(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:20)
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1060
`
`

`

`Attorney Bucket No. NlillN'silul /l)2l_lS 3 l 3663—20l 3
`Serial No. l3/l89,7l4
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`oil.1 ln addition, l am the sole inventor of an issued patent (US. 7,763,7l7) and the inventor
`
`of two other patent applications currently under examination. One focus of my patent and
`
`patent applications is a method for isolating lipids from lrrill.
`
`EN
`
`l serve on the Editorial Board for the. Journal Qquaatz‘c Food Product Tecizz’zology and as a
`
`peer—reviewer for several food science journals, such as Food Chemistry and the Journal of
`
`Agricultural rind Food Chen/zisny.
`
`i am a professional member of the institute. of Food,
`
`Technologists (“ET”), the American Chemical Society, the World Aquaculture Society, and
`
`Gamma Sigma Delta, an honorary society of agricultural scientists.
`
`1 served as a Chair of the
`
`Division of Aquatic Food Products of the HST tor the ZOlQ—lell terrn. For the past l0 years
`
`l have also taught food science—related courses at West Virginia University, many of which
`
`enroll over 300 students annually. My curriculum vitae is attached as Appendix A.
`
`in December of 20l l,
`
`l was engaged by counsel
`
`for Neptune Technologies and
`
`Bioressources, lnc. (“Neptune”) to review US Patent 8,030,348 (“the "348 patent”) and its
`
`substantive prosecution history, the Corrected Request for Reexamination tiled by Alter
`
`Biornarine (“Alter”), listed as USSN. 95/00l,7l4, including the lileclaration of Mr. Bjorn
`
`Ole Haugsgierd and the Declaration of Dr. Thomas Gundersen, and supporting materials, and
`
`to provide my expert scientific opinion regarding whether Gundersen and l’laugsgierd
`
`accurately followed the process disclosed in patent publication WO 00/23546 (“Beaudoin l”)
`
`and CA 2,25l,2(i5 (“Beaudoin ll”) and therefore whether the data presented by Alier
`
`accurately characterized the krill extract obtained by Beaudoin. Also, l was asked to express
`
`my opinion on why intact phospholipids hearing omega 3 fatty acids, such as those found in
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`l<rill oil extracts, are superior to other forms ofomega 3 fatty acids, such as the triglyceride—
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`bound forrns seen in fish and algal oils, as well as free fatty acids.
`
`LA
`
`l have had no prior direct involvement with either Neptune or Alrer.
`
`l am being compensated
`
`at my customary hourly rate for my time spent on developing, forming, and expressing the
`
`facts and opinions in this declaration.
`
`l have no personal interest in the ultimate outcome of
`
`1 See Gigliotti er a]. “Extraction and Characterisation of Lipids from Antarctic Krill (Ezrpizausia superba)” Food
`Chemistry 125(3): 10284036 (April, 2301 l), Appendix ll.
`
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`Attorney lllocket No. NlillNéilGl /02US 315663—208
`Serial No. l3/l89fi7l4
`
`the reexamination proceedings involving the ‘348 patent or any continuation applications
`
`derived from the ‘348 patent.
`
`6.
`
`l have carefully read the information provided and also conducted my own search of
`
`relevant, peermreviewed scientific literature Below l provide my expert scientitie opinion.
`
`Gnndersen and Hanvsferd Bid Not Accnratel ' leetllcate Beautloin l or Beandoin Hi
`
`7.
`
`ln my opinion? Gundersen and, liaugsgierd did not accurately reproduce the methodology for
`
`total lipid extraction from l<rill that is disclosed in Beandoin l or ll. Specifically Gundersen
`
`did not sufficiently heat the krill oil samples in a manner that was appropriate to replicate
`
`Beaudoin l or ii, and llaugsgierd did not accurately replicate the extraction method of
`
`Beandoin l or ll because he added a significant step to the Beandoin protocol. For at least
`
`these reasons, it is my opinion that Hangsgierd and, Gundersen failed to opirie on the specific
`
`process of Beaudoin l or ll and therefore failed to characterize the lrrill extract actually
`
`produced by Beaudoin l or ll.
`
`Gentlemen Did Not Appropriately Heat the Samples.
`
`8. Gundersen conducted the last step of the krill oil extraction procedure (which was partially
`
`conducted by llaugsgjerd).
`
`in doing so, Gundersen applied heat in a manner inconsistent
`
`with Beandoin l or ll to the lrrill oil extracted by Haugsgjerd. Specifically, Gunderseii
`
`alleges that he conducted, a heat treatment at l25°C for l5 minutes or at 70°C for 5 minutes,
`
`in an attempt
`
`to reproduce Beaudoin l and ll (see Gundersen Declaration,
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`liixhihit 2,
`
`Analytical Report second of two pages numbered, lf, between page 5 and, page 7').2 However,
`
`in his attempt to heat
`
`the oil” (iiundersen placed, a heat hiocl:
`
`inside the oven of a gas
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`chrornatograph set to either 700C or 125°C for at least one hour (see Grindersen Declaration!
`
`Analytical Report second page numbered l, between page 5 and page 7). A, vial of krill oil
`
`extract was then heated using the heat block for l5 minutes at lZSOC or 5 minutes at 700C
`
`(see Guildersen Declaration“, Exhibit 2, Analytical Report second of two pages numbered l,
`
`between page 5 and page 7). After Gnndersen heated the Vials, they were allowed to cool on
`
`4 l respectfully note that the confusion regarding page numbers in the Gundersen declaration stems from the
`declaration apparently being submitted either out of order or with incorrect pagination.
`
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`Attorney lloeket No. Nlillhléillll /l)2l_lS 315663—208
`Serial No. l3/l89,7l4
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`a laboratory bench to roorn tei'nperature (see Gundersen De. “laration, Exhibit 2, Analytical
`
`Report second of two pages nurnhered 1, between page 5 and page 7:).
`
`9.
`
`in my opinion, this heat treatment did not allow the oil
`
`to be heated to the temperature
`
`disclosed by Beaudoin l or ll for the time specified by Beaudoin l or ll due to slow heat
`
`transfer to the oil from the heat block.
`
`(:i'ilndersen’s heating method was mediated primarily
`
`by airmliduid convection and not conduction.
`
`it is a wellmestablished fact that conduction
`
`results in rnuch quiche. heat trai'isfer than convection.3 in simple terms, heated air contains
`
`relatively fewer molecules that can transfer heat from one object to another, as compared to
`
`heated liquids, such as oils as in a heated oil hath. Theretbre,
`
`the transfer of heat via
`
`con notion is much slower than conduction;
`
`thus,
`
`the samples heated as descrihed by
`
`Gundersen were not maintained at the temperature of lZSCC for l5 minutes or 70'3C for 5
`
`minutes.
`
`ll).
`
`A simple analogy allows illustration of this complex phenomenon. Consider placing cne’s
`
`hand in a standard kitchen oven set at a moderate temperature, say 400°F {which is about
`
`2000C). Qne could easily hold one’s hand in this oven for a period, of time before
`
`experiencing physical discomfort or injury.
`
`lf one were to place onels hand in a pot of
`
`boiling water (Zia, around, l000C), however, one would immediately experience a burning
`
`sensation. This common scenario is explained by the difference between heat transfer hy a
`
`slower method, convection (tie, the stove in the analogy), versus a faster method, conduction
`
`(Le. , the hot ling pot of water in the analogy).
`
`ll.
`
`Accordingly, when Dr. (hindersen placed the extracted krill oil in a heat hloclr, he relied on
`
`heat transfer by convection to allegedly heat the oil to lZSDC (or 70°C), Lilre the hand in the
`
`oven described above, the oil saniples themselves did not reach and maintain a temperature
`
`of lZSOC for l5 minutes.
`
`ln contrast, during the prosecution of US. Patent 8,030,348, the
`
`applicant submitted data obtained after heating for l5. minutes at
`
`lZSOC by placing the
`
`3 See, eg, Singh and l’leldrnan, Introduction to Food Ezrzgineering (3rd ed), New York, NY; Academic Press, 2008
`n . ZZZ—27‘}, A endix C‘ l’leldrnan and Lund Handbook of E9041 Envincering, New York, NY: Marcel Dehlter,
`A}?
`/
`1
`E3};
`9
`,
`9
`u
`‘
`O,
`.
`n.
`‘
`l992 (pp. 247-59), Appendix D, both at which are lnndarnental teed engineering textbooks.
`
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`

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`Attorney Bracket No. NliPNéiltll /02US 3 l 3663—201 3
`Serial No. 13/189,714
`
`extracted oil in an oil bath, which, in my opinion, accurately *e—created Beaudoiri EA Using
`
`this appropriate heat transfer niethod, mediated by conduction, the oil reached iZSOC and
`
`therefore experienced a. fall l5 ininnte exposure to this temperature.
`
`l2.l also note that the proper use of a, heat block to heat an oil extract effectively has been
`
`described in the literature. For example, in Herman and Groves} the authors conduct an
`
`experiment in which they therinally stress lipid emulsions containing phospholipids and
`
`observe hydrolysis of the tatty acids off of the phospholipids li‘on'i this heating, Specifically,
`
`they descrihe, at page 775:
`
`“Thermal stress was applied hv filling heath:U block chambers (Dry Baths, Fisher
`
`Scientific, ltasca, lL; 60 chambers per block, each 12 mm diameter and 50 rnrn deep) with oil
`
`and immersing the 2~mL stmponles containing the emulsion at the desired ternperattne,
`
`covering the blocks with aluminum foil to minimize thermal fluctuation” (emphasis added).
`
`Such a protocol would allow effective heat transfer to the sarnpies because it relies on
`
`conduction through hot oil, as was performed in obtaining the data presented in the
`
`prosecution ot‘US. 8,030,348. Gundersen did not follow this known protocol,
`
`l3, in rny expert opinion, the iriell'ective heating applied by Gnndersen had a significant ett‘ect
`
`on the extent of hydrolysis of the ester bonds connecting fatty acids (tag DHA and EPA) to
`
`the glycerol haclrhone of the phospholipids. Accordingly, Gnndersen only allegedly
`
`ohserved a residual mass spectrometry signal of phospholipids hearing Dl—lA and EPA (or
`
`EPA/EPA or DEA/DEA).
`
`l4. Further,
`
`1 also note that Gundersen provides an unclear trend as to the eflect of heating.
`
`Cornparing the llPliC—lvlfi data presented in Appendix A Gundersen appears to detect the
`
`same intensity peaks for non-heated, heated to 60°C or 709C, and heated to 1259C (see, 6.3.,
`
`chroniatograrns labeled 9308~l, P3084, and 39308—3)
`
`This thither underscores the
`
`ineffective heating approach used, by (lundersen.
`
`4 As noted in ‘14 above, I reviewed the office Action response tiled on May 31, 201i in the prosecution ofthe US,
`Patent 8,030,348
`
`5 Herman and Groves, “The influence or" Free Fatty Acid Formation on the pll of Phospholipidstabilized
`Triglyceride Ertntlsions,” Pharmaceutical Research, l0(5): 774-76 (1993), Appendix E.
`
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`Attorney Bracket No. NEPNétlm /l)21.l$ 315663—208
`Serial No. l3/l89,7l4
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`Haugsgierd and Sanderson flirted an Experimental Step Not Disclosed in Beautioin I or
`
`Beuztdoin H.
`
`l5.
`
`Further, it is my opinion as an expert on krill oil extraction that not only did l€la'ugsgierrl and
`
`Gundersen fail to establish that the oils were sufficiently heated to replicate Beaudoin l or ii,
`
`l-laugsgjerd also performed an initiations! experimental step in his extraction procedure that
`
`deviated from Beaudoin l and. ll. Specifically, Haugsgjerd reports the following steps to
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`generate Fractions lla and llh (llaugsgjerd Declaration, ll 3, Gundersen Becla‘ation, Exhibit
`
`2, Analytical Report second of two pages numbered l, between page 5 and page 7):
`
`6*
`
`a
`
`a
`
`extracting with either ethanol or ethyl acetate;
`
`filtering solvent and evaporating under reduced pressure;
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`“flushfingl samples with nitrogen gas” and “storlingl at "ZGC until further analysis?
`
`and
`
`a
`
`sending
`
`sainples
`
`front
`
`lI-iaugsgjerd
`
`to
`
`(Ilundersen
`
`and
`
`having
`
`(Ilundersen
`
`unsuccessfully attempt to heat to l 250C for 15 minutes.
`
`l note that the Beaudoin l or ii protocol, as successfully replicated to generate the data
`
`presented in the prosecution of US. 8,030,348, did not irwolye flushing oil tractions with
`
`nitrogen gas and freezing at 430%) before heating. 0n the contrary, in both the disclosed
`
`Beaudoin process and the experiments conducted to generate the data presented in the
`
`prosecution of US. 8,030,348,
`
`following, evaporation to partially reniove solvent,
`
`the
`
`fractions were immediately heated {see Response to Office Action oi‘April 29, 20K) in US.
`
`lit/485,094, Appendix 1;, Beaudoin l, pages 7 and l0), There is no mention in Beaudoin l or
`
`ll of either storing samples at ~200C or under nitrogen gas.
`
`16.
`
`in my opinion, Haugsgierd’s deviation from the process disclosed in Beaudoin l or ll is very
`
`significant. By treezing the sample before heating,
`
`llaugsgjerd further suppressed any
`
`hydrolysis of ester bonds found on the phospholipids being analyzed.
`
`it is my opinion that
`
`after Beaudoin completed the penultimate step of removing the solvent by rotary
`
`evaporation, he heated to remove the residual “volatile matter and humidity” from fraction 5
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`Attorney lloeket No. NEPNéilGl /l)2US 3 l 3663—20l 3
`Serial No. l3/l89,7l4
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`and traction ll {see Beaudoin l, page l0), Note that in Table iii of Beaudoin l “volatile
`
`matter and moisture levels” of l
`
`0/0 and 6.80/25, respectively, for fractions l and ll are reported.
`
`These levels of solvent and humidity would have rendered those oils crude for Beaudoins
`
`purposes of determining total lipids generated by his procedure, and, therefore prompted him
`
`to “get rid of traces of solvents {by} hrielly heatiing] (to about lZSDC‘, for about l5 min.) the
`
`oil under nitrogen” {see Beautioin 5, pages 7 and, l0),
`
`'l'hls heating inevitably led to the
`
`hydrolysis of ester bonds. As a result, the phospholipids were degraded and consequently
`
`released free latty acids such as EPA and DHA.
`
`it is my opinion that llaugsgierd suppressed
`
`this inevitable hydrolysis by storing his samples at —20°C. This is a step not taught by
`
`Beaudoin l or ii, which causes one to question Why llaugsgerd did this,
`
`it is also my
`
`opinion that had l-laugsgjerd heated the samples in an oil hath or with a heating jaeltet after
`
`removing the solvents by rotary evaporation and then stored. the final product under nitrogen
`
`at 420%? for subsequent evaluation by Gundersen, (luridersen would not have allegedly
`
`observed even residual amounts of phospholipids carrying two of DHA and/or EPA. I note,
`
`as evideneejbr this opinion, the paper Cited in ll i2 above in which the authors thermally
`
`stressedphosphohpid emulsions in heating blocks containing at! and described hydrolysis of
`
`ester bonds in phosphohpids. As an inevitable consequence (5f'thz’s thermal hydrolysis, flee
`
`fairly? acids were releasedfi‘mn the phosphoihflcfs.
`
`17. in summary, it is my opinion that, due to inadequate sample heating and, the addition of an
`
`experimental step to the extraction protocol, Haugsgierd and Gunderscn did not accurately
`
`reproduce the methodology of oil extraction from krill as disclosed in lileaudoin l or ii, and
`
`therefore the mass spectrometric data, presented by Gnnderscn fails to accurately characterize
`
`the krill oil actually produced, by Beaudoin l or ll,
`
`
`intact P’hos hell litiS Containine (line ta 3 Polyunsaturated Fart ' Acids Possess Desirable
`
`
`l’ro erties Not (Eliserved in (Ether Forms of Li ids Sliifll as 'l‘rielyeerides (Iontainin
`
`
`{lineea 3 l’elvansaturated Fatty Aeids.
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`Attarney Bracket Ne. NEPNéQGl r’llfllS 3 l 3663—20l 3
`Serial N0. l3/l89,7l4
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`l8.
`
`l have been asked to express tn y opinion, based on, the peer reviewed literature, including my
`
`lahcratory’s published research, on the superior properties of omega 3 fatty acids in
`
`phcspholipid fcriii as compared tc ether tbrrns (or lipid classes), such as triglycerides.
`
`Feasible Farms Qmecga 3 Fatty Acids and Their Presence in l/Zirimrs Extracts.
`
`l9. The ‘348 patent teaches extracts,
`
`including l<rill extracts, and mere specifically,
`
`intact
`
`phcspholipids hearing omega 3 pclyunsaturated fatty acids, such as EPA and Dl—lA. These
`
`compositions are distinguishable from fish oil and algal oil, as fish and algal oil extracts
`
`leatnre EPA and DHA bound to triglycerides. My lahcra‘tcry recently ccriducted a
`
`comparative study among krill cil, fish oil, and algal eil.‘S Thin—layer—chrcmatography (TLC)
`
`analysis clearly den'icnstrated that krill oil contained significant arncnrits cf phospholipids.
`
`While the amount of detected phospholipids in fish oil and algal oil was negligible. Further,
`
`TLC experiments shcwed that lerill cil ccntained negligible amounts of triglycerides, while
`
`fish Oil and algal oil contained significant amounts.
`
`. Further, the Kassis at all study shcwed that there are high amounts of EPA and, Dl-lA in krill
`
`oil {47% of total fatty acids) and these omega 3 pclyunsatnrated fatty acids are primarily
`
`esteril'ied in phcsphclipids. On the other hand, EPA and DHA in fish cil and algal oil are net
`
`significantly esterified in phosphelipids, but are largely esterilied iii triglycerides.
`
`. 'l‘herefcre, krill oil, unlike fish cil and algal oil contains significant amounts of omega 3 fatty
`
`acids esterified iii phcsphclipids.
`
`Phospizclz’pids Have .Szrperiar Piiysz'aiogical Absorption Prqfir’es.
`
`‘7 "l
`1... Based en the recently develcpihg research in this field, it appears that the chemical “carrier”
`
`ct" cmega 3 polyunsaturated, fatty acids in krill extracts, 22a, phospholipids, prcyides superior
`
`physiological absorption profiles, especially as compared tc triglyceride “carriers.”
`
`23.
`
`l’hcspholipids are ampthhilic and triglycerides are not. The chemical structure of an
`
`amphiphilic ccnipeimd contains fimcticn al grcups that allow simultaneous water~ and, lipid—
`
`° See Kassis at (1.7., “Characterization of Lipids and Antioxidant Capacity of Novel ' utraceutical Egg Products
`Developed with Ornega-S-Rich Oils” J Sci Food Agr 92(l); 66-573 (2012), Appendix I
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`Attorney Becket No. NEPNQGl /l)2US 3 l 3663—201 3
`Serial No. l3/l89,714
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`soluhility due to the presence of hydrophilic and hydrophobic n'ioieties. The hydrophilic
`
`rnoiety in krill phospholipids is present at the sn—3 position, where the nitrogenucontaining
`
`rnoiety (cg, the choline in phosphatidylcholine) 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 phospholipids is present at the sn-l and Sir—2 positions in the form of the long
`
`hydrocarbon chains of omega 3 polynnsaturated fatty acids.
`
`in contrast, the triglycerides of
`
`fish oil and algal oil are not arnphiphilie, as their sn—l, sn~2, and sn~3 positions are occupied
`
`with hydrophobic fatty acids that have long hydrocarbon chains. Accordingly, triglycerides
`
`are lipid—soluble but not water—soluble. By way of analogy, phospholipids (such as those of
`
`the ‘348 patent) are litre ambidextrous people, having the ability to write with both the right
`
`and left hand, while triglycerides (_such as those of fish oil and algal oil) are lilre right— or left"
`
`handed people, having the ability to write with only a single hand.
`
`. This duality of phospholipids has irnplications for absorption in the body. Vt’hen lipids (Le,
`
`typical oils and fats composed of triglycerides, such as fish oil and algal oil} are ingested,
`
`because. of their water insolubility and lower density than water, they float on top of and form
`
`a layer that is separate front the digestive juices in the stomach and small intestine.
`
`'l‘hese
`
`digestive juices contain lipolytic enzymes that digest
`
`lipids, Therefore, hefore y’ater—
`
`insoluble lipids (La,
`
`triglycerides) can be digested and absorbed,
`
`they first have to he
`
`emulsified by bile. However, ainphiphilic compounds such as phospholipids (like those in
`
`the “348‘ patent) skip the ernulsification process because they are water soluble.
`
`ln fact,
`
`phospholipids enhance eniulsification by bile and,
`
`thereby, aid in digestion of water—
`
`insoluhle oils,’
`
`l\) l}!
`
`, Phospholipids and triglycerides are also digested by different enzyrnes, and many authors
`
`have attributed this difference to the enhanced digestion and absorption of phospholipids.
`
`Lingual, gastric, and pancreatic lipases initiate. digestion of triglycerides by cleaving ester
`
`bonds preferentially at external positions, sn~l and 3:445, yielding a 2-rnonoglyceride and two
`
`free fatty acids; while phospholipases, responsible for digestion of phospholipids, cleave at
`
`7 See O’Doherty et all, “Role of Lurnirral Lecithin in intestinal Fat Absorption” Lipids 8; 249—55 (M73), Appendix
`G.
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`Attnrney Becket No. NEPNéilm /l)2l_lS 315663—208
`Serial Ne. l3/l89,7l4
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`the center position, sa—2, and yield a l~lysephospholipid and a tree fatty acid.8 Carnielli et ai.
`
`U998} suggests that cleavage at
`
`the center position results in enhanced digestinn.9
`
`Additionally, triglycerides in fish oil containing eniega 3 fatty acids are relatively resistant to
`
`digestion by human lipase.m li‘nrther, Carnielli er al. and Morgan at 11!.“ have shown that
`
`phosphclipids containing omega 3 polyunsaturated fatty acids are digested and abscrbed
`
`better than triglycerides.
`
`26. Digested and ahserhed lipids have to he delivered, via the blood stream, tc destination
`
`organs. However, hecause the digested lipids (except phospholipids) are water insolnhle,
`
`they are packaged in inicelles for abserptien and subsequently in chylctnicrons before they
`
`enter the bleed stream and reach the destinatidn ergans. Chylornicrens are specialized
`
`spherical, delivery vehicles for digested lipids which are distributed in the blood stream.
`
`it is
`
`important tn emphasize that the surface of chylnniicrons is coated with phospholipids that
`
`provide water selnhility in the blood, while triglycerides are buried in the interior.
`
`l\)7. Aniate er al. {200D suggests that lipids tellnwing their digesticn and absorption are re“
`
`esteril'ied to the same chemical fill‘l’l’l (Le, eithe‘ triglycerides in“ phesphnlinids) that they
`
`were in prior to digestion and ahsorption (226., the same chemical farm as they were in the
`
`diet).12 Chylernierons in the bleed, strearn exchange their components with, liigli~density~
`
`lipeproteins (HDLs, also referred te as “good chelesternl”).j 3
`
`8 See Mattsnn at (12. “The Digestion and Absnmtinn cl" Triglycerides” J Bid] Chem 239: 27 2,-7til964), Aepentlix
`ll; ’l‘se at all, “Evidence for Separate Pathways of Chylornicrnn and Very Lew-Density Lipnprntein Assembly and
`Transport by Rat Small intestine" Am J Physiei 247: GSQ9—Gél0 (1984), Appendix 1,
`,
`.. '
`,
`. ,
`'
`(“v
`t
`u“
`l
`'\
`V
`’
`'
`45
`,
`'
`,
`l
`‘ ‘
`'
`,
`u“
`,
`v
`‘
`,
`l
`5 (,arnrellr at az’.
`intestinal absorption of, iong~charn polyunsaturated ratty acids in preterm inlants red breast milk or
`lbrrnula” Am J (3/27; Nair 67; 97—l03 (Willi), Apgendix J.
`
`10 Bettina) e: at, “Resistance of Certain Lengchain Polyunsaturated Fatty Acids of Marine Oils to Pancreatic Lipase
`Hydrolysis” lipids: 2, 489—93 (“967), Appendix K; Hernell at a]., “Does the Bile Salt~Stlmnlated Lipase ot‘lluman
`Mill: Have a Role in the Use of the Milk Long/Chain Pnlyunsatnrated Fatty Acids?” J Pediatr (fiastmenteml Nair
`l6; 426—31tl993), Apnendix L.
`
`infants Fed Formula Milk Cnntaining Long—Chain
`(“Fatty Acid Balance Studies lri Terni
`l Mnrgan er, a].
`Polyunsaturated Fatty Acids” Acta Paediatr 87;
`l36—42 (1998'), Appendix M.
`
`12 Atnate e: aZ. “Feeding, lni"ant Piglets Formula with long-Chain Polytinsattn‘ated Fatty Acids as 'l‘riacylglycernls or
`Phosphdllplds Influences the Distrilsntidn (if these Fatty Acids in Plasma Lipnprnleln Fractinns” JCNmr 13' l: 1250--
`55 (2001), Annendix N.
`
`13 See Mattson at all “The Digestinn and Absnrptinn of Triglycerides" J Biol Chem 239(9): 2772—77 (1964),
`Appendix ll.
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`28. if the lipids in the diet are in the phospholipid forni (such as in krill oil) and since
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`phospholipids coat the exterior of chyloniicrons, they have a higher likelihood of affecting
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`the blood lipid panel (1'. 6 total blood cholesterol, total blood triglycerides, HDLs, and, low"
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`tlensity—lipoproteins or LIELs cornrnonly referred, to as “had cholesterol”) and thus, affecting
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`cardiovascular disease,
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`than triglycerides. Therefore,
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`the.
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`transport of phospholipids to
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`destination organs is also more easily facilitated as compared to triglycerides which are
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`buried in the interior of chyloniicrons and are water—insoluble (is, insoluble in blood).
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`Arnate at of. (200” confirmed this concept in piglets that were fed egg phospholipids
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`containing DHA. The piglets had higher concentrations of BSA in high density lipoproteins.
`
`29. One could use a simple. analogy to illustrate this complex concept.
`
`imagine driving a car
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`with two passengers that must reach the National Mall in Washington DC. in rush hour. The
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`delivery of the two passengers to the National Mall will be significantly delayed if the driver
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`takes the typical, always congested, highways of the DC. area. However, if the driver takes
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`the i—lGVdane, the two passengers will he able to reach the National Mall more efficiently.
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`The two passengers are analogous to the two essential omega 3 polyunsaturated fatty acids,
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`EPA and DltlA,
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`that have to reach a destination organ (the National Mall). The slow
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`highways in the DC. area are like. the triglyceride absorption mechanism and the HOV-lane
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`is like the phospholipid absorption mechanism.
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`it
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`is clear that
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`the llOVdane (£1.42
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`phospholipids} will allow more efficient delivery of the two passengers (17.6., EPA and 138A)
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`to the National Mall (126., destination organs) when cornpared to the typical, slow highways
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`(216., triglycerides) in the DC area.
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`Omega 3 Polynrtsaiumted Fatty A (32‘sz in Plzospholzpid Form Have Shperior Medical Egrecls.
`
`30. A, number of specific health benefits in humans are associated with EPA and lilllA.
`
`Although these health benefits do not intrinsically depend on whether EPA and DHA are
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`provided in the diet as phospholipids or triglycerides, as described above. the delivery of
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`these two essential omega—3 polyunsaturated fatty acids to the destination organs is more
`
`efficient yia phospholipids. Therefore, the extent of the health benefits has been shown to be
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`greater when EPA and Dl-lA are hound in phospholipids instead of triglycerides.
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`31. Two inajer classes of health benefits associated with dietary intake of EPA and DHA are
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`anti—inflammatory and, cardiovascular benefits“ These health benefits are based on how EPA
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`and DHA are metabolized in the human body. As EPA is rnetabolizedi it cenipetitively
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`inhibits two enzymes (cycleoxygenase and 5—lipexygenase}.
`
`if these enzymes are not
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`inhibited,
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`they catalyze production of eieesannids that initiate inflammation. However,
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`adequate supply efEPA results in reduced production of eicesanoidsfl thereby having an anti~
`
`inflannnatery
`
`effect, which ultimately
`
`reduces
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`platelet
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`aggregation
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`as well
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`as
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`vasoconstriction. As a final result“, the vasodilatinn is stintelated.14 it is apparent that EPA
`
`reduces the development of atherosclerotie plaques, and as such, improves cardiovascular
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`health.
`
`There.
`
`is anether
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`set at antimint'lannnatery compounds
`
`(B-series
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`resolvins,
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`deeosatrienes, and, neureprnteetins) associated with {El-{A metabolism“ These compounds
`
`have direct antininflaniniatory properties unlike the indirect effect of EPA via competitive
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`enzyme inhibition These Di’lAalerived compounds clear inflammation sites of eellnlar
`
`debris, suppress pro-inflammatory interleukins, and also have neureprotective properties.j5
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`Similar to EPA9 it is apparent that adequate supply ef Di’lA improves cardim’ascular health
`
`. As mentioned above, the extent of the health benefits efoniega 3 polyunsaturated fatty acids
`
`is greater when these molecules are esterified as intact phespholipids, as compared to being
`
`esterified to triglycerides. My krill research group recently published a review article
`
`describing,
`
`in part,
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`the health benefits associated with consumption of krill oil, which
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`contains intact phosphohpids bearing omega 3 polyunsaturated fatty acids, EPA and DHA.16
`
`This article was featured as a Special Article on the cever of the February 2307 issue of
`
`Nutrition Reviews As described therein, the effect of krill oil and fish oil en hyperlipideinia
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`was investigated using> human subjects diagnesed with mild to high blood cholesterol and
`
`H Simopoules, “Omega-3 Fa Ly
`(2002), Appendix O.
`
` Acids in inflammation and Antoiimriune Diseases” J Am Coil Nair 21(6): 4951-505
`
`15}long at (217., “Novel Docosatrienes and i7S—resolvins Generated from Docosahexaenoic Acid in Murine Brain,
`q x.
`l’lurnan Blood, and Glial Cells. Autacoids in Anti~1nflannnaiion” J'Bz'ol Chem 4,1,};(17): 1467’7- ?7 (2003}, Appendix
`F u
`
`:6 See Ton et all, “Krill for Human Consumption: Nutritional Value and Potential Health Benefits” Ai’tttr Rev 65(2):
`,7,
`63— ’.7 (2007’), Appendix Q.
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`total triglycerides}7 Krill oil (_12—3 g/day) reduced total blood, triglycerides by 273894;“, while
`
`fish oil had no effect. Krill oil and fish oil both reduced blood cholesterol, but krill oil
`
`resulted in higher reduction {up to 18%) than iish oil. Krill oil reduced LDLs by up to 39%?
`
`whereas lish oil had no effect.
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`in addition, lrrill oil increased l—lDlts by up to 60%, While lish
`
`oil had no effect.
`
`in infantsa DEA was absorbed better from egg phospliolipids than algal
`
`triglycerides despite a 2.5 times higher dose of 138A in algal triglycerides18 in rats, krill
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`protein concentrate (KEG) containing BSA in phospholipids resulted in better accretion of
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`DltlA in the brain and liver compared to triglycerides.19 This is important because DHA is
`
`indispensable and critical for proper brain development and the liver is the main organ that
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`processes tatty acids including Dl’lA alter absorption and digestion Consumption of EPA
`
`and DHA from krill oil resulted in an increased, l-lDies/triglycerides ratio compared to fish oil
`
`even though the DHA concentration in fish oil was more than 2 times higher than in the krill
`
`oil adn'iinistered.20 li‘urther,
`
`l<rill oil resulted in reduction of several symptoms commonly
`
`associated with premenstrual syndrome and dysinenorrhea to a greater degree than fish oil.2i
`
`This was attributed to more efficient delivery of EPA/Dim esteril'ied in phospholipids in
`
`krill oil as opposed to triglycerides in fish oil.
`
`The Eritrea? ofre “348 Parent, Bur Na Baltimore ’5 Oil, Whale! Likely Possess the A have Efiectr.
`
`33. The ‘3-48 patent teaches intact phospholipids containing EPA and DHA (see. Tables 9 and it)
`
`of the ‘348 patent} These phosnholipids are not heat damaged (see Column ll; of the ‘348
`
`patent). Therefore, in my opinion, the extract of the ‘348 patent would posses the superior
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`phospholipidmassociated physiological elects observed in the articles discussed above.
`
`in
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`contrast, the Beandoin oil, containing far fewer intact phospliolipids and being heat damaged,
`
`17 See Tou er a]. (citing Bunea et al, “Evaluation of the Effects of Neptune Krill Oil on the Clinical Course of
`Hyperlinideniia” A Item Med Rev 9; 420—28 (2004)., Appendix R.
`
`18 Cainielli e? at, “intestinal Absorption of Long~Chain Polyunsattn‘ated Fatty Acids in Preterm infants Fed Breast
`Milli: or Formula” Am J Clin Nutr 67: 97403 @998), Appendix J
`
`1'9 Bridges at all, “Determination of Digestihility, Tissue Deposition, and Metabolism of the Orncga—S’ Fatty Acid
`Content of Krill Protein Concentrate in Growing Rats” Jign’c Food Chem 58; 330—7 (2010), Annettdi‘x Si
`
`20 Ulven er al, “i‘vletabolic Effects of Krill Oil are Essentially Similar to Those of Fish Oil But at lower Dose of
`EPA and DHA, in Health Volunteers” Lipids 46; 37-46 (Elli l)? Agreendlx T
`2]
`
`Sanipalis e! (117,, “Evaluation of the Effects oi" Neptune Krill OilTM on the Management of Premenstrual Syndrome
`and Dysmenorrheal” Alien: M’ea’ Rev 8; l7l~9 (2003)» Appendix U.
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`would not possess the extent of these beneficial characteristics. Thi s is 1oolstered hy the fact
`
`that Beaudoin discloses that the fractions l and ll. before heating, have free fatty acid
`
`
`amounts ol‘23.7 i l.l% and 20.3 :: 0.3%5 respectively (see Beaudoin I, Table l4). That is to
`
`say, almost one quarter of the Beaudoin oil
`
`is species that are m2: matched to the
`
`phospholipld carrier befbre heating. After exposing the oil to lZSCC for l5 minutes, the
`
`amount of phospholipids that are intact is less, as seen in the experiments conducted in the
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`prosecution of the ‘348 patent and supported by the literature.
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`34. Specifically, heat—induced hydrolysis will occur upon this heating. The ex