`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1060
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`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
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`of two other patent applications currently under examination. One focus of my patent and
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`patent applications is a method for isolating lipids from lrrill.
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`EN
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`l serve on the Editorial Board for the. Journal Qquaatz‘c Food Product Tecizz’zology and as a
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`peer—reviewer for several food science journals, such as Food Chemistry and the Journal of
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`Agricultural rind Food Chen/zisny.
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`i am a professional member of the institute. of Food,
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`Technologists (“ET”), the American Chemical Society, the World Aquaculture Society, and
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`Gamma Sigma Delta, an honorary society of agricultural scientists.
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`1 served as a Chair of the
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`Division of Aquatic Food Products of the HST tor the ZOlQ—lell terrn. For the past l0 years
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`l have also taught food science—related courses at West Virginia University, many of which
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`enroll over 300 students annually. My curriculum vitae is attached as Appendix A.
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`in December of 20l 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 US Patent 8,030,348 (“the "348 patent”) and its
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`substantive prosecution history, the Corrected Request for Reexamination tiled by Alter
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`Biornarine (“Alter”), listed as USSN. 95/00l,7l4, including the lileclaration of Mr. Bjorn
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`Ole Haugsgierd and the Declaration of Dr. Thomas Gundersen, and supporting materials, and
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`to provide my expert scientific opinion regarding whether Gundersen and l’laugsgierd
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`accurately followed the process disclosed in patent publication WO 00/23546 (“Beaudoin l”)
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`and CA 2,25l,2(i5 (“Beaudoin ll”) and therefore whether the data presented by Alier
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`accurately characterized the krill extract obtained by Beaudoin. Also, l was asked to express
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`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.
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`LA
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`l have had no prior direct involvement with either Neptune or Alrer.
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`l am being compensated
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`at my customary hourly rate for my time spent on developing, forming, 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 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
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`the reexamination proceedings involving the ‘348 patent or any continuation applications
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`derived from the ‘348 patent.
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`6.
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`l have carefully read the information provided and also conducted my own search of
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`relevant, peermreviewed scientific literature Below l provide my expert scientitie opinion.
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`Gnndersen and Hanvsferd Bid Not Accnratel ' leetllcate Beautloin l or Beandoin Hi
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`7.
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`ln my opinion? Gundersen and, liaugsgierd did not accurately reproduce the methodology for
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`total lipid extraction from l<rill that is disclosed in Beandoin l or ll. Specifically Gundersen
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`did not sufficiently heat the krill oil samples in a manner that was appropriate to replicate
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`Beaudoin l or ii, and llaugsgierd did not accurately replicate the extraction method of
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`Beandoin l or ll because he added a significant step to the Beandoin protocol. For at least
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`these reasons, it is my opinion that Hangsgierd and, Gundersen failed to opirie on the specific
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`process of Beaudoin l or ll and therefore failed to characterize the lrrill extract actually
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`produced by Beaudoin l or ll.
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`Gentlemen Did Not Appropriately Heat the Samples.
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`8. Gundersen conducted the last step of the krill oil extraction procedure (which was partially
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`conducted by llaugsgjerd).
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`in doing so, Gundersen applied heat in a manner inconsistent
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`with Beandoin l or ll to the lrrill oil extracted by Haugsgjerd. Specifically, Gunderseii
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`alleges that he conducted, a heat treatment at l25°C for l5 minutes or at 70°C for 5 minutes,
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`in an attempt
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`to reproduce Beaudoin l and ll (see Gundersen Declaration,
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`liixhihit 2,
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`Analytical Report second of two pages numbered, lf, between page 5 and, page 7').2 However,
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`in his attempt to heat
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`the oil” (iiundersen placed, a heat hiocl:
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`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!
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`Analytical Report second page numbered 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 lZSOC or 5 minutes at 700C
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`(see Guildersen Declaration“, Exhibit 2, Analytical Report second of two pages numbered l,
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`between page 5 and page 7). After Gnndersen heated the Vials, they were allowed to cool on
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`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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`a laboratory bench to roorn tei'nperature (see Gundersen De. “laration, Exhibit 2, Analytical
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`Report second of two pages nurnhered 1, between page 5 and page 7:).
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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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`disclosed by Beaudoin l or ll for the time specified by Beaudoin l or ll due to slow heat
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`transfer to the oil from the heat block.
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`(:i'ilndersen’s heating method was mediated primarily
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`by airmliduid convection and not conduction.
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`it is a wellmestablished fact that conduction
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`results in rnuch quiche. heat trai'isfer than convection.3 in 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 as oils as in a heated oil hath. Theretbre,
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`the transfer of heat via
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`con notion is much slower than conduction;
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`thus,
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`the samples heated as descrihed by
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`Gundersen were not maintained at the temperature of lZSCC for l5 minutes or 70'3C 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 cne’s
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`hand in a standard kitchen oven set at a moderate temperature, say 400°F {which is about
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`2000C). Qne could easily hold one’s hand in this oven for a period, of time before
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`experiencing physical discomfort or injury.
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`lf one were to place onels hand in a pot of
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`boiling water (Zia, around, l000C), however, one would immediately experience a burning
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`sensation. This common scenario is explained by the difference between heat transfer hy a
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`slower method, convection (tie, the stove in the analogy), versus a faster method, conduction
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`(Le. , the hot ling pot of water in the analogy).
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`ll.
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`Accordingly, when Dr. (hindersen placed the extracted krill oil in a heat hloclr, he relied on
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`heat transfer by convection to allegedly heat the oil to lZSDC (or 70°C), Lilre the hand in the
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`oven described above, the oil saniples themselves did not reach and maintain a temperature
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`of lZSOC for l5 minutes.
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`ln contrast, during the prosecution of US. Patent 8,030,348, the
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`applicant submitted data obtained after heating for l5. minutes at
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`lZSOC by placing the
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`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
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`9
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`O,
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`n.
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`l992 (pp. 247-59), Appendix D, both at which are lnndarnental teed engineering textbooks.
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`Attorney Bracket No. NliPNéiltll /02US 3 l 3663—201 3
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`extracted oil in an oil bath, which, in my opinion, accurately *e—created Beaudoiri EA Using
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`this appropriate heat transfer niethod, mediated by conduction, the oil reached iZSOC and
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`therefore experienced a. fall l5 ininnte exposure to this temperature.
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`l2.l also note that the proper use of a, heat block to heat an oil extract effectively has been
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`described in the literature. For example, in Herman and Groves} the authors conduct an
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`experiment in which they therinally stress lipid emulsions containing phospholipids and
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`observe hydrolysis of the tatty acids off of the phospholipids li‘on'i this heating, Specifically,
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`they descrihe, at page 775:
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`“Thermal stress was applied hv filling heath:U block chambers (Dry Baths, Fisher
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`Scientific, ltasca, lL; 60 chambers per block, each 12 mm diameter and 50 rnrn deep) with oil
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`and immersing the 2~mL stmponles containing the emulsion at the desired ternperattne,
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`covering the blocks with aluminum foil to minimize thermal fluctuation” (emphasis added).
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`Such a protocol would allow effective heat transfer to the sarnpies because it relies on
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`conduction through hot oil, as was performed in obtaining the data presented in the
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`prosecution ot‘US. 8,030,348. Gundersen did not follow this known protocol,
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`l3, in rny expert opinion, the iriell'ective heating applied by Gnndersen had a significant ett‘ect
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`on the extent of hydrolysis of the ester bonds connecting fatty acids (tag DHA and EPA) to
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`the glycerol haclrhone of the phospholipids. Accordingly, Gnndersen only allegedly
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`ohserved a residual mass spectrometry signal of phospholipids hearing Dl—lA and EPA (or
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`EPA/EPA or DEA/DEA).
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`l4. Further,
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`1 also note that Gundersen provides an unclear trend as to the eflect of heating.
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`Cornparing the llPliC—lvlfi data presented in Appendix A Gundersen appears to detect the
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`same intensity peaks for non-heated, heated to 60°C or 709C, and heated to 1259C (see, 6.3.,
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`chroniatograrns labeled 9308~l, P3084, and 39308—3)
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`This thither underscores the
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`ineffective heating approach used, by (lundersen.
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`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
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`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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`Haugsgierd and Sanderson flirted an Experimental Step Not Disclosed in Beautioin I or
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`Beuztdoin 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 l€la'ugsgierrl and
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`Gundersen fail to establish that the oils were sufficiently heated to replicate Beaudoin l or ii,
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`l-laugsgjerd also performed an initiations! experimental step in his extraction procedure that
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`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
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`2, Analytical Report second of two pages numbered l, between page 5 and page 7):
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`6*
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`a
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`a
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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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`“flushfingl samples with nitrogen gas” and “storlingl at "ZGC until further analysis?
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`and
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`a
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`sending
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`sainples
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`front
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`lI-iaugsgjerd
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`to
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`(Ilundersen
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`and
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`having
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`(Ilundersen
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`unsuccessfully attempt to heat to l 250C for 15 minutes.
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`l note that the Beaudoin l or ii protocol, as successfully replicated to generate the data
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`presented in the prosecution of US. 8,030,348, did not irwolye flushing oil tractions with
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`nitrogen gas and freezing at 430%) before heating. 0n the contrary, in both the disclosed
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`Beaudoin process and the experiments 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 immediately heated {see Response to Office Action oi‘April 29, 20K) in US.
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`lit/485,094, Appendix 1;, Beaudoin l, pages 7 and l0), There is no mention in Beaudoin l or
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`ll of either storing samples at ~200C or under nitrogen gas.
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`16.
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`in my opinion, Haugsgierd’s deviation from the process disclosed in Beaudoin l or ll is very
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`significant. By treezing the sample before heating,
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`llaugsgjerd further suppressed any
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`hydrolysis of ester bonds found on the phospholipids being analyzed.
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`it is my opinion that
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`after Beaudoin completed the penultimate step of removing the solvent by rotary
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`evaporation, he heated to remove the residual “volatile matter and humidity” from fraction 5
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`and traction ll {see Beaudoin l, page l0), Note that in Table iii of Beaudoin l “volatile
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`matter and moisture levels” of l
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`0/0 and 6.80/25, respectively, for fractions l and ll are reported.
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`These levels of solvent and humidity would have rendered those oils crude for Beaudoins
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`purposes of determining total lipids generated by his procedure, and, therefore prompted him
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`to “get rid of traces of solvents {by} hrielly heatiing] (to about lZSDC‘, for about l5 min.) the
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`oil under nitrogen” {see Beautioin 5, pages 7 and, l0),
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`'l'hls heating inevitably led to the
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`hydrolysis of ester bonds. As a result, the phospholipids were degraded and consequently
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`released free latty acids such as EPA and DHA.
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`it is my opinion that llaugsgierd suppressed
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`this inevitable hydrolysis by storing his samples at —20°C. This is a step not taught by
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`Beaudoin l or ii, which causes one to question Why llaugsgerd did this,
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`it is also my
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`opinion that had l-laugsgjerd heated the samples in an oil hath or with a heating jaeltet after
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`removing the solvents by rotary evaporation and then stored. the final product under nitrogen
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`at 420%? for subsequent evaluation by Gundersen, (luridersen would not have allegedly
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`observed even residual amounts of phospholipids carrying two of DHA and/or EPA. I note,
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`as evideneejbr this opinion, the paper Cited in ll i2 above in which the authors thermally
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`stressedphosphohpid emulsions in heating blocks containing at! and described hydrolysis of
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`ester bonds in phosphohpids. As an inevitable consequence (5f'thz’s thermal hydrolysis, flee
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`fairly? acids were releasedfi‘mn the phosphoihflcfs.
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`17. in summary, it is my opinion that, due to inadequate sample heating and, the addition of an
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`experimental step to the extraction protocol, Haugsgierd and Gunderscn did not accurately
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`reproduce the methodology of oil extraction from krill as disclosed in lileaudoin l or ii, and
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`therefore the mass spectrometric data, presented by Gnnderscn fails to accurately characterize
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`the krill oil actually produced, by Beaudoin l or ll,
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`intact P’hos hell litiS Containine (line ta 3 Polyunsaturated Fart ' Acids Possess Desirable
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`l’ro erties Not (Eliserved in (Ether Forms of Li ids Sliifll as 'l‘rielyeerides (Iontainin
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`{lineea 3 l’elvansaturated Fatty Aeids.
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`l8.
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`l have been asked to express tn y opinion, based on, the peer reviewed literature, including my
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`lahcratory’s published research, on the superior properties of omega 3 fatty acids in
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`phcspholipid fcriii as compared tc ether tbrrns (or lipid classes), such as triglycerides.
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`Feasible Farms Qmecga 3 Fatty Acids and Their Presence in l/Zirimrs Extracts.
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`l9. The ‘348 patent teaches extracts,
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`including l<rill extracts, and mere specifically,
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`intact
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`phcspholipids hearing omega 3 pclyunsaturated fatty acids, such as EPA and Dl—lA. These
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`compositions are distinguishable from fish oil and algal oil, as fish and algal oil extracts
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`leatnre EPA and DHA bound to triglycerides. My lahcra‘tcry recently ccriducted a
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`comparative study among krill cil, fish oil, and algal eil.‘S Thin—layer—chrcmatography (TLC)
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`analysis clearly den'icnstrated that krill oil contained significant arncnrits cf phospholipids.
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`While the amount of detected phospholipids in fish oil and algal oil was negligible. Further,
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`TLC experiments shcwed that lerill cil ccntained negligible amounts of triglycerides, while
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`fish Oil and algal oil contained significant amounts.
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`. Further, the Kassis at all study shcwed that there are high amounts of EPA and, Dl-lA in krill
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`oil {47% of total fatty acids) and these omega 3 pclyunsatnrated fatty acids are primarily
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`esteril'ied in phcsphclipids. On the other hand, EPA and DHA in fish cil and algal oil are net
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`significantly esterified in phosphelipids, but are largely esterilied iii triglycerides.
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`. 'l‘herefcre, krill oil, unlike fish cil and algal oil contains significant amounts of omega 3 fatty
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`acids esterified iii phcsphclipids.
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`Phospizclz’pids Have .Szrperiar Piiysz'aiogical Absorption Prqfir’es.
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`‘7 "l
`1... Based en the recently develcpihg research in this field, it appears that the chemical “carrier”
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`ct" cmega 3 polyunsaturated, fatty acids in krill extracts, 22a, phospholipids, prcyides superior
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`physiological absorption profiles, especially as compared tc triglyceride “carriers.”
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`23.
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`l’hcspholipids are ampthhilic and triglycerides are not. The chemical structure of an
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`amphiphilic ccnipeimd contains fimcticn al grcups that allow simultaneous water~ and, lipid—
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`° 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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`soluhility due to the presence of hydrophilic and hydrophobic n'ioieties. The hydrophilic
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`rnoiety in krill phospholipids is present at the sn—3 position, where the nitrogenucontaining
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`rnoiety (cg, the choline in phosphatidylcholine) provides positive charges and the phosphate
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`bridge provides negative charges. These positive and negative charges interact with charges
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`on the water dipoles, thereby, resulting in phospholipid solubility in water. The hydrophobic
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`moiety in krill phospholipids is present at the sn-l and Sir—2 positions in the form of the long
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`hydrocarbon chains of omega 3 polynnsaturated fatty acids.
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`in contrast, the triglycerides of
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`fish oil and algal oil are not arnphiphilie, as their sn—l, sn~2, and sn~3 positions are occupied
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`with hydrophobic fatty acids that have long hydrocarbon chains. Accordingly, triglycerides
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`are lipid—soluble but not water—soluble. By way of analogy, phospholipids (such as those of
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`the ‘348 patent) are litre ambidextrous people, having the ability to write with both the right
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`and left hand, while triglycerides (_such as those of fish oil and algal oil) are lilre right— or left"
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`handed people, having the ability to write with only a single hand.
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`. This duality of phospholipids has irnplications for absorption in the body. Vt’hen lipids (Le,
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`typical oils and fats composed of triglycerides, such as fish oil and algal oil} are ingested,
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`because. of their water insolubility and lower density than water, they float on top of and form
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`a layer that is separate front the digestive juices in the stomach and small intestine.
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`'l‘hese
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`digestive juices contain lipolytic enzymes that digest
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`lipids, Therefore, hefore y’ater—
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`insoluble lipids (La,
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`triglycerides) can be digested and absorbed,
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`they first have to he
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`emulsified by bile. However, ainphiphilic compounds such as phospholipids (like those in
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`the “348‘ patent) skip the ernulsification process because they are water soluble.
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`ln fact,
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`phospholipids enhance eniulsification by bile and,
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`thereby, aid in digestion of water—
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`insoluhle oils,’
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`l\) l}!
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`, Phospholipids and triglycerides are also digested by different enzyrnes, and many authors
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`have attributed this difference to the enhanced digestion and absorption of phospholipids.
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`Lingual, gastric, and pancreatic lipases initiate. digestion of triglycerides by cleaving ester
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`bonds preferentially at external positions, sn~l and 3:445, yielding a 2-rnonoglyceride and two
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`free fatty acids; while phospholipases, responsible for digestion of phospholipids, cleave at
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`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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`the center position, sa—2, and yield a l~lysephospholipid and a tree fatty acid.8 Carnielli et ai.
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`U998} suggests that cleavage at
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`the center position results in enhanced digestinn.9
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`Additionally, triglycerides in fish oil containing eniega 3 fatty acids are relatively resistant to
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`digestion by human lipase.m li‘nrther, Carnielli er al. and Morgan at 11!.“ have shown that
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`phosphclipids containing omega 3 polyunsaturated fatty acids are digested and abscrbed
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`better than triglycerides.
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`26. Digested and ahserhed lipids have to he delivered, via the blood stream, tc destination
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`organs. However, hecause the digested lipids (except phospholipids) are water insolnhle,
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`they are packaged in inicelles for abserptien and subsequently in chylctnicrons before they
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`enter the bleed stream and reach the destinatidn ergans. Chylornicrens are specialized
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`spherical, delivery vehicles for digested lipids which are distributed in the blood stream.
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`it is
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`important tn emphasize that the surface of chylnniicrons is coated with phospholipids that
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`provide water selnhility in the blood, while triglycerides are buried in the interior.
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`l\)7. Aniate er al. {200D suggests that lipids tellnwing their digesticn and absorption are re“
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`esteril'ied to the same chemical fill‘l’l’l (Le, eithe‘ triglycerides in“ phesphnlinids) that they
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`were in prior to digestion and ahsorption (226., the same chemical farm as they were in the
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`diet).12 Chylernierons in the bleed, strearn exchange their components with, liigli~density~
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`lipeproteins (HDLs, also referred te as “good chelesternl”).j 3
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`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
`‘ ‘
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`,
`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.
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`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.
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`29. One could use a simple. analogy to illustrate this complex concept.
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`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.
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`30. A, number of specific health benefits in humans are associated with EPA and lilllA.
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`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
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`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}.
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`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~
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`inflannnatery
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`effect, which ultimately
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`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
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`reduces the development of atherosclerotie plaques, and as such, improves cardiovascular
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`health.
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`There.
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`is anether
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`set at antimint'lannnatery compounds
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`(B-series
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`resolvins,
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`deeosatrienes, and, neureprnteetins) associated with {El-{A metabolism“ These compounds
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`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
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`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
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`. As mentioned above, the extent of the health benefits efoniega 3 polyunsaturated fatty acids
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`is greater when these molecules are esterified as intact phespholipids, as compared to being
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`esterified to triglycerides. My krill research group recently published a review article
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`describing,
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`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
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`This article was featured as a Special Article on the cever of the February 2307 issue of
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`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
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`H Simopoules, “Omega-3 Fa Ly
`(2002), Appendix O.
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` Acids in inflammation and Antoiimriune Diseases” J Am Coil Nair 21(6): 4951-505
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`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
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`: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
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`fish oil had no effect. Krill oil and fish oil both reduced blood cholesterol, but krill oil
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`resulted in higher reduction {up to 18%) than iish oil. Krill oil reduced LDLs by up to 39%?
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`whereas lish oil had no effect.
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`in addition, lrrill oil increased l—lDlts by up to 60%, While lish
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`oil had no effect.
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`in infantsa DEA was absorbed better from egg phospliolipids than algal
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`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
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`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
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`and DHA from krill oil resulted in an increased, l-lDies/triglycerides ratio compared to fish oil
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`even though the DHA concentration in fish oil was more than 2 times higher than in the krill
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`oil adn'iinistered.20 li‘urther,
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`l<rill oil resulted in reduction of several symptoms commonly
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`associated with premenstrual syndrome and dysinenorrhea to a greater degree than fish oil.2i
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`This was attributed to more efficient delivery of EPA/Dim esteril'ied in phospholipids in
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`krill oil as opposed to triglycerides in fish oil.
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`The Eritrea? ofre “348 Parent, Bur Na Baltimore ’5 Oil, Whale! Likely Possess the A have Efiectr.
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`33. The ‘3-48 patent teaches intact phospholipids containing EPA and DHA (see. Tables 9 and it)
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`of the ‘348 patent} These phosnholipids are not heat damaged (see Column ll; of the ‘348
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`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.
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`in
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`contrast, the Beandoin oil, containing far fewer intact phospliolipids and being heat damaged,
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`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
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`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
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`say, almost one quarter of the Beaudoin oil
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`is species that are m2: matched to the
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`phospholipld carrier befbre heating. After exposing the oil to lZSCC for l5 minutes, the
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`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