(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2008/0021000 A1
`(43) Pub. Date:
`Jan. 24, 2008
`Chen et al.
`
`US 20080021000Al
`
`(54) MIXTURES OF AND METHODS OF USE
`FOR POLYUNSATURATED FATTY
`ACID-CONTAINING PHOSPHOLIPIDS AND
`ALKYL ETHER PHOSPHOLIPIDS SPECIES
`
`(76) Inventors:
`
`Su Chen, Malta, NY (US); Hung
`KWong, Malta, NY (US)
`
`Correspondence Address:
`LAW OFFICE OF MICHAEL A. BLAKE
`112 BROAD STREET
`MILFORD, CT 06460
`
`(21) Appl. No.:
`
`11/458,451
`
`(22) Filed:
`
`Jul. 19, 2006
`
`Publication Classi?cation
`
`(51) Int. Cl.
`(2006.01)
`A61K 31/685
`(2006.01)
`C07F 5/06
`(52) US. Cl. .......................................... .. 514/78; 554/76
`(57)
`ABSTRACT
`Mixtures of natural phosphatidylcholine species, natural
`lysophosphatidylcholine species, phosphatidylserine spe
`cies, phosphatidylethanolamine species, l-hydroxy-Z-acyl
`phosphatidylcholine species, l-hydroxy-2-acyl-phosphati
`dylserine
`molecular
`species,
`1 -hydroxy-2 -acyl
`phosphatidylethanolamine molecular species, l-O-alkyl-2
`hydroxy
`phosphatidylcholine
`species,
`l-O-alkyl-2
`docosaheaxnoyl phosphatidylcholine species l-O-alkyl-2
`docosahexaenoyl phosphatidylserine species, and l-O
`alkyl-Z-docosahexaenoyl
`phosphatidylethanolamine
`species, Methods using the above disclosed mixtures in
`mammals to treat Various conditions.
`
`RIMFROST EXHIBIT 1072 page 0001
`
`

`

`Patent Application Publication
`
`Jan. 24, 2008 Sheet 1 0f 3
`
`US 2008/0021000 A1
`
`RIMFROST EXHIBIT 1072 page 0002
`
`

`

`Patent Application Publication
`
`Jan. 24, 2008 Sheet 2 0f 3
`
`US 2008/0021000 A1
`
`100 (A) I
`
`4.05
`
`
`
`
`
`Relative Intensity (“/n)
`
`733
`
`820
`
`17: 0
`
`269
`
`423
`
`22:,6_
`
`327
`
`.
`
`__
`
`'
`
`v
`
`i
`
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`
`"|"1| '
`300
`
`'
`
`l
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`.400‘
`
`i
`
`l‘
`
`l
`l'
`500
`
`1
`
`H
`
`600
`
`700
`
`800
`
`100 ‘
`
`'
`
`(B)
`
`377
`
`
`
`
`
`Relative intensity ("/o)
`
`705
`
`0 -'
`
`I‘
`
`'
`
`‘
`
`I
`
`l
`
`200
`
`'
`
`300
`
`400
`
`500
`
`600
`
`‘700
`
`800
`
`mass lcharge
`
`Fig. 3
`
`RIMFROST EXHIBIT 1072 page 0003
`
`

`

`Patent Application Publication
`
`Jan. 24, 2008 Sheet 3 0f 3
`
`US 2008/0021000 A1
`
`1
`
`Pl. 0 m m d e p "a n .w
`In“ 0 ml.
`01 D C
`t w I.
`
`+ ] a N M h [
`:m
`n .w
`J. H 0 C D... L m. D.
`‘I... ‘III
`
`
`
`
`
`531
`
`0 Mw
`+ m
`
`
`
`W 1
`
`4_ 0
`
`
`M W ESL mama-main 9.5a?“ m
`I'll‘
`
`100
`
`200
`
`300
`
`400
`
`500
`
`600
`
`(B)
`100 m
`
`CID production spectmm of [M+Na]+ ion of I-DIL‘I-Z-OH-LPC
`
`5.31.
`
`U Ill. 1 i
`
`100 '
`
`200 i
`
`300
`
`400
`
`masslcharge
`
`Fig. 4
`
`600
`
`RIMFROST EXHIBIT 1072 page 0004
`
`

`

`US 2008/0021000 A1
`
`Jan. 24, 2008
`
`MIXTURES OF AND METHODS OF USE
`FOR POLYUNSATURATED FATTY
`ACID-CONTAINING PHOSPHOLIPIDS AND
`ALKYL ETHER PHOSPHOLIPIDS SPECIES
`
`FIELD OF THE INVENTION
`
`[0001] The present invention relates to (1) the extraction
`and puri?cation of neW mixtures of phosphatidylcholine and
`alkyl ether phosphatidylcholine species as Well as lysophos
`phatidylcholine species, Which are obtained from the liver of
`saltWater ?shes, With the structural characteriZation of (i)
`having a mixture of acyl and enriched alkyl fatty chains
`linked to the sn-1 position of the glycerol backbone, and (ii)
`having enriched 00-3 polyunsaturated fatty acid chains, in
`particular docosahexaenoic acid (DHA), linked to the sn-2
`position or the sn-1 position of the glycerol backbone; (2)
`the preparation of disclosed mixtures of phosphatidylcho
`line, phosphatidylserine and phosphatidylethanolamine spe
`cies as Well as lysophosphatidylcholine, lysophosphati
`dylserine and lysophosphatidylethanolamine species from
`the pho spholipid species mixtures extracted from the liver of
`saltWater ?shes by enZymatic reactions; and (3) their use as
`carriers of 00-3-polyunsaturated fatty acids to the brain for
`the prevention and alleviation of neurodegenerative and
`neurological diseases Which may be caused by the de?
`ciency of 00-3 polyunsaturated fatty acids, in particular
`DHA.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Omega-3 (or 00-3) polyunsaturated fatty acids, in
`particular docosahexaenoic acid (DHA), are especially
`important during prenatal brain development and mainte
`nance of human brain function. Compared With saturated
`fatty acid, polyunsaturated fatty acid With multiple double
`bonds Within molecule, in particular DHA, causes carbon
`carbon chains to become more curved. The more kinked the
`fatty acid is, the more space it Will take up, When it is built
`into cell membrane phospholipids, in order to keep neuronal
`membrane functions. This is the main reason that Why the
`brain requires a large amount of nutritionally essential
`polyunsaturated fatty acids, especially DHA, because DHA
`and DHA-containing molecular species of phospholipids
`may contribute to important brain functions including signal
`transduction and information processing [Akbar et al.,
`Docosahexaenoic acid: a positive modulator of Akt signal
`ing in neuronal survival. Proc. Natl. Acad. Sci. U.S.A. 102:
`10858 (2005)]. Alteration of neuronal membrane DHA
`containing phospholipid species can not only in?uence
`crucial intracellular and intercellular signaling but also alter
`many membrane physical properties such as ?uidity, phase
`transition temperature and bilayer thickness. The de?ciency
`of DHA markedly affects neurotransmission, membrane
`bound enZyme and ion channel activities leading to brain
`aging, AlZheimer’s disease, Parkinson’s disease, schiZo
`phrenia and peroxisomal disorders. For example, a study
`indicated that the concentration of DHA in patients With
`AlZheimer’s disease is signi?cantly decreased [Conquer, et
`al., Fatty acid analysis of blood plasma of patients With
`AlZheimer’s disease, other type of dementia, and cognitive
`impairment, Lipids, 35:1305 (2000)]. The studies of Garcia
`et al. [Garcia et al., Effect of docosahexaenoic acid on the
`synthesis of phosphatidylserine in rat brain in misrosomes
`and C6 gliome cells. J. Neurochem. 70: 24 (1998); Kim et
`
`al., Inhibition of neuronal apoptosis by docosahexaenoic
`acid (22:6n-3): Role of phosphatidylserine in anti-apoptotic
`effect. J Biol. Chem. 275:35215 (2000)] found out the neW
`role of DHA and phosphatidylserine in neuronal apoptosis,
`indicating that exogenous DHA may enhance phosphati
`dylserine accumulation in apoptotic Neuro-2A cells leading
`to the protection of neuronal cells from apoptotic death. The
`studies strongly suggest that one of supporting roles for
`anti-apoptosis of neurons is supplying DHA to the brain.
`[0003] Because the human body cannot synthesiZe 00-3
`polyunsaturated fatty acids, in particular DHA, exogenous
`introduction of DHA to human has been applied. There are
`a feW products available for use as brain nutrients, such as
`?sh oils (DHA-containing neutral lipids) and similar prod
`ucts.
`[0004] Although these products contain DHA and other
`omega-3 polyunsaturated fatty acids, experiments have
`demonstrated that only a very small amount of DHA can be
`found in the brain after administering a large amount of
`these products. But an early study shoWed that DHA
`containing lysophospholipid in albumin, rather than the
`forms of free DHA and other esteri?ed DHA, is preferred in
`the uptake of DHA in the brain of young rats When an in
`vitro model of blood-brain barrier is used [Thies et al.,
`Unsaturated fatty acids esteri?ed in 2-acyl-1-lysophosphati
`dylcholine bound to albumin are more e?iciently taken up by
`the young rat brain than unesteri?ed form. J. Neurochem. 59:
`1110 (1992)].
`[0005] Interestingly, a study reported that dietary phos
`pholipid With DHA-containing molecular species as supple
`mentation is much more ef?cient than soybean phospholipid
`for ensuring a normal level DHA in the brain during the
`period of brain development in rats [Bourre and Dumont,
`Neurosci. Lem, 335:129 (2002)] because DHA species are
`absent in the latter. The result suggests that DHA-containing
`phospholipid species are effective forms to be used as DHA
`carriers to brain.
`[0006] Phosphatidylcholine (PC), phosphatidylserine (PS)
`and phosphatidylethanolamine (PE) as Well as lysophos
`phatidylcholine (Lyso PC), lysophosphatidylserine (Lyso
`PS) and lysophosphatidylethanolamine (Lyso PE) are natu
`rally occurring phospholipid classes, existing in mixture
`forms of the molecular species. The structural diversity of
`the molecular species of phospholipids has been described in
`detail [Chen, Lipids, 28, 85 (1997); Chen et, al. Biomed.
`Mass Spectrom. 21, 655 (1992)]. Biochemical and biophysi
`cal functions of phospholipids are Well documented and
`appear to be determined by the fatty acid composition of the
`lipids.
`[0007] Ether phospholipids are usually found in animal
`tissues and human cells as minor components, existing
`together With molecular species of diacyl phospholipids
`carrying the same polar head group. It is Well knoWn that
`there are tWo predominant types of ether bonds in the
`phospholipid. One form is represented by the plasmalogens
`(With 1-alk-1'-enyl fatty chain linked to the sn-1 position of
`the glycerol backbone), Which is the most abundant subclass
`of phospholipids in most tissues. The other form is alkyl
`phospholipids that contain l-O-alkyl fatty chain(s) linked to
`the sn-1 position of the glycerol backbone. Although mix
`tures of phospholipids and ether phospholipids have been
`found in animals and humans [Diagne, et. al., Studies on
`ether phospholipids, Biochim. Biophys. Acla, 793, 221
`(1984)], little has been described regarding the presence of
`
`RIMFROST EXHIBIT 1072 page 0005
`
`

`

`US 2008/0021000 A1
`
`Jan. 24, 2008
`
`phospholipid species mixtures containing 1-O-alkyl-2-DHA
`molecules, as Well as DHA-containing lysophospholipids in
`aquatic animals.
`[0008] Investigation of PC metabolism in human [Galli et
`al., Prolonged retention of doubly labeled phosphatidylcho
`line in human plasma and erythrocytes after oral adminis
`tration. Lipids, 27: (1992)] indicated that a major portion of
`PC species can be found as intact molecules in plasma after
`oral administration of labeled phospholipid species. The
`result is supported by an animal experiment, suggesting that
`more than 80% of PC, Which is recovered from the intestinal
`lymph of rats, is still intact after oral administration of
`phospholipids [Ikeda et al., Absorption and transport of base
`moieties of phosphatidylcholine and phosphatidylethanola
`mine in rats, Biochim. Biophys. Acla, 921; 245 (1987)].
`[0009] However, fatty acid chains of phospholipids can be
`further hydrolyZed by lecithin-cholesterol acyltransferase in
`plasma after molecular species are incorporated into high
`density lipoprotein, and folloWed by phospholipase A1 and
`phospholipase A2 hydrolyses in the liver. Recently, several
`papers reported the possibility of transporting high density
`lipoprotein across the blood-brain barrier using an in vitro
`model [BalaZs et al., Uptake and transport of high-density
`lipoprotein (HDL) and HDL-associated ot-tocopherol by an
`in vitro blood-brain barrier model. J. Neurochem. 89: 939
`(2004)] and the importance of endothelial lipase in the
`metabolism of high density lipoprotein associated phospho
`lipids at the blood-brain barrier [Ma et al., Endothelial lipase
`is a major genetic determinant for high-density lipoprotein
`concentration, structure, and metabolism. Proc. Natl. Acad.
`Sci. USA. 100: 2748 (2003)], demonstrating that endothe
`lial lipase (i) exhibits primarily the activity of phospholipase
`A1 leading to the hydrolysis of sn-1 fatty acid chains from
`the molecular species of high density lipoprotein phospho
`lipids, and (ii) is inactive in the hydrolysis of ether phos
`pholipid molecular species [Gauster et al., Endothelial lipase
`release saturated and unsaturated fatty acid of high density
`lipoprotein phosphatidylcholine. J Lipid Res. 46: 1517
`(2005)].
`[0010] An early study also shoWed that after the admin
`istration of lysophospholipids, these lipid species do not
`enhance fatty acid chain retention in mucosa and may
`survive from the hydrolysis of lecithin-cholesterol-acyl
`transferase in plasma, as Well as other phospholipases in the
`liver [Viola et al., Absorption and distribution of arachido
`nate in rats receiving lysophospholipids by oral route, J.
`Lipid Res. 34, 1843 (1993)].
`[0011] Based on these published results, it is clear to see
`that ether phospholipid species, such as 1-O-alkyl-2-acyl
`molecular species, and lysophospholipid molecular species
`are more stable in vivo lipid metabolism, compared With
`related acyl species. Because ether phospholipid and lyso
`phospholipid species can be survived from blocking due to
`the hydrolysis of phospholipase A1 and phospholipase
`Al-like enZymes in vivo metabolism [Shamburek et al.,
`Disappearance of tWo major phosphatidylcholine from
`plasma is predominantly via LCAT and hepatic lipase, Am J.
`Physiol. 271: E1073 (1996); plasma 1-palmitoyl-2-linoleoyl
`phosphatidylcholine. Evidence for extensive phospholipase
`A1 hydrolysis and hepatic metabolism of the products, J.
`Biol. Chem. 266: 18002 (1991)], DHA-containing ether
`phospholipid species and DHA-containing lysophospholipid
`species can be delivered smoothly into the brain as carriers
`of DHA, resulting in the uptake of free DHA after brain
`
`phospholipase hydrolyses of the phospholipids species
`[Ross et al., Characterization of a novel phospholipase A2
`activity in human brain. J. Neurochem. 64, 2213 (1995)].
`Interest is focused on the preparation of phospho
`[0012]
`lipid species containing 1-O-alkyl-2-DHA molecules and
`DHA-containing lysophospholipid species. HoWever,
`obtaining molecular species mixtures containing enriched
`DHA-containing ether phospholipids and -lysophospholip
`ids species from aquatic animals is poorly understood
`[Chapelle, Plasmalogen and O-alkyl glycerophospholipids
`in aquatic animals, Comp. Biochem. Physiol. 88, 1 (1987)].
`Additionally, the chemical synthesis of these phospholipids
`species is both dif?cult and expensive, and thus a large-scale
`preparation of the molecular species for use as potential
`brain health supplementation is not available. Thus there is
`a need for a method of a large-scale preparation of DHA
`containing phospholipids and -lysophospholipids species for
`use as potential brain health supplementations.
`
`SUMMARY OF THE INVENTION
`
`[0013]
`The disclosed invention relates to a mixture of
`natural phosphatidylcholine species as shoWn in Formula 1:
`
`(sn-l position) CH2 — R1
`
`(Sn-2 position) CH—R2
`
`(Formula 1)
`
`0
`
`on
`
`CH3
`
`CH3
`
`[0014] Where R1 is a mixture of acyl and alkyl fatty
`chains, linked to the sn-1 position, selected from the group
`consisting of COOCISH31 (acyl fatty chain; palmitic acid;
`16:0); COOCl6H33 (acyl fatty chain; margaric acid; 17:0);
`COOCUH35 (acyl fatty chain; stearic acid; 18:0); OCl6H33
`(alkyl fatty chain; O-16:0); OCISH37 (alkyl fatty chain;
`O-18:0); and OCISH35 (alkyl fatty chain; O-18:1); and
`[0015] Where R2 consists of a mixture of acyl fatty chains,
`linked to the sn-2 position, and selected from the group
`consisting of COOCBH27 (acyl fatty chain; myristic acid;
`14:0); COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCISH29 (acyl fatty chain; palmitoleic acid; 16:1);
`COOCUH33 (acyl fatty chain; oleic acid; 18:1); COOC19H31
`(acyl fatty chain; arachidonic acid; 20:4); COOClgH29 (acyl
`fatty chain; eicosapentaenoic acid; 20:5 (00-3)); COOCZIH33
`(acyl fatty chain; docosapentanoic acid; 22:5); and
`COOCZIH31 (acyl fatty chain; docosahexaenoic acid; 22:6
`((11-3))
`[0016]
`The disclosed invention also relates to a mixture of
`natural lysophosphatidylcholine species as shoWn in For
`mula 2:
`
`RIMFROST EXHIBIT 1072 page 0006
`
`

`

`US 2008/0021000 A1
`
`Jan. 24, 2008
`
`(Formula 2)
`
`(Formula 4)
`
`(sn-l position) CH2— R
`
`(sn-Z position) CH—OH
`
`0
`
`on
`
`CH3
`
`cn3
`
`(sn-l position) CH2—R1
`
`(sn-Z position) CH—R2
`
`O
`
`(sn-3 position) CH2 — O— P — O — CH2— CH— NHZ
`
`OH
`
`[0017] Where R is a mixture of acyl and alkyl fatty chains,
`linked to the sn-1 position, and selected from the group
`consisting of COOCBH27 (acyl fatty chain; myristic acid;
`14:0); COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCISH29 (acyl fatty chain; palmitoleic acid; 16:1);
`COOCUH35 (acyl fatty chain; stearic acid;
`18:0);
`COOCUH33 (acyl fatty chain; oleic acid; 18:1); COOCUH31
`(acyl fatty chain; linoleic acid; 18:2); COOClgH31 (acyl
`fatty chain; arachidonic acid; 20:4); COOClgH29 (acyl fatty
`chain; eicosapentaenoic acid; 20:5 (00-3)); COOCZIH31 (acyl
`fatty chain; docosahexaenoic acid; 22:6 (00-3)); OC14H29
`(alkyl fatty chain; O-14:0); OCl6H33 (alkyl fatty chain;
`O-16:0); OC16H31 (alkyl fatty chain; O-16:1); OCISH37
`(alkyl fatty chain; O-18:0); and OCISH35 (alkyl fatty chain;
`O-18:1).
`[0018] The disclosed invention additionally, relates to a
`mixture of phosphatidylserine species as shoWn in Formula
`3:
`
`(sn-l position) CH2 — R1
`
`(sn-Z position) CH—RZ
`
`(F ormula 3 )
`
`Where R1 is a mixture of acyl and alkyl fatty chains,
`[0022]
`linked to the sn-1 position, selected from the group consist
`ing of COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCMH33 (acyl fatty chain; margaric acid; 17:0);
`COOCUH35 (acyl fatty chain; stearic acid; 18:0); OCl6H33
`(alkyl fatty chain; O-16:0); OCISH37 (alkyl fatty chain;
`O-18:0); and OCISH35 (alkyl fatty chain; O-18:1); and
`Where R2 consists of a mixture of acyl fatty chains,
`[0023]
`linked to the sn-2 position, and selected from the group
`consisting of COOCBH27 (acyl fatty chain; myristic acid;
`14:0); COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCISH29 (acyl fatty chain; palmitoleic acid; 16:1);
`COOCUH33 (acyl fatty chain; oleic acid; 18:1); COOClgH31
`(acyl fatty chain; arachidonic acid; 20:4); COOC19H29 (acyl
`fatty chain; eicosapentaenoic acid; 20:5 (00-3)); COOCZIH33
`(acyl fatty chain; docosapentanoic acid; 22:5); and
`COOCZIH31 (acyl fatty chain; docosahexaenoic acid; 22:6
`((11-3))
`[0024]
`In addition, the disclosed invention relates to a
`mixture of 1-hydroxy-2-acyl-phosphatidylcholine species as
`shoWn in Formula 5.
`
`on
`
`NH2
`
`(Formula 5)
`
`[0019] Where R1 is a mixture of acyl and alkyl fatty chains,
`linked to the sn-1 position, selected from the group consist
`ing of COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCMH33 (acyl fatty chain; margaric acid; 17:0);
`COOCUH35 (acyl fatty chain; stearic acid; 18:0); OCl6H33
`(alkyl fatty chain; O-16:0); OCISH37 (alkyl fatty chain;
`O-18:0); and OCISH35 (alkyl fatty chain; O-18:1); and
`[0020] Where R2 consists of a mixture of acyl fatty chains,
`linked to the sn-2 position, and selected from the group
`consisting of COOCBH27 (acyl fatty chain; myristic acid;
`14:0); COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCISH29 (acyl fatty chain; palmitoleic acid; 16:1);
`COOCUH33 (acyl fatty chain; oleic acid; 18: 1); COOClgH31
`(acyl fatty chain; arachidonic acid; 20:4); COOClgH29 (acyl
`fatty chain; eicosapentaenoic acid; 20:5 (00-3)); COOCZIH33
`(acyl fatty chain; docosapentanoic acid; 22:5); and
`COOC H31 (acyl fatty chain; docosahexaenoic acid; 22:6
`(00-3)).
`[0021] The disclosed invention, also, relates to a mixture
`of phosphatidylethanolamine species as shoWn in Formula
`4:
`
`21
`
`(sn-l position) CH2 — OH
`
`(sn-Z position) CH—R
`
`0
`
`on
`
`CH3
`
`cn3
`
`Wherein R is a mixture of acyl fatty chains linked
`[0025]
`to the sn-2 position, selected from the group consisting of
`COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCUH33 (acyl fatty chain; oleic acid; 18:1); COOClgH31
`(acyl fatty chain; arachidonic acid; 20:4); COOClgH29 (acyl
`fatty chain; eicosapentaenoic acid; 20:5 (00-3)); COOCZIH33
`(acyl fatty chain; docosapentanoic acid; 22:5); and
`COOCZIH31 (acyl fatty chain; docosahexaenoic acid; 22:6
`((11-3))
`[0026]
`Additionally, the disclosed invention relates to a
`mixture of 1-hydroxy-2-acyl-phosphatidylserine molecular
`species as shoWn in Formula 6:
`
`RIMFROST EXHIBIT 1072 page 0007
`
`

`

`US 2008/0021000 A1
`
`Jan. 24, 2008
`
`[0032] The disclosed invention, in addition, relates to a
`mixture of l-O-alkyl-2-docosaheaxnoyl phosphatidylcho
`line species as shoWn in Formula 9:
`
`(Formula 6)
`
`(sn-l position) CH2 — OH
`
`(sn-2 position) CH—R
`
`0
`
`on
`
`NH2
`
`[0027] wherein R is a mixture of acyl fatty chains linked
`to the sn-2 position, selected from the group consisting of
`COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCUH33 (acyl fatty chain; oleic acid; 18:1); COOC19H31
`(acyl fatty chain; arachidonic acid; 20:4); COOClgH29 (acyl
`fatty chain; eicosapentaenoic acid; 20:5 (00-3)); COOCZIH33
`(acyl fatty chain; docosapentanoic acid; 22:5); and
`COOCZIH31 (acyl fatty chain; docosahexaenoic acid; 22:6
`((11-3))
`[0028] Furthermore, the disclosed invention relates to a
`mixture of l-hydroxy-2-acyl-phosphatidylethanolamine
`molecular species as shoWn in Formula 7:
`
`(sn-l position)
`
`CH2 — R
`
`(sn-2 position)
`
`OH—COOC2iH3i
`
`on
`
`(Formula 9)
`
`cn3
`
`cn3
`
`[0033] Where R is a mixture of alkyl fatty chains linked to
`the sn-1 position, selected from the group consisting of
`OCMH29 (alkyl fatty chain; O-l4:0); OCl6H33 (alkyl fatty
`chain; O-l6:0); OCl6H31 (alkyl fatty chain; O-l6:l);
`OClsH37 (alkyl fatty chain; O-l8:0); and OCISH35 (alkyl
`fatty chain; O-l8:l).
`[0034] In addition, the disclosed invention relates to a
`mixture of l-O-alkyl-2-docosahexaenoyl
`phosphati
`dylserine species as shoWn in Formula 10:
`
`(sn-l position)
`
`CH2—OH
`
`(sn-2 position) OH—R O
`
`OH
`
`(Formula 7)
`
`(Formula 10)
`
`(sn-l position)
`
`CH2—R
`
`(sn-2 position)
`
`OH—COOC2iH3i
`
`[0029] Wherein R is a mixture of acyl fatty chains linked
`to the sn-2 position, selected from the group consisting of
`COOCISH31 (acyl fatty chain; palmitic acid; 16:0);
`COOCUH33 (acyl fatty chain; oleic acid; 18:1); COOC19H31
`(acyl fatty chain; arachidonic acid; 20:4); COOClgH29 (acyl
`fatty chain; eicosapentaenoic acid; 20:5 (00-3)); COOCZIH33
`(acyl fatty chain; docosapentanoic acid; 22:5); and
`COOCZIH31 (acyl fatty chain; docosahexaenoic acid; 22:6
`(00-3)).
`[0030] Also, the disclosed invention relates to a mixture of
`l-O-alkyl-2-hydroxy phosphatidylcholine species as For
`mula 8:
`
`on
`
`NH2
`
`[0035] Where R is a mixture of alkyl fatty chains linked to
`the sn-1 position, selected from the group consisting of
`OCMH29 (alkyl fatty chain; O-l4:0); OCl6H33 (alkyl fatty
`chain; O-l6:0); OCl6H31 (alkyl fatty chain; O-l6:l);
`OClsH37 (alkyl fatty chain; O-l8:0); and OCISH35 (alkyl
`fatty chain; O-l8:l).
`[0036] Furthermore, the disclosed invention relates to a
`mixture of l-O-alkyl-2-docosahexaenoyl phosphatidyletha
`nolamine species as shoWn in Formula 11.
`
`(Formula 1 l)
`
`(sn-l position)
`
`CH2 — OH
`
`(sn-2 position)
`
`QH — R O
`
`on
`
`(Formula 8)
`
`(sn-l position)
`
`CH2—R
`
`CH3
`
`cn3
`
`(sn-2 position)
`
`OH—COOC2iH3i
`
`0.....
`
`OH
`
`[0031] Wherein R is a mixture of alkyl fatty chains linked
`to the sn-1 position, selected from the group consisting of
`OCMH29 (alkyl fatty chain; O-l4:0); OCl6H33 (alkyl fatty
`chain; O-l6:0); OCl6H31 (alkyl fatty chain; O-l6:l);
`OClsH37 (alkyl fatty chain; O-l8:0); and OCISH35 (alkyl
`fatty chain; O-l8:l).
`
`[0037] Where R is a mixture of alkyl fatty chains linked to
`the sn-1 position, selected from the group consisting of
`OCMH29 (alkyl fatty chain; O-l4:0); OCl6H33 (alkyl fatty
`chain; O-l6:0); OCl6H31 (alkyl fatty chain; O-l6:l);
`OClsH37 (alkyl fatty chain; O-l8:0); and OCISH35 (alkyl
`fatty chain; O-l8:l).
`
`RIMFROST EXHIBIT 1072 page 0008
`
`

`

`US 2008/0021000 A1
`
`Jan. 24, 2008
`
`In addition, the disclosed invention relates to a method of
`using the above disclosed mixtures in mammals to treat
`various conditions.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0038] Surprisingly, the applicants have discovered that it
`is possible to obtain PC species mixtures With enriched
`1-O-alkyl-2-DHA molecules and Lyso PC species mixtures
`With enriched DHA-containing molecule from the liver of
`saltWater ?shes by chemical extraction, folloWed by column
`puri?cation. For example, the PC species mixture from
`common dolphin liver contains approximately 50% of sn-2
`(uu-3)-polyunsaturated fatty acid molecules, including
`approximately 25% of 1-O-alkyl-2-DHA molecules; for
`another example, the Lyso PC species mixture from shark
`liver contains approximately 33% of DHA-containing mol
`ecule. The collection of raW liver materials in a large
`quantity is readily available from marine animals.
`[0039] A second aspect of the present invention is that it
`is possible to prepare disclosed mixtures of PS and PE
`species by the transphosphatidylation [Comfurius et, al.
`Enzymatic synthesis of phosphatidylserine on small scale by
`use of a one-phase system, J. Lipid Res. 31, 1719 (1990)]
`using the special starting material combinations that consist
`of (i) phospholipase D, (ii) L-serine or ethanolamine and (iii)
`the PC species mixtures extracted from the liver of saltWater
`?shes.
`[0040] Athird aspect of the present invention relates to the
`preparation of disclosed sn-2-DHA-Lyso PC, -Lyso PS and
`-Lyso PE species mixtures by lipase hydrolyses of mixtures
`of the liver PC species, as Well as transphosphatidylated ?sh
`liver-PS and -PE species [Slotboom et al., Hydrolysis of
`phosphoglycerides by Puri?ed lipase preparations II. Prepa
`ration of unsaturated 2-monoacyl choline phosphoglycer
`ides, Chem. Phys. Lipids, 4, 30 (1970)].
`[0041] A fourth aspect of the present invention is involved
`in the preparation of disclosed mixtures of 1-O-alkyl-2
`DHA-PC, -PS and -PE species using 1-O-alkyl-2-hydroxy
`PC species mixtures [Paltauf et al., In Method in Enzymol
`ogy, 197, 141 (1991)] as the starting material, Which are
`prepared from the liver PC species and/or the liver Lyso PC
`species mixtures.
`[0042] Compared With available phospholipid product
`Lecithin that contains soybean PC species mixtures, neWly
`discovered and puri?ed PC and Lyso PC species mixtures
`are alternative supplementations. Furthermore, the PC and
`Lyso PC may be used as brain nutrients for the delay of brain
`aging. HoWever, Lecithin containing PC species mixtures
`lack DHA molecular species.
`[0043] A mixture of bovine cortex phosphatidylserine
`(BCPS) species, Which contains approximately 8% of sn-2
`DHA molecular species, Was registered as a drug in Europe
`for both cognitive de?cits in the old and the alleviation of
`AlZheimer’s disease [Crook et al., Effect of phosphatidyl
`serine in AlZheimer’s Disease, Psychopharmacology Bulle
`[in 28, 61(1992); Pepeu, et al., A revieW of phosphati
`dylserine pharmaceutical and clinical
`effects:
`Is
`phosphatidylserine a drug for aging brain? Pharmacol. Res.
`33, 51 (1996)]. Unfortunately, the risk of bovine spongiform
`encephalopathy made the use of the BCPS extracted from
`bovine brain potentially dangerous.
`[0044] Compared With the BCPS, disclosed mixtures of
`transphosphatidylated ?sh liver-PS and -PE species, as Well
`
`as sn-2-DHA-Lyso PS and -Lyso PE and alkyl-DHA-PC,
`-PS and -PE species contain much more sn-2-DHA molecu
`lar species (over 40%, even 100%), and are considered to be
`of much more therapeutic interest in the alleviation and
`treatment of neurodegenerative and neurological diseases,
`Which are associated With dysfunctions of neurotrophins and
`neurotrophin receptors caused by the de?ciency of (00-3)
`polyunsaturated fatty acids, in particular DHA.
`[0045] Compared With commercially available phospho
`lipids product transphosphatidylated soybean PS species
`mixtures, disclosed phospholipid species mixtures are quali
`?ed for use as substitutes of the BCPS for the treatment of
`neurodegenerative and neurological diseases caused by
`DHA de?ciency, because DHA-containing phospholipid
`species are absent in currently available transphosphatidy
`lated soybean PS species mixtures (http://WWW.cfsan.fda.
`gov/~dms/dsltr36.html).
`[0046] Although the preparation of a mixture of transpho
`sphatidylated ?sh brain-PS species has been reported (Euro
`pean patent: EPO 819 760 Al), the disadvantages of the
`preparation include: (i) the collection of ?sh brain material
`in a large quantity is difficult, and thus the product prepa
`ration in industrial scale is limited; (ii) Lyso PC has less than
`5% of ?sh brain phospholipids, and DHA-containing Lyso
`PC is almost absent; and (iii) l-O-alkyl-DHA phospholipids
`species are almost absent in ?sh brain.
`[0047] Although the preparation of a mixture of transpho
`sphatidylated ?sh skin-PS has been also reported,
`[HosokaWa et al., Conversion to docosahexaenoic acid
`containing phosphatidylserine from squid skin lecithin by
`phospholipase D-mediated transphosphatidylation, J. Agric.
`Food. Chem. 48, 4550 (2000)], the collection of ?sh skin as
`starting materials in a large quantity is much more dif?cult,
`and thus an industrial scale preparation of the product is
`almost impossible. Furthermore, l-O-alkyl-DHA phospho
`lipids species and DHA-containing lysophospholipids are
`almost absent in ?sh skin as Well.
`[0048] Materials and procedures described in this disclo
`sure have made it possible to prepare a series of disclosed
`mixtures of phospholipid species in a large scale, and even
`in an industrial scale. The resulting products can be used as
`the preventive and therapeutic reagents for brain and mental
`health.
`
`EXAMPLE 1
`
`Preparation of Crude Lipids Extracted from the
`Liver of SaltWater Fishes
`
`[0049] About tWo sharks Were obtained in a ?sh market
`near the city of Medan in Indonesia, and the livers (approxi
`mately 1,170 grams (FIG. 1; see beloW) and 550 grams of
`the Wet liver material, respectively) Were transported in ice
`and then passed through a hand meat grinder. Tiny liver
`fragments Were made With an electronic blender and Were
`then mixed With about 20 volumes of cold acetone standing
`for four hours. After removing the acetone, the liver frag
`ments, noW the pellets Were dried under nitrogen, and then
`the dried pellets Were homogeniZed With about 10 volumes
`(W/v) of ethanol/ethyl acetate (1/ 1; v/v) stirring overnight.
`After evaporation of the ethanol/ethyl acetate, about 172
`grams of crude lipids extracted from 1,170 grams of the Wet
`liver material Were obtained.
`[0050] About 50 grams of the crude lipids Were mixed
`With about 10 volumes (W/v) of acetone, and then the
`
`RIMFROST EXHIBIT 1072 page 0009
`
`

`

`US 2008/0021000 A1
`
`Jan. 24, 2008
`
`solution Was stirred at about 300 C. for about 90 min. The
`mixture Was ?ltered, and the acetone extract Was kept at
`about —20° C. for overnight. The mixture solution Was
`rapidly ?ltered and led to the precipitation of crude phos
`pholipids (approximately 23.4 grams), consisting of about
`80% of PC, about 15% of Lyso PC and about 5% of PE and
`others. The crude lipids and phospholipids are also harvested
`from the livers of frozen common dolphin (FIG. 2; about
`770 grams of the Wet liver material), tuna (about 720 grams
`of the Wet liver material) and salmon (about 810 grams of
`the Wet liver material), Which Were provided by a Fishing
`Company located in the province of Fujian in China, using
`the procedure described above.
`
`EXAMPLE 2
`
`Puri?cation of PC and Lyso PC from Crude Phos
`pholipids
`[0051] Mixtures of PC and Lyso PC species Were puri?ed
`from about 10 grams of crude phospholipids by silica gel
`
`chromatography using an axial pressure in an about 250
`mL-column equilibrated With chloroform/methanol (95/5);
`gradient elution Was performed With mixtures of chloro
`form/methanol 90/10 (v/v), 80/20 (v/v), 70/30 (v/v), and
`60/40 (v/v). PC species mixture Was eluted from the column
`With a mixture of chloroform/methanol/Water 50/ 50/ 1 (v/v/
`v), monitored by thin layer chromatography (TLC); and
`Lyso PC species mixture Was eluted from the column With
`a mixture of chloroform/methanol/Water 50/50/5 (v/v/v),
`monitored by TLC. The structures and percentage of puri?ed
`PC and Lyso PC species mixtures, Which Were obtained by
`intensities of their protonated molecules of mass spectrom
`etry [Brugger et al., Proc. Natl. Acad. Sci. USA, 94, 2339
`(1997); Liebisch et al., Clin. Chem. 48, 2217 (2002)], are
`shoWn beloW.
`
`(sn-l position) CH2_COOC15H31, COOC1sH33, COOC17H35, OC1sH33, OC1sH37, OC1sH35
`coocunp, cooc l5H3 1, COOC15H29,COOC17H33
`
`(Sn-2 position)
`
`(Sn-3 position)
`
`9
`
`c
`
`cooc 19H29 (EPA), COOC19H31, COOC21H33,COOC21H31 (DHA)
`CH3
`
`0
`
`OH
`
`CH3
`
`[M+H]+ Molecular Species*
`
`Dolphin (%)
`
`Shark(%)
`
`Tuna (%)
`
`Salmon (%)
`
`18:O*22:6
`834.6
`822.6 aa18:O*22:5***
`820.6
`17:O*22:6
`818.6 aa18:1*22:6
`806.6
`16:O*22:6
`794.6 aa18:1*20:4
`aa16:O*22:5
`792.6 aa16:O*22:6
`aa18:1*20:5
`16:O*20:4
`782.6
`16:O*20:5
`780.6
`16:O*18:1
`760.6
`746.6 aa16:O*18:1
`734.6
`16:O*16:0
`732.6
`16:O*16:1
`720.6 aa16:O*16:0
`718.6 aa16:O*16:1
`706.6
`16:O*14