`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1053
`
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:20)
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1053
`
`
`
`Reexamination U.S.S.N. 95/001,774
`Declaration of Dr. Jacek Jaczynski
`
`oil.1 In addition, I am the sole inventor of an issued patent (US. 7,763,717) and the inventor
`
`of two other patent applications currently under examination. One focus of my patent and
`
`patent applications is a method for isolating lipids from krill.
`
`3.
`
`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.
`
`I am a professional member of the Institute of Food
`
`Technologists (“IFT”), the American Chemical Society, the World Aquaculture Society, and
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`Gamma Sigma Delta, an honorary society of agricultural scientists.
`
`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
`
`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.
`
`4.
`
`In December of 2011,
`
`I was engaged by counsel
`
`for Neptune Technologies and
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`Bioressources, Inc. (“Neptune”) to review US. Patent 8,030,348 (“the ‘348 patent”) and its
`
`substantive prosecution history, the Corrected Request for Reexamination filed by Aker
`
`Biomarine (“Aker”), listed as U.S.S.N. 95/001,714, including the Declaration of Mr. Bjorn
`
`Ole Haugsgjerd and the Declaration of Dr. Thomas Gundersen, and supporting materials, and
`
`to provide my expert scientific opinion regarding whether Gundersen and Haugsgjerd
`
`accurately followed the process disclosed in patent publication WO 00/23 546 (“Beaudoin I”)
`
`and CA 2,251,265 (“Beaudoin II”) and therefore whether the data presented by Aker
`
`accurately characterized the krill extract obtained by Beaudoin. Also, I was asked to express
`
`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—
`
`bound forms seen in fish and algal oils, as well as free fatty acids.
`
`5.
`
`I have had no prior direct involvement with either Neptune or Aker.
`
`I am being compensated
`
`at my customary hourly rate for my time spent on developing, forming, and expressing the
`
`facts and opinions in this declaration.
`
`I have no personal interest in the ultimate outcome of
`
`the reexamination proceedings involving the ‘348 patent.
`
`_
`
`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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`2
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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.
`
`I have carefully read the information provided and also conducted my own search of
`
`relevant, peer—reviewed scientific literature. Below I provide my expert scientific opinion.
`
`Gundersen and Haugsgjerd Did Not Accurately Replicate Beaudoin I or Beaudoin II.
`
`7.
`
`In my opinion, Gundersen and Haugsgjerd did not accurately reproduce the methodology for
`
`total lipid extraction from krill that is disclosed in Beaudoin I or 11. Specifically, Gundersen
`
`did not sufficiently heat the krill oil samples in a manner that was appropriate to replicate
`
`Beaudoin I or II, and Haugsgjerd did not accurately replicate the extraction method of
`
`Beaudoin I or II because he added a significant step to the Beaudoin protocol. For at least
`
`these reasons, it is my opinion that Haugsgjerd and Gundersen failed to opine on the specific
`
`process of Beaudoin I or II and therefore failed to characterize the krill extract actually
`
`produced by Beaudoin I or II.
`
`Gundersen Did Not Appropriately Heat the Samples.
`
`8. Gundersen conducted the last step of the krill oil extraction procedure (which was partially
`
`conducted by Haugsgjerd).
`
`In doing so, Gundersen applied heat in a manner inconsistent
`
`with Beaudoin I or II to the krill oil extracted by Haugsgjerd. Specifically, Gundersen
`
`alleges that he conducted a heat treatment at 125°C for 15 minutes or at 70°C for 5 minutes,
`
`in an attempt
`
`to reproduce Beaudoin I and II (see Gundersen Declaration, Exhibit 2,
`
`Analytical Report second of two pages numbered 1, between page 5 and page 7).2 However,
`
`in his attempt to heat the oil, Gundersen placed a heat block inside the oven of a gas
`
`chromatograph set to either 70°C or 125°C for at least one hour (see Gundersen Declaration,
`
`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
`
`(see Gundersen Declaration, Exhibit 2, Analytical Report second of two pages numbered 1,
`
`between page 5 and page 7). After Gundersen heated the vials, they were allowed to cool on
`
`a laboratory bench to room temperature (see Gundersen Declaration, Exhibit 2, Analytical
`
`Report second of two pages numbered 1, between page 5 and page 7).
`
`'
`
`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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`Declaration of Dr. Jacek Jaczynski
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`9.
`
`In my opinion, this heat treatment did not allow the oil to be heated to the temperature
`
`disclosed by Beaudoin I or II for the time specified by Beaudoin I or 11 due to slow heat
`
`transfer to the oil from the heat block. Gundersen’s heating method was mediated primarily
`
`by air-liquid convection and not conduction.
`
`It is a well-established fact that conduction
`
`results in much quicker heat transfer than convection.3 In simple terms, heated air contains
`
`relatively fewer molecules that can transfer heat from one object to another, as compared to
`
`heated liquids, such as oils as in a heated oil bath. Therefore, the transfer of heat via
`
`convection is much slower than conduction;
`
`thus,
`
`the samples heated as described by
`
`Gundersen were not maintained at the temperature of 125°C for 15 minutes or 70°C for 5
`
`minutes.
`
`10.
`
`A simple analogy allows illustration of this complex phenomenon. Consider placing one’s
`
`hand in a standard kitchen oven set at a moderate temperature, say 400°F (which is about
`
`200°C). One could easily hold one’s hand in this oven for a period of time before
`
`experiencing physical discomfort or injury.
`
`If one were to place one’s hand in a pot of
`
`boiling water (i.e., around 100°C), however, one would immediately experience a burning
`
`sensation. This common scenario is explained by the difference between heat transfer by a
`
`slower method, convection (i.e., the stove in the analogy), versus a faster method, conduction
`
`(i. e. , the boiling pot of water in the analogy).
`
`ll.
`
`Accordingly, when Dr. Gundersen placed the extracted krill oil in a heat block, he relied on
`
`heat transfer by convection to allegedly heat the oil to 125°C (or 70°C). Like the hand in the
`
`oven described above, the oil samples themselves did not reach and maintain a temperature
`
`of 125°C for 15 minutes.
`In contrast, during the prosecution of US. 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
`
`this appropriate heat transfer method, mediated by conduction, the oil reached 125°C and
`
`therefore experienced a full 15 minute exposure to this temperature.
`
`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.
`
`4 As noted in 1H above, I reviewed the office Action response filed on May 31, 2011 in the prosecution of the US.
`Patent 8,030,348.
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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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`12. I also note that the proper use of a heat block to heat an oil extract effectively has been
`
`described in the literature. For example, in Herman and Groves,5 the authors conduct an
`
`experiment in which they thermally stress lipid emulsions containing phospholipids and
`
`observe hydrolysis of the fatty acids off of the phospholipids from this heating. Specifically,
`
`- they describe, at page 775:
`
`“Thermal stress was applied by filling‘heating block chambers (Dry Baths, Fisher
`
`Scientific, Itasca, IL; 60 chambers per block, each 12 mm diameter and 50 mm deep) with oil
`
`and immersing the 2-mL ampoules containing the emulsion at the desired temperature,
`
`covering the blocks with aluminum foil to minimize thermal fluctuation” (emphasis added).
`
`13.
`
`14.
`
`Such a protocol would allow effective heat transfer to the samples because it relies on
`
`conduction through hot oil, as was performed in obtaining the data presented in the
`
`prosecution of US. 8,030,348. Gundersen did not follow this known protocol.
`
`In my expert opinion, the ineffective heating applied by Gundersen had a significant effect
`
`on the extent of hydrolysis of the ester bonds connecting fatty acids (e. g. DHA and EPA) to
`
`the glycerol backbone of the phospholipids. Accordingly, Gundersen only allegedly
`
`observed a residual mass spectrometry signal of phospholipids bearing DHA and EPA (or
`
`EPA/EPA or DHA/DHA).
`
`Further, I also note that Gundersen provides an unclear trend as to the effect of heating.
`
`Comparing the HPLC-MS data presented in Appendix A, Gundersen appears to detect the
`
`same intensity peaks for non—heated, heated to 60°C or 70°C, and heated to 125°C (see, e.g.,
`
`chromatograms labeled P308-l, P308—2, and P308-3).
`
`This further underscores the
`
`ineffective heating approach used by Gundersen.
`
`Haugsgjerd and Gundersen Added an Experimental Step Not Disclosed in Beaudoin I or
`
`Beaudoin II.
`
`15. Further, it is my opinion as an expert on krill oil extraction that not only did Haugsgjerd and
`
`Gundersen fail to establish that the oils were sufficiently heated to replicate Beaudoin I or II,
`
`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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`Declaration of Dr. Jacek Jaczynski
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`Haugsgjerd also performed an additional experimental step in his extraction procedure that
`
`deviated from Beaudoin I and 11. Specifically, Haugsgjerd reports the following steps to
`
`generate Fractions 11a and IIb (Haugsgjerd Declaration, 11 3, Gundersen Declaration, Exhibit
`
`2, Analytical Report second of two pages numbered 1, between page 5 and page 7):
`
`o
`
`0
`
`0
`
`extracting with either ethanol or ethyl acetate;
`
`filtering solvent and evaporating under reduced pressure;
`
`“flush[ing] samples with nitrogen gas” and “stor[ing] at —20C until further analysis,”
`
`and
`
`o
`
`sending
`
`samples
`
`from Haugsgjerd to Gundersen
`
`and
`
`having Gundersen
`
`unsuccessfully attempt to heat to 125°C for 15 minutes.
`
`I note that the Beaudoin I or II protocol, as successfully replicated to generate the data
`
`presented in the prosecution of US. 8,030,348, did not involve flushing oil fractions with
`
`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
`
`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 US.
`10/485,094, Appendix I; Beaudoin 1, pages 7 and 10). There is no mention in Beaudoin I or
`
`II of either storing samples at -20°C or under nitrogen gas.
`
`l6.
`
`In my opinion, Haugsgj erd’s deviation from the process disclosed in Beaudoin I or II is very
`
`significant. By freezing the sample before heating, Haugsgjerd further suppressed any
`
`hydrolysis of ester bonds found on the phospholipids being analyzed.
`
`It is my opinion that
`
`after Beaudoin completed the penultimate step of removing the solvent by rotary
`
`evaporation, he heated to remove the residual “volatile matter and humidity” from fraction I
`
`and fraction II (see Beaudoin I, page 10). Note that in Table 13 of Beaudoin I “volatile
`
`matter and moisture levels” of 10% and 6.8%, respectively, for fractions I and II are reported.
`
`These levels of solvent and humidity would have rendered those oils crude for Beaudoin’s
`
`purposes of determining total lipids generated by his procedure, and therefore prompted him
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`Declaration of Dr. Jacek Jaczynski
`
`to “get rid of traces of solvents [by] briefly heat[ing] (to about 125°C, for about 15 min.) the
`
`oil under nitrogen” (see Beaudoin I, pages 7 and 10). This heating inevitably led to the
`
`hydrolysis of ester bonds. As a result, the phospholipids were degraded and consequently
`
`released free fatty acids such as EPA and DHA. It is my opinion that Haugsgjerd suppressed
`
`this inevitable hydrolysis by storing his samples at —20°C. This-is a step n_ot 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
`
`removing the solvents by rotary evaporation and til—CI; stored the final product under nitrogen
`
`at -20°C for subsequent evaluation by Gundersen, Gundersen would not have allegedly
`
`observed even residual amounts of phospholipids carrying two of DHA and/or EPA. I note,
`
`as evidence for this opinion, the paper cited in 11 12 above in which the authors thermally
`
`stressed phospholipid emulsions in heating blocks containing oil and described hydrolysis of
`
`ester bonds in phospholipids. As an inevitable consequence of this thermal hydrolysistree
`
`fatty acids were releasedfrom the phospholipids.
`
`17. In summary, it is my opinion that, due to inadequate sample heating and the addition of an
`
`experimental step to the extraction protocol, Haugsgjerd and Gundersen did not accurately
`
`reproduce the methodology of oil extraction from krill as disclosed in Beaudoin I or II, and
`
`therefore the mass spectrometric data presented by Gundersen fails to accurately characterize
`
`the krill oil actually produced by Beaudoinl or II.
`
`Intact Phospholipids Containing Omega 3 Polyunsaturated Fatty Acids Possess Desirable
`
`Properties Not Observed in Other Forms of Lipids Such as Triglycerides Containing
`
`Omega 3 Polyunsaturated Fatty Acids.
`
`18. I have been asked to express my opinion, based on the peer reviewed literature, including my
`
`laboratory’s published research, on the superior properties of omega 3 fatty acids in
`
`phospholipid form as compared to other forms (or lipid classes), such as triglycerides.
`
`Possible Forms of Omega 3 Fatty Acids and Their Presence in Various Extracts.
`
`7
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`19.
`
`The ‘348 patent teaches extracts,
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`Reexamination U.S.S.N. 95/001 ,774
`
`Declaration of Dr. Jacek Jaczynski
`including krill extracts, and more specifically,
`intact
`
`phospholipids bearing omega 3 polyunsaturated fatty acids, such as EPA and DHA. These
`
`compositions are distinguishable from fish oil and algal oil, as fish and algal oil extracts
`
`feature EPA and DHA bound to triglycerides. My laboratory recently conducted a
`
`comparative study among krill oil, fish oil, and algal oil.6 Thin—layer—chromatography (TLC)
`
`analysis clearly demonstrated that krill oil contained significant amounts of phospholipids,
`
`while the amount of detected phospholipids in fish oil and algal oil was negligible. Further,
`
`TLC experiments showed that krill oil contained negligible amounts of triglycerides, while
`
`fish Oil and algal oil contained significant amounts.
`
`20.
`
`21.
`
`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
`
`esterified in phospholipids; On the other hand, EPA and DHA in fish oil and algal oil are not
`
`significantly esterified in phospholipids, but are largely esterified in triglycerides.
`
`Therefore, krill oil, unlike fish oil and algal oil contains significant amounts of omega 3 fatty
`
`acids esterified in phospholipids.
`
`Phospholipids Have Superior Physiological Absorption Profiles.
`
`' 22.
`
`Based on the recently developing research in this field, it appears that the chemical “carrier”
`
`of omega 3 polyunsaturated fatty acids in krill extracts, i. e, phospholipids, provides superior
`
`physiological absorption profiles, especially as compared to triglyceride “carriers.”
`
`23.
`
`Phospholipids are amphiphilic and triglycerides are not. The chemical structure of an
`
`amphiphilic compound contains filnctional groups that allow simultaneous water— and lipid—
`
`solubility due to the presence of hydrophilic and hydrophobic moieties. The hydrophilic
`
`moiety in krill phospholipids is present at the sn-3 position, where the nitrogen—containing
`
`moiety (e.g., the choline in phosphatidylcholine) provides positive charges and the phosphate
`
`bridge provides negative charges. These positive and negative charges interact with charges
`
`'
`
`on the water dipoles, thereby, resulting in phospholipid solubility in water. The hydrophobic
`
`6 See Kassis et (1]., “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—1 and sn-2 positions in the form of the long
`
`hydrocarbon chains of omega 3 polyunsaturated fatty acids.
`
`In contrast, the triglycerides of
`
`fish oil and algal oil are not amphiphilic, as their sn-l, sn-2, and sn-3 positions are occupied
`
`with hydrophobic .fatty acids that have long hydrocarbon chains. Accordingly, triglycerides
`
`are lipid-soluble but not water—soluble. By way of analogy, phospholipids (such as those of
`
`the ‘348 patent) are like ambidextrous people, having the ability to write with both the right
`
`and left hand, while triglycerides (such as those of fish oil and algal oil) are like right— or left—
`
`handed people, having the ability to write with only a single hand.
`
`24.
`
`This duality of phospholipids has implications for absorption in the body. When lipids (i.e.,
`
`typical oils and fats composed of triglycerides, such as fish oil and algal oil) are ingested,
`
`because of their water insolubility and lower density than water, they float on top of and form
`
`a layer that is separate from the digestive juices in the stomach and small intestine. These
`
`digestive juices contain lipolytic enzymes that digest
`
`lipids. Therefore, before water-
`
`insoluble lipids (i.e.,
`
`triglycerides) can be digested and absorbed,
`
`they first have to be
`
`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—
`
`insoluble oils.7
`
`25.
`
`Phospholipids and triglycerides are also digested by different enzymes, and many authors
`have attributed this difference to. the enhanced digestion and absorption of phospholipids.
`
`Lingual, gastric, and pancreatic lipases initiate digestion of triglycerides by cleaving ester
`
`bonds preferentially at external positions, sn—l and sn—3, yielding a 2—monoglyceride and two
`
`free fatty acids; while phospholipases, responsible for digestion of phospholipids, cleave at
`
`the center position, 371-2, and yield a l-lysophospholipid and a free fatty acid.8 Carnielli et al.
`
`(1998) suggests that cleavage at
`
`the center position results in enhanced digestion.9
`
`7 See O’Doherty et al., “Role of Luminal Lecithin in Intestinal Fat Absorption” Lipids 8: 249-55 (1973), Appendix
`G.
`
`8 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—G610 (1984), Appendix I.
`
`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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`Additionally, triglycerides in fish oil containing omega 3 fatty acids are relatively resistant to
`
`digestion by human lipase.lo Further, Carnielli et al. and Morgan et al.” have shown that
`
`phospholipids containing omega 3 polyunsaturated fatty acids are digested and absorbed
`
`better than triglycerides.
`
`V
`
`26.
`
`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,
`
`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
`
`spherical, delivery vehicles for digested lipids which are distributed in the blood stream. It is
`
`important to emphasize that the surface of chylomicrons is coated with phospholipids that
`
`provide water solubility in the blood, while triglycerides are buried in the interior.
`
`27.
`
`28.
`
`Amate et al. (2001) suggests that lipids following their digestion and absorption are re—
`
`esterified to the same chemical form (i.e., either triglycerides or phospholipids) that they
`
`Were in prior to digestion and absorption (i.e., the same chemical form as they were in the
`
`diet).12 Chylomicrons in the blood stream exchange their components with high-density—
`
`_1ipoproteins (HDLs, also referred to as “good cholesterol”).13
`
`If the lipids in the diet are in the phospholipid form (such as in krill oil) and since
`
`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-
`
`density—lipoproteins or LDLs commonly referred to as “bad cholesterol”) and thus, affecting
`cardiovascular disease,
`than triglycerides. Therefore,
`the transport of phospholipids to
`
`1° Bottino et £11., “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.
`
`'2 Amate et a1. “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,
`
`I3_See Mattson et a]. “The Digestion and Absorption of Triglycerides” J Biol Chem 239(9): 2772-77 (1964),
`Appendix H.
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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 (i.e.,
`
`insoluble in blood).
`
`Amate et at. (2001) confirmed this concept in piglets that were fed egg phospholipids
`
`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 DC. 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 DC. area. However, if the driver takes
`
`'
`
`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,
`
`that have to reach a destination organ (the National Mall). The slow
`
`highways in the DC. 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 Efi‘ects.
`
`- 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'
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`these two essential omega-3 polyunsaturated fatty acids to the destination organs is more
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`efficient via phospholipids. Therefore, the extent of the health benefits has been shown to be
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`greater when EPA and DHA are bound in phospholipids instead of triglycerides.
`
`31.
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`Two major 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 metabolized, it competitively
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`inhibits two enzymes (cyclooxygenase and 5—lipoxygenase).
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`If these enzymes are not
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`inhibited, they catalyze production of eicosanoids that initiate inflammation. However,
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`adequate supply of EPA results in reduced production of eicosanoids, thereby having an anti-
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`inflammatory effect, which ultimately reduces platelet aggregation and adherence as well as
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`vasoconstriction. As a final result, the vasodilation is stimulated.14 It is apparent that EPA
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`reduces the development of atherosclerotic plaques, and as such, improves cardiovascular
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`health.
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`There is another
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`set of anti—inflammatory compounds
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`(D—series
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`resolvins,
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`docosatrienes, and neuroprotectins) associated with DHA metabolism. These compounds
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`havedirect anti-inflammatory properties unlike the indirect effect of EPA via competitive
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`enzyme inhibition. These DHA—derived compounds clear inflammation sites of cellular
`15
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`debris, suppress pro-inflammatory interleukins, and also have neuroprotective properties.
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`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 a'review 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.16
`
`This article was featured as a Special Article on the cover of the February 2007 issue of
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`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
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`total triglycerides.” Krill oil (23 g/day) reduced total blood triglycerides by 27—28%, 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 fish oil. Krill oil reduced LDLs by up to 39%,
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`whereas fish oil had no effect. In addition, krill oil increased HDLs by up to 60%, while fish
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`oil had no effect.
`
`In infants, DHA was absorbed better from egg phospholipids than algal
`
`‘4 Simopoulos, “Omega-3 Fatty Acids in Inflammation and Autoimmune Diseases” J Am Coll Nutr 21(6): 495-505
`(2002), Appendix 0.
`
`15 Hong et al., “Novel'Docosatrienes and l7S—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.
`17 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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`triglycerides despite a 2.5 times higher dose of DHA in algal triglycerides.18 In rats, krill
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`protein concentrate (KPC) containing DHA in phospholipids resulted in better accretion of
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`DHA 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 fatty acids including DHA after absorption and digestion. Consumption of EPA
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`and DHA from krill oil resulted in an increased HDLs/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 administered.20 Further, krill oil resulted in reduction of several symptoms commonly
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`associated with premenstrual syndrome and dysmenorrhea to a greater degree than fish oil.21
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`This was attributed to more efficient delivery of EPA/DHA esterified in phospholipids in
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`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 heat'damaged (see Column 18 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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`phospholipid—associated physiological effects observed in the articles discussed above.
`
`In
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`contrast, the Beaudoin oil, containing far fewer intact phospholipids and being heat damaged,
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`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
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`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
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`prosecution of the ‘348 patent and supported by the literature.
`
`18 Camielli 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.
`
`'9 Bridges et al., “Determination of Digestibility, Tissue Deposition, and Metabolism of the Omega—3 Fatty Acid
`Content of Krill Protein Concentrate in Growing Rats” J Agric Food Chem 58: 2830-7 (2010), Appendix S.
`
`20 Ulven et al., “Metabolic 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 (2011), Appendix T
`
`21 Sampalis et al., “Evaluation of the Effects of Neptune Krill OilTM on the Management of Premenstrual Syndrome
`and Dysmenorrheal” Altern Med Rev 8: 171—9 (2003), Appendix U.
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`34. Specifically, heat-induced hydrolysis will occur upon this heating. The extracted krill oils
`
`produced by Beaudoin contained 10% moisture and volatiles. This moisture would be-
`
`sufficient to allow a heat~induced hydrolytic reaction in phospholipids. The literature is
`
`replete with examples of such; for instance,
`
`in Herman and Groves, referenced above,
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`hydrolysis of the fatty acids from phospholipids occurred upon thermal stress during
`
`sterilization at 121°C in a soy oil—based emulsion.
`
`35. Accordingly, it is my opinion that the intact, non—heat damaged phospholipids disclosed and
`
`claimed in the ‘348 patent would p