Attorney‘ Boeliet No. Pl\l—0lll/ll2?US 3l3€i(i3—2i}l3
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`l’A'l‘EN'l‘
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`lN 'l‘Hl7j UNl'"l‘l7jl) S'l‘A'l‘lili§ l’A.'l‘El\i'l” ANS 'l‘RAl)El\/EARK OFEKCE
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`in Re Application oi‘:
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`S./~\.l\/ll’Al_.lS, Fotlni
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`Confirrnation l\lo.:
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`l767
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`Serial No.:
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`l3./l89,7l4
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`Group Art Unit:
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`M329
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`Filed:
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`July’ 25, 20ll
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`Examiner:
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`PULANTSKY, Gregg
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`FOR:
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`NATURAL
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`l°i/l’Al?;ll‘€'E
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`SQURCE
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`l’HflSl’HflLll”l BS
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`Ci} llv’l l3’l?§lS ENG
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`PQl.tYUNS/X7l‘[lRA'l" E B FATTY ACRES AND T HE {R AP?l_4lCATl(3NS
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`Mail Stop Qeelaration
`Cornrnissioner for Patents
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`PO. Box ll-‘l50
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`Alexandria, VA 223 l3—l'-’l50
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`DlT.Cl_.ARATlGN OF JACEK tlACZYl‘\'SKl Tt’l:l.B. UNDER 37 C.F.R.
`‘ l.l3?.
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`l, lacek Jaczynski, declare as follows:
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`1.
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`l am a tenured Associate Professor of Food Science and Technology at West Virginia
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`University; Davis College of Agi'icultu1‘e, Natural
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`l{esources, and Design; Division of
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`Anilnal and Nutritional Sciences. My appointnient is 50% research and 50% teaching.
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`l
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`have been a professor at West Virginia llniversity since 2002.
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`SM)
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`l earned a l’h.E). in Food Science and Teclinology in 2002 frorn Oregon State University,
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`Seafood Research and Education Center.
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`linlnediately following my doctoral worlr, l joined
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`West Virginia University
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`a faculrty l’,‘(l,f:Il'll;)€lI, For the past l4 years l have been actively
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`pursuing scientific research specializing in aquatic foods, with an emphasis on krill.
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`l have
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`pulolishecl
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`l5 book chapters and over 50 pee.r—reviewed articles on food science and
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`technology’, many in high impact journals as indexed by Journal Citation Reports.® For
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`exainple, one of my peer~revie.wed publications directly concerns solvent extraction of krill
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`000001
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`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
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`ENZYMOTEC - 1060
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`Attorney lllocket No. Nl3Pl\li—i)l)l /l)IZUS 3 l 3ti€S3—2{ll 3
`Serial No. 13//189fi‘;I'14
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`oil} ln ailtlition, l am the sole inventor of an issued patent (US. '7,763,7l7) and the inventor
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`of two other patent applications currently under exarnination. One ‘focus of my patent and
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`patent applications is a niethotl for isolating lipids from krill.
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`[M
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`l serve on the Etlitorial Board for the Journal Qfi4a;i2/télflic Food Product Tecizzrzologjr and as a
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`peer—reviewer for several food science journals, such as Food Cizenzistzj- and the J'om*rzaZ of
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`Agriczttiturai (md Food Chemistry.
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`I am a professional meniher of the lnstitute of Food
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`Technologists (“ll:”'T”), the Anierican Chemical Society, the World Aquaculture Society, and
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`Gamma Signia Delta, an honorary society of agricultural scientists.
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`l servecl as a Chair of the
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`Division of Aquatic Food Products of the lFT for the 2(ll€)—2l}ll
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`teiin. For the past ll) years
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`1 have also taught food science—related. courses at West Virginia University, many of which
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`enroll over 300 stutients annually. My eurriculuin vitae is attached as Appentllx A.
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`ln December of Ztll l,
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`l was engaged by counsel
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`for Neptune Technologies and
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`Bioressources, lnc. (“Neptune”) to review U Patent 8,l)3(),348 (“the ‘348 patent”) and its
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`sulsstantiye prosecution historyg, the Coirectetl RC§}ll,CSl for R€:CX’<l.1’l’ll1‘lail0ll
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`tiletl by Al<;er
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`Biornarine (“Alger”), listed as U.S.S.N. 95/(}0l,7l4, includiiig the l:lf3Cl£il'£il.l0‘(l of Mr. Bjorn
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`Ole Hatrgsgierd and the Declaration of Dr. Thornas Gunclersen, and supporting materials, and
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`to provide my expert scientific opinion regarding whether Guntiersen and l'iaugsgier'tl
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`accurately followecl the process disclosecl in patent publication W0 O0/’23546 (“Beaucloin 1”)
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`and CA 2;25l52{i5 (“Beaudtoin ll”) and therefore whether the data presented by Alger‘
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`accurately characterized the krill extract obtained hy lfieaudoin. Also, 3 was asked to €‘f.?<pl'eSS
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`my opinion on why intact phospholipicls hearing omega. 3 fatty acids, such as those found in
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`krill oil extracts, are superior to other forms ofornega 3 fatty acids, such as the triglyceride»
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`bound forms seen in fish and algal oils, as well as free fatty acicls.
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`Li‘!
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`l have hacl no prior clirect involvement with either Neptune or Aker.
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`1 am being cornpensatecl
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`at my custornary hourly rate for my time spent on developing, forrnirig, and expressing the
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`facts and, opinions in this declaration.
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`l have no personal interest in the ultimate outcome of
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`1 See Gigliotti er a1’. “l3x=.raction and Cliaracterisation of Lipids frorn Antarctic Krill (Eztpizausia supetré-as)” Food
`C}zerm..:‘zI'y lZ5(3): lO2?}C~lO36 (April, 2011), Apperltllx E.
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`Attorney lllocket No. Nl3Pl\l—ill)l /'l)2l_.lS 3 l 36€53—2(ll 3
`Serial No. 13//189fi‘;I'14
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`the reexaininatioh proeeedirlgs involving the ‘348 patent or any eorltinuation applications
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`derived from the ‘348 patent.
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`6.
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`l have carel‘"ully read the irtformation pr'ovitled and also conducted my own Search of
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`relevant, peer-reviewed scieritiric literature. Below l provide my expert scientitie opinion.
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`Galitlerseri and Haasterd Bid Not Acctrratel ' Relleate Beaudoiri l or Beaadoira ll.
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`7.
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`in my opinion, Gurrdersen arid Hatugsgje.rd did not accurately reproduce the methodology for
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`total lipid extraction from lerill that is disclosed in Beaudoiri l or ll. Speeitiealrly, Gundersen
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`did not sufficiently heat the krill oil samples in a rnanner that was appropriate to replicate
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`Beaudoih l or ii, and l'laugsg}'erd did not accurately replicate the extractiort method of
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`Beaudoih i or ll because he added a significant step to the Beaudoih protocol. For at least
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`these reasons, it is my opinion that Haugsgierd arid Guriderscii failed to opirie on the spe.citic
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`process of Beaudoih l or ll and therefore failed to characterize the l{l'lll extract actually
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`produced hy Beaudoin l or H.
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`Gwzciersien D211’N0tAppr0priaifeZy Heat the .S'amples.
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`8. Guoderseh conducted the last step of the hrill oil extraction procedure {which was partially
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`conducted by lrilaugsgjerdl.
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`lh doing so, Guhderserr applied heat in a ntaruter irleorlsistent
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`with Beaudoirr l or ll to the lrrill oil extracted by Haugsgjerd. Specifically, Gundersen
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`alleges that he conducted a heat treatrneht at l2S°C for l5 minutes or at 70°C for S tnirlutes,
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`in an attempt
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`to reproduce Beaudoih l and ll {See (luoolersen Declaratiorl, Exhibit 2,
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`Analytical Report second of two pages riurribered 1, betweerr page 5 arid page 7)? However,
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`in his attempt to heat
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`the oil, (Eundersen placed a heat hloel:
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`ihsitle the oven of a gas
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`chrorriatograph set to either 70°C or 125°C for at least one hour ( Guiidcrserr Declaration,
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`Ahalrytieal Report second page nutnbered l, between page 5 and page 7). A vial of krill oil
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`extract was then heated using the heat block for l5 minutes at il25"C' or 5 rnirlutes at 70°C
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`(see Guhderseh Declaration, EXl'lll)l,l 2, Analyticatl Report second of two pages numbered l,
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`between page 5 and page 7). After Guridersert heated the Vials, they were allowed to cool on
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`2 i respectfully note that the confusion regarding page rrurrrhers iii the Gumlerseir declaratiori sterns from the
`declaration apparently being submitted either out of order or with incorrect pagiriatiorr.
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`Attorney Becket No. NE3l?l\li—i)0l /'l)2l_.lS 3 l 36€S3—2{ll 3
`Serial No. l3/l89,7l4
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`a laboratory bench to room temperature ( Gnndersen De “laration, Exhibit 2, Analytical
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`Report second of two pages nninhered l, hetween page 5 and page 70).
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`9.
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`in my opinion, this heat treatment did not allow the oil
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`to be heated to the temperature
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`diselesed by Beandoin l or ll for the time. specified by Beandoin l or ll due to slow heat
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`transfer to the oil l‘l'0I’l‘l the heat block.
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`(3nnderseh”s heating method was mediated primarily
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`by airmliqnid convection and not eonduetien.
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`it is a wellmestablished faet that eonduetien
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`results in much qnieke. heat transl’er than C0llV’€Cfli01’l.3
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`ln simple terms, heated air contains
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`relatively fewer molecules that can transfer heat from one object to another, as compared to
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`heated liquids, such
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`oils
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`in a heated oil hath. Therefore,
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`the transfer of heat via
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`eon eetion is much slower than conduction;
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`thus,
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`the samples heated as described by
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`Gnndersen were not inaintained at the temperature. of l25°C for l5 minutes or 70°C for 5
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`minutes.
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`ll).
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`A simple analogy allows illustration of this complex phenomenon. Consider placing ohe’s
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`hand in a standard l<;itchen oven set at a moderate temperature, say 4{}0°F (which is about
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`200°C). Qne could easily hold one’s hand in this even for a period of time belhr'e
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`experiencing physical discornfoi‘t or ininiy.
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`lf one were to place ones hand in a pot of
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`boiling water (118,, around l0(l°C}, however, one would inirnediately experience a hnrning
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`sensation. This common scenario
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`explained hy the difference between heat transfer hy a
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`slower inethed, convection (zle, the stove in the analogy), Versus a faster method, conduction
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`(fie. , the boiling pot of Water in the analogy).
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`ll.
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`Accordingly, when Dr. (Eundersen placed the extracted krill oil in a heat hloelr, he relied on
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`heat transfer by eonveetion to allegedly heat the oil to l?.5°C (or 70°C). Like the hand in the
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`oven described above, the oil samples themselves did not reach and maintain a ternperature
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`of l25°C for l5 minutes.
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`in contrast, during the prosecution of US. Patent 8,030,348, the
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`applicant submitted data obtained after heatirig for lfi minutes at
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`l'.735°'C by planing the
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`3 See‘, e,g,, Singh and lleldrnan, 1ntrooz'uc{io.n Z0 1~“:;2oa' Ezrzgineerirzg (3rd ed), New York, NY: Academic Press, 2008
`(pp. 222-27), Appendix C; Heldrnan and Land, Handbook :;2f1~*:;2oa' Engiz';eeI'ing, New York, NY: Marcel l)6l{l{t’::1”,
`i992 (pp. 247-59), Appeiidiir D, both of which are lhndarnental food engineering =.e_Ktbo0l<s.
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`Attmaiey lllaeket Ne. Nl3l?l\1—00l /GIZUS 3 1 3663-201 3
`Serial N0. 13//189fi‘;I'14
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`f.’.X't1”EtCl'f.’.(l oil in an oil bath, which, in my opinion, aecihately re—<:i"ea,ted Beaudein
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`Using
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`this apprepriate heat transfer inetlled, mediated by cenductien, the oil reaelled ll25"C' and
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`therefeie experienced a. full 15 minute expesure te this teinperatme.
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`12.l alse note that the proper use of a heat bleelg te }:1%a't an ell extract effectively has been
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`described in the literature. For example, in 1-lenvnan and Greves,5 the authors cendnct an
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`experiinent in which they tlierlnally stress lipid enihlsiens containing phospholipids and
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`observe hydrolysis efthe fatty acids all’ (if the phesplielipids li'en'i this heaticiig. Specifically,
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`they describe, at page 775:
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`“‘"l“hei*mal stress was applied hv filling heatinv hlaek chambers (Dry Baths, Fisher
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`Scieniific, ltasca, IL; 60 Cl1é1l11l3Cl‘S pe1‘l3l0c1<, each
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`mm diameter and 50 mm deep) with all
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`and lmnierslngv the 2~mL ampimles containing the emulsion at the desired temperature,
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`covering the blocks with aluminuni foil to minimize thermal lluctuatien” (emphasis added).
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`Such a protocol would allow efteetive heat ‘l‘.‘(‘a,,'(1Sff.’.1” to the samples heeause it: relies en
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`conduction threugh hot oil, as was perfonned in Obtaining the data presented in the
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`pmsecuttien et‘U.S. 8,030,348. Gundeirsen did not follow this l{l10VV‘(l'p1‘0l70C0l.
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`13. ln my expert: epinion, the iiieffective heating applied hy Gnndersen had a sighilieant etteeet
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`en the extent of hyczimlysis at‘ the ester bends eenneeting fatty acids (eg. DHA and LEELPA) to
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`the glycerol haeldieiie of the phespholiplds.
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`Accenlliiglyg, Gnndeisen only allegedly
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`ehserved a residual niass speetminetry signal at‘ phesplieliplds hearing DHA and EPA (or
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`EPA/El*A or DHA/DHA).
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`14. Furtliei‘,
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`I alse note that Gundersen pmvides an unclear trend as to the efleet of heating.
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`Comparing the llPl_.C—l&/lS data presented in Appendix
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`Gunclersen appears to detect the
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`intensity peaks for non-heated, heated te 60°C 01‘ 70°C, and heated to 125°C (see, e.g.,
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`cllmniategrarns labeled l3‘308~l, P308—2, and P308—3)t
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`This furthcer underseeres the
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`inelifeetiyte heating, appmaeli used l?yC!Ll.lli"l61'S6l’l.
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`4 As netted in
`Patent 8,030,318.
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`21l')()'Vt3, I reviewed the office Action response tiled (in May 31, 2011 in the pmseciltieil ofthe US,
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`5 Herman and Graves, “The lnlluence of Free 13 aity Acid lhrniaiinn on the pll ef Pllesplielipid-stabilized
`’1"riglyeeride E.n1ulsie1'ls,” Hzarrrzaceuricai Resecarc-h, l0(5): 774-76 (1993), Appendix E.
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`Attorney lllocket No. Nl3Pl\l—i)l)l /l)IZl_.lS 3 l 36€53—2(ll 3
`Serial No. 13//189fi‘;I'14
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`}iattg.sgjerl:i am’ Gzmdersen .4z:it:z,’eti an E}.’,[):’3i‘I/°':Vi'Z(*fi’Zftil17' Step Not iDis'ci0sea’ in Beaudoin I or
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`Beaztdoirz H.
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`l5.
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`Further, it is my opinion as an expert on krill oil extraction that not only did E-Ilatugsgjerd and
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`Gundersen fail to establish that the oils were sufficiently heated to replicate Beaudoin l or H,
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`E-laugsgjerdt also perforrned an z,zt:t’d2'z‘[0:/262! experimental step in his extraction procedure that
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`deviated from Beaudoin l and ll. Specificatlly, Haugsgjenl reports the following steps to
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`generate Fractions lla and llh (llaugsgjerd Declaration, ‘El 3, Gundersen llleclaration, lixliihit
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`2, Analytical Report second of two pages numbered l, between page 5 and page 7):
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`e
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`8
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`is
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`extracting with either ethanol or ethyl acetate;
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`filtering solvent and evaporating under reduced pressure;
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`“llush[ing] saniples with nitrogen gas” and “storlingl at -206‘ until further analysis;”
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`and
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`8
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`sending
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`samples
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`from l%latugsg.jei'd
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`to Gundersen
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`and
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`having Gundersen
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`unsuccessfully attempt to heat to at 25°C for l5 minutes.
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`i note that the Beaudoin l or ill protocol, as successfully replicated to generate the data
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`presented in the prosecution of US, 8,630,348, did
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`involve flushing oil tractionis with
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`nitrogen gas and freezing at —2tl°C before heating. On the contrary, in both the disclosed
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`Beaudoin process and the CXp€:I‘ll’l’1f3lliS
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`conducted to generate the data presented in the
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`prosecution of US. 8,030,348,
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`following evaporation to partially reniove solvent,
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`the
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`fractions were inrniediately heated
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`Response to Oflice Action of April 219, Zllltl in US.
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`ll}/485,094, Appendix l; Beaudoin l, pages
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`and l0).
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`'l‘here is no rnention in Beaudoin l or
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`ll of either storing samples at -ZGOC or under nitrogen gas.
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`l6.
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`ln my opinion, Haugsgjerd’s deviation from the process disclosed in Beaudoin l or ii is very
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`Slgl1i,l,lC3.1ll. By treezinig the sample before ll,f:9tlll’lg,
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`liaugsgjerd fitrther suppreswti any
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`hydrolysis of ester bonds found on the phosplrolipids heing analyzed.
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`lt my opinion that
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`after Beaudoin conipleted the penultirnate step of renioving the solvent by rotary
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`evaporation, he heated to remove the residual “volatile matter and huinidity” from fraction E
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`Atterney llmeket No. Nl3Pi\l—i)t)i /t)IZUS 3 l 36€S3—2()l 3
`Serial No. l3/l89,7l4
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`and i’ractiert ll {see Beautlein L page l0). Note that in Table l3 of Beautleiti l “Volatile
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`matter ancl moisture levels” of l % and 6.8%, respectively, fer fractions l ancl ll are reported.
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`These levels of solvent and hurriidity would have i”f311(.lf31"f:d these oils crude for Beaudoin°s
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`purposes of determining total lipids generateel hy his procetlure, all(i,li1€T6ft3T€
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`prernpted him
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`te “get rid of traces of solvents {by} ‘orietly i’lt.’.a‘([i1lg] (to aheut lIZ.5°C, for aheut 15 min.) the
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`oil uhtier nitregen” {see Beattdein E, pages 7 and l0). This heating ihevitahiy led to the
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`hydrolysis of ester bonds. As a result, the phosplioiipids were degraded and eoiisequently
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`released free fatty acids such as EPA anti DHA,
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`lt i s my opinion that leiatuigsgierd suppressed
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`this inevitable hydrolysis by storing his samples at —2(l°C.
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`"l‘liis is a step not tattght by
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`Beaudoin l or it, which causes one to question why tilaugsgerd did this.
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`it is also my
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`opinion that haol E-laugsgjei‘d heatetl the samples in an oil bath or with a heating jaehiet after‘
`
`removing the selyents by rotary CV’<l.pOl'a,liOll and then stored the final prociuct under nitrogen
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`at
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`-2t}"(ff for sul>seqi.:ent evaluation hy Guhelei'sen, Gundersen would not have allegedly
`
`ebserved even residual amounts of pliospholipids carrying two of DHA arid/or EPA. I note,
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`as evz'dem':e_;‘br this apinirm,
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`the: paper c:I°te:a’ in it I2 above in wizz'c:.IZz the a2,tz‘lz(.rrs rlzerrna/’!'ja
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`stressedphosjphoizlriid emuisiorzs in hearing blocks‘ conrainirzg‘ 02'! and a’escrz'bet<'Z h.y.:2’r0Zysz'.s of
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`ester bonds in ph0spiz0Zz'pz'z:is,
`
`/is an .i7rzevz'z'czb!'e <:(m..seqz4ence (:fz‘lzz’s rlzeimai :"1yt:£i"olysz's,_fi"ee
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`fatty acids were rele<2sedfi‘0in: the pi2osphr)iz'pids'.
`
`l7. in summary, it is my opinion that, due to inaoleqttate sample heating and the addition cf an
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`expeiirriental step to the exti'action p1‘OtGCOi, Haugsgjeiti and Guntlersen did not accurately
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`r'epr'otiuee the methodology of oil extraction item kriii as tiiseioseti ih i368.E.,t€i0il'i
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`l or ii, ahol
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`therefore the mass spectrornetric data presentetl hy Gundersen fails to aecui*ately' characterize
`
`the krill oil actually protluced by Bf.’.Ei1l(,i()i,l’l
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`i or H.
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`intact Rios hell aids Containiiict Grne ta 3 Pelyansatiiratetl Fatt ' Aeirls Pessess Desirable
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`l"ro erties Net (lhseryetl in (Ether E-‘nrrns of Egi iris Sash as 'l‘r'ilyeerirles Cnntaiiiin
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`Gmea 3 Paiyunsatnratefi Fatty Acids.
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`Attnrney lltneket Ne. l\lEl,)l\lFlll)l /l)2US 3 l 36€53—2(ll 3
`Serial N0. l3/l89,7l/-l
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`18. l have been asked to express rn y opinion, based on the peer reviewed literature, including in y
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`la'beratery’s published research, on the superier preperties of omega 3 fatty acids in
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`}_:‘il’lOSpliOlipi(.l ferin
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`ennipared te ether forms (or lipid classes), such as triglycei*ides.
`
`Possible Farms Qfflmega 3 Fatty zlciris rmd Their Presence in Various Extracts.
`
`l9. The ‘348 patent teaches extracts,
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`including lrrill extracts, and innre specifically,
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`intact
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`phesphelipicls bearing omega 3 polyunsaturated fatty acids, such as EPA and Dl-lA_ These
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`eeinpesitiens are clistiiiguishahle front fish oil and algal nil,
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`fisli and algal Oil extracts
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`feature
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`and DHA bound to ti'iglycerid,es. My laheratmry recently enndueted a.
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`coinparative study arming krill eil, fish oil, and algal oil.“(’ "l‘liin—layer—chrornategraphy (TLC)
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`analysis clearly den'ienstrated that lcrill nil containetl siigriiitieant arneunts ef phostphelipids,
`
`while the arneunt ef detected phesplielipids in fish oil and algal ell was negligible. Further‘,
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`TLC expei*irnents showed that krill nil eentained negligible amounts nf triglycerides, while
`
`fish nil and algal nil contained significant amounts.
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`20. l7urthei', the Kassis
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`(15. study shewed that there are high arneunts ell’ El’/~\. and Dll-lA in l<rill
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`oil (47% of total fatty acids) and these omega 3- pelyunsaturated fatty acids are primarily
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`esterilied in pliesplinlipids. On the other hand, l?.l’A and DHA in iisli (iii and algal oil are net
`
`l:~
`3’
`l3
`P
`l‘
`.:>
`3
`E» .,
`si‘7nifit::antl ' esterifiecl in hos holi ,ids, but are lar<>‘el I esterifiecl in tri ‘rlveericles.
`
`2l. '7l‘liei‘efC:re, krill nil, unlike fish eil and algal nil contains significant amounts Of omega 3 fatty
`
`acids esterified in plinsphelipids.
`
`Phospizalipiciis Have .Sziperz’0i" Pizysioiagicrzl Absorption Prqfiles.
`
`22. Based en the recently deyeleping researeli in this field, it appears that the elieniieal “carrier”
`
`of emega 3 pelyunsaturated fatty acids in lrrill e>rti‘aets, 225., phesplielipids, provides superior‘
`
`physielegieal abserpti01ipi‘0files, especially
`
`cenipared te ti'iglyceride “earriers.”’
`
`23. Phesplinlipids are aziuplzzlnlziiic and triglycerides are net. The chemical structure of an
`
`ainirnhitphiliie eenipeund centaiiis funetien all gmups that allow Sim1rli!(Ifl60l4S water~ and lipid-
`
`\.’{)
`A
`.
`Navel ’Nutra(:eutieal Ego l‘r<)duets
`6 See Kassis at aft‘ “Charaeteri7.at,:i0n of Li,ni<ls and Antirmidant Calaeiiy
`Developed with Omega-3--Rirtli Oils” J Sci Food /gr 9’?.(l,); 66--73 (2012,), Apgieaitlix
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`Attorney lllocket No. Nl3Pl\i—ill)l /'l)IZl_lS 3 l 3(i€i3—2(ll 3
`Serial No. l3/l 89,7l/-l
`
`solubility due to the presence of hydrophilic and hydrophobic moieties, The hydrophilic
`
`moiety in krill phospholipids
`
`present at the sn—3 position, where the nitrogen-containing
`
`moiety ( the choline in phosphatidylchollne) provides positive charges and the phosphate
`
`bridge provides negative charges. These positive and negative charges interact with charges
`
`on the water dipoles, thereby, resulting in phospholipid solubility in water. The hydrophobic
`
`moiety in krill phospholiplds is present at the srz-l and 571-2 positions in the forth of the long
`
`hydrocarhon chains of omega 3 polyunsaturated fatty acids.
`
`in contrast, the triglycerides of
`
`tish oil and algal oil are not ainpliipliilie, as their sn—l., are-2, and siz~3 positions are occupied
`
`with hydrophobic fatty acids that have long hydrocarbon chains. Accordingly, triglycerides
`
`are lipid—sohible but not water—soln‘ole. By way of analogy, phospholipids (such as those of
`
`the ‘348 patent) are lilce alnhidextrotis people, having the aliility to write with hotli the right
`
`and left hand, while triglycerides (such as those of fish oil and algal oil) are like right— or left-
`
`handed people, having the ability to write with only a single hand.
`
`. This duality of phospholipids has implications for absorption in the ‘body. When lipids (La,
`
`typical oils and fats composed of triglycerides, such as fish oil and algal oil} are ingested,
`
`because of their water irisolnhil.ity and lower density than tvater, they iloat on top of and iorrn
`
`a layer that is separate from the digestive juices in the stomach and small intestine. These
`
`digestive juices contain lipolytic en'.2:yrnes
`
`that digest
`
`lipids, Therefore, hetore water-
`
`insohihle lipids (iya,
`
`triglycerides) can he digested and alisorhed,
`
`they first have to he
`
`emulsified by bile. However, arnphiphihc compounds such as phospholipids (like those in
`
`the ‘348 patent) skip the erniilsltication process because they are water soluble.
`
`in fact,
`
`phospholipids enhance. Cl’1‘ltllSlil(3‘d‘tl01l hy bile and,
`
`thereby, aid in digestion of water-
`
`insoluhle oils.,7
`
`l\) Lil
`
`. Phospholipids and itriglycerides are also digested by different enzyrnes, and many authors
`
`have attributed this difference to the enhanced digestion and ahsorption of phospholipids.
`
`Lirigual, gastric, and pancreatic lipases initiate digestion of triglycerides by cleaving ester
`
`bonds preferentially at external positions, stz~l and .3‘:/245, yielding a fZ~inonoglyceride and two
`
`free fatty acids; while phospliolipases, responsible for digestion of phospholipids, cleave at
`
`7 See O’Doherty at at, “Role of Ltirninal Lecithin in lntestinal Fat Absorption” Lz’piaZs 8; 249-55 (N73), Appendix
`G.
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`Serial No. l3/l 89,7l/-l
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`the Cf.’.l1lC€I'lI?0S:ilit_‘tll,
`
`.m—2, and yield a l~lys0phospholipitl and a free fatty acid.8 Carttielli at a/’.
`
`U998} suggests that cleavage at
`
`the center position results in enhanced digestion?
`
`Atiditioriallyg, triglycerides in fish oil eohtairiirig omega 3 fatty acids are relatively resistant to
`
`tligestioh by hurnan lipase.” Further, Carrtieili at at’. and Morgan et a!.” have shown that
`
`phospliolipids eoiitaiiiirig omega 3 polyunsaturated fatty acids are digested and absorbed
`
`better than triglyceihies.
`
`26. Digested and absorbed ltipitls have to he delivered, via the blood stream, to destination
`
`organs. However, hecause the digested lipids (except phospholipids) are water irisoluhle,
`
`they are packaged in rtticelltes for absorption and subsequently in Cl’lyl,OlIllCl'0,'(lS before they
`
`enter the blood stream and reach the destination organs. Chyloinicrons are specialized
`
`spherical, delivery vehicles for digested lipids which are distributed in the blood stream.
`
`it is
`
`important to emphasize that the surface of chylornicrons is coated with phospholipids that
`
`provide water solubility in the blood, while triglycerides are hutrietl in the interior.
`
`l\)7. Airiate at al. (2001) suggests that lipids following tlieir digestion and absorption are re—
`
`esterified to the same chemical fotrn (£16., either triglycerides or phtostpholipids) that they
`
`were in prior to digestion and ahsorption (ale, the same chemical form as they were in the
`.
`12
`‘
`.
`.
`.
`.
`.
`.
`diet).
`{,ilyl0t’l’l,tQl'0Il.‘§ in the blood stream exeltange their corhportehts with liigli~derisity~
`
`lipoproteiiis (HDLs,, also referred to as “good cholesterol“").j 3
`
`8 See Mattsori ez ai. “lite Digestion and Absorption of 'l‘r1glyt:erities” J B2322! Cizem 239: 2772.-711964), Appentliit
`ii; Tso et‘ all, “E.Videriee for Separate Pathways of Chylomicroii and Very Low-Density Lipoproteiri Assembly and
`T1‘2tIf‘iS}30!l hy Rat Small lritestiiie" Am J Pizysioi 247: G599-Gélll (i984), Appendix 1.
`
`9 Cariiielli en‘ .11’. “liitestiiial absorption ofloiig~chair1 polyniisatniated fatty
`formula” Am J C/2'1’: ]‘»/Mr 67; 97-lG3 (1998), Appendix J.
`
`in preterm infants
`
`breast milk or
`
`10 Bottino er al, “Resistance of Certain Loiigehaiii l’oly1tnsat1ti‘ated Fatty Acids of Marine Oils to l’ai1creatie Lipase
`Hydrolysis” Z.»z‘pz‘ds 2,
`(1967), Agipeiidix K; H€)1‘l1€li
`et al., “Does the
`Salt-Stiintilated Lipase ofliumah
`Millr Have a Role in the U of the Milk Loi1g~Chai1i Polyunsaturated Fatty Acit"1s'?” J .F'ed.iatr tfiasstmzznterol /‘v’u.,"r
`‘:6; 4263 l (1993), Apgientiix L.
`
`(“Fatty Acid Balance Studies lit Term infants Fed Formula Milk Coiitaining l_.ong—Cl1ain
`l\/{organ er a].
`l
`Polyurtsaturated Fatty Acids” Acza Paediatr 87; E3642 (1998), Apgieridix M.
`
`12 /—‘xn1ate ex ai. “Fee(l:i1i_g Infant Piglets Forirtula. with Long--Cliaiii Polyttiisatiuratied Fatity .— cicis as Triacylglycerols or
`Phospliolipids lnfluetices the Distrihutittrt of these Fatty Acids in Plasirta. Lipoproteiii Fi‘actio1is”’ .}'Nz.m~ 13 l: 1250--
`(l2tlOl), Agxpentlix N.
`
`B See lvlattsori et‘ all “The Digestion and Absorption of 'frigiycerides" J Bibi Cizem 239(9): 2772-77 (i964),
`Appendix H.
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`Attorney llloeket No. Nl3l?l\li—ill)l /'l)IZUS 3 l 3tio3—2(ll 3
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`28. lf the lipids in the diet are in the pll0Sl3l'l0ll,pl(l form (such as in lrrill oil) and sirree
`
`pliospholipids coat the exterior" of ehylornierons, they have a higher likelihood of affecting
`
`the blood lipid panel (22 total blood cholesterol, total blood triglycerides, HDLs, and low-
`
`tlensity—lipoproteihs or l,l)Ls cornrnonly refer‘r'ed to as “had cholesterol”) and thus, affecting
`
`cardiovascular disease,
`
`than triglycerides. Therefore,
`
`the transport of phospholipids to
`
`destination organs is also more easily facilitated as eornpared to triglycerides which are
`
`buried in the interior of ehylornierons and are water—insoluhle (ire,
`
`insoluble in blood).
`
`Ainate er of. (2l)0l) eontirrned this concept in piglets that were fed egg pliosplrolipids
`
`containing DHA. The piglets had higher concentrations of DHA in high density lipoproteins.
`
`29. One Could use a siniple analogy to illustrate this eornplex eoneept.
`
`lnnagine driving a car
`
`with two passengers that must reach the National Mall in Washington DC. in rush hour. The
`
`delivery of the two passengers to the National Mall will he signllieahtly delayed if the driver‘
`
`takes the. typical, always congested, highways of the DC. area. However, if the driver takes
`
`the l-l()V»lane, the two passengers will he alile to reach the Natiorial Mall rnore ellieiently.
`
`The two passengers are analogous to the two essential ornega 3 polyunsaturated fatty acids,
`
`EPA and DHA,
`
`that have to reach a destination organ (the National Mall). The slow
`
`highways in the DE. area are like the triglyceride absorption nieehanisni and the HOV-lane
`
`is like the phospliolipid absorption rneeliariisni,
`
`lt
`
`is clear that
`
`the l:lOV~lane
`
`phospholipids} will allow more efficient delivery of the two passengers (116,, EPA and TEE-ISA)
`
`to the National Mall (i.€., destination organs) when eornpared to the typical, slow highways
`
`(216,, triglycerides) in the DC area.
`
`1.)
`.
`Onzeea 3 P0iv:/msatzrzrated F625‘: ‘ml (:z‘ds in Pizos iiolz” 217a’ Form Have
`
`.1.
`ierior Medz”r:at' .€i‘,f’et;:'z‘s.
`
`30. A riuniher' of speeitie health henetitts in liunians are associated with
`
`and lZll::lA.
`
`Although these health benefits do not intrinsically depend on whether EPA and DlrlA are
`
`provided in the diet as phospholipids or triglycerides, as deseril;ied above, the delivery of
`
`these two essential oniega—3 polyunsaturated fatty acids to the destination organs is more
`
`efficient via pliospliolipids. Therefore, the arterzt of the health benefits has been shown to be
`
`greater‘ when EZPA and Dll-TA are hound in pliosplioliplds instead of tr'iglycerldes.
`
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`3l3ifi63~2{ll3
`Atterney Baeltet No. NEPN—O€)l/€)2US
`Serial No. l3/l 89,7 l4
`
`3l.
`
`Two major classes of health benefits associated with dietary intake of EPA and DHA are
`
`ahti—iriflarhmator'y and cardiovascular heneiits. These health l)€‘l”l€'lll.S
`
`are based on how EPA
`
`and DHA are irietaholized in the huiriarr body. As EPA is rrietahdlized, it competitively
`
`inhibits two enzymes (eyclooxygeriase and bllipoxygehasep}.
`
`ll" these errzyrnes are hot
`
`inhibited,
`
`they catalyze production of eicosanoids that initiate ihflaihniatioii. However,
`
`adequate supply ol’l?.l’.A results in reduced production. of eicosarieids, therehy havirlg ah anti~
`
`iriflarrnhatory
`
`effect, which ultimately
`
`reduces
`
`platelet
`
`aggregation
`
`as Well
`
`as
`
`Vasoceustrictiop. As a final result, the va,sodil.atioh is Selill’l'1E.llEJtlit”:(‘l,.M lt is apparent that l?;l’.A
`
`reduces the development of atherescleretic plaques, and as such, irhprpves eardiotvaseullar
`
`health.
`
`There is another
`
`set of f,t1“lll~lI1ll£i1Tlll1EllDI‘y eoriipouhds
`
`(D~series
`
`resolvihs,
`
`doeosatriehes, and heuroprotectins) associated with D}-{A metaholisni. These eornpeuhds
`
`have direct a.r1ti—irrflarhriiat0i*y properties uiilihe the indirect eftect of EPA via competitive
`
`enzyme i1’llll,l}llTl£‘.dl. These Dl*lA~derived Compounds clear ll'll'lEt1’l’l.'(};li~3.'ll01’l sites of cellular
`
`debris, suppress pro—iiiflarnrnatory iriterleul<ihs, and also have heuroproteetive properties.”
`
`Siniilar to l3PA, it is apparent that adequate supply of DHA improves eatrditwascular health.
`
`. As mentioned aheve, the exfem of the health herietits oforhega 3 polyunsaturated tatty acids
`
`greater when these molecules are esteritied as intact phospholipids, as compared to heing
`
`esteritied ta triglycerides. My krill research group recently published a review article
`
`describing,
`
`in part,
`
`the health hehetits associated with consumption of ltrill oil, which
`
`eeiitaihs ihtaet phesphohpids hearing diriega 3 polyunsaturated fatty acids, EPA and DEA.”
`
`This article was t"eatured as a Special Article on the cever of the February 2007 issue of
`
`/Vzlez‘.rz'z‘z'0n Reviews‘.
`
`described therein, the effect of krill oil and fish oil on hyperlipideniia
`
`was ipvestigaited using human subjects diagnosed with mild to high blood cholesterol arid
`
`14 Simopoulos, “Omega.--3 Fa LA" Acids in lutlamn1atio'u and Autoimmune Diseases” J .4171 C02? Nmr 2l(6): 495--505
`.r.,,
`(zooai Appendix O.
`
`l5 lie-rig er ¢z.'7., “Novel Docosatrieries and l’.7S—resolviris Generated from Docosaliexaenoic Acid in Marine Brain,
`llurhan Blood, and Glial Cells. Aulacoids in 1'-lxI1l,l~lI1l"l2l,II1II1Etll0I1”J'Bi0Z C,i’7.s2m 27tE€(:l'.7’p}:
`l46'.7”.7— ?7 (2003), Appendix
`F .
`
`:6 See Tou es’ aZ., “Krill for lluman Consurriptioh: Nutritional Value
`63—'.'/‘.7 (2t)0'?’}, Appendix Q.
`
`lflrtential Health Benefits” Nufr Rev 65(2):
`
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`Attorney lllocket No. Nl3l?l\li—€lt)l
`Serial No. l3/l89,7l/l
`
`total triglycei'ides,l‘7 lririlsl oil ('2-3 g/day) reduced total E)—i@fl(i' ll'lgl,yC€l‘l('l6S by 27-28‘?/o, while
`
`fish oil had no effect. Krill oil and fish oil hotli reduced blood cholesterol, hut krill oil
`
`resulted in higher reduction {up to l8%) than fish oil. Krill oil retlticetl LDLS by up to 39%,
`
`whereas llsh oil had no effect.
`
`in atldltion, krill oil increased l-l E3148 by up to 60%, while llsh
`
`oil had no efiect.
`
`ln infantsi DEA was absorbed better trorn egg pliospliolipids than algal
`
`triglycerides despite a 2.5 times higher dose of El-lA in algal trlglycei'ides_l8 in rats, krill
`
`protein concentrate (Kllif) containing DEA in phospholipicls resulted in better accretion of
`
`l3l*lA in the brain and liver cotnparetl to t'riglyceriCles.1‘Q’ This
`
`irnportant because Dl:ilA is
`
`iriclispensable and critical for proper brain development and the liver is the main organ that
`
`processes fatty acids including Dl~lA after‘ absorption and digestion, Consuniptison of EPA
`
`and D}-{A liorn krill oil resulted in an incie