PA 905667
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`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
`
`i1
`
`{ PRIORITY DOCUMENT
`SUBMITTED OR TRANSMITTED IN
`COMPLIANCE WITH
`RULE 17.1(a) OR (b)
`
`§
`‘
`
`. L. JACKSON
`Certifying Officer
`
`Petition for Inter Partes Review
`Of U.S. Patent 8.278.351
`Exhibfl
`ENZYMOTEC - 1062
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`PTOISB/16 (am
`Approved for use tnrougi110l31I2OD2. OMB 0851-OD
`US. Patent and Trademark Oiiice; US. DEPARTMENT OF COMMERC
`Under the Paperwork Reduction Act or 1995. no person: are raqulnsa to respond to a collection (II lrttormatlon unless it uispiays a van: OMB mnlml numtter. C‘-I ?—
`
`PROVISIONAL APPLICATION FOR PATENT COVER SHEET
`This is a request for filing a PROVISIONAL APPLICATION FOR PATENT under 37 CFR 1.53(c).
`
`INVENTOR s
`
`'5'
`
`Residence
`and either state or Foreln Court
`Cl
`Laval, Quebec, Canada
`
`
`
`Given Name (firs: and middle [if anvi)
`TINA
`
`Family Name or Surname
`SAMPALIS
`
`s'nTSOTF
`
`
`
`
`
`
`
`
`‘-
`
`separately numbered sheets attached hereto
`E Additional inventors are being named an the
`TITLE OF THE INVENTION (280 characters max)
`NATURAL MARINE SO ROE PHOSPHOLIPIDS COMPRISING FLAVONOIDS,
`POLYUNSATURATED FATTY ACIDS AND THEIR APPLICATIONS
`
`
`
`
`
`
`
`Direct all correspondence to:
`CORRESPONDENCE ADDRESS
`Place Customer Number
`_...._.._..
`..,,,;,,,em,,,,‘,,,,§
`III cw<=mer~um»er C::'_":::J
`OR
`Type Customer Number here
`
` SMART 8. BIGGAR
`
`
`Firm or
`pr‘
`4 individual Name
`Add ass
`
`"
`1000 do la Gauchetiere St. W.
`M°'“'ea'- °"eb°° —
`514-954-1395
`"33 ‘W5 °“"“=
`Telephone 51‘*954‘1_‘-E90
`
`________ _ _» LICATION PARTS cIlet:kaIlrt1a_:§pg_I
`
`Specification Numb:-roIPa99s
`I
`21
`_ l
`E] cD(5)‘Numb,-3, C:
`
`Drawingisi Numberafsheets [E E] om(Spam) l:::
`
`D AppIIcat'lon Data sheet. See 37 GFR 1.76
`METHOD OF PAYMENT OF FILING FEES FOR THIS PROVISIONAL APPLICATION FOR PATENT (check one)
`
`>3
`Applicant claims small entity status. Sea 37 CFR 1.27.
`D A check or money order is enclosec to cover the filing fees
`
`
`N The Commissioner is hereby authorized to charge filing
`575-00
`‘
`fees or credit any overpayment to Deposit Account Numbe
`
`
`
`D 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 age-HG! OI I09
`
`
`
`United states Government.V
`No.
`
`
`D Yes. the name oi the us. Govomment agency and the Govamment contract numbararo: __
` __
`
`Respectfully suomi ed.
`
`
`
`or/27io1
`s‘GNAT”RE%
`REGISTRATIONNO.
`
`
`
`try
`
`
`‘_
`23“
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`_
`19 255°
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`FILING F55
`AMO “
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`
`
`-
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`We
`
`46474
`
`5 Meme
`TYPED or PRINTED NAME
`
`TELEPHONE
`613~232-2486
`USE ONLY FOR FILING A PROVISIONAL APPLICATION FOR PATENT
`Tllls_col_lection of information is required by 37 CFR 1.51. The information is used by the public to His (and by the PTO to [srocess a provisional
`appiloalton. Coniiplenuality is govemad by 35 u.s.c. 122 and 37 cm 1.14. This collecuon is estimated to take 8 hours 0 comp eta. inoluding
`gathering. prepanng. and submitting the cornplete provisional application to the PTO. Time will vary depending upon the Individual case. Any
`comments on the amount of time you require to complete this form andlor suggestions for reducing tt-its burden. should be sent to the Chief
`lnformauon Officer. U.s. Patent and Trademark Otfice. U.S. Department of Commerce. Wastlln ton. 0.0. 20231. Do NOT SEND FEES OR
`COMPLETED FORMS TO THIS ADDRESS. SEND TO: Box Provisional Apnllcafion. Assistant ommlssioner for Patents. Washington. DC.
`
`(:7apprapnata)
`Docket Number:
`
`861 SM
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`000002
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`From-SIB/Flco.
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`86187-1
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`NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING FLAVONOIDS,
`POLYUNSAEURATED FATTY ACIDS AND THEIR APPLICATIONS
`
`Field. of the Invention
`
`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
`
`ks-...
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`
`Phaaphollpids are can-rpleur lipids cnntaining phosphorus. The plrasphatldes. known
`as plrnsphollpids. are usually divided into groups on the basis of cnmpnunds from
`which they are derived.
`in addiflon in ma chains of fatty acids may contain
`phosphoric acid. glyceml and nit:-oganous bases such as challna. The pncsphuliplds
`considered must impnrtam are phaspnatidyidrclina. phcsphafldylethanalamina and
`phaaphatidylinasitol. Their nature as amphophilir: molecules pmvidas them with
`unique pllyalcachamical praparxies. Tlleirfuncxlcn as the principle components of cell
`msmhranes makers phcspholiplds essential for all vital cell processes. Thsy an
`widespread as secretary and structural aumpananls of the body and can mimic or
`enhance namral physiological processes.
`
`Phcsphollpld production may as either synthetic or thmugh an-action from natural
`tissues. The chief source of aummercial natural pfiuaphollpids is soybean. egg yolk
`and cows (brain and liver). Slncs an individual pg-laaphallpid may contain a'variatyuf
`fatty acid residues. it may be described as pure ally with lhis llmilaflon In mind.
`Naturally ucwrljng essential polyunsaturated fatty acids. can contribute to the
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`preparations or individually are 20:4 among the eicosanoids. kncwn as arachidonlc
`acid and 22:6 known as doccsahexanoic acid.
`
`Arachidonlc acid is a fatty acid that is found as part of the phosphclipid membrane,
`generally as part of pi-rosphatidylcholine and phosphatidylinosiioi. Adverse cellular
`stimuli will activate enzymes (phoaphoiipeea) that cleave arachidonic acid from the
`phosphoiipid backbone-in the cell membrane. Arachidonic acid. which serves as the
`precursor for prostagiandins and prostacyclin (Pee. Paiz) and thmrnbcmne (Tits)
`can then he metabolized by one of two inajor pathways: til! cycioaxycenana (COX)
`pathway or the iipoxygenasa
`pathway. The COX pathway products. PEG: and
`P591: can then be acted upon by thromboxane synthase (in platelets) orprostacyciin
`eynthase (in endothoiium) to form Txs or PGI2. respectively. Arachidonic acid can
`also he acted upon by 5-lipoxygenase. primarily in leukocytes. to form ieukotrienes
`{LTs). one or more of these metabolites can mediate all the signs and symptoms
`associated with arachidonic acid. i.e. inflammatory disease and pain.
`
`prostacyclin (P613), iouicotrlenes (E75). and thmmboxanes (Txs). These substances
`can either act as vasodilators or as vasoizonstfictors.
`PG!-2 is essential in vascular
`function since it inhibits platelet adhesion to the vascular endolhelium and has
`
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`acid is the symbol ofwhite matter in glucoside. which is quantitatively contained in
`naive tissue and white matter. The absence of nervonlc acid may result in cerebral
`lesion. fatigue. hypodynaniia. amentia. and senile dementia.
`
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`is monounsaturated,
`tertraeosenic acid in another name,
`Nervonic acid.
`non-oxldableldecomposed and absorptive.
`it is called a rare tonic as it is
`rare
`existent in nature. may be micro-obtained by compounded In cerebral chondrlosome.
`Therefore.
`the substance is far below the demand of human body.
`In foreign
`countries, nervonic acid mainly comes from shark brain and oil.
`
`'
`
`Flevonolds are polyphenolic compounds ubiquitous in nature. They are categorized
`into ieoflevonea, anthocyanidins, flevano.
`tlavonole. flavenes. citrus flavonoide.
`heeparidln. chalconee. oetechins, rutin, and flevenones. Essential iiavonolds. such
`as quercetin ln onions and genistain in soy are actually considered subcategories
`rather than independent categories. Over 4.000 flavonolds 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
`difierent from each other. Flavonoido have been shown to have antibacterial. anti-
`
`
`
`Flevonolds are found in a wide range of fruits and vegetables. For example.
`Queroatin (a flavonol in vegetables, fruit and onions), xanthohumol (a prenylated
`chalcone in beer). lsoxenthohumol (a prenylated fiavanone in beer), Genietein (an
`isoflavone in soy). Gnaleonerinenin (a nomprenyiated chaloone in citrus fruits) and
`Narineenin (a non-Prflnylated tlavanone in citrus fruics).
`in plants flavonoida have very well defined functions. First. the accumulation of
`Piumant in flower petals. seeds and leafs. Flowe . as pollinators, must attract pollen
`carriers. second. they protect plants from UV damage. by absorbing W at the
`epidennel layer. Third. they protect the plants against insane and pathogens.
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`maionyl-coA with one molecule p-oourmaroyi-CoA to form a C1: ifltlflfifidmfi
`naringonin chaioono, with a R storeoohomisny at the 2"‘ carbon. Chalcone
`iaornaraso. transforms the intermediate into the first rlavonoid of the pathway. 25-
`flavonona (naringenin). This inaction is part of all major flavonoid bioswlhoois
`oatmvays chaloone synihase and chalcone isomer-one form a complex ensuring the
`right staraochemintry (1995. Lysiar).
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`depends upon their hloohemioai structure, and mom spocifioaily. in: position oftno
`hydroxyl groups. Epioatacnin gallate. epigallocatoonin galiato. lutoolin and quarcotin
`exhibit the highest antioxidant activity, followed by opigaliooatecnin. some add.
`epioaiaechin, cateohin. rutin. and dihydroquoroofin. ii is worth noticing at this point that
`the oniy difference between quoroofin or lutoolin
`(the most potent) and
`dihydrnqueroatin
`{the least potent) is the doubio bond batwoan the second
`
`OH
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`The patent antioxidant auaivity of fievonolde seems to be‘ the most important function
`of flevonoide. responsible for many of the above mentioned health benefits.
`
`The flavonoids moat recognised byseienfists until today are:
`
`
`
`Cluercetln chaioone. is quercetln with an opened :3 ring and the oxygen found in the
`C-ring of queroetin convened into a hydrowl group. Queroetln is mainly found in tea
`and even more in green tea.
`
`Iia
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`
`Ollgomeric proenthooyenidine are oligomeno flevonoide. usually dimers and trirnenl.
`based on the fleven- 3- ol. or cateohin. molecule. sometimes attached to gellio acid.
`They are found in the bark of pine trees. in grape seeds and skins. In peanut skins.
`oranbeniee. tea. and other sources.
`
`§in.issz9.£1I£=_b_a.Emc_!
`
`Ginkgo bilobe extracts contain 24% ginkgo flavone glycesides end 6% terpenee.
`They are extracted from the eldest living tree species. Glnno Bliobe. scientific
`research auggeets that the beneficial constituent: of ginge hlloba extracts are
`quercefin and myrieefln.
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`Luteolin is a flavonoid 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. U.s. 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
`
`chromatography to produce high purity phosphatidyl choline and
`phosphatidyl ethanolamine. There is no disclosure of a
`phospholipid extract comprising a flavcnoid or nervonic acid.
`
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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.
`preferred zooplankton is Krill.
`
`A particularly
`
`the
`
`Krill can be found in any
`marine environment around the world.
`For example,
`the
`Antarctic Ocean (where the krill is auphasia superba),
`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,
`Japanese Coast,
`the Gulf of St.
`
`are krill habitats.
`
`Lawrence and the Bay of Fundy
`
`the Canadian West Coast,
`
`the
`
`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
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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 Meq/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-
`6o% w/w. A variety of types of phospholipids may be present in
`the extract. These include phosphatidyl ethanolamine,
`phosphatidyl inositol, pnosphatidyl serine, phospnatidyl
`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, docosahexaenoic 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.
`
`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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`Jul-27-01
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`14:43
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`From-S&B/F&Co.
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`86187-1
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`T-989
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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, al1—trans retinal): 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/100 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 activity.
`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 O0/23546, 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
`
`A
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`Jul-2T-DI
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`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
`
`..d
`I‘-tun‘-u
`
`I"I‘'‘‘‘'HH:f"‘li.!|'JP“):1l“"‘d"'ll,fl‘"!I
`
`
`
`
`
`
`
`
`
`
`41*‘112‘.5I.”'_§£"1!4'32}:521...32...?»i72.‘S..11...!
`
`i
`
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`
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`
`S %
`
`0000012
`
`

`
`Jll|'27-U1
`
`FI'0m"3&B/FRED,
`14:43
`86187-1
`
`+
`
`7.9“
`
`p_15/33
`
`F_mg
`
`11
`
`3!
`
`JH_._..l}:::l5l’".I|'2!"?111:1".Il“‘.|§~'|£:".gg11.’...{:11sa...,:o!...slM:....u.._».....
`
`
`
`
`
`
`
`
`
`
`..,,...,,..._,,‘W..:m,....,,,,,£3»2!»)!AL...
`
`
`
`11.....2...!Z.A!
`
`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 (§Lg;, pregelatinised maize starch, polyvinylpyrrolidone
`
`or hydroxypropyl methylcellulose): filters (§;g;,
`microcrystalline cellulose or calcium phosphate);
`
`lactose,
`lubricants
`
`(g;g;, magnesium stearate, talc or silica); disintegrants
`
`(E;E;] potato starch or sodium starch glycollate); or wetting
`
`agents (§;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 product for constitution with water or other suitable
`
`Such liquid preparations may be prepared
`vehicle before use.
`by conventional means with pharmaceutioally acceptable
`additives such as suspending agents (§;g;, sorbitol syrup,
`methyl cellulose or hydrogenated edible fats); emulsifying
`agents (g;g;,
`lecithin or acacia); non-aqueous vehicles (gggy,
`almond oil, oily esters or ethyl alcohol); and preservatives
`(g;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
`
`

`
`Jul-ZT-U!
`
`14:44
`
`F'°”'s‘B’F‘c°'
`
`86187-1
`
`+
`
`12
`
`T-989
`
`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 dystrophy).
`
`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.
`
`
`
`..'72“:éflé
`
`
`
`=.’===
`$3§'-~‘3«'
`5‘;
`
`
`
`
`
`4!‘“"41.11’4:‘-a.112...:iYL.,{L31
`
`:‘T.'3’a’
`
`0000014
`
`0000014
`
`

`
`Jul-Z?-01
`
`14:44
`
`From-5&8/F460.
`
`86187-1
`
`Brief Description of the Drawings
`
`13
`
`+
`
`r-ass
`
`P. was
`
`F-709
`
`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
`
`

`
`ur 4! a1
`
`14:59
`
`ID=5.l3 232 8449
`
`N
`
`Table 1. Fatty acid composition of will llpldt
`v
`I
`'
`-
`0
`
`w\,!
`Fatty Acid Composition
`014:0
`
`3 3.00
`
`
`
`
`
`,,g;,_,u;;;;;,;1;,_,M:11.’...s‘».’:i1i:..h.s:L..!1...I:a2'.....1L..i-'3...’!..._.-...,5...“_,.-.....,,‘,.-R...“,,,0.1511,;'_u-3;.fir51-4-uat-3:“11'-1.:gvgn35..M;
`
`
`
`
`
`
`
`3 0.01
`
`_>_ 0.3
`
`_>_ 20.00
`
`3 3.25
`
`g 1.00
`
`_>_ 10.00
`
`3 2.00
`
`2, 0.04
`
`_>_ 0.01
`
`_>_ 1.50
`
`_>, 0.05
`
`3 1.00
`
`_>, 0.05
`
`3 (3.05
`
`5 0.50
`
`3 0.01
`
`014:1
`
`618:0
`
`610:0
`
`C18.-1
`
`cu.-a
`
`O18:1
`
`c10:2n0
`
`C18:3n€ OLA
`
`c18:3n3 ALA
`
`018:4!!!
`
`620:0
`
`020:1
`
`C20.-2n6
`
`@0:3n8
`
`C20.-Ins
`
`c20:8n3
`
`c20:4n3
`
`C20:5n3 EPA
`622:0
`
`022:1
`
`c22.'2n6
`
`C22:-Inc
`
`022.608
`
`(:22.-5n3 DPA
`
`022.-ans DI-IA
`
`624:0
`
`cu.-1
`
`0000016
`
`0000016
`
`

`
`Jul-27-DI
`
`l4:44
`
`F rum-S!.B/F&Co,
`
`T-939
`
`P.l8/38
`
`F-709
`
`B
`
`Table 2. Fatty acld compuultian of total Illllfli
`
`Saturated (311009 llplcl)
`
`Monunnturatad (311009 llpld)
`
`" Polyunsaturated (911009 llpld)
`
`Omega-3 (911009 lipid)
`omega-0‘ (gnoog llpld)
`
`232.00
`
`321.00
`
`_>_ 45.00
`
`_>_40.0o
`
`__>_ 3.00
`
`all
`
`
`
`;ITJxI"’a:.1‘l::"..tl"‘l§55"}:m13...?!
`
`
`ii“....}1'.~‘sln,."
`.¥JL00'.::.:L.:21:;z,,.-3;
`
`
`"fl”‘g1l”‘lI‘nu’-wn,.a-11"‘);,3"-’r3='
`
`
`
`
`3.0!!.43....5l.‘L.2452:...-’.‘..J£
`
`Table 3. Lipid compaslflon, vitamins A and E and pigments of minute
`
`
`Ilonoglydarldes (MG) (an 009 eamplo)
`
`Triglycerides (TG) (911000 sample)
`
`Frau Fatty Acids (FFA) (911009 sample)
`
`Chalostarul (911009 sample)
`
`,>_0.7
`
`2 3.00
`
`_>_ 5.00
`
`5 2.00
`
`Total Phosphalipids (FL) (gI1009 sample)
`
`3 45.00
`
`Phusphatidyl Ethanolamine (PE) (9I100g sample)
`
`Phosphatldyl lnosltol (F!) (911003 sample)
`
`Phasphatidyl Sarine (PS) (an 009 sample)
`
`- Phusphatldyl choline (PG) (911009 sampln)
`
`Sphingomyelln (911 009 sample)
`
`Vltamln A (pgI100ml)
`
`Vitamin E (ugI100ml)
`
`Bun-carotene (11911 00ml)
`
`Azmxanlhln (mgI1 Doml)
`canthaxanthln (mgl100ml) H
`Flavonold (mgllauml)
`'
`
`32.50
`
`30.20
`
`3 0.20
`
`5-_ 35.00
`
`5-_ 0.50
`
`31.400
`
`3 15
`
`_>, 1,600
`
`3, 100
`
`_>:_ 100
`
`> 7-0.
`
`0000017
`
`0000017
`
`

`
`Jul-27-01
`i
`
`14:44
`From-S&E/F&Co.
`UOLBI-L
`
`4,
`
`lb
`
`T-989
`
`P.l9/38
`
`F-709
`
`Table H Fcuy udd acflnpoallinn of phcsphullpiiis in oumpla 504A.
`.
`Phunggnllfi FAQ0
`taam
`cu.-1
`cnuo
`cm.-1
`¢1l:lnl
`Giflfinfl
`cam
`¢1l:‘!
`cnuantmwuieauq
`cvuummlufldluda
`cuauma
`.
`(:10.-Sn.)
`cu.-otaa-ck.-aua
`czmflhmdcudm
`cram mm)
`622:8 as
`cu.-a
`¢2fl?n?
`824:‘! net‘ vatfic
`Total 56
` —cr—?-
`Total Ll Id
`II!!! In
`25.8
`
`0.85
`0.87
`1t23
`1.03
`9.06
`03$
`0-72
`5.08
`133
`0.66
`Q54
`11.53
`1.35
`16Jfl
`19-95
`4-39
`2.38
`353
`4.13
`991%)
`
`
`
`
`Hz-.2222:.55::35:33:1;
`‘$5;)-Nu_,..,,.....,...,.
`
`5
`
`
`
`0000018
`
`0000018
`
`

`
`Jui-Z?-Ul
`
`14:44
`
`From-SQB/F&Ca.
`
`T-989
`
`P.2U/38
`
`F-T09
`
`\'1
`
`
`
`
`
`musuxwfiuwmwaoammocmma»mawmwnaedumnwmwfioamwonmnocowuwwomfiouvwommuumwmomwmzmmcm.m
`
`
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`
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`Hunmflmm
`
`manna
`
`..u.uEflo._%oam5Em438.noE
`
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`mnwumm
`
`aanwumgmmocm
`
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`
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`
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`
`0000019
`
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`
`
`
`
`
`
`

`
`V
`Jul-27-01
`
`14:44
`
`From-5&8/Ftc0:
`
`86187-1
`
`\3
`
`*
`
`1-939
`
`P.21/38
`
`F-709
`
`Tank 6. am: companion and ulvnnt-nulduu nflho phonphallpkl mlxumo
`Zinc (mg/wag)
`>0.1
`
`solnnium (mu/9009)
`
`Sohmnt nsldua
`
`‘ < 2
`
`<25 ppm
`
`1'
`
`I
`
`.
`
`h
`
`I
`
`Poraxldo value rmsqlky)
`Ollstabilflylndax (cuarzo hours) (mEqIkg)
`Slponlflcatlanlndax
`Iodine inch: (36)
`
`4: 0.1
`<o.1
`70-180
`60 - 130
`
`
`
`0000020
`
`0000020
`
`

`
`Jill-'27-Di
`F
`
`14:44
`
`From-S68/FECO.
`
`3
`
`Uvaélnpucao SKIN cmcee
`
`+
`
`Vi
`
`T-989
`
`P.ZZ/39
`
`F-709
`
`--v.
`
`.
`952593255
`To evaluate the photoprotectlve potential of lrnll extract against UVB-induced skin
`cancer.
`
`81;lg? DE§_[§fl
`Prospective randomized control trial
`Statistical significance p<o.o5
`
`§1u91El:e§a
`Pro-clinical
`
`P
`
`i
`
`Type : Nude Mice
`Strain : C57BL6 Nude Congenic Mice - BGNU -T (heterozygotas)
`(Preference of specific type because of proven susceptibility to skin cancer).
`
`ov
`
`1-ac
`
`Number of nude mice = 96
`Randomization groups : 48 placebo :
`
`16 per os
`16 local application
`1 8 per es and local application
`
`
`
`
`
`"“‘llé;”'and}Amysin...-‘innit33....3i.._.;-nu)!,
`L..:""M",:
`
`
`EE.V!
`
`1
`
`S‘!
`
`$13
`
`
`
`48 krill extract: 16 per os
`16 local application
`16 per as and local application
`
`in order to establish efllcacy 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 zfat-free chow
`Week 2 — 20 : according to group
`
`0000021
`
`0000021
`
`

`
`J“"'27'°' W“
`
`F'°“"5WF‘°°v
`
`+
`
`T-989
`
`9.23/as
`
`F-709
`
`NTALD S
`
`'
`
`’ The mice will be divided in six groups as follows:
`Group A: fat~free chow with supplementation of ‘soy extract (20% of total calories)
`Group 8: fat free chow (1oo% of calories) + local application of soy extract 2
`times
`_
`per day
`Group 6: 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 (10096 of calories) 4- 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: W3 radiation using a fluorescent test lamp. emission spectrum 270
`— 400 nm.
`Week 3 - 20: liquid from blisters formed is examined for PGE2 levels
`week 3 - 20: mice are aneesthetlzed with ether and sacrificed when malignant
`tumours have fanned or at the end of the 20 weeks.
`Skin is examined by pathologist for signs of carcinogenesis.
`
`
`
`"it.I‘ %
`
`aa
`
`
`
`“i2"‘rP"!i:1!‘-":"1i:r?‘_ri1.111“mu1!an~ii~
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`0000022
`
`0000022
`
`

`
`Jul-27-U1
`3
`
`14:44
`
`From-8&3/Ftca.
`
`86187-1
`
`T-989
`
`P.24/38
`
`F-T09
`
`+
`
`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.
`
`
`
`3 §
`
`EH5isiima
`
`‘=1
`5‘m
`
`0000023
`
`0000023
`
`

`
`Jul-27-01
`
`14:44
`
`From-8&3/Ftco.
`
`T-989
`
`P. 25/38
`
`F-‘(D9
`
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`
`
`
`
`
`3c.¢§ana3n_.3!J.u.31:=.2§8«.52..lfil._.-cqfllnnvlzn.
`
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`
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`
`
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`
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`n:.uu_:§luu=.n.3..u¢3ua3.nn_i.u..a.cH.133nGalois
`
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`
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`
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`
`
`
`
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`

`
`Jul-27-01
`
`I4:44
`
`From-sae/Faco.
`
`41-939
`
`P.2s/as
`
`F-709
`
`86187-1
`
`95’:
`2.-.3 =
`$3
`
`
`
`
`
`
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`
`0000025
`
`

`
`Jul-Z7-D1
`I
`
`14:44
`
`From-SIB/F&Co.
`
`T-989
`
`P.ZT/38
`
`F-709
`
`86187-1
`
`
`
`
`
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