`
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`
`
`
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`
`
`
`
`
`SANDOZ INC.,
`APOTEX INC., APOTEX CORP.,
`EMCURE PHARMACEUTICALS LTD.,
`HERITAGE PHARMA LABS INC.,
`HERITAGE PHARMACEUTICALS INC.,
`GLENMARK PHARMACEUTICALS, INC., USA,
`GLENMARK HOLDING SA,
`GLENMARK PHARMACEUTICALS, LTD.,
`MYLAN LABORATORIES LIMITED,
`TEVA PHARMACEUTICALS USA, INC.
`FRESENIUS KABI USA, LLC, and WOCKHARDT BIO AG,
`
`Petitioners
`
`v.
`
`ELI LILLY AND COMPANY,
`
`Patent Owner.
`
`Case IPR2016-003181
`U.S. Patent 7,772,209
`
`
`
`
`
`
`
`
`
`
`
`Reply Declaration of Patrick J. Stover, Ph.D.
`
`
`
`
`1 Cases IPR2016-01429, IPR2016-01393, and IPR2016-01340 have been joined
`with the instant proceeding.
`
`1
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0001
`
`
`
`BACKGROUND ............................................................................................. 3
`I.
`QUALIFICATIONS AND EXPERIENCE ..................................................... 3
`II.
`III. MATERIALS CONSIDERED ........................................................................ 6
`IV. PERSON OF ORDINARY SKILL ................................................................. 7
`V.
`THERE IS NO SUPPORT FOR THE PURPORTED
`CONCERNS ABOUT FOLIC ACID AND VITAMIN B12
`TREATMENT RAISED BY DR. ZEISEL ..................................................... 8
`A.
`Studies By Dr. Sidney Farber Did Not Suggest That Folic
`Acid Was Contraindicated For Use With Antifolates ........................... 8
`Dr. Zeisel’s Opinions Rely On An Oversimplification Of
`Folate Metabolism ................................................................................. 9
`The Methyl Trap Would Not Have Presented Any
`Concern When Treating A Patient With Folic Acid And
`Vitamin B12 .......................................................................................... 14
`There Was No Precedent For Dr. Zeisel’s Proposal To
`Use Betaine To Address Toxicity in Cancer Patients ......................... 18
`VI. MASKING OF VITAMIN B12 DEFICIENCES GIVES
`REASON TO ADMINISTER VITAMIN B12 .............................................. 20
`VII. DR. ZEISEL DRAWS IMPROPER INFERENCES
`REGARDING THE RELATIONSHIP BETWEEN VITAMIN
`METABOLITES ............................................................................................ 22
`
`D.
`
`B.
`
`C.
`
`2
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0002
`
`
`
`I.
`
`BACKGROUND
`
`1. My name is Patrick J. Stover. I hold a Ph.D. degree in Biochemistry
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`and Molecular Biophysics from the Medical College of Virginia, Richmond, VA. I
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`am currently a Professor and Director of the Division of Nutritional Sciences at
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`Cornell University.
`
`2.
`
`I have been retained by counsel for Petitioner, Sandoz Inc.
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`(“Sandoz”), to investigate and opine about the nutritional and scientific principles
`
`underlying the vitamin pre-treatment regiments of claims 1-22 of U.S. Patent No.
`
`7,772,209 (“the ’209 patent”) in response to the Declaration of Dr. Steven H.
`
`Zeisel, dated September 30, 2016.
`
`3.
`
`I am being compensated at my usual hourly rate of $500.00 for the
`
`time I spend working on this matter. My compensation is not contingent upon the
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`substance of my testimony or the outcome of this matter.
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`4.
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`I understand that Dr. W. Archie Bleyer, MD, FRCP[GLASG], has
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`also submitted declarations and been deposed in connection with related challenges
`
`to U.S. Patent No. 7,772,209 brought by Neptune Generic LLC. Except as
`
`expressly noted below, I express no views regarding Dr. Bleyer’s opinions.
`
`II. QUALIFICATIONS AND EXPERIENCE
`5. My qualifications and experience are set forth in my curriculum vitae,
`
`which is attached as Appendix A. I have worked in the nutritional field for about
`
`3
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0003
`
`
`
`25 years starting as a graduate student at the Medical College of Virginia and a
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`postdoctoral fellow at the Department of Nutritional Sciences at the University of
`
`California at Berkeley. Currently, I am the Director of the Division of Nutritional
`
`Sciences at Cornell University, which is amongst the largest academic nutrition
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`units globally with about 74 academic faculty members. I am a member of the
`
`Cancer Center of Weill Cornell Medical College. I have an active research lab that
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`investigates the fundamental chemical, biochemical, genetic and epigenetic
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`mechanisms that underlie the relationships among nutrition, including the B-
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`vitamins, folate, and vitamin B12 and cancer amongst other diseases.
`
`6.
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`Folate- and vitamin B12-mediated one-carbon metabolism has been the
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`central focus of my career, in which I have helped elucidate some of the
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`fundamental functions of the enzymes in the folate pathway. The work that I
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`completed in earning my PhD defined some of the functions of the enzyme serine
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`hydroxymethyltransferase, which I discovered is a central enzyme in regulating
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`cellular folate metabolism.
`
`7.
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`In 1997, I was awarded the Presidential Early Career Award for
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`Scientists and Engineers by President William J. Clinton for my work involving
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`folate turnover and metabolism in cancer cells and pregnancy models. I discovered
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`a new enzyme which catabolizes (breaks down) folate, which helps explain the
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`rapid breakdown of folate in tumor cells and during pregnancy. In addition, I have
`
`4
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0004
`
`
`
`received several grant awards from the National Institutes of Health (NIH) to
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`investigate folate catabolism and also folate interactions in cancer.
`
`8.
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`I have served on numerous national and international nutritional
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`expert review panels relating to folic acid and vitamin B12 and their respective
`
`roles in neural tube defects, cancer, and neurodegeneration. I also have served on
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`the editorial boards of several leading nutrition based journals including the
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`American Journal of Clinical Nutrition, Annual Review of Nutrition, and
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`Nutritional Reviews.
`
`9.
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`I have published 72 peer-reviewed original research publications and
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`50 peer-reviewed review articles most of which directly pertain to one-carbon
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`metabolism and the folate pathways. Many of my publications are directly related
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`to the role of folate and folate requiring enzymes in various aspects of cancer. For
`
`example, I recently reported on gene expression differences in a folate enzyme in
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`cancer and its effect on the efficacy of various antifolates including lometrexol,
`
`pemetrexed, and LY309887 to cancer cells.
`
`10.
`
`I also have significant experience and expertise on the methyl trap.
`
`My post-doctoral advisor, Barry Shane, is one of the preeminent experts on the
`
`methyl trap. (Ex. 1108, Shane.) I also note that the author of the book on which
`
`Dr. Zeisel relies for his methyl trap arguments, Dr. Gerald Combs, has long been
`
`one of my colleagues at Cornell. (Ex. 2093, Combs.) My work often requires me
`
`5
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0005
`
`
`
`to consider the impact of the methyl trap. Indeed, I have a study currently in
`
`review for publication that demonstrates how vitamin B12 deficiency inhibits de
`
`novo thymidylate synthesis in the nucleus through the accumulation of 5-
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`methyltetrahydrofolate in the nucleus. This study provides a mechanism for the
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`methyl trap hypothesis.
`
`11. Given the foregoing experience, I consider myself as an expert in the
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`area of folate and vitamin B12 metabolism and nutritional requirements relating to
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`cancer and other disease states.
`
`III. Materials Considered
`12. A list of the documents I have considered in preparing this declaration
`
`is attached as Appendix B.
`
`13.
`
`I understand that exhibit numbers in this Declaration correspond to
`
`references relied upon either by Patent Owner Eli Lilly or used by Petitioner
`
`Sandoz. For the convenience of the Patent Trial and Appeal Board, counsel for
`
`Sandoz has assisted me in adding those exhibit numbers to this declaration. A
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`copy of the exhibit lists used in the proceeding are attached as Appendix C. For
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`clarity, I note that exhibits may be listed in Appendix C that I have not considered.
`
`14. This declaration is based upon information currently available to me.
`
`To the extent additional information becomes available, I reserve the right to
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`continue my investigation and study.
`
`6
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0006
`
`
`
`IV. Person of Ordinary Skill
`I have been informed by Counsel for Sandoz that a medical
`15.
`
`oncologist, Dr. Ron Schiff, is also testifying in these proceedings on behalf of
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`Sandoz. I have been informed that Dr. Schiff testified that “the person of ordinary
`
`skill in the art (‘POSA’) would have been a medical doctor experienced in
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`oncology with knowledge of and/or several years of experience regarding the use
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`of antifolates in the treatment of cancer and additional qualifications or experience
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`in the field of nutritional sciences involving vitamin deficiencies.” I further
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`understand that another oncologist, Dr. Bruce Chabner, is testifying in these
`
`proceedings on behalf of Lilly and agrees with Dr. Schiff’s definition of a POSA. I
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`am not an oncologist, but instead offer my opinions about the information known
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`in the nutritional sciences that would have been available to the POSA at the time
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`of the invention.
`
`16.
`
`I understand that Dr. Steven H. Zeisel has submitted a declaration in
`
`this matter, and like myself, Dr. Zeisel is a nutritional scientist. To the extent a
`
`POSA would consult a nutritional scientist, I am able to attest to the information
`
`that a nutritional scientist would provide to a POSA in June 1999, which Counsel
`
`for Sandoz has informed me is the relevant time frame. Further, I have sufficient
`
`relevant experience to offer testimony in response to Dr. Zeisel’s Declaration
`
`testimony as it relates to the nutritional science field related to folate, the
`
`7
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0007
`
`
`
`regulation of folate pools in cancer cells, and the interaction of folates and
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`antifolates (in particular pemetrexed) in cancer cells and normal cells.
`
`V. THERE IS NO SUPPORT FOR THE PURPORTED CONCERNS
`ABOUT FOLIC ACID AND VITAMIN B12 TREATMENT RAISED
`BY DR. ZEISEL
`
`A.
`
`Studies By Dr. Sidney Farber Did Not Suggest That Folic Acid
`Was Contraindicated For Use With Antifolates
`
`17. Dr. Zeisel testified that the POSA would be concerned that folic acid
`
`administration would be expected to decrease the efficacy of an antifolate, citing to
`
`the work conducted by Dr. Sidney Farber. (Ex. 1009, Farber; Ex. 2118, Zeisel
`
`Decl. ¶ 46.) This study described the effects of antifolates, including aminopterin,
`
`on childhood acute leukemia in a series of case reports in which several remissions
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`were achieved. The high level of toxicity from aminopterin was not lost on Dr.
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`Farber who reported “[t]oxic effects included stomatitis, with early ulceration” and
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`“[i]n an attempt to prevent this complication crude liver extract was employed, as
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`were folic acid and folic acid conjugates” and that “the toxic effects may make
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`continued use of the drug impossible.” (Ex. 1009, Farber at 792.) Thus, Dr. Farber
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`realized early on that an antifolate, such as aminopterin, was highly toxic and folic
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`acid regimens could ameliorate these toxicities. The doses of folic acid Dr. Farber
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`used to treat these toxicities (5 mg folvite, which is folic acid, and 1 unit of crude
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`liver extract) exceeded the recommended daily allowance for folic acid, a point on
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`which Dr. Zeisel agrees. (Ex. 1086, Zeisel Dep 146:21-24.) In addition, crude
`
`8
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0008
`
`
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`liver extracts have long been known to contain vitamin B12 useful for treating
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`pernicious anemia. (Ex. 1109, Cuthbertson at 708.)
`
`B. Dr. Zeisel’s Opinions Rely On An Oversimplification Of Folate
`Metabolism
`
`18. By June 1999, the role of folic acid in one carbon metabolism had
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`been the subject of extensive research for over 50 years. Thus, by June 1999,
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`much was known about the intracellular mechanisms and biochemistry involved in
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`folic acid metabolism as well as the mechanism by which particular antifolates
`
`impact folic acid metabolism. Based on the known features of these intracellular
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`mechanisms as of June 1999, Dr. Zeisel’s purported concerns about folic acid’s
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`impact on pemetrexed’s efficacy are not well-founded.
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`19. First, Dr. Zeisel’s opinion that folic acid would have been considered
`
`an “antidote” for pemetrexed fails to take into account the knowledge of
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`pemetrexed and folic acid’s binding affinities. (Ex. 2118, Zeisel Decl. ¶ 50.) I
`
`agree with Dr. Zeisel that it was known in June 1999 that “[a]ntifolates work
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`against cancer by competing with natural folates and inhibiting the activity of one
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`or more of the various enzymes in the folate pathway . . . .” (Ex. 2118, Zeisel Decl.
`
`¶ 25.) It was well-known by June 1999 that the binding affinity of an antifolate to
`
`its target versus the affinity of the natural substrate determines whether the
`
`antifolate will successfully out-compete the natural folate and thus be efficacious.
`
`9
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0009
`
`
`
`However, when asked about relative binding affinities of pemetrexed, Dr. Zeisel
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`responded that he hadn’t “looked more deeply into that literature because it wasn’t
`
`part of what I was asked to consider.” (Ex. 1086, Zeisel Dep. 24:12-23.)
`
`20.
`
`In the cell, folic acid is converted to other forms of folate, including
`
`5,10-methylenetetrahydrofolate. Had Dr. Zeisel considered the binding affinity of
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`pemetrexed for thymidylate synthase (TS), its primary target, versus 5,10-
`
`methylenetetrahydofolate, its natural substrate, it would have been apparent that it
`
`was known in June of 1999 that folic acid is not an “antidote” for pemetrexed.
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`(Ex. 2118, Zeisel Decl. ¶¶ 25, 50.) The measure of binding affinity for an
`
`antifolate like pemetrexed is called the inhibition constant (Ki). The relevant value
`
`for the natural substrate (here, 5,10 methylenetetrahydrofolate, which is a reduced
`
`form of folic acid), is called the Michaelis-Menten constant (Km), which is the
`
`concentration of substrate (5,10-methylenetetrahydrofolate) required to achieve
`
`half maximal velocity of the enzyme. For each constant, lower values indicate less
`
`of the substance is required to inhibit (Ki) or activate (Km) the target enzyme (here,
`
`TS). It was known in June 1999 that pemetrexed undergoes a process known as
`
`polyglutamation inside the body to become pentaglutamated pemetrexed, and that
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`in this form, pemetrexed was an extremely strong inhibitor of TS, with a Ki of 1.3
`
`± 0.3 nM. (Ex. 1021, Shih at abstract and 1118.) By contrast, it was known in
`
`June 1999, that the form of reduced folic acid that naturally binds to TS was
`
`10
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0010
`
`
`
`5,10 methylenetetrahydrofolate, which has a much higher Km of 3.0 µm.
`
`(Ex. 1021, Shih at 1118.) Thus, the relative affinity of the natural substrate (5,10
`
`methylenetetrahydrofolate) to the inhibitor (pemetrexed) was approximately 2,300
`
`(3,000 nM/1.3 nM). This means that approximately 2,300 times more 5,10
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`methylenetetrahydrofolate is required to activate TS than is required for
`
`pemetrexed to inhibit TS. Given this knowledge, pemetrexed would have been
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`expected to outcompete folic acid (in reduced form) for its primary target and thus
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`low levels of folic acid supplementation would not work as an antidote.
`
`21. This view is confirmed by the fact that it was also known in June 1999
`
`that human tumor cells grown in vivo have about approximately 0.2 µM and
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`0.5 µM of 5,10 methylenetetrahydrofolate. (Ex. 1110, Houghton at 3500.) This
`
`study by Houghton et al. demonstrated that during leucovorin supplementation the
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`pool of 5,10 methylenetetrahydrofolate was only modestly expanded from 1.4 µM
`
`to 1.8 µM. (Id.) These levels of reduced folate are still below the estimated Km
`
`reported for 5,10 methylenetetrahydrofolate (3.0 µM). Therefore, at these low
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`intracellular levels of 5,10 methylenetetrahydrofolate present in folate
`
`supplemented cancer cells, pemetrexed would have been expected to outcompete
`
`the natural substrate for TS. Further, unlike pemetrexed, it was known that
`
`polyglutamation of the natural substrate (5,10 methylenetetrahydrofolate) does not
`
`lead to a similar large reduction in Km at TS. (Ex. 1111, Lu at 6873.)
`
`11
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0011
`
`
`
`22. Second, Dr. Zeisel’s reliance on the fact that pemetrexed’s effects on
`
`both cancer and healthy cells work by the “same mechanisms” fails to take into
`
`account differences in the cells themselves. It was known in June 1999 that cancer
`
`cells waste folate through mechanisms of folate catabolism. Conversely, it was
`
`known that healthy cells to not have a high rate of catabolism. For example, tumor
`
`bearing mice have been shown to have a mere 5% increase in the weight of the
`
`tumour and a 50% increase in folate catabolism (Ex. 1112, Kelly at 305.)
`
`Consistent with these experimental findings in animals, Meenan et al. studied the
`
`difference of folate in tumor colon epithelial cells and adjacent normal cells and
`
`found that “[a] significant difference has been identified between the folate content
`
`of colon tumor epithelial cells and that of adjacent normal cells.” (Ex. 1113,
`
`Meenan at 1165.) This depletion is not simply due to the fact that the cancer cells
`
`divide rapidly, but rather is likely due to “abnormal rates of cell turnover or
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`abnormal cellular enzyme activity, such as methylene tetrahydrofolate reductase.”
`
`(Id. pg. 1166.) It makes sense that folic acid supplementation would have a greater
`
`impact on protecting healthy cells, which more efficiently use folate, than cancer
`
`cells, which catabolize a much higher proportion of the folate received. Notably,
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`Dr. Zeisel acknowledged that he had considered folate metabolism only generally,
`
`not specifically catabolism, in forming his opinions. (Ex. 1086, Zeisel Dep.
`
`156:15-157:5.)
`
`12
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0012
`
`
`
`23. Third, Dr. Zeisel fails to account for the differences in intracellular
`
`transport between pemetrexed and folic acid, which were known in June 1999.
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`Specifically, it was known in June 1999 that pemetrexed has a high affinity for the
`
`transporter mechanisms into cancer cells. Specifically, pemetrexed can be
`
`transported by either the reduced folate carrier or the folic acid receptors.
`
`(Ex. 1022, Westerhof at abstract.) As of June 1999, it was known that pemetrexed
`
`has approximately a 100-fold greater affinity for the reduced folate carrier
`
`compared to folic acid and approximately a 1.5 fold greater affinity for the folate
`
`receptor (also referred to as the membrane-associated folate binding protein (FBP))
`
`compared to folic acid. (Id. at 464-65) Thus, pemetrexed has a high affinity for
`
`multiple carriers that are responsible for the cellular transport of pemetrexed into
`
`cancer cells compared to folic acid. Given the relative affinity, pemetrexed could
`
`outcompete folic acid for absorption into cells and folic acid administration would
`
`not necessarily be expected to affect cancer and normal cells identically.
`
`24. The fact that orally administered folic acid is eventually converted to
`
`5-mTHF in the body (a useable form of folate), which has a higher affinity than
`
`folic acid for the reduced folate carrier, does not affect my opinion. By June of
`
`1999 it was understood that the conversion of folic acid primarily to 5-mTHF is a
`
`rate-limiting (slow) process. In addition, the folic acid converted to 5-mTHF also
`
`has to undergo additional conversion to 5,10-methylenetetrahydrofolate to compete
`
`13
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0013
`
`
`
`with pemetrexed at thymidylate synthase. In contrast, pemetrexed does not
`
`undergo pre-processing steps other than rapid polyglutamation (see above ¶ 20) to
`
`assert its effects on TS. Therefore, the levels of available reduced folate (5-mTHF)
`
`from routine levels of folic acid supplementation would not be sufficient to have
`
`any appreciable effect on pemetrexed anti-tumor activity.
`
`C. The Methyl Trap Would Not Have Presented Any Concern
`When Treating A Patient With Folic Acid And Vitamin B12
`I disagree with Dr. Zeisel’s claim that that the methyl trap would
`25.
`
`cause concerns about the viability of pre-administering vitamin B12 to a patient
`
`receiving pemetrexed. (Ex. 2118, Zeisel, ¶¶ 32, 34, 52-56.) There are several
`
`reasons for my disagreement.
`
`26. First, Dr. Zeisel’s concerns about the effect of vitamin B12 on
`
`pemetrexed efficacy are effectively the same as his concerns about the impact of
`
`folic acid on pemetrexed’s efficacy. (Ex. 1086, Zeisel Dep. 141:3-20.) For the
`
`reasons explained in paragraphs 18-21 above, it was known in June 1999 that there
`
`were differences between the intracellular mechanics of folate and pemetrexed.
`
`Also, there are differences between the folate catabolism (see above ¶ 22) that
`
`would give reason to expect that pemetrexed’s efficacy would not be eliminated or
`
`materially compromised by folate supplementation or release of “trapped” folate.
`
`14
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0014
`
`
`
`27. Second, the only types of patients with a vitamin B12 deficiency of
`
`sufficient severity that the patient would have an appreciable methyl trap are those
`
`with severe pernicious anemia, who have an underlying inability to absorb
`
`nutritional vitamin B12. Although I am not a practicing physician, I would expect
`
`that in most cases this type of severe pernicious anemia would be known by the
`
`patient due to a variety of symptoms that may include neurological issues like
`
`fatigue and numbness, in addition to diarrhoea and glossitis (tender bright red
`
`smooth tongue) and progressive neuropathy. By June 1999, pernicious anemia was
`
`typically diagnosed by diagnostic tests that affirm low serum levels of vitamin B12
`
`and a lack of intrinsic factor (i.e., the Schilling Test). (Ex. 1121, Karanad,
`
`generally.) If left untreated, severe pernicious anemia can be life threatening and
`
`was once considered an incurable disease. (Id. at 231.) Underlying pernicious
`
`anemia that is severe enough to trigger a methyl trap would require correction by
`
`vitamin B12 injection prior to initiation of cancer chemotherapy. Thus, to the
`
`extent a patient has 5-mTHF in the methyl trap, that trapped folate would have
`
`been released by the patient’s regular B12 injections, leaving no trapped 5-mTHF
`
`during chemotherapy.
`
`28. Third, Dr. Zeisel’s purported concern that triggering the methyl trap
`
`would trigger an unpredictable amount of folate is based on an exaggerated view as
`
`to how much folate would be trapped even in a severely vitamin B12 deficient
`
`15
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0015
`
`
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`patient. (Ex. 2118, Zeisel Decl. ¶ 56.) Generally, I agree with Dr. Zeisel on the
`
`theory of how a “methyl trap” of 5-methyltetrahydrofolate (5-mTHF) can occur in
`
`a vitamin B12 deficiency. (See Ex. 2118, Zeisel Decl. ¶ 32.) However, cellular
`
`levels of 5-mTHF are saturable and cells can only hold a certain amount of 5-
`
`mTHF. Once inside the cell, folate in the form of 5-mTHF must be converted to a
`
`polyglutamated form by folylpoly-γ-glutamate synthetase (FPGS) in order to be
`
`retained within the cell. However, 5-mTHF is a poor substrate for FPGS.
`
`Therefore, much of the 5-mTHF (particularly newly absorbed 5-mTHF from food)
`
`is not polyglutamated to a significant extent and it may travel freely in and out of
`
`the cell, i.e., it is not sequestered in the cell. As explained by a review article co-
`
`authored by my post-doctoral advisor Barry Shane:
`
`Although the tissue levels of 5-methyl-H4PteGlu [5-mTHF]
`initially increase, this compound is a very poor substrate for
`folylpolyglutamate synthetase and is metabolized only to short-
`chain-length polyglutamate derivatives that can leak out of the
`cell. The decreased ability to elongate the glutamate chain
`length of the folate molecule results in a decreased retention of
`folate by the tissue.
`
`
`(Ex. 1108, Shane at 129.)
`
`
`29.
`
`Indeed, the methyl trap was first observed as increased proportions of
`
`folate in the 5-mTHF form and elevated serum folate levels as 5-mTHF.
`
`16
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0016
`
`
`
`(Ex. 1108, Shane at 122.) It has been long-known that non-sequestered serum
`
`folate is eliminated relatively quickly (Ex. 1114, Krumdieck at abstract.) Thus,
`
`while the proportional levels of 5-mTHF are increased during the methyl trap, the
`
`total levels of reduced folate decreases because the unpolyglutamated 5-mTHF in
`
`the serum is eliminated by the body. Shane also explains that, “Although vitamin
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`B12 deficiency invariably increases the proportion of 5-methyl-H4PteGlun [5-
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`mTHF] in the livers of experimental animals, the absolute level of methylated
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`folate is often decreased as a result of the decreased total tissue folate levels in
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`these animals.” (Ex. 1108, Shane at 124.) Thus, even if the administration of
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`vitamin B12 were to release the trapped 5-mTHF, the resulting folate would not be
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`an overwhelming amount that would push overall folate levels abnormally high
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`and could possibly cause concern about unanticipated effects, such as impeding
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`antifolate efficacy.
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`30. My view is confirmed by considering how the methyl trap works in
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`patients with pernicious anemia, i.e., a patient with severe vitamin B12 deficiencies
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`that could potentially have pools of folate trapped in the form of 5-mTHF. Even
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`considering this 5-mTHF, such patients are actually deficient in total levels of
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`reduced folate. (Ex. 1108, Shane at 124.) Thus, the amount of folate liberated by
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`administration of vitamin B12 even to such a severely vitamin B12 deficient patient
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`would be minimal. Rather, vitamin B12 supplementation would restore normal
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`17
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`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0017
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`total levels of reduced folate and redistribute the trapped 5-mTHF to other forms.
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`It certainly would not “be tantamount to administering a potentially large, but
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`unquantifiable, dose of folic acid,” as alleged by Dr. Zeisel. (Ex. 2118, Zeisel
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`Decl. ¶¶ 56.) This is even more true of patients with subclinical vitamin B12
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`deficiencies who would be very unlikely to have a methyl trap scenario, and if so,
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`to a lesser extent.
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`31. Fourth, vitamin B12 is an innocuous vitamin with no known side
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`effects. In fact, it was common in June 1999, just as it is today, to administer doses
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`of vitamin B12 in amounts that greatly exceed the daily recommended intake
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`(“DRI”), given the potential long-term benefit and lack of any side-effects. I am
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`aware of no validated reports where administration of vitamin B12 has been shown
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`to promote tumor cell growth in human patients prior to June 1999. Thus, I do not
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`see any reason to expect that the methyl trap would be triggered by administering
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`vitamin B12 in such a way that it would promote tumor growth.
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`D. There Was No Precedent For Dr. Zeisel’s Proposal To Use
`Betaine To Address Toxicity in Cancer Patients
`
`32. Dr. Zeisel advocates lowering homocysteine through administration of
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`betaine as an alternative to administration of folic acid and vitamin B12. (Ex. 2118,
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`Zeisel Decl., ¶ 75.) I disagree with these sentiments. I highly doubt that an
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`oncologist would have considered betaine an option for reducing toxicity in
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`18
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`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0018
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`chemotherapy in June 1999 because even in the nutritional field, little was known
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`about betaine deficiencies at that time. In fact, I am not aware of any study prior to
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`June 1999 that demonstrated that a “betaine” deficiency exists in the population
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`and as Dr. Zeisel admits, “what portion of the population had low dietary betaine
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`and its precursor, intake, we didn’t know that until more recently.” (Ex. 1086,
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`Zeisel Dep. 38:23-39:7.)
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`33.
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`In June 1999, betaine was almost exclusively used in therapy for
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`patients with very rare genetic defects in the cystathionine beta synthase (CBS)
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`gene. The prevalence of this defect worldwide has been estimated at 1 in 344,000.
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`(Ex. 1115, Gaustadnes at 1513.) These individuals suffer from a condition known
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`as hyperhomocysteinemia (e.g., homocysteine levels as high as 299 µM), which
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`results from the lack of an enzyme responsible for breaking down homocysteine
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`called cystathionine beta synthase. (Ex. 1116, Kluijtmans at 59, 63.) I am not
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`aware of betaine’s use prior to June 1999 outside the context of patients suffering
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`from CBS defects and in my opinion, therefore, betaine would not have been
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`considered an alternative to more common methods used to reduce homocysteine
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`such as administering folic acid and vitamin B12 prior to that time.
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`34. Moreover, betaine is not a required nutrient like folate for life.
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`Treating folate- or vitamin-B12 deficiency in a way that ignores the deficiencies of
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`these essential nutrients places the patients at risk for related diseases including
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`19
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`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0019
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`cancer and neuropathies. There are also certain adverse effects of betaine
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`administration that are disrupting and unpleasant as it is known to cause extreme
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`body odor.
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`35. Finally, the same concerns discussed in paragraphs 36-40 below about
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`masking a B12 deficiency by treating with folic acid alone exist with respect to
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`betaine.
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`VI. MASKING OF VITAMIN B12 DEFICIENCES GIVES REASON TO
`ADMINISTER VITAMIN B12
`36. Dr. Zeisel contends that there would be no reason to administer
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`vitamin B12 to a patient suffering from cancer in order to prevent masking given
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`the rarity of such vitamin B12 deficiencies. (Ex. 2118, Zeisel Decl. ¶¶ 33, 76-78.)
`
`While I agree that vitamin B12 deficiencies are rare, they are devastating when they
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`occur and thus it was understood in June 1999 (just as it is today) that steps should
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`be taken to prevent masking such deficiencies, i.e., the administration of folic acid
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`without vitamin B12 should be avoided (see, e.g., Ex. 1020, Brattström at 1278S;
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`explaining that erroneously attempting to lower homocysteine by folate alone in a
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`vitamin B12 deficiency would exacerbate neuropathy).
`
`37. Dr. Zeisel points to the United States’ program for the fortification of
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`the grain supply in the late 1990s with only folic acid (not vitamin B12) as evidence
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`that there would have been no concern about masking. (Ex. 2118, Zeisel Decl.
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`20
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`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1091-0020
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`¶ 78.) What Dr. Zeisel omits is that the United States deliberately chose a
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`relatively low amount of folic acid that is equal to the daily recommended intake of
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`folic acid (400 µg) because the view was that such an amount would reduce the
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`risk of masking any vitamin B12 deficiency. Moreover, as Dr. Zeisel admitted at
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`his deposition, the concern about masking a B12 deficiency with this amount has
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`remained in some circles and proposals are thus periodically made to fortify grain
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`with vitamin B12 as well. (Ex. 1086, Zeisel Dep. 101:5-19.) Thus, far from
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`evidencing a lack of concern about masking a vitamin B12 deficiency, the history of
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`the U.S. fortification of the grain supply to the masking indicates that masking was
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`a concern in the June 1999 timeframe.
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`38. Dr. Zeisel also fails to recognize that masking would have been
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`particularly concerning for cancer patients being treating with pemetrexed, which
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`inhibits both DHFR and/or TS. In cells from both healthy individuals and patients
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`with pernicious anemia, inhibition of DHFR and/or TS reduces by about 50% the
`
`ability of lymphocytes to process vitamin B12 to methylcobalamin (a useable form
`
`of vitamin B12). (Ex. 1117, Quadros at 615.) Thus, treating a B12 deficient patient
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`with a DHFR/TS inhibitor could cause an even more severe B12 deficiency that is
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`potentially lethal. It would thus be important t