Attorney Docket No. NEPN-003/00US 313663-2015 (AKBM-32174)
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`PATENT
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
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`In Re Patent of:
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`SAMPALIS, Fotini
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`Confirmation No.:
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`1897
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`Control No.£
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`95/001,774
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`Group Art Unit:
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`3991
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`Filed:
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`FOR:
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`October 19, 2011
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`Examiner:
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`CAMPELL, Bruce R.
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`INTER PARTES REEXAM OF U.S. PATENT 8,030,348: NATURAL MARINE
`SOURCE PHOSPHOLIPIDS COMPRISING POLYUNSATURATED FATTY
`ACIDS AND THEIR APPLICATIONS
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`Mail Stop Declaration
`Commissioner for Patents
`PO. Box 1450
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`Alexandria, VA 22313-1450
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`DECLARATION OF JACEK JACZYNSKI, PH.D. UNDER 37 C.F.R. § 1.132 I
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`I, Jacek Jaczynski, declare as follows:
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`1.
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`I am a tenured Associate Professor of Food Science and Technology at West Virginia
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`University; Davis College of Agriculture, Natural Resources, and Design; Division of
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`Animal and Nutritional Sciences. My appointment is 50% research and 50% teaching.
`have been a professor at West Virginia University since 2002.
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`I
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`2.
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`I earned a Ph.D. in Food Science and Technology in 2002 from Oregon State University,
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`Seafood Research and Education Center.
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`Immediately following my doctoral work, I joined
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`West Virginia University as a faculty member. For the past 14 years I have been actively
`pursuing scientific research specializing in aquatic foods, with an emphasis on krill.
`I have
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`published 15 book chapters and over 50 peer-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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`example, one of my peer—reviewed 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 - 1053
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`Reexamination U.s.s.N. 95/001,774
`Declaration of Dr. Jacek Jaczynski
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`oil.1 In addition, I am the sole inventor of an issued patent (U.S. 7,763,717) 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 krill.
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`3.
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`I serve on the Editorial Board for the Journal ofAquatic Food Product Technology 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 and Food Chemistry.
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`I am a professional member of the Institute of Food
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`Technologists (“IFT”), 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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`I served as a Chair of the
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`Division of Aquatic Food Products of the IFT for the 2010-2011 term. For the past 10 years
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`I 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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`4.
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`In December of 2011,
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`I was engaged’ by counsel
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`for Neptune Technologies and
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`Bioressources, Inc. (“Neptune”) to review U.S. Patent 8,030,348 (“the ‘348 patent”) and its
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`substantive prosecution history, the Corrected Request for Reexamination filed by Aker
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`Biomarine (“Aker”), listed as U.S.S.N. 95/001,714, including the Declaration of Mr. Bjorn
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`Ole Haugsgjerd 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 Haugsgjerd
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`accurately followed the process disclosed in patent publication W0 00/23 546 (“Beaudoin I”)
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`and CA 2,251,265 (“Beaudoin II”) and therefore whether the data presented by Aker
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`accurately characterized the krill extract obtained by Beaudoin. Also, I was asked to express
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`my opinion on why intact phospholipids bearing omega 3 fatty acids, such as those. found in
`krill oil extracts, are superior to other forms of omega 3 fatty acids, such as the triglyceride-
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`bound fonns seen in fish and algal oils, as well as free fatty acids.
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`5.
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`I have had no prior direct involvement with either Neptune or Aker.
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`I 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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`I have no personal interest in the ultimate outcome of
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`the reexamination proceedings involving the ‘348 patent.
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`_
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`1 See Gigliotti et al. “Extraction and Characterisation of Lipids from Antarctic Krill (Euphausia superba)” Food
`Chemistry 125(3): 1028-1036 (April, 2011), Appendix B.
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`Reexamination U.S.S.N. 95/001,774
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`Declaration of Dr. Jacek Jaczynski
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`6.
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`I have carefully read the information provided and also conducted my own search of
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`relevant, peer—reviewed scientific literature. Below I provide my expert scientific opinion.
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`Gundersen and Haugsgjerd Did Not Accurately Replicate Beaudoin I or Beaudoin II.
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`7.
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`In my opinion, Gundersen and Haugsgjerd did not accurately reproduce the methodology for
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`total lipid extraction from krill that is disclosed in Beaudoin I or II. 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 I or II, and Haugsgjerd did not accurately replicate the extraction method of
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`Beaudoin I or II because he added a significant step to the Beaudoin protocol. For at least
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`these reasons, it is my opinion that Haugsgjerd and Gundersen failed to opine on the specific
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`process of Beaudoin I or II and therefore failed to characterize the krill extract actually
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`produced by Beaudoin I or II.
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`Gundersen 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 Haugsgjerd).
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`In doing so, Gundersen applied heat in a manner inconsistent
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`with Beaudoin I or II to the krill oil extracted by Haugsgjerd. Specifically, Gundersen
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`alleges that he conducted a heat treatment at 125°C for 15 minutes or at 70°C for 5 minutes,
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`in an attempt
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`to reproduce Beaudoin I and II (see Gundersen Declaration, Exhibit 2,
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`Analytical Report second of two pages numbered 1, between page 5 and page 7).2 However,
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`in his attempt to heat the oil, Gundersen placed a heat block inside the oven of a gas
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`chromatograph set to either 70°C or 125°C for at least one hour (see Gundersen Declaration,
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`Analytical Report second page numbered 1, between page 5 and page 7). A vial of krill oil
`extract was then heated using the heat block for 15 minutes at 125°C or 5 minutes at 70°C
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`(see Gundersen Declaration, Exhibit 2, Analytical Report second of two pages numbered‘ 1,
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`between page 5 and page 7). After Gundersen heated the vials, they were allowed -to cool on
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`a laboratory bench to room temperature (see Gundersen Declaration, Exhibit 2, Analytical
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`Report second of two pages numbered 1, between page 5 and page 7).
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`'
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`2 I 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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`Reexamination U.S.S.N. 95/001,774
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`Declaration of Dr. Jacek Jaczynski
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`9.
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`In my opinion, this heat treatment did not allow the oil to be heated to the temperature"
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`disclosed by Beaudoin I or II for the time specified by Beaudoin I or II due to slow heat
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`transfer to the oil from the heat block. Gundersen’s heating method was mediated primarily
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`by air-liquid convection and not conduction.
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`It is a well-established fact that conduction
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`results in much quicker heat transfer 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 bath. Therefore, the transfer of heat via
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`convection is much slower than conduction;
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`thus,
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`the samples heated as described by
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`Gundersen were not maintained at the temperature of 125°C for 15 minutes or 70°C for 5
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`minutes.
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`10.
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`A simple analogy allows illustration of this complex phenomenon. Consider placing one’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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`200°C).
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`One 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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`If one were to place one’s hand in a pot of
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`boiling water (i.e., around 100°C), however, one would immediately experience a burning
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`sensation. This common scenario is explained by the difference between heat transfer by a
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`slower method, convection (i. e., the stove in the analogy), versus a faster method, conduction
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`(i. e. , the boiling pot of water in the analogy).
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`ll.
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`Accordingly, when Dr. Gundersen placed the extracted krill oil in a heat block, he relied on
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`heat transfer by convection to allegedly heat the oil to 125°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 temperature
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`of 125°C for 15 minutes.
`In contrast, during the prosecution of U.S. Patent 8,030,348, the
`applicant submitted data obtained after heating for 15 minutes at 125°C by placing the
`extracted oil in an oil bath, which, in my opinion, accurately re—created Beaudoin 1.4 Using
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`this appropriate heat transfer method, mediated by conduction, the oil reached 125°C and
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`therefore experienced a full 15 minute exposure to this temperature.
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`3 See, e.g., Singh and Heldman, Introduction to Food Engineering (3rd ed.), New York, NY: Academic Press, 2008
`(pp. 222-27), Appendix C; Heldman and Lund, Handbook of Food Engineering, New York, NY: Marcel Dekker,
`1992 (pp. 247-59), Appendix D, both of which are fundamental food engineering textbooks.
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`4 As noted in fi[4 above, I reviewed the office Action response filed on May 31, 2011 in the prosecution of the U.S.
`Patent 8,030,348.
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`Declaration of Dr. Jacek Jaczynski
`12. I 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,5 the authors conduct an
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`Reexamination U.S.S.N. 95/001,774
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`experiment in which they thermally stress lipid emulsions containing phospholipids and
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`observe hydrolysis of the fatty acids off of the phospholipids from this heating. Specifically,
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`- they describe, at page 775:
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`“Thermal stress was applied by fillingrheating block chambers (Dry Baths, Fisher
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`Scientific, Itasca, IL; 60 chambers per block, each 12 mm diameter and 50 mm deep) with oil
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`and immersing the 2-mL ampoules containing the emulsion at the desired temperature,
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`covering the blocks with aluminum foil to minimize thermal fluctuation” (emphasis added).
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`13.
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`14.
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`Such a protocol would allow effective heat transfer to the samples 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 of U.S. 8,030,348. Gundersen did not follow this known protocol.
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`In my expert opinion, the ineffective heating applied by Gundersen had a significant effect
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`on the extent of hydrolysis of the ester bonds connecting fatty acids (e. g. DHA and EPA) to
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`the glycerol backbone of the phospholipids. Accordingly, Gundersen only allegedly
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`observed a residual mass spectrometry signal of phospholipids bearing DHA and EPA (or
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`EPA/EPA or DHA/DHA).
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`Further, I also note that Gundersen provides an unclear trend as to the effect of heating.
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`Comparing‘ the HPLC-MS 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 70°C, and heated to 125°C (see, e. g.,
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`chromatograms labeled P308-l, P308—2, and P308-3).
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`This further underscores the
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`ineffective heating approach used by Gundersen.
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`Haugsgjerd and Gundersen Added an Experimental Step Not Disclosed in Beaudoin I or
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`Beaudoin 1].
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`IS.
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`Further, it is my opinion as an expert on krill oil extraction that not only did Haugsgjerd and
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`Gundersen fail to establish that the oils were sufficiently heated to replicate Beaudoin I or II,
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`5 Herman and Groves, “The Influence of Free Fatty Acid Formation on the pH of Phospholipid-stabilized _
`Triglyceride Emulsions,” Pharmaceutical Research, 10(5): 774-76 (1993), Appendix E.
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`Reexamination U.S.S.N. 95/001,774
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`Declaration of Dr. Jacek Jaczynski
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`Haugsgjerd also performed an additional experimental step in his extraction procedure that
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`deviated from Beaudoin I and II. Specifically, Haugsgjerd reports the following steps to
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`generate Fractions Ila and Ilb (Haugsgjerd Declaration, 1] 3, Gundersen Declaration, Exhibit
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`2, Analytical Report second of two pages numbered 1, between page 5 and page 7):
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`0
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`0
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`0
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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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`“flush[ing] samples with nitrogen gas” and “stor[ing] at -20C until further analysis,”
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`and
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`0
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`sending
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`samples
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`from Haugsgjerd to Gundersen
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`and
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`having Gundersen
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`unsuccessfully attempt to heat to 125°C for 15 minutes.
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`I note that the Beaudoin I or II protocol, as successfully replicated to generate the data
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`presented in the prosecution of U.S. 8,030,348, did Q involve flushing oil fractions with
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`nitrogen gas and freezing’ at —20°C before heating. On the contrary, in both the disclosed
`Beaudoin process and the experiments conducted to generate the data presented in the
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`the
`prosecution of US. 8,030,348, following evaporation to partially remove solvent,
`fractions were immediately heated (see Response to Office Action of April 29, 2010 in U.S.
`l0/485,094, Appendix I; Beaudoin 1, pages 7 and 10). There is no mention in Beaudoin I or
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`II of either storing samples at -20°C or under nitrogen gas.
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`l6.
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`In my opinion, Haugsgj erd’s deviation from the process disclosed in Beaudoin I or II is Very
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`significant. By freezing the sample before heating, Haugsgjerd 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 1
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`and fraction II (see Beaudoin 1, page 10). Note that in Table 13 of Beaudoin I “volatile
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`matter and moisture levels” of 10% and 6.8%, respectively, for fractions I and II are reported.
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`These levels of solvent and humidity would have rendered those oils crude for Beaudoin’s
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`purposes of determining total lipids generated by his procedure, and therefore prompted him
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`Declaration of Dr. Jacek Jaczynski
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`to “get rid of traces of solvents [by] briefly heat[ing] (to about 125°C, for about 15 min.) the
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`oil under nitrogen” (see Beaudoin I, pages 7 and 10). This 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 fatty acids such as EPA and DHA. It is my opinion that Haugsgjerd suppressed
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`this inevitable hydrolysis by storing his samples at -20°C. Thisuis a step Q taught by
`Beaudoin I or II, which causes one to question why Haugsgjerd did this.
`It is also my
`opinion that had Haugsgjerd heated the samples in an oil bath or with a heating jacket after
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`removing the solvents by rotary evaporation and @ stored the final product under nitrogen
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`at -20°C for subsequent evaluation by Gundersen, Gundersen 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 evidence for this opinion, the paper cited in 1] 12 above in which the authors thermally
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`stressed phospholipid emulsions in heating blocks containing oil and described hydrolysis of
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`ester bonds in phospholipids. As an inevitable consequence of this thermal hydrolysis,'.free
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`fatty acids were releasedfrom the phospholipids.
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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, Haugsgjerd and Gundersen did not accurately
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`reproduce the methodology of oil extraction from krill as disclosed in Beaudoin I or II, and
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`therefore the mass spectrometric data presented by Gundersen fails to accurately characterize
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`the krill oil actually produced by Beaudoinl or II.
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`Intact Phospholipids Containing Omega 3 Polyunsaturated Fatty Acids Poss'essiDesirable
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`Properties Not Observed in Other Forms of Lipids Such as Triglycerides Containing
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`Omega 3 Polyunsaturated Fatty Acids.
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`18. I have been asked to express my opinion, based on the peer reviewed literature, including my
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`laboratory’s published research, on the superior properties of omega 3 fatty acids in
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`phospholipid form as compared to other forms (or lipid classes), such as triglycerides.
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`Possible Forms of Omega 3 Fatty Acids and Their Presence in Various Extracts.
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`19.
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`The ‘348 patent teaches extracts,
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`Reexamination U.S.S.N. 95/001,774
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`Declaration of Dr. Jacek Jaczynski
`including krill extracts, and more specifically,
`intact
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`phospholipids bearing omega 3 polyunsaturated fatty acids, such as EPA and DHA. These
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`compositions are distinguishable from fish oil and algal oil, as fish and algal oil extracts
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`feature EPA and DHA bound to triglycerides. My laboratory recently conducted a
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`comparative study among krill oil, fish oil, and algal oil.6 Thin-layer-chromatography (TLC)
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`analysis clearly demonstrated that krill oil contained significant amounts of 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 showed that krill oil contained negligible amounts of triglycerides, while
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`fish oil and algal oil contained significant amounts.
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`20.
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`21.
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`Further, the Kassis et al. study showed that there are high amounts of EPA and DHA in krill
`oil (47% of total fatty acids) and these omega 3 polyunsaturated fatty acids are primarily
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`esterified in phospholipids; On the other hand, EPA and DHA in fish oil and algal oil are not
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`significantly esterified in phospholipids, but are largely esterified in triglycerides.
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`Therefore, krill oil, unlike fish oil and algal oil contains significant amounts of omega 3 fatty
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`acids esterified in phospholipids.
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`Phospholipids Have Superior Physiological Absorption Profiles.
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`' 22.
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`Based on the recently developing research in this field, it appears that the chemical “carrier”
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`of omega 3 polyunsaturated fatty acids in krill extracts, i. e., phospholipids, provides superior
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`physiological absorption profiles, especially as ‘compared to triglyceride “carriers.”
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`23.
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`Phospholipids are amphiphilic and triglycerides are not. The chemical structure of an
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`amphiphilic compound contains fiinctional groups that allow simultaneous water- and lipid-
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`solubility due to the presence of hydrophilic and hydrophobic moieties. The hydrophilic
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`moiety in krill phospholipids is present at the sn-3 position, where the nitrogen—containing
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`moiety (e.g., 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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`'
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`on the water dipoles, thereby, resulting in phospholipid solubility in water. The hydrophobic
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`6 See Kassis et al., “Characterization of Lipids and Antioxidant Capacity of Novel Nutraceutical Egg Products
`Developed with Omega-3-Rich Oils” J Sci Food Agr 92(1): 66-73 (2012), Appendix F.
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`Declaration of Dr. Jacek Jaczynski
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`moiety in krill phospholipids is present at the sn—l and sn-2 positions in the form of the long
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`hydrocarbon chains of omega 3 polyunsaturated fatty acids.
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`In contrast, the triglycerides of
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`fish oil and algal oil are not amphiphilic, as their sn-1, 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 like 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 like right— or left-
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`handed people, having the ability to write with only a single hand.
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`24.
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`This duality of phospholipids has implications for absorption in the body. When lipids (i.e.,
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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 from the digestive juices in the stomach and small intestine. These
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`digestive juices contain lipolytic enzymes that digest
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`lipids. Therefore, before water-
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`insoluble lipids (i.e.,
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`triglycerides) can be digested and absorbed,
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`they first have to be
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`emulsified by bile. However, amphiphilic compounds such as phospholipids (like those in
`the ‘348 patent) skip the emulsification process because they are Water soluble.
`In fact,
`phospholipids enhance emulsification by bile and,
`thereby, aid in digestion of water-
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`insoluble oi1s.7
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`25.
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`Phospholipids and triglycerides are also digested by different enzymes, and many authors
`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 sn—3, yielding a 2—monoglyceride and two
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`free fatty acids; while phospholipases, responsible for digestion of phospholipids, cleave at
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`the center position, sn-2, and yield a l-lysophospholipid and a free fatty acid.8 Camielli et al.
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`(1998) suggests that cleavage at
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`the center ‘position results in enhanced digestion.9
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`7 See O’Doherty et al., “Role of Luminal Lecithin in Intestinal Fat Absorption” Lipids 8: 249-55 (1973), Appendix
`G.
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`3 See Mattson et al. “The Digestion and Absorption of Triglycerides” J Biol Chem 239: 2772—7(l964), Appendix
`H; Tso et al., “Evidence for Separate Pathways of Chylomicron and Very Low-Density Lipoprotein Assembly and
`Transport by Rat Small Intestine” Am J Physiol 247: G599—G6l0 (1984), Appendix I.
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`9 Carnielli et al. “Intestinal absorption of long-chain polyunsaturated fatty acids in preterm infants fed breast milk or
`formula” Am J Clin Nutr 67: 97-103 (1998), Appendix J.
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`Declaration of Dr. Jacek Jaczynski
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`Additionally, triglycerides in fish oil containing omega 3 fatty acids are relatively resistant to
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`digestion by human lipase.1° Further, Carnielli et al. and Morgan et al.” have shown that
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`phospholipids containing omega 3 polyunsaturated fatty acids are digested and absorbed
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`better than triglycerides.
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`I
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`26.
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`Digested and absorbed lipids have to be delivered, via the blood stream,
`to destination
`organs. However, because the digested lipids (except phospholipids) are water insoluble,
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`they are packaged in micelles for absorption and subsequently in chylomicrons before they
`enter the blood stream and reach the destination organs. Chylomicrons are specialized
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`spherical, delivery vehicles for digested lipids which are distributed in the blood stream. It is
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`important to emphasize that the surface of chylomicrons is coated with phospholipids that
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`provide water solubility in the blood, while triglycerides are buried in the interior.
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`27.
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`28.
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`Amate et al. (2001) suggests that lipids following their digestion and absorption are re-
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`esterified to the same chemical form (i.e., either triglycerides or phospholipids) that they
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`were in prior to digestion and absorption (i.e., the same chemical form as they were in the
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`diet).12 Chylomicrons in the blood stream exchange their components with high-density-
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`_lipoproteins (HDLS, also referred to as “good cholesterol”).13
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`If the lipids in the diet are in the phospholipid form (such as in krill oil) and since
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`phospholipids coat the exterior of chylomicrons, they have a higher likelihood of affecting
`the blood lipid panel (i. e., total blood cholesterol, total blood triglycerides, HDLs, and low-
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`density-lipoproteins or LDLs commonly referred to as “bad cholesterol”) and thus, affecting
`cardiovascular disease,
`than triglycerides. Therefore,
`the transport of phospholipids to
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`'0 Bottino et al., “Resistance of Certain Longchain Polyunsaturated Fatty Acids of Marine Oils to Pancreatic Lipase
`Hydrolysis” Lipids 2, 489-93 (1967), Appendix K; Hemell et al., “Does the Bile Salt-Stimulated Lipase of Human
`Milk Have a Role in the Use of the Milk Long-Chain Polyunsaturated Fatty Acids?” J Pediatr Gastroenterol Nutr
`16: 426-31(1993), Appendix L,
`'
`
`(“Fatty Acid Balance Studies In Term Infants Fed Formula Milk Containing Long-Chain
`” Morgan et al.
`Polyunsaturated Fatty Acids” Acta Paediatr 87: 136-42 (1998), Appendix M.
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`'2 Amate et al. “Feeding Infant Piglets Formula with Long-Chain Polyunsaturated Fatty Acids as Triacylglycerols or
`Phospholipids Influences the Distribution of these Fatty Acids in Plasma Lipoprotein Fractions” J Nutr 131: 1250-
`55 (2001), Appendix N.
`
`13_See Mattson et al. “The Digestion and Absorption of Triglycerides” J Biol Chem 239(9): 2772-77 (1964),
`Appendix H.
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`Declaration of Dr. Jacek Jaczynski
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`destination organs is also more easily facilitated as compared to triglycerides which are
`
`buried in the interior of chylomicrons and are water-insoluble (z'.e.,
`
`insoluble in blood).
`
`Amate et at. (2001) confirmed this concept in piglets that were fed egg phospholipids
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`containing DHA. The piglets had higher concentrations of DHA in high density lipoproteins.
`
`29..
`
`One could use a simple analogy to illustrate this complex concept.
`
`Imagine driving a car
`
`with two passengers that must reach the National Mall in Washington D.C. in rush hour.‘ The '
`
`delivery of the two passengers to the National Mall will be significantly delayed if the driver
`
`takes the typical, always congested, highways of the D.C. area. However, if the driver takes
`
`'
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`the HOV-lane, the two passengers will be able to reach the National Mall more efficiently.
`
`’ The two passengers are analogous to the two essential omega 3 polyunsaturated fatty acids,
`
`EPA and DHA,
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`that have to reach a destination organ (the National Mall). The slow
`
`highways in the D.C. area are like the triglyceride absorption mechanism and the HOV-lane
`is like the phospholipid absorption mechanism.
`It is clear that
`the HOV-lane (i. e.,
`
`phospholipids) will allow more efficient delivery of the two passengers (i. e., EPA and DHA)
`
`to the National Mall (i.e., destination organs) when compared to the typical, slow highways
`
`(i. e., triglycerides) in the DC area.
`
`Omega 3 Polyunsaturated Fatty Acids in Phospholipid Form Have Superior Medical Ejfects.
`
`- 30.
`
`A number of specific health benefits in humans are associated with EPA and DHA.
`
`Although these health benefits do not intrinsically depend on whether EPA and DHA are
`provided in the diet as phospholipids or triglycerides, as described above, the delivery of'
`
`these two essential omega-3 polyunsaturated fatty acids to the destination organs is more
`
`efficient via phospholipids. Therefore, the extent of the health benefits has been shown to be
`
`greater when EPA and DHA are bound in phospholipids instead of triglycerides.
`
`31.
`
`Two major classes of health benefits associated with dietary intake of EPA and DHA are
`
`anti-inflammatory and cardiovascular benefits. These health benefits are based on how EPA
`
`and DHA are metabolized in the human body. As EPA is metabolized, it competitively
`
`inhibits two enzymes (cyclooxygenase and 5—lipoxygenase).
`
`If these enzymes are not
`
`inhibited, they catalyze production of eicosanoids that initiate inflammation. However,
`
`adequate supply of EPA results in reduced production of eicosanoids, thereby having an anti-
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`Declaration of Dr. Jacek Jaczynski
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`inflammatory effect, which ultimately reduces platelet aggregation and adherence as well as
`
`vasoconstriction. As a final result, the vasodilation is stimulated.” It is apparent that EPA
`
`reduces the development of atherosclerotic plaques, and as such, improves cardiovascular
`
`health.
`
`There is another
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`set of anti-inflammatory compounds
`
`(D-series
`
`resolvins,
`
`docosatrienes, and neuroprotectins) associated with DHA metabolism. These compounds
`
`have.direct anti-inflammatory properties unlike the indirect effect of EPA via competitive
`
`enzyme inhibition. These DHA—derived compounds clear inflammation sites of cellular
`15
`
`debris, suppress pro-inflammatory interleukins, and also have neuroprotective properties.
`
`Similar to EPA, it is apparent that adequate supply of DHA improves cardiovascular health.
`
`32. As mentioned above, the extent of the health benefits of omega 3 polyunsaturated fatty acids
`is greater when these molecules are esterified as intact phospholipids, as compared to being
`
`esterified to triglycerides. My krill research group recently published avreview article
`
`describing,
`
`in part,
`
`the health benefits associated with consumption of krill oil, which
`
`contains intact phospholipids bearing omega 3 polyunsaturated fatty acids, EPA and DHA.”
`
`This article was featured as a Special Article on the cover of the February 2007 issue of
`
`Nutrition Reviews. As described therein, the effect of krill oil and fish oil on hyperlipidemia
`
`was investigated using human subjects diagnosed with mild to high blood cholesterol and
`
`total triglycerides.” Krill oil (2-3 g/day) reduced total blood triglycerides by 27-28%, 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 fish oil. Krill oil reduced LDLs by up to 39%,
`
`whereas fish oil had no effect. In addition, krill oil increased HDLs by up to 60%, while fish
`
`oil had no effect.
`
`In infants, DHA was absorbed better from egg phospholipids than algal
`
`14 Simopoulos, “Omega-3 Fatty Acids in Inflammation and Autoimmune Diseases” J Am Coll Nutr 21(6): 495-505
`(2002), Appendix 0.
`
`‘5 Hong et al., “Novel'Docosatrienes and 17S-resolvins Generated from Docosahexaenoic Acid in Murine Brain,
`Human Blood, and Glial Cells. Autacoids in Anti-Inflammation” J Biol Chem 278(17): 14677-87 (2003), Appendix
`P.
`
`'6 See Tou et al., “Krill for Human Consumption: Nutritional Value and Potential Health Benefits” Nutr Rev 65(2):
`63-77 (2007), Appendix Q.
`I7 See Tou et al. (citing Bunea et al., “Evaluation of the Effects of Neptune Krill Oil on the Clinical Course of
`Hyperlipidemia” Altern Med Rev 9: 420-28 (2004), Appendix R.
`
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`Declaration of Dr. Jacek Jaczynski
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`triglycerides despite a 2.5 times higher dose of DHA in algal triglycerides.” In rats, krill
`
`protein concentrate (KPC) containing DHA in phospholipids resulted in better accretion of
`
`DHA in the brain and liver compared to triglycerides.” This is important because DHA is
`
`indispensable and critical for proper brain development and the liver is the main organ that
`
`processes fatty acids including DHA after absorption and digestion. Consumption of EPA
`
`and DHA from krill oil resulted in an increased HDLs/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 administered.” Further, krill oil resulted in reduction of several symptoms commonly
`
`associated with premenstrual syndrome and dysmenorrhea to a greater degree than fish oil.”
`
`This was attributed to more efficient delivery of EPA/DHA esterified in phospholipids in
`
`krill oil as opposed to triglycerides in fish oil.
`
`The Extract ofthe ‘348 Patent, But Not Beaudoin ’s Oil, Would Likely Possess the Above Eflects.
`
`33. The ‘348 patent teaches intact phospholipids containing EPA and DHA (see Tables 9 and 10
`
`of the ‘348 patent). These phospholipids are not heatdamaged (see Column 18 of the ‘348
`
`patent). Therefore, in my opinion, the extract of the ‘348 patent would posses the superior
`
`phospholipid-associated physiological effects observed in the articles discussed above.
`
`In
`
`contrast, the Beaudoin oil, containing far fewer intact phospholipids and being heat damaged,
`
`would not possess the extent of these beneficial characteristics. This is bolstered by the fact
`
`that Beaudoin discloses that the fractions 1 and II, before heating, have free fatty acid
`
`amounts of 23.7 i 1.1% and 20.3 :1: 0.3%, respectively (see Beaudoin I, Table 14). That is to
`
`say, almost one quarter of the Beaudoin oil
`
`is species that are not attached to the
`
`phospholipid carrier before heating. After exposing the oil to 125°C for 15 minutes, the
`
`amount of phospholipids that are intact is less, as seen in the experiments conducted in the
`
`prosecution of the ‘348 patent and supported by the literature.
`
`18 Camielli et al., “Intestinal Abs