preparations, or pharmaceutical agent. However, there is no
`disclosure concerning any pharmaceutical, nutraceutical, or
`cosmetic use of a composition comprising a flavonoid.
`
`SUMMARY OF THE INVENTION
`
`In one aspect, the invention provides novel phospholipids,
`wherein the two fatty acids chains of the phospholipid are
`occupied by eicosapentanoic acid (EPA) and docosahexanoic
`acid (DHA) simultaneously, within the same molecule, i.e.: a
`phospholipid of the general formula (I):
`
`(I)
`
`0
`II
`H2C-0-c
`0
`II
`I
`0
`C-0-CH
`II
`I
`H2C-O-P-O-X
`I o-
`
`000009
`
`

`

`US 8,278,351 B2
`
`3
`wherein X represents a moiety normally found in a phospho-
`lipid.
`According to a further aspect of the present invention there
`is provided a composition, comprising:
`(a) a phospholipid of the general formula (I),
`
`4
`There is also provided a composition comprising the above
`noted phospholipid and flavonoid derived from a marine or
`aquatic biomass. The composition and the components are
`useful in the prevention or treatment of a variety of disease
`states and for the aesthetic enhancement of an animal, includ-
`(I)
`
`0
`II
`H2C-O-C
`0
`II
`I
`0
`C-0-CH
`II
`I
`H2C-0-P-0-X
`I o-
`
`15 ing human, body. Commercial packages containing the com-
`position are also within the invention.
`
`The novel phospholipids and the novel