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`H. L. JACKSON
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`Certifying Officer
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`Petition for Inter Panes Review
`Of U.S. Patent 8,278,351
`Exhibil
`ENZYMOTEC - 1062
`
`
`
`PA 905667
`
`
`
`
`
`r
`
`'
`
`United States Patent and Trademark Office
`
`October 03, 2002
`
`THIS IS TO CERTIFY THAT ANNEXED HERETO IS A TRUE COPY FROM
`THE RECORDS OF THE UNITED STATES PATENT AND TRADEMARK
`
`PRIORITY DOCUMENT
`SUBMITTED OR TRANSMITTED IN
`COMPLIANCE WITH
`RULE 17.1(a) OR (b)
`
`II I
`
`I
`I
`
`FILING DATE: July 27, 2001
`
`
`
`
`
`000001
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`

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`PROVISIONAL APPLICATION FOR PA TENT COVER SHEET
`to
`3m E
`This is a request for filing a PROVISIONAL APPLICATION FOR PATENT under 37 CFR 1.53(c).
` INVENTOR s
`S
`.
`Residence
`and either State or Forei-n Coun
`Cl
`Laval, Quebec, Canada
`
`Given Name (first and middle [If anvi)
`TINA
`
`Family Name or Surname
`SAMPALIS
`
`
`
`c.
`
`
`
`
`
`
`
`separately numbered sheets attached hereto
`E Additional invenlars are being named on the
`
`
`TITLE OF THE INVENTION (280 characters max)
`
`
`NATURAL MARINE SO ROE PHOSPHOLIPIDS COMPRISING FLAVONOIDS,
`POLYUNSATURATED FAT” ACIDS AND THEIR APPLICATIONS
`
`
`Direct all correspondence to:
`CORRESPONDENCE ADDRESS
`
`
`D Cu m
`Bar Code Labelhare
`Place CustomerNumber
`5“, 3, Number I
`I _._._.__.
`
`
`Type Customer Number hero
`OR
`
` SMART 8- BIGGAR ‘~ individual Name
`W Firm or
`
`
`
`Add ess
`
`
`
`1000 do la Gauchetiere St. W.
`"
`
`”wreak Quebec %—m_
`
`H33 4W5 Canada
`Telephone 514'954‘1500
`514'954'1 395
`
`ENCLOSEDflAPELlCATION PARTS [check all that neg!
`_
`
`Specificalion Number«Atom
`I
`21
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`[:1 CD“). Number
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`
`DraMnQIS) NumberafShoots
`[1"]
`E] om(Specify) E:::
`
`
`I] AppIication Data Sheet. See 37 CFR 1.76
`METHOD or PAYMENT or FILING FEES FOR THIS PROVISIONAL APPLICATION FOR PATENT (check one)
`
`lly
`
`
`
`-
`51
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`V
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`45474
`88187-1
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`I
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`000002
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`
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`
`Applicant claims small entity status. Son 37 CFR 1.27.
`*3
`D A check or money order is endosec to cover the filing fees
`N The Commissioner is hereby authorized to charge filing
`‘
`fees or credit any overpayment to Deposit Account Numbe
`‘
`E]
`Payment by credit card. Form PTO-2038 is attached.
`The Invention was made by an agency of the United States Government or under a contract with an agency 0‘ "‘9
`United States Government.
`No.
`
`
`
`_
`19 255°
`
`FILING FEE
`AMO u
`
`
`
`
`575-00
`
`
`
`
`
`D Yes. the name oi the us. Govommant agency and the Government contract numberara:
` .—
`
`
`
`
`07/27I01
`
`
`
`Respectful/y SUDMI ed.
`Date
`REGISTRATION No.
`SIGNATURE
`s Koeni
`(:Tappropn‘ato)
`TYPED “PRINTED ”AME
`613 232 2386
`Docket Number:
`
`TELEPHONE
`'
`USE ONLY FOR FILING A PROVISIONAL APPLICATION FOR PATENT
`Thlspoliecrlon or information is required by 37 CFR 1.51. The information is used by the public to file (and by the PTO to [arouse a provisional
`application. Conflplenuailty is govemod by 35 U.S.C. 122 and 37 OFR g.14. This collection is _estimaied to take 8 hours 0 comp eta. including
`gathering. preparing. and Submitting the complete provisional application to the PTO. Tlmo Will vary depending upon the individual case. Any
`comments on the amount of time you requtre to complete this form and/or suggesdons for reducing this burden. should be sent to the Chief
`lnformauon Officer. us. Patent and Trademark Office. 0.5. Department of Commerce. Washin ton. 0.0. 20231. Do NOT SEND FEES OR
`COMPLETED FORMS TO THIS ADDRESS. SEND TO: Box Provisional Application. Assismnt ommlssioner for Patents. Washington. DC.
`
`000002
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`

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`86187—1
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`P989. ”my F-mg
`
`NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING FLAVONOIDS,
`POLYUNSAEURATED FATTY ACIDS AND THEIR APPLICATIONS
`
`Field of the InventionM
`
`The present invention is directed to nutraceutical,
`pharmaceutical or cosmetic compositions, particularly to
`phospholipid compositions derived from natural marine or
`aquatic sources.
`
`Background of the Invention
`
`Phosphollpids are complex lipids containing phosphorus. The phosphatldos. lmcwn
`as phnsohcllpids. are usually divided into groups on the basis of compounds from
`which they are derived.
`in addih’on lo Mo chains of fatty acids they contain
`phasphorlc acid. glycerol and nitrogenous bases such as chcllno. The phosphcllplds
`considered most important are phosphalidylchclina. phosphatloylethanolamino and
`phoephatldyllnositcl. Their nature as amphophilic molecules provides them with
`unique phyelcochamical prom. Theirfunctlon as the pot-triple components of cell
`membranes makes phospholiplds essential for all vital cell processes. They are
`widespread as secretary and strucuaral components of the body and can mimic or
`enhance neural physiological processes.
`
`Pheaphollpld production may be either synthetic or through Mouton from mural
`tissues. The chief source of commercial natural phosphcllpide is soybean. egg yolk
`and cows (brain and liver). Since an individual phospholloid may contain a'varletycf
`fatty acid residues. lt may be dasalbed as puns only with this limitation In mind.
`Naturally ccwrrjng essential polyunsamratod fatty acids. can contribute to the
`activation of cellular metabolism The main fatty acid found in phosphollpid products
`is nucleic acid. pmm in soybean at more than 85%. The longest chain
`polyunsaturated fatty acids found in commercial available phosphnllpida either as
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`preparations or individually are 20:4 among the elcosenoids. known as araohidonlc
`acid and 22:6 known as docosahoxanoic acid.
`
`Arachidonlc acid is a fatty acid that is found as part of the phospholipid membrane.
`generally as part of phosphatidylcholine and phosphatidyllnositol. Adverse cellular
`stimuli will activate enzymes (phospholipeee) that cleave arachldonlc acid from the
`phospholipid backbone-in the cell membrane. Arachidonic acid. which Serves as the
`precursor for prootaolendins and prostacyclin (Fae, Pan) and thmrnboxeno (TXe)
`can then he metabolized by one of two major pathways: the cyciooxygenana (COX)
`pathway or the ilpoxygonase
`pathway. The COX pathway products, PEG: and
`PGH; can then be acted upon by thromboxane syndtase (in platelets) orprostacyclln
`synthase (in endothelium) to form TXs or PGlz. respectively. Arachidonic acid can
`also be acted upon by 5-iipoxygenase. primarily in leukocytes, to form iouirotrleneo
`(Us). One or more of these metabolites can mediate all the signs and symptoms
`associated with arachidonic acid. Le. inflammatory disease and pain.
`
`Platelets. lwkocytea. smooth muscle. and endothelium can produce vesoective
`substances. products of arachldonic acid metabolism such as proateglendins (P63).
`prostacyciin (P612). ioulrotrlenes (LTs). and thromboxanes (Dis). These substances
`can either act as vasodilators or as vasoocnstrictors. PGlz is essential in vascular
`function since it inhibits platelet adhesion to the vascular endotheliurn and has
`significant vesodllatation qualities. Dari-raped endothelial cells cannot produce PGlz.
`making the vessel more susceptible to thrombosis and vasospasm. Thromboxanes
`and leukotrlenes serve a vascular function during inflammation. generally producing
`vasomnotrlction. Proataglandins have a mailer role during inflammation. and also
`play a more subtle role in normal new regulation. moot notabiy‘as modulators of
`other connol mechanisms. Prostagiandino have both vasooenstrictor and vasodllator
`activities. Leukotrieneo and pmstaglandlns can also increase the ondotheliai
`membrane penneabillty thus promoting edema during Inflammation. Arachldonlc
`acid is naturally present in most phosphoilpld mixtures or emulsions available today.
`Nervonic acid (024:1) is also called selecholeic acid or tertracoecnlc acid. Nervonic
`acid is the symbol ofwhite matter in giucoelde, which is quantitatively contained in
`nerve tissue and white matter. The absence of nervonlc acid may result in cerebral
`leeion. fatigue. hypodynamia. amentle. and senile dementia.
`
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`
`is monounsaturated,
`teflraecsenic acid in another name,
`Nervonic acid,
`nen-oxidabieldeeomposed and absorptive.
`it is called a rare tonic as it is
`rare
`existent in nature. may be micro-obtained by compounded in cerebral ehondrleeome.
`Therefore.
`the substance is far below the demand of human body.
`in foreign
`countries, nervonic acid mainly eomes from shark brain and oil.
`
`'
`
`Flevonoids are polyphenoilc compounds ubiquitous in nature. They are categorized
`into ieoflavonea. anthoeyanidins. havens. flavonole. flevonee. citrus fiavonnide.
`heeperidin. chaiconee. cetechins, mtin. and flevenones. Eeeential flavor-roles. such
`as quercetin in onions and genietein in soy are actually considered subcategories
`rather than independent categories. Over 4.000 flavonoids have been identified in
`fruits. vegetables and beverages (tea, coffee. beer: wine and fruit drinks). Even
`though they have a similar molecular structure between them, their functions are
`different from each other. Fiavonoide have been shown to have antibacterial. anti-
`lnfiammatory. antialiergio. antimutagenic. antivirai. antineeplestic. anti-thrombotic.
`and vaeodilatory activity. Quereetin has been proven to block the "earbitoi pathway‘
`which is directly associated with diabetes as well as to prevent LDL-choieeteroi
`nxidetive damage. which is essential for the“maintenance of a healthy cardiovascular
`system.
`
`Fiavonoids are found in a wide range of fruits and vegetables. For example.
`Quereetin (a flavonol in vegetables, fruit and onions), Xanthonumol (a pnenylated
`chaloone in beer). isoxenthohumoi (a prenylated fiavanone in beer). Genietein (an
`Bottavane in any). Chaleenarinuenin (a non-prenyteted chalcone in citrus fruits) and
`Naringenin (a non-prenyiated flavanone in citrus fruits).
`in plants flavoncide have very Well defined functions. First. the accumulation of
`pigment in flower petals. seeds and leafs. Fiewe . as pollinators, must attract pollen
`earners. Second, they protect plants from UV damage. by absorbing W at the
`epidermal layer. Third. they protect the plants against insects and pathogens.
`
`show to be the limiting step in this pathway (197:1. Greasy). Another key enzyme of
`the flavonord pathway is the chaieone eynthase. It condenses three molecules of
`
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`maionyi-CDA with one molecule p-eeurmaruyi-COA to form a Cu intermediate.
`nafingenin ehaieone, with a R stereeehemisuy at the 2" carbon. Chaiacme
`immense. transforms the intermediate into the first newneid of the pathway. 25-
`flavanane (naringenin). This reaction is part of all major flavoneid bieswthuie
`Mmaye Chainene enthuse and chaieene isomer-aria farm a complex ensuring the
`right Bismuthemiatry (1995. Lysmf).
`
`depends upon their biochemimi structure. and more specifically. the position oftne
`hydmxyl groups. Epiatecnin gallate. epigalleeateenin senate. iuteelin and queruetin
`exhibit the highest antioxidant activity. followed by epigalleeateenin. game add.
`epicaiieehin, catechin. man. and dihydroquercefin. it is warm noticing at this point that
`the uniy difference between quercefin or luteolin
`(the most potent) and
`dihydrnquereetin
`{the least patent) is the double bond between the second
`
`OH
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`The potent antioxidant activity of fievonolde seems to be' the most important function
`of tievonoide. responsible for many of the above mentioned health benefits.
`
`The flavonoids moat recognised by'soientiets until today are:
`
`WW2
`
`Quercetln cheieone. is quercetin with an opened :3 ring and the oxygen found in the
`coring of quercetin convened into a hydrowl group. Queroetln is mainly found in tea
`and even more in green too.
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`Oligomeric proenthooyenldine are alignment: flevonoide, usually dimers and timers.
`based on the fleven- 3- ot. or catechii. molecule. sometimes attached to gellic acid.
`They are found in the bark of pine trees. in grape seed: and skins. in peanut skins.
`cranberries. tea. and other sources.
`
`Wm
`
`Ginkgo biiobe extracts contain 24% ginkgo flavone glycoeldes end 6% terpenee.
`They are extracted from the eldest living tree species. Ginno Bilobe. Scientific
`research nuggeats that the beneficial constituent: of since hiloba extracts are
`queroefin and myn'cefln.
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`Luteolin
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`P.10/38
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`F-709
`
`Luteolin is a flavoncid found in the same foods as
`
`apigenin (vegetables and fruits). Scientific research has
`
`shown that luteolin and quercetin can inhibit platelet
`activating factor and suppress the inflammatory response
`induced by allergens.
`
`Flavonoids have been Studied for the last 60 years.
`Their antioxidant activity is accepted as a scientific fact.
`Epidemiological, clinical, and laboratory research on
`flavonoids demonstrates the use of flavonoids in the prevention
`and/or treatment of cardiovascular disease, cancer,
`inflammatory conditions, asthma, peridontal disease, liver
`disease, cataracts and macular degeneration. Until today there
`has never been a flavonoid extracted from anything other than a
`plant, vegetable, fruit or algae.
`
`United States Patent No. 5,434,183 issued on July 18,
`1995 describes a phospholipid emulsion derived from marine
`and/or synthetic origin comprising polyunsaturated fatty acids
`and having anti-inflammatory and immunosuppressive effects and
`which promotes normal brain or retinal development and
`function. 0.5. 5,434,183 does not disclose the presence of
`flavonoids or nervonic acid (a mono—unsaturated fatty acid)
`the composition.
`
`in
`
`JP 2215351, published on August 28, 1990, discloses a
`method for extracting and purifying phospholipids from fresh
`krill. Krill is lyophilized and then extracted with ethanol to
`
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`

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`
`Jul-2H)!
`
`14:43
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`From-S&B/F&Cu.
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`86187-1
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`Summary of the Invention
`
`There is provided an extract comprising phospholipids
`derived from a marine or aquatic biomass.
`The extract is
`useful in the prevention or treatment of a variety of disease
`states and for the aesthetic enhancement of an animal,
`including human, body.
`
`There is also provided a novel flavonoid compound
`comprising an aglycone moiety and two or more glucosides,
`the
`novel flavonoid being derived from the phospholipid extract.
`
`Detailed Description of the Invention
`
`The phospholipid extract of the present invention may
`be extracted from a variety of marine or aquatic biomass
`sources. Preferred sources of the phospholipid composition are
`crustaceans,
`in particular, zooplankton.
`A particularly
`preferred zooplankton is Krill. Krill can be found in any
`marine environment around the world.
`For example,
`the
`the
`Antarctic Ocean (where the krill is euphasia superba),
`the
`Pacific Ocean (where the krill is eupbasia.pacifica),
`Atlantic Ocean and the Indian Ocean all contain krill habitats.
`In particular,
`the coastal regions of Mauritius Island and/or
`Reunion Island off Madagascar,
`the Canadian West Coast,
`the
`Japanese Coasr,
`the Gulf of St. Lawrence and the Bay of Fundy
`are krill habitats.
`
`The phospholipid extract of the present invention is
`preferably a product of initial processing of the biomass. As
`such,
`the phospholipids are extracted from the biomass grease
`as opposed to the oil,
`the oil being a product of subsequent
`processing steps of a biomass.
`Since the phospholipid extract
`is derived from the biomass grease,
`the viscosity of the
`
`000009
`
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`
`Jul-ZT-Ol
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`
`phOSpholipid extract tends to be higher than extracts from
`biomass oils.
`The extract has a very high natural stability
`with a peroxide value of zero or approaching zero and a good
`Oil Stability Index of less than about 0.2 Meg/kg after 20
`hours.
`
`Phospholipids are generally present in the extract in
`an amount of at least 40% w/w, preferably at least 45% w/w.
`More preferably,
`the amount of phospholipid is from about 45-
`60% w/w. A variety of types of phospholipids may be present in
`the extract. These include phosphatidyl ethanolamine,
`phosphatidyl inositol, phosphatidyl serine, phosphatidyl
`choline and sphingomyelin.
`
`The phospholipid extract preferably further comprises
`a number of other components.
`The extract may also comprise
`fatty acids, antioxidants and/or metals.
`
`Fatty acids found in the phospholipid extract may be
`saturated, monounsaturated or polyunsaturated fatty acids.
`Polyunsaturated fatty acids are particularly preferred,
`the
`omega-3 and omega-6 fatty acids being most preferred.
`In
`particular, doccsahexaenoic acid (DHA), eicosapentaenoic acid
`(EPA), myristic acid, myristoleic acid,
`lignoceric acid,
`linolenic acid, alpha linolenic acid, nervonic acid,
`linoleic
`acid, oleic acid, stearic acid, palmitic acid and palmitoleic
`acid are present in significant quantities. Arachidonic acid
`content of the extract is generally very low to non-existent
`despite the presence of phosphatidyl inositol and phosphatidyl
`serine. Other lipid components that may be present include
`monoglycerides, triglycerides and/or cholesterol.
`
`Free fatty acids are present in the extract in an
`amount of at least 4% w/w and preferably at least 5% w/w.
`Polyunsaturated fatty acids,
`in particular omega~3 fatty acids,
`
`~
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`w/w, and even more preferably at least 45% w/W, of the total
`lipids in the extract.
`DHA and EPA are generally the largest
`component of the fatty acids and preferably account for at
`least 35% w/w, more preferably at least 37%, of the total lipid
`content of the extract.
`
`Antioxidants present in the extract may include
`vitamin A (for example, all—trans retinol), vitamin E (for
`example, alpha—tocopherol), beta—carotene, astaxanthin (mainly
`esterified but non-esterified may be present), canthaxanthin
`and/or flavonoids. Antioxidants are preferably present in the
`extract in an amount of at least 200 mg/lOO ml.
`
`The extract of the present invention may comprise a
`novel flevonoid. This novel flavonoid appears to be similar in
`structure to 6,8-di~C-glucosylluteolin but comprises an
`aglycone moiety and two or more glucosides.
`The novel
`flavonoid possesses more and stronger double bonds than
`previously known flavonoids, has more hydroxyl groups and
`possesses more potent antioxidant aetivity.
`The molecular
`weight of the novel flavonoid is 510.
`The flavonoid may be a
`part of the mixture or may be actually attached to a
`phospholipid component of the extract.
`
`The metals present in the extract are preferably zinc
`and selenium- Zinc is preferably present in an amount of at
`least 0.05 mg/lOOg of extract while selenium is generally
`present in an amount of less than 3 mg/lOOg of extract.
`
`Extraction of the phospholipid composition is
`
`in commonly owned PCT publication number WO 00/23546, published
`on April 27, 2000,
`the disclosure of which is incorporated
`herein by reference.
`The extraction is generally carried out
`by successive acetone and alcohol treatments.
`For the
`extraction of the instant application,
`the preferred treatment
`
`0000011
`
`0000011
`
`

`

`Jul-ZT-Dl
`
`From-SEB/FECO,
`14:43
`86187-1
`
`+
`
`T-QBD
`
`P.l4/?8
`
`F-709
`
`u
`'‘L‘iiil
`
`1)“):Mm"d'“llJim}!
`
`
`31...?2173;.1M
`3““.Il'‘‘"”SELF"..3L...:5!1!M:a:..,.
`
`:—
`i
`3M!
`5,122...
`
`
`i-
`a..r
`
`flail
`45.,
`
`10
`
`involves the use of 100% acetone in the first extraction
`
`followed by extraction with a 95%/5% ethyl acetate/ethanol
`
`mixture-
`
`The procedure produces two successive lipid fractions
`
`and a dry residue enriched in protein,
`enzymes .
`
`including active
`
`Preferably, freshly harvested and finely divided
`marine and aquatic animal material is subjected to acetone
`
`extraction, for at least about two hours and preferably
`
`overnight. However, extraction time is not critical to the
`
`yield of lipid extracted. Particle sizes of less than 5mm are
`
`preferred.
`
`The eXtraction is preferably conducted under an
`
`inert atmosphere and at a temperature of about 5 degrees
`
`Celsius or less.
`
`The mixture may be agitated during extraction
`
`and a volume ratio of about 6:1 of acetone to biomass is
`
`generally most preferred.
`
`The solubilized lipid fraction is separated from the
`
`solid starting material by known techniques, for example, by
`filtration, centrifugation or sedimentation. Filtration is
`
`preferred.
`
`The residue is optionally washed with acetone to
`
`recover more lipid and the acetone removed by flash evaporation
`
`or spray drying. Water residue is allowed to separate from the
`
`lipid extract at low temperature.
`
`'The solid residue left on the filter from the initial
`
`extraction is suspended and extracted with 95/5 ethyl
`acetate/ethanol, preferably two volumes (original volume of
`
`The filtrate is evaporated yielding a second
`material).
`fraction of lipids. Extraction period is not critical although
`it is preferred to extract for about 30 minutes at a
`
`temperature below about 5 degrees Celsius.
`
`0000012
`
`0000012
`
`

`

`)
`Jul-27-01
`
`From-SiB/FlCn.
`14:43
`86187-1
`
`+
`
`T-989
`
`P.15/38
`
`F-709
`
`r
`
`11
`
`ma
`
`.9“.
`Me
`
`
`
`.1
`
`“'"Jln’m'11%|:‘0‘?“
`
`‘inm;
`n9"5....o
`vuq,
`{EliIa
`m.
`mt
`
`
`
`“ml!.II'
`
`
`
`.27.»lid!
`
`'~n"‘hm"mull
`
`.1“.fin.“Mum.“J.
`
`flum5524..1121322
`
`The phospholipid extract of the present invention may
`
`be used with or without other additives. Preferably, no other
`
`additives are used. However, if other additives are used,
`
`nutraceutical formulations may be made by methods known in the
`
`art.
`
`For example,
`
`the compositions of the present invention
`
`may be formulated in a conventional manner using one or more
`
`pharmaceutically acceptable carriers. Thus,
`
`the extract may be
`
`formulated for oral administration.
`
`For oral administration,
`
`the nutraceutical compositions may take the form of, for
`
`example, tablets or capsules prepared by conventional means
`
`with pharmaceutically acceptable excipients such as binding
`
`agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone
`
`or hydroxypropyl methylcellulose); filters (e.g.,
`
`lactose,
`
`microorystalline cellulose or calcium phosphate);
`
`lubricants
`
`(e.g., magnesium stearate, talc or silica); disintegrants
`
`(e.q., potato starch or sodium starch glycollate); or wetting
`
`agents (e.g., sodium lauryl sulphate).
`
`The tablets may be
`
`coated by methods well known in the art. Liquid preparations
`for oral administration may take the form of, for example,
`solutions, syrups or suspensions, or they may be presented as a
`
`dry producr for constitution with water or other suitable
`
`Such liquid preparations may be prepared
`vehicle before use.
`by conventional means with pharmaceutically acceptable
`additives such as suspending agents (g4g;, sorbitol Syrup,
`methyl cellulose or hydrogenated edible fats); emulsifying
`agents (§;g;,
`lecithin or acacia); non-aqueous vehicles (§;gL,
`almond oil, oily esters or ethyl alcohol); and preservatives
`(e.g., methyl or propyl p—hydroxybenzoates or sorbic acid).
`
`The phospholipid extract of the present invention may
`be used in the treatment or prevention of a variety of disease
`states including: liver disease; chronic hepatitis; steatosis;
`liver fibrosis; alcoholism; malnutrition: chronic parenteral
`nutrition; phospholipid deficiency: lipid peroxidation;
`
`0000013
`
`0000013
`
`

`

` Eu
`
`{5:3
`”a
`Nuii,
`
`.0114“
`
`.u'11%:1!!.
`etam:er’12..-1L3!
`
`5"Am!.11.
`is:
`E5
`
`Jul-ZT-UI
`
`14:44
`
`From-SQB/F&Co.
`
`86187-1
`
`+
`
`12
`
`T-SBQ
`
`P 16/38
`
`F-709
`
`disarrhythmia of cell regeneration; destabilization of cell
`membranes; coronary artery disease caused by
`hypercholesterolemia; high blood pressure; menopausal or post-
`menopausal conditions; cancer; hypertension; aging; benign
`prostatic hypperplasia; kidney disease; edema; skin diseases;
`gastrointestinal diseases; peripheral vascular system diseases
`(e.g.
`leg ulcers); pregnancy toxemia; and neurodegenerative and
`psychiatric diseases (e.g Parkinson’s, Alzheimer’s, autism,
`attention deficit disorder,
`learning disorders, mood disorders,
`bipolar depression, multiple sclerosis, muscular dystrdphy).
`
`The extracts are also useful for targeting tumors and
`can be used in conjunction with radioisotopes for diagnosing
`central nervous system tumors.
`The extract can also be used to
`reduce local fat deposits and reducing visible cellulite.
`The
`extract can also be used in aesthetics such as breast
`enlargement by acting on the lobular tissue of the breast and
`by increasing hydration of the breast.
`
`0000014
`
`0000014
`
`

`

`Jul-2?-01
`
`14:44
`
`From-SfiB/FACO.
`
`86187—1
`
`w
`
`+
`
`13
`
`Brief Description of the Drawings
`
`T-989
`
`P-lT/38
`
`F-709
`
`
`
`
`’9-.m
`"“1:ran}:gm".
`
`H
`
`(11”
`
`Figure 1 is a comparison of the mass spectrum of the
`novel flavonoid of the present invention with 6,8-di—C-
`glucosylluteolin.
`
`Figure 2 is an HPLC profile of the flavonoid fraction
`of the'bomposition of the present invention eluted with
`methanol.
`The HPLC shows an aglycone peak and two or more
`glucosides.
`
`Figures 3A to 3K are mass spectra (molecular
`analysis) of fatty acids attached to phospholipids in the
`composition of the present invention.
`
`Examples
`
`Materials and MEthods
`
`For analysis of lipids, samples were dissolved in
`solvent and standards were added. Lipid classes were isolated
`using silica gel and quantified. Fatty acid composition of
`total lipids and individual phospholipids was determined by gas
`chromatography.
`Pigments were measured by reversed phase high
`performance liquid chromatography.
`
`0000015
`
`0000015
`
`

`

`I 4! u
`
`1
`
`:
`
`14 59
`
`u
`
`ID=513 282 8440
`
`H
`
`Table 1. Fatty acid composition of 002.! llplds
`..-..
`
`31,2
`Fatty Acid Composition
`014:0
`014:1
`010:0
`616:0
`010:1
`010:0
`618:1
`010:2»:
`010:3“ OLA
`3 magnum
`0:
`010:403
`;3
`020:0
`:3
`020:1
`:3-
`020:2n5
`0213:3110
`020mm
`1220:2113
`0211:4113
`020:5n3 EPA
`022:0
`022-1
`022:2n0
`6220016
`6225118
`022:5»: DPA
`022:0»: DHA
`024:0
`024:1
`
`3 3.00
`z 0.01
`3 0.3
`a 20.00
`33.25
`3 1.00
`3 10.00
`32.00
`30.04
`3 0.01
`3 1.50
`3 0.05
`3 1.00
`3 0.05
`3 0.05
`5 0.50
`3 0.01
`30.20
`3 25.00
`3 0.01
`3 1.50
`30.03
`30.01
`_>_ 0.01
`30.50
`3 12.00
`3 0.01
`> 0.05
`
`.
`-
`EE
`
`'
`
`._
`
`~
`
`-
`
`0000016
`
`0000016
`
`

`

`Jul-Zl-DI
`
`14:44
`
`Fram-sia/Fmo.
`
`+
`
`T-QBQ
`
`RIB/38
`
`F-709
`
`.--
`5F“):
`
`“=
`
`“”51Jim”.1l'”5i
`
`‘ll.1!““1!.41‘
`.1"1-0“)!mu-
`”a.-
`.:1"““nfind!.43...
`i
`
`
`“ll‘”aim}:1,23
`
`I
`513331
`
`:3;
`
`Table 2. Fatty acid compualflan of total lipid:
`
`Satin-319:! (311009 lipid)
`Monunnturatad (311009 lipid)
`. Polyunsaturated (911009 lipid)
`Omega-3 (911009 lipid)
`Omega-5‘ (911003 lipid)
`
`232.00
`2 21.00
`a 45 .00
`340.00
`,_>_ 3.00
`
`Table 3. Lipid composition, vlhmlns A and E and pigment- nf minim
`
`
`Monoglydarldes (MG) (311 009 aampio)
`
`Triglycorldos (TG) (911000 sample)
`Fm Fatty Acids (FFA) (911009 sample)
`Cholestaml (911009 sample)
`
`z 0.7
`
`2. 3.00
`_>_ 5.00
`52.00
`
`Total Phosphnlipids (PL) (911009 sample)
`
`145.00
`
`Phasphatidyl Ethanolamine (PE) (9I100g sample)
`
`a 2.50
`
`. Phosphatldyl Inositol (Pl) (911 003 sample)
`
`Phasphatidyl Sarina (PS) (911009 sample)
`
`- Phasphatldyl Choline (PG) (911009 sampln)
`Sphingomyalln (911 009 sample)
`
`Vitamin A (pg/100ml)
`Vitamin E (pg/100ml)
`
`Bun-Garment: (1.1911 00ml)
`
`Wanlhln (mg/100ml)
`
`.
`
`Canthaxmthln (mgl100ml)
`
`Flavonold (mg/100ml)
`
`'
`
`a 0.20
`
`a 0.20
`
`g 35.00
`a 0.50
`
`a 1.400
`_>_ 15
`
`3, 1 .600
`
`z, 100
`
`z; 100
`
`2. 7.!)
`
`'
`
`0000017
`
`0000017
`
`

`

`Frum-S&E/F&Co.
`Jul-ZT-m ”=44
`i
`8 61,3 [—1,
`
`+
`
`lb
`
`T4”
`
`P. 19/39
`
`F-TU9
`
`-0
`:55
`j=;~.f
`h:
`in
`:5
`i:
`55-
`:2g
`:7“:
`a = :
`
`1.:
`3::
`
`Table H Fatty udd comm of phasphullpi‘ds in 33mph 50“.
`W '-l
`Mongolia lg}:m
`:
`cum
`‘
`014:1
`=
`cu-o
`'
`arm
`0:1qu
`.
`swans
`'.
`aim-o
`,_
`- C1!”
`i
`619:2“ (Ah-III: «In
`,
`g mmW.“
`'1
`sum:
`1 mm -
`mm (ad-2k:m
`5
`622:1!me
`exam (am;
`5
`1
`can»
`I
`cum
`cum»?
`624:1 nervank
`lam,“ ,.
`_
`Talal Ll Id
`
`_
`
`0.85
`0.87
`14.23
`1 .03
`9.06
`0.5
`0.72
`5.08
`1 .83
`0.56
`0.84
`11.53
`1.35
`16.81
`18.95
`4.39
`2.38
`3.53
`4.1a
`99.99
`25.8
`
`.
`
`.
`
`.
`“III I.
`
`0000018
`
`0000018
`
`

`

`Jui-ZY-Ul
`
`14
`
`M4
`
`Frum-SlB/F&Ca.
`
`T-989
`
`P.20/38
`
`F-709
`
`\‘1
`
`
`flamaNumfio
`:N.mHU
`
`ficEflofimofi5Em133now...
`
`Q!
`l!)H
`
`II!IIII
`
`l
`Ias53new.Mac5&8mmcam...cm80gE;mam.20I
`
`38IEEI":8go35'
`
`8.oagmfimofi
`
`333.335
`
`wcflaocu
`
`qflfifiamofi
`
`Hofimofi
`
`Hauflmfimofi
`
`mnwzmaoamnum
`
`mamaonu
`
`dmgumfimofi
`
`Hofimofi
`
`inflgamofi
`
`mamEmHocmguw
`
`
`
`
`
`«02¢wavwmwaoammonmmaymcwmwfimfioumvflmflofimwonmHocogfiwomfiou3.0mhum—mmmomwmzfimcfl.m2.1an
`
`
`
`
`
`.74.me
`
`0000019
`
`0000019
`
`
`
`
`
`
`

`

`V
`Jul-27-01
`
`14:44
`8 6187-1
`
`From-S&B/F£Co.
`
`+
`
`‘8
`
`T-989
`
`PIE/38
`
`F-709
`
`Tum. a. final companion and «Mat-midn- nflhoWk“ mm
`Zinc (ma/1mm
`>01
`
`Salaam (ma/1009)
`
`3mm Malaya
`
`' < 2
`
`Q5 ppm
`
`EE:
`:
`
`T I
`
`.
`
`h
`
`I
`
`WHO value (MW
`Oflsubimylndax (mam hours) (mEq/kg)
`Summation India:
`Iodine mm (36)
`
`4: 0.1
`«2.1
`70 - 180
`60 - 130
`
`
`
`0000020
`
`0000020
`
`

`

`Jul-ZY-Ul
`!
`
`14:44
`
`Fram-SGB/FECO.
`
`+
`
`T-QBQ
`
`P.22/38
`
`F-709
`
`)
`
`UVBélnoucED SKIN emcee
`
`H
`
`”w.
`
`Oygcnggg
`To evaluate the photoprctective potential of krill extract against UVB-induced skin
`cancer.
`
`81!19v DE§1§E
`Prospective randomized control trial
`Statistical significance p<0.05
`
`wP
`
`ro-clinical
`
`P
`
`|
`
`Type : Nude Mice
`Strain : CS7BL6 Nude Congenic Mice - BGNU -T (heterozygotes)
`(Preference of specific type because of proven susceptibility to skin cancer).
`
`ii: We.
`E“;
`Number of nude mice = 96
`Randomization groups : 48 placebo :
`
`
`
`.iiilaffix”.£2
`
`$3
`
`
`
`::
`
`16 per os
`16 local application
`18 per es and local application
`
`48 krill extract: 16 per os
`16 local application
`16 per as and local application
`
`in order to establish efficacy of knil extract for the prevention of skin cancer. the
`test will be conducted as a randomized double blind controlled trial (both the
`pathologist and the research assistant will be blind). Half of the mice will be
`treated orally or topically or both with extract containing 100% by weight of krill
`extract and the other half will undergo the same method of treatment with a
`placebo. The groups will be divided as follows:
`
`Nutrition: Week 1 :fat-frae chow
`Week 2 — 20 : according to group
`
`0000021
`
`0000021
`
`

`

`"
`
`__:
`:15
`
`Jul-27-fli
`X
`
`14:44
`
`From-SlB/FliCo.
`
`‘i'
`
`10
`
`T-QEB
`
`P.23/38
`
`F-709
`
`NTALD S
`
`'
`
`’ The mice will be divided in six groups as l'olicwe:
`Group A: fat-free chow with supplementation of soy extract (20% of total calories)
`Group 8: fat free chow (100% of calories) ¢ local application of soy extract 2
`times
`_
`per day
`Group 0: fat free chow with supplementation of soy extract (20% of total calories)
`4- local application of soy extract 2 times per day
`Group D: fat-free chow with supplementation of krill extract (20% of total calories)
`Group E: fat free chow (100% of calories) + local application of krill extract 2
`times per day
`Group F: fat-free chow with supplementation of krill extract (20% of total calories)
`+ local application of krill extract 2 times per day
`
`Week 2 - 20: uxgoradiaticn uslng a fluorescent test lamp. emission spectrum 27o
`—
`nm.
`Week 3 - 20: liquid from blisters fanned is examined for PGEZ levels
`Week 3 . 20: mice are anaesthetized with other and sacrificed when malignant
`tumours have formed or at the and of the 20 weeks.
`Skin is examined by pathologist for signs of carcinogenesis.
`
`0000022
`
`
`.I“1in.111“'"fl1!11’"t'i.umgiiuu
`
`it.I'"s
`
`a i
`
`iZZiiii31
`
`323..
`J"'1'
`
`32ml;
`“an«M:_03am.
`
`~13“
`
`0000022
`
`

`

`v
`Jul-27-01
`
`From-SéB/F‘Ca,
`14:44
`86187—1
`
`T-989
`
`P.24/38
`
`F-709
`
`+
`
`21
`
`Abstract
`
`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.
`
` m u
`
`.5"...3,...”1‘"W.1M5£51,“,1:
`
`m
`
`0000023
`
`0000023
`
`

`

`Jul-ZY-M 14:44
`
`From-saB/Fmo,
`
`T-989
`
`P. 25/38
`
`F-‘(DD
`
`
`
`.JosiavndualsaufiaiflwégaflmrzEiseflflgoid..