WO 2004/047554
`
`.
`
`PCT/EP2003/013299
`
`-~16-
`
`iv) optionally other additives selected from the group consisting of polymers,
`
`nutritional components, stabilisers, preservatives, and antioxidants.
`
`The water dispersible concentrate may be utilised in food for human nutrition, or as a
`
`supplement administered in a suitable unit dosage form with or without biologically active
`
`compounds. It may also be used to prepare lipid aggregates embedded in feed particles to
`
`contain nutritional components for feeding fish livestock or fish larvae. Preferably, the water
`
`dispersible concentrate is prepared using a purified, waxy phospholipid composition with at
`
`least 40 % by weight of PC purified by ethanol extraction of a particulate or powdered
`marine phospholipid/marine protein, amino acid and minerals composition.
`|
`The invention further describes a method to prepare a water dispersible MPL concentrate
`
`from MPL compositions obtained by fluid and ethanol extraction.
`
`The invention further provides a method of preparing lipid aggregates which involves
`
`dispersing a lipid concentrate (B) comprising:
`
`i) 25 wt% to 75 wt% of an MPL composition comprising PC or blend of PC and the
`
`monoacy] derivative prepared by enzyme hydrolysis in an amountof at least 40 wt%
`
`as the major component and minor amounts of other components, including PE, PI,
`
`SPM, PS and their monoacyl derivatives,
`
`ii) 15 wt% to 75 wt% of glycerol or another polyol, or sugar
`
`iii) 1 wt% to 50 wt% water,
`
`iv) optionally, other additives selected form the group consisting of polymers,
`
`nutritional components, stabilisers, preservatives, and antioxidants;
`
`in water or an aqueous medium for embedment and inclusion in feed particles or addition to
`
`food for human consumption.
`
`The fatty acid profile of the phospholipids is characterised by at least 30 wt% of HUFAs,
`
`chiefly DHA and EPA based onthetotal fatty acid content present. Preferably it should
`
`comprise between 40 wt% and 60 wt% omega-3 fatty acids.
`
`RIMFROST EXHIBIT 1024 page 2026
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`page 2026
`
`

`

`WO 2004/047554
`
`PCT/EP2003/013299
`
`-17-
`
`Hydrophilic medium
`
`Suitable hydrophilic medium are non toxic polyols such as glycerol, propylene glycol,
`polyglycerols and mixtures thereof. The preferred polyol is glycerol.For human use ethanol
`may be suitable. Alternative polyols which maybe usedin place of or in addition to glycerol
`are aqueous sugar solutions comprising at least 25 wt% of e.g, glucose, sucrose, soluble
`
`maltodextrins, or polyhydric alcohols like mannitol and sorbitol etc.
`
`Polymers
`
`Natural gums, particularly sodium alginates which can cross link with Ca++ ions are
`
`preferred as matrix forming material for the microparticles. Alternative materials are
`proteins such as gelatine, poly peptides and peptides such as casein and soya proteins.
`
`Stabilisers
`
`These include buffers, osmotic components, antioxidants and anti microbials commonly used
`in fish feeds and supplements.
`
`For fish feed applications a purified, waxy MPL composition after ethanol reatment, is
`hydratedin the hydrophilic medium, preferably without using elevated temperatures to
`
`prepare a homogeneouswater dispersible gel like concentrate. Preferably, permitted
`antioxidants and preservatives may be added. On mixing with aqueous medium, the
`concentrates readily disperse into a variety of lipid aggregates below 10 p average diameter,
`
`preferably below 1p measuredbylaser light diffraction. The concentrate further comprising
`nutritional components and alginates may be embedded in micro particle feed compositions.
`The amount of MPL concentrate that may be incorporated and embeddedin the
`multicomponent feed composition may be between 10 wt% to 75 wt%, preferably 20 wt% to
`40 wt%. After dehydration or lyophilisation (where appropriate) between 25 wt% to 75 wt%
`
`of water soluble nutritional components such as protein hydrolysates based on the total
`phospholipid content may be retained in the microparticles. The high capacity is due to the
`
`RIMFROST EXHIBIT 1024 page 2027
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`
`

`

`WO 2004/047554
`
`PCT/EP2003/013299
`
`-~18-
`
`unexpected properties of the marine phospholipid concentrates for containment of both
`water and oil soluble nutritional components in compositions in a destabilising environment
`like sea water.
`
`It should be understood that the invention also includes MPL compositions wherein the
`mixture comprises marine diacyl phospholipids and their monoacyl derivatives. The mixture
`is prepared by enzyme hydrolysis using phospholipase A2 or Al ona phospholipid
`substrate which maybeeither a particulate MPL composition or a waxy composition
`according to the method described in EP 1011634. The amountof diacyl phospholipid to
`monoacy phospholipid in the enzyme hydrolysed MPL composition may be between 1:10 to
`
`20:1.
`
`There is described, characterised and defined marine phospholipid (MPL) compositions
`which may be used either as such in powderform orfor preparing purified waxy lipid
`compositions which may be used to prepare improved water dispersible MPL concentrates,
`as nutritional supplements and/or ingredients for functional foods as well as the delivery
`and containmentof nutritional components in multicomponentfish feed compositions. The
`MPL compositions in powder form and as water dispersible concentrates may be used in
`unit dosage forms as supplements to supply highly bioavailable omega-3fatty acids for
`human use. The dosage forms may also contain biologically active compoundsin
`combination with the MPL compositions. There is also described MPL concentrates
`
`comprising standardised and clearly defined phospholipids for embedmentin feed
`microparticles, which are nutritionally essential in larvae feed.
`
`Example 1
`
`A dried marine raw material consisting 1000 g of freeze dried capelin roe with a total lipid
`content of 35 wt% is extracted in a batch extraction vessel with 40 kg/h carbon dioxide at 300
`bar and 40° C for 4 hours to remove 180 g of a clear orange-brownoil containing 18 wt% of
`cholesterol and cholesterol esters. The particulate MPL composition in the form of a coarse
`powder which is recovered containsa total lipid content of about 21 wt% , of which the
`
`RIMFROST EXHIBIT 1024 page 2028
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`
`

`

`WO 2004/047554
`
`PCT/EP2003/013299
`
`-19-
`
`major proportion (about 17 wt%) is polar phospholipids and 4 wt% are neutrallipids.
`About 70 wt% of the phospholipids is phosphatidylcholine comprising at least 30 wt%
`HUFAS. About 70 wt% of the composition is proteinaceous material including about 8 wt%
`minerals and trace elements. The composition has a mild fishy taste and odour with a
`
`peroxide value below 3 and may be used as such or mixed with suitable excipients in
`
`functional food applications. A unit dose of 300 mg of the powder maybe filled into hard
`gelatine capsules for oral use as nutritional supplement. The powder may also be converted
`into tablets, Optionally the MPL composition may contain biologically active compounds
`
`In place of capelin roe, herring or cod roe may be used. An alternative dried starting material
`that may be usedin this example is from krill.
`
`Example 2
`
`820 g of the powder composition from Example 1 is extracted three times successively using
`a5:1,3:1,anda2:1ratio of ethanol (containing 6 % of water) to powderto extract the
`
`phospholipids. About 160 g of a waxy material comprising phospholipids as the major
`component with minor amounts of neutral lipids is obtained. A typical purified composition
`
`comprises:
`
`Polar lipids:
`
`PC content:
`
`82 wt%
`
`72 Wt%
`
`esterfied HUFAs:
`
`33 wt% (of PC based. on total lipids)
`
`non polar (neutral) lipids:
`
`13 wt%
`
`free fatty acids:
`
`Cholesterol/ esters
`
`Iodine value:
`
`minerals, (ash)
`
`ethanol:
`
`water:
`
`5 wt%
`
`4 wt%
`
`112 (range 110 -130)
`
`2wt%
`
`2.2 wt%
`
`1.9 wt%
`
`RIMFROST EXHIBIT 1024 page 2029
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`page 2029
`
`

`

`WO 2004/047554
`
`PCT/EP2003/013299
`
`-20-
`
`The MPL composition may be blended with 50 wt% of an oil which maybe a fish oil,
`vegetable oil or medium chain glyceride such as Miglyol, to prepare a fluid lipophilic
`composition. Alternatively, it may also be hydrated overnight in glycerol at room
`temperature to prepare a hydophlic composition . The liquid compositions maybefilled
`into soft gelatine capsules for oral use or it may be used in fish and larvae feeds as in
`
`Example 1.
`
`Example 3
`
`*MPL with 72 wt% PC
`
`Glycerol 90%
`
`50 wt%
`
`50 wt%
`
`*MPL with about 40 % PC mayalso be used.
`The MPL is hydrated overnightin a solution of glycerol at room temperature to prepare a
`
`viscous gel like MPL concentrate.
`
`Example 4
`
`The MPL concentrate from Example 3 is mixed with up to 25 wt% lipophilic nutritional
`components which may befish triglycerides and anti oxidants such as Vit E, ascorbyl
`palmitate., t-butylated hydroxytoluene, t-butylated hydroxyanisole, ascorbic acid or
`ethoxyquin. Additionally 25 wt% to 50 wt% of hydrophilic fish feed components such asfish
`protein hydrolysates may be added. 2 wt% sodium alginate in aqueous solution may be
`added to the MPL concentrate as shown in the example. The resultant aqueous suspension of
`lipid aggregates associated with nutritional components and alginate is used to prepare feed
`microparticles by cross linking the alginate in the composition in a bath containing Ca ++
`ions to prepare microparticles as described in WO0027218. The microparticles are recovered
`and dried. A typical composition is illustrated below.
`
`*MPL ( with about 70 wt% PC)
`Glycerol (70 %)
`
`20.0 wt%
`10.0 wt%
`
`RIMFROST EXHIBIT 1024 page 2030
`RIMFROST EXHIBIT 1024
`page 2030
`
`

`

`WO 2004/047554
`
`PCT/EP2003/013299
`
`Water (containing 2% Sod alginate)
`
`Fish oil
`
`(lipophilic nutrional component)
`Fish protein hydrolysate
`
`(hydrophilic nutritional component)
`
`-21-
`
`19.5 wt%
`
`10.0 wt%
`
`40.0 wt%
`
`mixed antioxidants
`
`0.5 wt%
`
`(tocopherol, ascorbylpalmitate)
`
`* waxy MPL composition with a minimum PC content of about 40 wt% may also be used.
`
`There is described marine phospholipids (MPL) compositions suitable for human,
`feed and aquaculture purposes comprising a dry composition comprising nutritional
`components selected from the group consisting of marine phospholipids, marine proteins
`and amino acid blends obtainable by fluid extraction of a dried marine raw material. The
`
`compositions have low amounts of neutrallipids and particularly low amounts of
`cholesterol and cholesterol esters. They may be used either as such in powder form and for
`preparing purified MPL compositions by ethanol extraction. Both forms may be used as
`nutritional supplements, ingredients for functional foods either on their own or in
`combination with biologically active compounds and to optimise the delivery and
`containmentof nutritional components in multicomponent fish feed compositions.
`
`There is further described a blend of the purified, ethanol extracted MPL compositions
`mixed with marine, vegetable, microbial or medium chain triglyceride oils to prepare liquid
`
`preparations for hard or soft gel encapsulation.
`There is further described MPI. concentrates comprising purified and clearly
`
`defined phospholipid types dispersed in hydrophilic medium which are nutritionally
`essential in larvae feed. Still further there is described MPL concentrates in unit dosage forms
`
`as supplements to supply highly bioavailable omega-3 fatty acids for human use, optionally
`with biologically active compounds.
`
`RIMFROST EXHIBIT 1024 page 2031
`RIMFROST EXHIBIT 1024
`page 2031
`
`

`

`WO 2004/047554
`
`PCT/EP2003/013299
`
`-22.
`
`CLAIMS:
`
`1.
`
`2.
`
`3.
`
`A.
`
`A marine phospholipid (MPL) composition suitable for human, feed or aquaculture
`applications comprising a dry composition comprising nutritional components
`selected from the group consisting of marine phospholipids, marine proteins and
`amino acid blends obtainable by fluid extraction of a dried marine raw material.
`
`The composition according to claim 1, wherein the dry composition is present in
`
`particulate or powdered form.
`
`The composition according to claim 1, wherein the dry composition comprises a
`major amountof polar and a lower amount of non-polar (neutral) lipids.
`
`The composition according to claim 1, wherein the dry composition comprises
`I)
`10 wt% to 30 wt% polar and neutrallipids; and
`Tl)
`70 wt% to 90 wt% marine proteins and aminoacid blends.
`
`5.
`
`The composition according to claim 1, wherein the dry composition comprises
`
`lh
`Il}
`
`15 wt% to 30 wt% polar and neutrallipids;
`70 wt% to 85 wt% marine proteins, amino acid blend and minerals.
`
`6.
`
`7.
`
`8.
`
`The composition according to claim 4, wherein the total lipid content consists of 70
`wt% to 95 wt% polarlipids and 5 wt% to 30 wt% consist of neutrallipids.
`
`The composition according to claim 4, wherein the total lipid content consists of 80
`wt% to 95 wt% polar lipids and 5 wt% to 20 wt% consist of neutral lipids.
`
`The composition according to claim 4, wherein 40 wt% to 80 wt% of the polar lipids
`consist of phosphatidylcholine and their monoacyl derivative, wherein said
`phosphatidylcholine and their monoacyl derivative are esterified with 30 wt% to 60
`
`wt% HUFAs.
`
`RIMFROST EXHIBIT 1024 page 2032
`RIMFROST EXHIBIT 1024
`page 2032
`
`

`

`WO 2004/047554
`
`PCT/EP2003/013299
`
`~ 23 -
`
`The composition according to claim 4, wherein 20 wt% to 60 wt% of the polarlipids
`consist of phospholipids selected from the group consisting of phosphatidyl
`ethanolamine, phosphatidyl inositol, phosphatidyl serine, sphingomyelin and the
`monoacyl derivatives thereof.
`
`10.
`
`The composition according to claim 4, which is prepared by meansof ethanol
`
`extraction to achieve a waxy constitution.
`
`11.
`
`12.
`
`The composition according to claim 10 comprising
`i)
`70 wt% to 95 wt% of polar and neutrallipids,
`
`ii)
`
`5 wt% to 30 wt% of neutral lipids.
`
`The composition according to claim 11 blended with 30 wt% to 60 wt% ofa fish oil or
`any other edible neutral oil for use in hard or soft gelatine capsules.
`
`13.
`
`A homogeneous water dispersible MPL concentrate comprising,
`
`i)
`ii)
`
`25 wt% to 75 wt% marine phospholipids;
`15 wt% to 75 wt% of ethanol or at least one polyol or mixtures thereof
`
`iii) water to make 100%; and,
`iv) optionally further additives selected from the group consisting of polymers,
`nutritional components, stabilizers, preservatives and antioxidants.
`
`14.
`
`15.
`
`A composition accordingto claims 1, 4, 10 and 11 mixed homogeneously with
`phospholipids selected from the group consisting of soya, plant, egg, semi-synthetic,
`enzyme modified phospholipids and optionally biologically active compounds.
`
`A method for further processing the MPL composition according to claim 1, which
`comprises separating the marine proteins and amino acid blends from the dry
`
`composition.
`
`RIMFROST EXHIBIT 1024 page 2033
`RIMFROST EXHIBIT 1024
`page 2033
`
`

`

`WO 2004/047554
`
`PCT/EP2003/013299
`
`- 24 ~
`
`16.
`
`A method for further processing the water dispersible MPL concentrate according to
`
`claim 13, which comprises dispersing the concentrate in water and administering the
`
`dispersion to fish and/or larvae.
`
`17.
`
`18.
`
`19.
`
`A process for preparing a waxy marine phospholipid (MPL) composition suitable for
`human or aquaculture applications, which process comprises the step of preparing a
`dry composition comprising nutritional components selected from the group
`consisting of marine phospholipids, marine proteins ,amino acid and minerals blend
`
`by subjecting a dried marine raw material to fluid extraction.
`
`A process for preparing a marine phospholipid composition according to claim 1,
`which comprises preparing the dry composition by subjecting the dried marine raw
`
`material to fluid extraction with acetone.
`
`A process for preparing a marine phospholipid composition accordingto claim 1,
`which process comprises preparing the dry composition by subjecting the dried
`marine raw material to fluid extraction with supercritical gas.
`
`20.
`
`A process for preparing a water dispersible MPL concentrate from an MPL
`
`composition involving fluid and ethanol extraction.
`
`RIMFROST EXHIBIT 1024 page 2034
`RIMFROST EXHIBIT 1024
`page 2034
`
`

`

`INTERNATIONAL SEARCH REPORT
`
`ona! Application No
`INP
`PCT/EP 03/13299
`
`: A. CLASSIFICATION OF SUBJECT MATTER
`PIPC 7
`=©A23K1/18
`A23K1/00
`A23L1/305
`C11B1/10
`
`A23K1/16
`
`A23D9/013
`
`A23L1/30
`
`According to International Patent Classification (IPC) or to both nationalclassification and IPC
`| B. FIELDS SEARCHED
`Minimum documentation searched (classification system followed byclassification symbols)
`IPC 7
`A23K A23D A23L C11B
`
`:
`
`Documentation searched other than minimum documentation to the extent that such documentsare included in the fields searched
`
`Electronic data base consulted during the international search (nameof data base and, where practical, search terms used)
`
`WPI Data, EPO-Internal, PAJ, FSTA, BIOSIS, MEDLINE
`
`C., DOCUMENTS CONSIDERED TO BE RELEVANT
`
`
`
`Category °}Citation of document,with indication, where appropriate, of the relevant passages Relevant to claim No.
`
`DATABASE WPI
`Section Ch, Week 198611
`Derwent Publications Ltd., London, GB;
`Class BO5, AN 1986-074743
`XP002276083
`& SU 1 175 486 A (ATLANTIC FISHING
`OCEANOG), 30 August 1985 (1985-08-30)
`abstract
`
`X
`
`X
`
`.
`
`Further documentsare listed in the continuation of box C.
`
`Patent family members are listed in annex.
`
`-/--
`
`I
`
`DATABASE WPI
`Section Ch, Week 199435
`Derwent Publications Ltd., London, GB;
`Class B05, AN 1994-283548
`XP002276084
`& JP 06 212186 A (AGENCY OF IND SCI &
`TECHNOLOGY), 2 August 1994 (1994-08-02)
`abstract
`
`° Special categories of cited documents:
`P
`nes
`"A" documentdefining the general state of the art which is not
`consideredto be of particular relevance
`"E" earlier document but published on orafter the international
`*X" documentofparticular relevance; the claimed invention
`filing date
`cannot be considered novel or cannot be considered to
`involve an inventive step when the documentis taken alone
`*L" document which may throw doubtson priority claim(s) or
`whichis cited to establish the publication date of another
`"Y" documentof particular relevance; the claimed invention
`citation or other special reason (as_ specified)
`cannot be considered to involve an inventive step when the
`documentis combined with one or more other such docu-
`"©" documentreferring to an oral disclosure, use, exhibition or
`other means
`ments, such combination being obvious to a person skilled
`intheart.
`“P" document published prior to the international
`filing date but
`"&" document memberof the samepatentfamily
`later than thepriority date clairned
`Date of the actual completion of the international search
`
`“T" jater document published after the internationalfiling date
`or priority date and notin conflict with the application but
`cited to understand the principte or theory underlying the
`invention
`
`Date of mailing of the international search report
`
`5 April 2004
`
`Name and mailing address of the ISA
`European Patent Office, P.B. 5818 Patentlaan 2
`NL — 2280 HV Rijswijk
`Tel. (431-70) 340-2040, Tx. 31651 epo ni,
`Fax; (+31-70) 340-3016
`
`Form PCT/SA/210 (second sheet) (January 2004)
`
`23/04/2004
`Authorized officer
`
`Muller,
`
`RIMFROST EXHIBIT 1024 page 2035
`RIMFROSTExpiary 1024
`page 2035
`
`

`

`C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
`
`
`
`Category °|Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
`
`inyGPionar Application No
`PCT/EP 03/13299
`
`INTERNATIONAL SEARCH REPORT
`
`
`
`DATABASE WPI
`Section Ch, Week 199040
`
`
`Derwent Publications Ltd., London, GB;
`
`
`Class B04, AN 1990-302732
`
`
`XP002276087
`
`
`& JP 02 215351 A (TAIYO FISHERY CO LTD),
`
`
`28 August 1990 (1990-08-28)
`
`
`abstract
`.
`
`
`& PATENT ABSTRACTS OF JAPAN
`
`
`vol. 014, no. 513 (C-0777),
`
`
`9 November 1990 (1990-11-09)
`
`
`& JP 02 215351 A CTAIYO FISHERY CO LTD),
`
`
`28 August 1990 (1990-08-28)
`
`
`abstract
`
`
`
`DATABASE WPI
`Section Ch, Week 199740
`
`
`Derwent Publications Ltd., London, GB;
`
`
`Class BO5, AN 1997-431382
`
`
`XP002276085
`
`
`& JP 09 194362 A (BIZEN KASEI KK),
`
`
`29 July 1997 (1997-07-29)
`
`
`abstract
`
`
`
`DATABASE WPI
`Section Ch, Week 198716
`
`
`Derwent Publications Ltd., London, GB;
`
`
`Class BO5, AN 1987-111742
`
`
`XP002276086
`
`
`& JP 62 056497 A (NISSHIN OIL MILLS LTD),
`
`
`12 March 1987 (1987-03-12)
`
`
`abstract
`
`
`
`
`
`
`
`
`WO 01 50884 A CHALLDORSSON OLAFUR
`;HARALDSSON GUDMUNDUR G (IS); HJALTASON
`BALDUR) 19 July 2001 (2001-07-19)
`cited in the application
`page 4 -page 10; claims 14-22,27-39;
`examples
`
`ET AL)
`
`
`
`
`
`
`
`
`
`
`US 6 372 460 B1 (BEHRENS PAUL W
`16 April 2002 (2002-04-16)
`
`
`claims; examples 1,2C,3
`
`
`
`WO 02 092540 A (BANZHAF WULF ;KOHN GERHARD
`(DE); ABRIL JESUS RUBEN (US); MARTEK BI)
`
`
`21 November 2002 (2002-11-21)
`
`
`page 8,
`line 19 -page 9,
`line 24; example
`
`
`
`1 U
`
`S 5 434 183 A (LARSSON-BACKSTROEM CARIN)
`18 July 1995 (1995-07-18)
`column 4,
`line 33 -column 5,
`claims 1-8,16; examples 1,2
`
`
`
`
`
`
`
`
`line 24;
`
`
`
`
`Form PCTASA/210 (continuation of second sheet} (January. 2004)
`
`RIMFROST EXHIBIT 1024 page 2036
`RIMFROSTEXPY 1024
`page 2036
`
`

`

`INTERNATIONAL SEARCH REPORT
`
`Patent document
`cited in search report
`
`Publication
`date
`
`
`
`15-05-2002
`30-03-1995
`08-01-1993
`10-12-1992
`23-05-2002
`26-09-2002
`05-07-1995
`16-11-2002
`02-12-1992
`17-03-2003
`14-09-1994
`25-02-1994
`31-08-1993
`10-12-1992
`16-01-2001
`ieneeercreicretentcenereAeaeSSESP
`
`In@rionar Application No
`PCT/EP 03/13299
`
`
`Patentfamily
`member(s)
`
`Publication
`date
`
`1175486
`
`30-08-1985
`
`1175486 Al
`
`30-08-1985
`
`2034924 C
`7065066 B
`
`' 28-03-1996
`| 12-07-1995
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`acoresaenceaSeSnUSUDSPtSASNSESyESSSSSftSY
`
`1875089
`26-09-1994
`5086796
`14-12-1993
`eseeaASSSSSPAGSaaSASSSPSYS
`
`0150884
`
`19-07-2001
`
`24-07-2001
`2394201
`‘ 19-07-2001
`2397216
`05-03-2003
`1400868
`23-10-2002
`1250058
`19-07-2001
`0150884
`24-06-2003
`2003519501
`, 21-08-2002
`20023375
`03-07-2003
`2003124218
`aeaeaaAAISSSPhSDSAASSYA SSSYPYS
`
`6372460
`
`16-04-2002
`
`2002123111
`05-09-2002
`748220
`30-05-2002
`22-02-1999
`8603498
`03-05-2000
`0996740
`24-06-2003
`134242
`21-08-2001
`2001512029
`31-03-2000
`20000491
`11-02-1999
`9906585
`eeceeeaneSSASSYSSNHSHS SNSSESYSSSAVP Sy
`
`03-03-2004
`1392623
`21-11-2002
`02092540
`eeeeeeAcalSASSSSSSSSSESENGSentSt
`
`5434183
`
`18-07-1995
`
`216227
`657969
`1910192
`2109611
`69232564
`69232564
`0660708
`2174833
`921477
`3387498
`6508123
`242697
`100524
`9221335
`
`Form PCTASA/210 {patent family annex) (January 2004)
`
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`(19) 0
`
`D)) sorcerer
`
`European Patent Office
`Office européen des brevets
`
`VATAAT
`
`EP 0 973 532 B1
`
`(11)
`
`(12)
`
`EUROPEANPATENT SPECIFICATION
`
`(45) Date of publication and mention
`Oo Oe oan alinp08 /36
`709.2005
`Bulletin
`2005
`(21) Application number: 98904353.4
`
`(22) Dateoffiling: 23.02.1998
`
`(51) Int cl7; A61K 35/78, A61K 35/12,
`A61K 35/56, AO1N 61/00,
`AO1N 63/00, A61K 7/48,
`A61P 35/00
`
`(86) International application number:
`PCT/IL1998/000085
`
`(87) International publication number:
`WO 1998/036761 (27.08.1998 Gazette 1998/34)
`
`(74) Representative: Desaix, Anne etal
`Ernest Gutmann - Yves Plasseraud S.A.
`
`3, rue Chauveau-Lagarde
`75008 Paris (FR)
`
`(56) Referencescited:
`US-A- 5 104 668
`
`¢ PATENT ABSTRACTSOF JAPAN vol. 006, no.
`191 (C-127), 30 September 1982 & JP 57 102810
`A (ISONO JIRO;OTHERS: 01), 26 June 1982,
`¢ LEANDRO SASTREETAL.: "PURIFICATION
`AND PROPERTIES OF A POLYADENYLATE
`POLYMERASE FROM ARTEMIA DORMANT
`EMBRYOS"BIOCHIMICA ET BIOPHYSICA
`
`ACTA, vol. 661, 1981, pages 54-62, XP002064843
`*« P. A. NAGAINIS ET AL.: "EVIDENCE FOR THE
`PRESENCE OF AN ACID PROTEASE AND
`PROTEASE INHIBITORS IN DORMANT
`EMBRYOS OF ARTEMIA SALINA."
`
`DEVELOPMENTAL BIOLOGY, vol. 68, no. 1,
`January 1979, pages 259-270, XP002064844
`
`(54) ANTI-PROLIFERATIVE PREPARATIONS
`ANTIPROLIFERATIVE ZUBEREITUNGEN
`
`PREPARATIONS ANTI-PROLIFERATIVES
`
`(84) Designated Contracting States:
`DE FR GB
`
`(30) Priority: 23.02.1997 IL 12029197
`23.02.1997 IL 12029297
`
`16.07.1997 IL 12132097
`
`(43) Date of publication of application:
`26.01.2000 Bulletin 2000/04
`
`(73) Proprietor: I.B.R.-lsraeli Biotechnology Research
`Ltd.
`
`62301 Tel Aviv (IL)
`
`(72) Inventors:
`¢ SOUDANT, Etienne
`F-94260 Fresnes (FR)
`¢ BEZALEL, Lea
`84359 Beer Sheva(IL)
`¢ ZIV, Meira
`Rehovot 76353 (IL)
`¢ PERRY, Inon
`62301 Tel Aviv (IL)
`
`
`
`Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
`notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
`a written reasoned statement. It shall not be deemed to have beenfiled until the opposition fee has been paid. (Art.
`99(1) European Patent Convention).
`
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`EP0973532B1
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`EP 0 973 532 B1
`
`Description
`
`FIELD OF THE INVENTION
`
`[0001] The present invention concerns extracts of cells or tissue or their supernatants which can inhibit proliferation
`of cells or tissue. The invention also provides compositions comprising such extracts as well as pharmaceutical, cos-
`metic and agricultural uses of the compositions and extracts.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Dormancy is a phenomena which is found in representatives of the plant kingdom as well as the animal
`kingdom.
`[0003] The germination of various grains and seeds comprising the necessary propagation organs is delayed under
`certain circumstances and yet the grains or seeds are capable of germinating after various periods of time. The period
`of time in which germination of such seeds may be delayed varies and depends both on intrinsic properties of the seed
`as well as on the nature and extremity of the environmental conditions. Seeds have been shown to be in dormancy
`for a few days, a year, several years and even for more than several centuries (as was discovered lately in the case
`of some nympheaceae and seeds of trees of the Leguminosae family (Shen-Miller, J., ef al., American Journal of
`Botany, 82:1367-1380, 1995)).
`[0004]
`Insome cases, the capability to develop dormancylies in the embryo envelopes. In such acase, the separation
`of the envelopes from the embryo, result in its immediate germination.
`[0005]
`In other cases, chemical growth inhibitors capable of preventing germination are presentin the embryo itself
`and thus even a bare embryo may remain dormant (such asin the case of Rosaceae plants such as Kerria, Peach, etc.).
`[0006]
`Inplants, a state of dormancy may be found in the whole plant orin one or more ofits parts. Dormant plants
`are plants which have two main metabolic states in their growth cycle.
`In their dormant state, the planis' metabolism
`is extremely low, and the plant growth processis significantly inhibited although differentiation of certain cells may
`occur. In their active state, the plants' metabolism rate is higher, the cells divide and differentiate and thereis significant
`growth of various parts of the plant.
`In some cases, the whole plant enters the dormant state. Such is the case in
`Narcissus plants in which during the dormant state the only remaining viable part is the bulb which is in its dormant
`state. In other cases, some parts of the plants may be active while other parts may be in darmancy such as, for example,
`is the case of apple trees.
`[0007] Substances capable of inhibiting germination have also been shownto be presentin the juice of fleshy fruits
`or in other plant organs which producejuice. Examples are tomatoes, grapes, kiwi, watermelon and grapefruit wherein
`pips present in the fruit do not germinate although their surroundings are suitable for germination due to the water
`within the fruit.
`
`Several plant-derived substances having an effect on cell proliferation have been reported. For example,
`[0008]
`European Patent Application No. 0351514 describes compositions comprising both naturally derived as well as syn-
`thetically prepared sphingolipids which have growth inhibitory activity on various kinds of cells. Another well known
`plant-derived substance having an anti-mitotic effect on various kinds of human cells is the substance colchicine (Sam-
`son, F.E., A. Rev. Pharmac. Toxic 16:143 (1976)). The Narcissus alkaloid, pretazettine, was shownto have a cytotoxic
`effect on Rausher virus-carrier cells as well as anti-leukemic activity in leukemic mice although the predominantactivity
`of the substance was shown to be an antiviral activity (Furusawa, E. ef al, Chemotherapy, 26:36-45, (1980) and Fu-
`rusawa, E. ef al., Proc. Soc. Exp. Biol. Med., 152:186-191, (1976). Ulex europaeus seed extracts were shown to com-
`prise a non glycoprotein lectin capable of reversibly inhibiting growth of certain lymphocytes as well as to inhibit the
`growth of various reticulo endothelial tumor cell lines (Pirofsky, B., et af, Vox-Sang, 42:295-303, (1982) and Pirofsky,
`B., et al., J. Biol. Response Mod., 2:175-185, (1983). Root extract of Panex ginseng was shown to decrease DNA
`synthesis measured by [H3]-thymidine incorporation of V 79 Chinese hamster lung cells. Another substance, Narcicla-
`sine obtained from bulbs of various Narcissus varieties was shown, amongstother ofits activities, to inhibit growth of
`wheat kernel radicals (Ceriotti, G., ef al., Tumors 53:359-371 (1967)). Bulbs of Pancratium iittoral collected in Hawaii
`were found to contain a product designated pancratistatin capable of inhibiting growth of various neoplastic cell lines
`in vitro (Pettit, G.R., et af., J. Nat. Prod. 49:995-1002 (1986)).
`[0009] The Japanese Patent Application JP 55177865 (Kosugi Kikuo, filed on December 16, 1980) reports the use
`of an extracted solution from bulbs of cluster-amaryllis of amaryllidaceous plant or narcissus in the preparation of a
`cosmetic composition to prevent the aging of the skin and the occurrence of the stain caused bythe influence of the
`sunlight, and to exhibit beautifying action.
`[0010] Against this, many plant extracts having an opposite effect on cells, i.e., capable of augmenting their prolif-
`eration were also described such as, for example, the methanolic extract from the root of Scutellaria baicalensis georgi
`were shownto significantly augment the cellular activity of fibroblasts (Chung, C.P., ef a/., Planta-Med, 61:150-153
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`(1995)). Gibberellin-like growth substances were found in six different plant species having bulbs (Staby, G.L., Hort.
`Science, 399-400 (1970)). Several cytokinins which were found in roots that developed from Narcissus bulbs had an
`effect on bulb growth of the plants in which they were detected (Vanstaden, J.V., Pflanzenphysiol., 86:323-30 (1978)).
`[0011] The phenomena of dormancy may also be found in the animal kingdom, for example, in the small crustacean
`Artemia salina (Finamore and Clegg In: The Cell Cycle, Academic Press Ed., 249-278, 1969). The natural environment
`of this marine crustacea is usually briny ponds. After fertilization, the early stages of development of artemia involve
`the formation of a blastula which then becomesa gastrula. Under severe environmental conditions such as dehydration
`(drought), the gastrula is capable of forming a cyst wherein the whole organism enters a dormancy phase. The dormant
`artemia gastrula (commonly miscalled “artemia eggs") are capable of remaining in their dormant state for many years.
`When the encysted gastrula are rehydrated, the various metabolic activities of the artemia are resumed and protein
`synthesis can be seen after about 10 minutes. However, DNA synthesis and cell division are absentuntil about after
`60 hours (Le Gal, Y, In: Biochimie Marine, (Ed. Masson) p. 176, 1988).
`[0012] Various plant derived compositions (such as retinoic acid (US 5,438,073) and a-hydroxy acids (Ditre, C.M.,
`et al, J. Am. Acad Dermatol., 34:187-195, 1996)) as well as animal derived extracts have been proposedfor use in
`the cosmetic field for stimulating the proliferation and renewal of epidermal cells. Such compositions were considered
`to be useful in the cosmetic field where it is accepted that the natural renewal process of epidermis is slowed down
`with aging. It is believed that removal of the outer surface with simultaneous stimulation of growth of new cells in the
`inner layers of the epidermis to divide and migrate to the outer surface, will result in skin renewal and in a younger skin
`appearance. However, it is also known and has been recently shownthat the increase in cell division is a crucial factor
`in converting normal cells into premalignant or malignant cells (Ames, B.N. et a/., Environ. Health Perspect 101:35-44
`(1993)).
`
`[0013] Itis also believed today that normal human and animal cells haveafinite capacity to replicate. It has been
`shown that the number of mitotic events that cultured normal animal cells can undergo appe