FJ. Schweigert Supplementary Declaration 26 June 2013
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`Cyan.|PR.One and Cyan.|PR.Two
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`CYAN EXHIBIT 1039
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`DECLARATION OF FLORIAN J. SCHWEIGERT
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`Containing Claims Charts for Exhibits 1010 and 1014
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`I, Florian J. Schweigert, declare as follow:
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`1.
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`2.
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`I am a citizen of the Federal Republic of Germany, and resident of Berlin, Germany.
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`Since 1996 to the present, I have been employed by the University of Potsdam as (full)
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`Professor of Physiology, and Chair of Physiology and Pathophysiology of Nutrition at the
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`Institute of Nutritional Science, Faculty of Sciences, University of Potsdam, Potsdam,
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`Germany.
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`From 1993 to 1996, I was employed as (fiJll) Professor of Nutrition Physiology, Dept. of
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`Physiology, University of Leipzig, Leipzig, Germany.
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`From 1988-1990, I was a Research Fellow in Medicine in the Channing Laboratories,
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`Harvard Medical School, Boston, Mass.
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`From 1985 to 1993, I was employed in various research and teaching positions, as shown on
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`Exhibit A, annexed hereto.
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`My graduate degree credentials are:
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`1986
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`Ph.D. (Dr. med. vet.) in Nutritional Physiology is from the Veterinary Faculty,
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`Department of Physiology, Biochemistry and Nutritional Physiology, Munich, Germany.
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`1983
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`D.V.M., Veterinary Faculty, Munich, Germany.
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`Other degrees, honors, and fellowships are shown on Exhibit A, annexed hereto.
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`From 2004 until 2009, I was an Expert Member of the Working Group on Carotenoids of
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`the European Food Safety Authority (colloquially known as the “FDA. of the EU”).
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`Exhibit B annexed hereto recites 146 of my peer-reviewed publications (out of over 155)
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`and 31 of my other publications in the fields of vitamin A and carotenoids, eye damage,
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`injury, disease, and therapy, antioxidants and the eye, especially the retina, and other areas
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`related to nutrition and disease.
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`10.
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`11.
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`I have made over 200 presentations in national and international conferences on vitamin A
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`and carotenoids, free radicals, eye damage, injury, disease, and therapy, antioxidants and the
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`eye, especially the retina, and other areas related to nutrition and disease.
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`I am being compensated at my normal consulting rate for my work. My compensation is not
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`dependent on and in no way affects the substance of my statements in this Declaration.
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`FJ. Schweigert Supplementary Declaration 26 June 2013
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`12. I have no financial interest in Petitioner or the owner of the ‘533 patent.
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`13. I have reviewed and understand the specification, claims, and file history of US. Patent No.
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`5,527,533 (“ ‘533 Patent”), including the Declarations filed in the ‘533 patent. Iunderstand
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`that ‘533 patent is considered to have been filed on 27 October 1994 (“Critical Date”) for
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`the purposes of determining whether a reference will qualify as prior art.
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`14. I have reviewed the following references, all of which were published before the Critical
`
`Date:
`
`Berson, E., “Nutrition And Retinal Degenerations: Vitamin A, Taurine, Ornithine, and
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`Phytanic Acid,” Retina: Vol.2, Issue 4, pp 236-255 (Fall 1982)
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`Carter-Dawson, L., Kuwabara T., O'Brien P.J., and Bieri, J .G., “Structural and
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`Biochemical Changes in Vitamin A-Deficient Rat Retinas”. Invest. Ophthalmol. Vis.
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`Sci. 18: 437-446, (1979).
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`Dowling, J .E. and Gibbons, I.R., “The effect of vitamin A deficiency on the fine structure
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`of the retina”, in The Structure of the Eye, Smelser C.K., editor. New York, Academic
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`Press, Inc., p. 85-99 (1961).
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`Dowling, J .E. and Wald, G., “Vitamin A deficiency and night blindness”. Proc Nat Acad
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`Sci USA 44:648, (1958).
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`Dowling, J .E. and Wald, G., “The Biological Function of Vitamin A,” Proc Nat Acad Sci
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`USA, May, 46(5) 587—608 (1960).
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`Goto, H. Wu, G-S., Gritz, D.C., Atalia, L.R.A., and Rao, N.A., “Chemotactic activity of the
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`peroxidized retinal membrane lipids in experimental autoimmune uveitis”, Current Eye
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`Res, V0.10, No. 11, 1009-1014 (1991).
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`Grangaud, Rene', “Astaxanthin Research, New Vitamin A Factor”, 69 pp.(Editions Desoer,
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`Liege, 1951), English translation.
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`Grangaud, Rene', “Recherches sur l’Astaxanthine, Nouveau Facteur, Vitaminique A”, 69
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`pp. (Editions Desoer, Liege, 1951), in French.
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`Grangaud, Rene', Massonet, Rene'e, Conquy The'rese, and Ridolfo, Jacqueline,
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`“Transformation of Astaxanthin to Vitamin A in the Albino Rat: Neoformation in vivo
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`and in vitro”, Comptes Rendus Hebdomadaires des Seances de l'Academie des
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`Sciences, Vol. 252, pp. 1854-1856 (1961b), English translation.
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`FJ. Schweigert Supplementary Declaration 26 June 2013
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`Cyan.|PR.One and Cyan.|PR.Two
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`Grangaud, Rene, Massonet, Renee, Conquy Therese, and Ridolfo, Jacqueline,
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`“Transformation de l'astaxanthine en Vitamine A chez le Rat albinos: ne'oformation in
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`Vivo et in Vitro”, Comptes Rendus Hebdomadaires des Seances de l'Academie des
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`Sciences, Vol. 252, pp. 1854-1856 (1961b), in French.
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`Grangaud, R., and Massonet, R., “Antixerophthalmic effect of the esters of astaxanthin”,
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`Comptes Rendus Hebdomadaires des seances de la Societe de biologie et de ses filiales,
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`Vol. 148, pp. 1392-1394 (1954), English translation.
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`Grangaud, R., and Massonet, R., “Activite antixe'rophtalmique des esters de l'astaxanthine”,
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`Comptes Rendus Hebdomadaires des seances de la Societe de biologie et de ses filiales,
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`Vol. 148, pp. 1392-1394 (1954), in French.
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`Grangaud, Rene, and Massonet, Renee, “Antixerophthalmic Activity of the Carotenoid
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`Pigment of the Aristeomorpha foliacea (Penzeidze)”, Comptes Rendus Hebdomadaires
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`des Seances de l'Academie des Sciences, Vol. 230, pp. 1319-1321 (March 27, 1950),
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`English translation.
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`Grangaud, Rene, and Massonet, Renee, “Activite antixe'rophtalmique du pigment
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`carote'no'i'de d'Aristeomorpha foliacea (Penzeidze)”, Comptes Rendus Hebdomadaires
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`des Seances de l'Academie des Sciences, Vol. 230, pp. 1319-1321 (March 27, 1950) , in
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`French.
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`Grangaud, Rene, and Massonet, Renee, “The Action of Shrimp Oil (Penaeus foliaceus) on
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`the Vitamin A Deficient White Rat”, Comptes Rendus Hebdomadaires des Seances de
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`l'Academie des Sciences, Vol. 227, pp. 568-570 (1948), English translation.
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`Grangaud, Rene, and Massonet, Renee, “Action de l'huile de Crevette (Penaeus foliaceus)
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`sur le Rat blanc carence en Vitamine A”, Comptes Rendus Hebdomadaires des Seances
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`de l'Academie des Sciences, Vol. 227, pp. 568-570 (1948), in French.
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`Hayes, K.C., “Retinal degeneration in monkeys induced by deficiencies of Vitamin E or A,”
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`Invest. Ophthalmol. Vis. Sci., vol. 13 no. 7, 499-510 (July, 1974).
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`Herisset, Armand, “Antioxidant properties of carotenoids and their derivatives”.Comptes
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`Rendus Hebdomadaires des Seances de l’Acade'mie des Sciences, V253, pp. 47-49
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`(July — December) 1946, English translation.
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`FJ. Schweigert Supplementary Declaration 26 June 2013
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`Cyan.|PR.One and Cyan.|PR.Two
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`Herisset, Armand, “Proprie'te's antioxygenes des carotenoides et de leurs derives”.Comptes
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`Rendus Hebdomadaires des Se'ances de l’Acade'mie des Sciences, v.253, pp. 47-49
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`(July — December) 1946, in French.
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`Kurashige, M. et al., "Inhibition of Oxidative Injury of Biological Membranes by
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`Astaxanthin", Physiol. Chem. Phys. and Med. NMR, 22, pp. 27-38 (1990).
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`Massonet, Renee, “Research into the Biochemistry of Astaxanthin”, 146 pp.(F.Fontana,
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`Algiers, 1960), English translation.
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`Massonet, Renee, “Recherches sur la Biochemie de l’Astaxanthine,”, 146 pp. (F.Fontana,
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`Algiers, 1960, in French.
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`Massonet, R., Conquy, T., and Grangaud, R.R. “The Study of Astaxanthin Transformation
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`into Vitamin A in the Albino Rat: in vitro Experiments”, Ann. Nutrit. Alimentation,
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`Vol. 19 pp. pages C655-C658 (1965)), English translation.
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`Massonet, R., Conquy, T., and Grangaud, R.R. “Etude de la transformation de
`
`l'astaxanthine en vitamine A chez le Rat albinos: Experiences ‘in vitro’”, Ann. Nutrit.
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`Alimentation, Vol. 19 pp. pages C655-C658 (1965)), in French.
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`Massonet, R., Conquy, T., and Grangaud, R., “Transformation of astaxanthin to vitamin A
`
`by ocular tissue of the rat in vitro”, Comptes Rendus Hebdomadaires des seances de la
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`Societe de biologie et de ses filiales, Vol. 155, pp. 747-750 (1961a), English translation.
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`Massonet, R., Conquy, T., and Grangaud, R., “Transformation invitro de l'astaxanthine en
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`vitamine A par le tissu oculaire du Rat”, Comptes Rendus Hebdomadaires des seances
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`de la Societe de biologie et de ses filiales, Vol. 155, pp. 747-750 (1961a) , in French.
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`Reading, V.M., Weale, R.A., Aberration, C., Malinow, M.R., “The Effect of Deficiency of
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`Vitamins E And A on the Retina”, Nutrition Reviews, Volume 38, Issue 11, pages 386—
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`389 (Nov. 1980).
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`Schiedt et al., “Recent progress on carotenoid metabolism in animals”, Pure& Appl Chem,
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`Vol.63, No. 1 pp 89-100 (1991).
`
`US. Patent No. 5,310,764 (“Treatment of age related macular degeneration with B-
`
`carotene”), to Baranowitz, et al., issued 10 May 1994.
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`Zigler, J .S. and Hess H.H., “Cataracts in the Royal College of Surgeons Rat: Evidence for
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`Initiation by Lipid Peroxidation Products”, EXp. Eye. Res., 41:67-76 (1985).
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`FJ. Schweigert Supplementary Declaration 26 June 2013
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`Cyan.|PR.One and Cyan.|PR.Two
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`15.
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`I have reviewed and understand the Grangaud thesis in French and English (Ex. 1002 and
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`1003), the Massonet thesis in French and English (Ex. 1004 and 1005), the six Massonet et
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`al. journal articles in French and English (Exs. 1008-1019), the three Dowling et al. journal
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`articles (Exs. 1024-1026), the file history of the ‘533 patent (Ex. 1006), and US. Patent No.
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`(“USPAT”) 5,310,764 (Ex. 1021), and the description of those publications in the Petition
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`for Inter Partes Review and think each description set forth in Sections III(C), IV, and V
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`accurately summarizes the disclosure of the relevant Exhibit.
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`16.
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`I have reviewed and understand the claim charts in the Petition for Inter Partes Review,
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`which claims charts are a condensed version of the claims charts in this Declaration. In my
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`opinion, a person of ordinary skill in the art would agree that each chart identifies and
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`discusses representative subject matter from the Exhibits cited in a given claims chart and (i)
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`teaches each and every claim limitation of claims 1, 3, and 8-27 of the’533 patent as to the
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`claims charts for Ground 1 in Cyan.IPR.One and in Cyan.IPRTwo (see Claims Charts for
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`claims 25 and 27 for more detail on the absence of astaxanthin in the brain and spinal cord),
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`and (ii) renders obvious each of claims 1-27 of the’533 patent as to the claims charts for
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`Ground 2 in Cyan.IPR.One and in Cyan.IPRTwo.
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`17.
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`18.
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`19.
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`“Cyan.IPR.One” refers to the Petition for Inter Partes Review filed by Cyanotech to
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`challenge USPAT 5,527,533 and that cites Grangaud’s thesis (Ex. 1002) as the base
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`reference in Ground 1 thereof. “Cyan.IPRTwo” refers to the Petition for Inter Partes
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`Review filed by Cyanotech to challenge USPAT 5,527,533 and that cites Massonet’s thesis
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`(Ex. 1004) as the base reference in Ground 1 thereof.
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`In my opinion, a person of ordinary skill in the art would find the Grangaud thesis, the
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`Massonet thesis, the Massonet et al. journal articles, the Dowling et al. journal articles, and
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`USPAT 5,310,764 recited in the Exhibits List of Cyan.IPR.One and of Cyan.IPR.Two to be
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`enabling disclosures of the subject matter each discusses.
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`After searching on the terms “astaxanthin” or “vitamin A” in Chemical Abstracts, for
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`instance, a diligent searcher would have easily been able to locate and retrieve the cited
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`publications prior to the Critical Date, determine the author’s name, and search on the
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`authors’ names to retrieve more prior art, e.g., Grangaud’s thesis (Ex. 1002), Massonet’s
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`thesis (Ex. 1004), or any of the journal articles in the Exhibits List of Cyan.IPR.One or
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`Cyan.IPRTwo.
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`FJ. Schweigert Supplementary Declaration 26 June 2013
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`Cyan.|PR.One and Cyan.|PR.Two
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`20.
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`The words “treating , damage ,
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`injury”, and “disease” have commonly accepted
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`77
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`cc
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`7:
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`(C
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`meanings in the field of ‘533 patent (i.e., the pharmaceutical/medical arts) with regard to
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`“treating an individual suffering from” damage, injury, or disease. Stedman’s Medical
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`Dictionary, 28th Edition (2006) (Philadelphia, Wolters Kluwer Health), attached as EX.
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`1040, provides definitions appropriate for the ‘533 patent of the terms “treating”, “damage”,
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`“injury”, and “disease”:
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`“Treating” means “To manage a disease by medicinal, surgical, or other measures, to
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`care for a patient medically or surgically.”.
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`“Damage” means “Harm, diminution, or destruction of an organ, body part, system, or
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`fiJnction”
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`“Injury” means “1. The damage or wound of trauma. 2. Lesion.” .
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`“Disease” means a “1. An interruption, cessation, or disorder of a body, system, or organ
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`structure or fill’lCthl’l. SYN: illness, morbus, sickness. 2. A morbid entity ordinarily
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`characterized by two or more of the following criteria: recognized etiologic agent(s),
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`identifiable group of signs and symptoms, or consistent anatomic alterations”.
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`Substantially similar definitions for such terms are found in other medical dictionaries, such
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`as Dorland’s Medical Dictionary (Elsevier).
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`21.
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`22.
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`There are two classes of carotenoids: xanthophylls (e. g, lutein, zeaxanthin, canthaxanthin,
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`and astaxanthin), and other carotenes (e. g., (x-, [5-, and y-carotene). See attached EX. 1032
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`(molecular skeletons of xanthophylls and B-carotene). Xanthophylls lutein, zeaxanthin, and
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`astaxanthin are much stronger antioxidants than (x-, [5-, and y-carotenes and other carotenes.
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`Before explaining in detail how Grangaud and Massonet performed and published the same
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`methods as claimed in the ‘533 patent decades before the Critical Date, I first point out
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`where the ‘533 patent is scientifically in error:
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`Today, almost two decades after the Critical Date, there is no evidence that astaxanthin is
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`transported into, much less accumulates, in the brain and spinal cord, or in any part of the
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`central nervous system other than the retina. If astaxanthin accumulated in the brain and
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`spinal cord, those organs would be pigmented, just as the macula lutea in the human retina is
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`pigmented by the xanthophylls lutein and zeaxanthin, and the corpus luteum in the human
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`ovary is pigmented by the carotene B-carotene.
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`The statement in the ‘533 patent that “In addition, astaxanthin has a protective effect on
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`FJ. Schweigert Supplementary Declaration 26 June 2013
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`Cyan.|PR.One and Cyan.|PR.Two
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`23.
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`24.
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`25.
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`the central nervous system in general, especially damage to the brain and spinal cord caused
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`by free radicals.” (Ex. 1001, 14:60-62), has no support in the ‘533 patent (including the file
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`history thereof) as to the brain and spinal cord, is scientifically erroneous, and cannot be
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`supported even today (excluding damage to the retina; embryologically, the retina is an
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`outgrowth of the developing brain, and is therefore is part of the central nervous system).
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`Therefore, claims 25 and 27 of the ‘533 patent are scientifically erroneous.
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`Astaxanthin is one of the strongest antioxidants known, in addition to benefiting from the
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`antioxidant properties of astaxanthin, in the rat retina astaxanthin is converted into vitamin
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`A (Exs. 1008 and 1010), an essential vitamin.
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`Transport of astaxanthin from the bloodstream into a tissue requires specialized “binding
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`proteins” that are present in the retina and a few other animal tissues. Suppression of free
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`radicals necessarily occurs if astaxanthin is present in animal tissue that contains free
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`radicals, such as retinal tissue exposed to bright light. Irradiating the retina with bright light
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`creates excited states of oxygen that characterize peroxyl radicals (R000) andsinglet oxygen
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`(102) radicals.
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`Any administration of astaxanthin (other than topical) necessarily results in blood-based
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`transport of astaxanthin to the retina. The only blood-based access to the eye in vertebrates
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`is through the retinal and uveal capillary networks that service the retina (including the
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`retinal pigment epithelium (“RPE”)), and the iris and ciliary body, respectively. Retinal
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`tissue contains binding proteins that preferentially transport xamhophyll carotenoids, like
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`lutein, zeaxanthin, canthaxanthin, and astaxanthin, from the retinal capillary network into
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`retinal tissue, but disfavor transport into retinal tissue of carotene carotenoids, like B-
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`carotene. Transport of astaxanthin in the bloodstream requires specialized “binding
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`proteins”. Transport of astaxanthin from the bloodstream into a tissue, and accumulation of
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`astaxanthin in a given type of tissue, requires specialized “binding proteins” that are present
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`in some, but not all, animal tissue.
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`26.
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`Astaxanthin’s inherent mode of action in vertebrate tissue, including retinal tissue, is as a
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`strong antioxidant and free radical scavenger. Suppression of free radicals, such as peroxyl
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`and singlet oxygen radicals, and of free radical-induced damage necessarily occurs if
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`astaxanthin is present in animal tissue that contains free radicals, such as retinal tissue
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`exposed to bright light.
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`FJ. Schweigert Supplementary Declaration 26 June 2013
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`Cyan.|PR.One and Cyan.|PR.Two
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`27.
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`28.
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`29.
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`30.
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`Xerophthalmia (“dry eye disease”) is the first plainly visible sign of vitamin A deficiency in
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`rats (symptoms of “night blindness” precede visible signs of xerophthalmia).
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`Xerophthalmia is secondary to retinal damage, injury, and disease, i.e., retinal damage,
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`injury, and disease from vitamin A deficiency occurs first (and causes night blindness), then
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`xerophthalmia manifests at a later stage in the cornea and surrounding areas.
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`Xerophthalmia is caused by severe vitamin A deficiency. Rats and other vertebrates
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`become diseased, go blind, and die from continued vitamin A deficiency.
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`Infliction of
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`vitamin A deficiency is an injury and causes stunted growth as well as retinal, corneal, and
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`other injury and diseases.
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`The layered structure of the retina is shown in Ex. 1032. “Inner” retinal layers refer to
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`layers closer to the center of the ocular globe. “Outer” retinal layers refer to layers closer to
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`the sclera (outer surface) of the ocular globe. The retina is largely comprised of various
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`types of neurons, e.g., ganglion, photoreceptor rods, and photoreceptor cones.
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`Although photoreceptor cells are one of the “bottom” or “outer” layers of the retina,
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`photoreceptor cell membranes are especially vulnerable to oxidation, due to an unusual
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`combination of conditions: a high concentration of oxygen and mitochondria, the presence
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`of light energy, and a high proportion of polyunsaturated fatty acids (“PUFA”) in their
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`membranes. In general, the potential for free radical-induced cellular damage may be greater
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`in the retina than in any other tissue because the transparency of ocular structures allows
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`light-induced generation of free radicals (especially peroxyl radicals of PUFAs) in addition
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`to free radicals produced by normal oxidative metabolism.
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`31.
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`All oxygen-consuming tissues produce small amounts of highly reactive free radicals by
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`univalent reduction of oxygen, an alternative pathway of oxygen reduction through the
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`cytochrome system. The high density of mitochondria (which use oxygen in synthesizing
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`ATP, but create free radicals when oxygen is prematurely reduced) in the photoreceptor
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`cells increases the production of free radicals. Free radical production increases
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`dramatically when bright light strikes the photoreceptor cell membranes. Aerobic cells
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`(which use mitochondria for glycolysis and ATP production) have evolved many “free
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`radical scavengers”. A vitamin A-deficient retina, with inadequate amounts of endogenous
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`free radical scavengers, such as retinol (the alcohol form of vitamin A), cannot neutralize
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`Cyan.|PR.One and Cyan.|PR.Two
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`32.
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`33.
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`34.
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`even normal amounts of free radicals produced in retinal tissue, much less free radicals
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`produced by photic insult, ischemia, or high intraocular pressure.
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`Membrane lipid peroxidation is one of the most prominent forms of cellular damage
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`induced by conditions of oxidative stress (e. g., free radical barrage). The retina contains
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`several enzymatic free radical scavengers (e.g., superoxide dismutase, and catalase) for
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`neutralizing free radicals as well as a host of endogenous antioxidant compounds, including
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`(among others) vitamin E, ascorbate, taurine, glutathione, various vitamin A compounds,
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`and various carotenoids.
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`Astaxanthin suppresses free radicals, such as peroxyl radicals (R000) and singlet oxygen
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`(102) radicals, and thereby suppresses free radical-induced damage in tissues into which
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`astaxanthin is transported and accumulates.
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`Light impinging on the retina penetrates the “inner” layers of the retina (some of which is
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`called the inner retinal thickness or “IRT” in the ‘533 patent), the middle layer (including
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`the outer nuclear layer, or “ONL” in the ‘533 patent) of the retina, and passes through the
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`photoreceptor cell membrane (made primarily of PUFAs) to excite rhodopsin in the rods
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`(monochrome vision) and photopsins in the cones (color vision) of photoreceptor cells.
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`Rhodopsin consists of the protein moiety opsin and a reversibly covalently bound cofactor,
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`retinal (the aldehyde form of vitamin A).
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`35.
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`PUFAs exposed to light energy readily form peroxyl radicals that attack and destroy
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`photoreceptor cell membranes and other membranes and structures in the retina unless the
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`peroxyl radicals are neutralized by a free radical scavenger, such as an antioxidant. In the
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`absence of effective free radical scavenging, a barrage of peroxyl radicals is released from
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`the photoreceptor layer that can travel through the eye causing damage, injury, and disease
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`in the middle and inner layers of the retina, and even travel across the vitreous humor to the
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`anterior parts of the eye. For instance, peroxyl radicals from degenerated retina are a
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`primary or contributing cause of cataracts (Zigler, 1985) and of uveitis (Goto, 1991).
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`36.
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`The role of vitamin A in the chemistry of vision was elucidated by George Wald in the
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`period from the mid-1930s to the mid-1960s, which led to his Nobel Prize in 1967. Wald,
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`John Dowling, and IR. Gibbons published in the late 1950s and early 1960s the results of
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`their extensive research on degeneration of the retina caused by vitamin A deficiency
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`(Dowling and Wald (195 8), Dowling and Wald (1960), Dowling and Gibbons (1961).
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`FJ. Schweigert Supplementary Declaration 26 June 2013
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`Cyan.|PR.One and Cyan.|PR.Two
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`Those publications (Ex. 1024, 1025, and 1026, respectively) contain numerous micrographs
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`showing the reduction of the ONL and IRT, and graphs of the reduction of rhodopsin levels,
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`in photoreceptor cells lacking adequate vitamin A. See, e.g., Fig. 1 of Ex. 1024 (Dowling
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`and Wald, 1958) (reduction of rhodopsin levels), Figs. 2, 13, and 15 of Ex. 1025 (Dowling
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`and Wald, 1960) (reduction of ONL and IRT), Figs. 2 and 10 of Ex. 1026 (Dowling and
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`Gibbons, 1961) (reduction of ONL and IRT). Later publications (Reading (1980), Zigler
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`(1985)) explained that the degeneration of the photoreceptor cell membranes, reduction in
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`thickness of the retina, and reduced rhodopsin levels were due to attack by free radicals,
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`especially attack by peroxidized lipids emitted from disintegrating outer segments of
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`photoreceptor rods.
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`37.
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`38.
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`39.
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`40.
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`Vitamin A has three molecular forms relevant to this discussion: an acid form, “retinoic
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`acid”, required for normal growth, but not active in the chemistry of vision, an aldehyde
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`form, “retinal”, that combines with opsin to create rhodopsin, and an alcohol form,
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`“retinol”, which is an antioxidant. Retinal and retinol are enzymatically interconvertible,
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`but the enzymatic conversion to retinoic acid is irreversible.
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`The absence of vitamin A in the retina of a vitamin A deficient rat would necessarily result
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`in the absence of vitamin A as an antioxidant and as a component of rhodopsin, rendering
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`the retina vulnerable to free radical attack, e.g., during photic insult and during reperfiJsion
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`following retinal ischemia or high intraocular pressure (the experiments conducted in the
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`‘533 patent), and causing collapse of the rods in the photoreceptor cell layer by the inability
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`to synthesize rhodopsin (which is vital to rod structure).
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`In Fig. 15 of Ex. 1025 (Dowling and Wald, 1960), a retina (middle micrograph and
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`electroretinogram (“ERG”)), degenerated by 6.5 months of vitamin A deficiency with severe
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`damage to the photoreceptor cell layer, was restored to normal structure and fill’lCthl’l (Fig.
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`15, right micrograph and ERG) by administration of vitamin A.
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`Figs. 2a to 2d, and 10a to 10c, of Ex. 1026 (Dowling and Gibbons, 1961) also irrefiJtably
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`establish the protective, and therapeutic, effects of vitamin A (as both an antioxidant and as
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`a component of rhodopsin). Again, the retinal degeneration, including the ONL and IRT, as
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`shown in Figs. 2d and 10b, is far more severe than reported in the ‘533 patent. As in Ex.
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`1025 (Dowling and Wald, 1960), in Ex. 1026 (Dowling and Gibbons, 1961), vitamin A
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`protected the eye from retinal degeneration (group receiving vitamin A, Figs 2a and 10a)
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`and healed the degeneration and resultant disease when administered therapeutically (Figs.
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`2d and 10c) after free radical-induced retinal injury in a different group.
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`41.
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`If adequate vitamin A is available and photoreceptor cells are undamaged, rhodopsin levels
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`are normal. The reduction of ONL, IRT, and rhodopsin levels in the ‘533 patent reflect the
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`free radical scavenging mechanisms being temporarily overwhelmed by a free radical
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`barrage and consequent damage to the photoreceptor cells. The much more pronounced
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`reduction of rhodopsin levels in EX. 1025 (p. 588, Dowling and Wald, 1960) and Fig. 10 of
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`EX. 1026 (Dowling and Gibbons, 1961), compared with the minor, short term, drop in
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`rhodopsin levels in the ‘533 patent, reflect the combination of vitamin A deficiency and
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`more severe photoreceptor cell damage in the Dowling et al. references.
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`42.
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`Fig. 15 of EX. 1025 (Dowling and Wald, 1960) and Fig. 10 of EX. 1026 (Dowling and
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`Gibbons, 1961) are notable for showing not only the retinal degeneration (including
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`reduction of ONL and IRT) in rat retina caused by vitamin A deficiency, but prevention of
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`such degeneration by vitamin A, and reconstruction of degenerated rat retinal layers by
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`administration of vitamin A after retinal injury (in different test groups). This protection
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`against retinal degeneration (in the control group that received vitamin A) and “treating” of
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`retinal degeneration in the vitamin A deficient group that subsequently received vitamin A,
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`is biochemically, prophylactically (in the case of prevention) and therapeutically (in the case
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`of damage, injury, and disease) equivalent to the administration of astaxanthin to rats (since
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`astaxanthin is an antioxidant and is also converted in rat retina to vitamin A (Massonet et al.
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`(EX. 1008, (1965) and EX. 1010 (1961b)).
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`43.
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`Therefore, if astaxanthin is administered before an event that would otherwise cause a free
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`radical barrage (e.g., vitamin A deficiency, photic insult, reperfiJsion after ischemia or high
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`intraocular pressure), astaxanthin is necessarily transported to the retina and scavenges
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`(neutralizes) free radicals before they can cause damage. If astaxanthin is administered after
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`free radical-induced injury of the retina, astaxanthin is converted in rat retina into vitamin A,
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`which is then used to reconstruct the retina (explained in Dowling et al., 1958, 1960, and
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`1961), assuming that irreversible damage of the cornea (from xerophthalmia) and retina has
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`not occurred.
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`44.
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`Whether the retinal degeneration arises from vitamin A deficiency or from photic insult or
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`reperfusion following ischemia or high intraocular pressure, the biochemical, histological,
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`and pathological mechanism is the same: if the photoreceptor cell membranes (particularly
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`the rod outer segments) are exposed to light energy without adequate free radical
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`scavenging, the result is a free radical barrage of peroxidized fatty acids and singlet oxygen
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`that cause retinal degeneration and reduction of ONL, IRT, and rhodopsin levels. Even in
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`rats with normal vitamin A levels, intense photic energy or reperfiJsion (after ischemia or
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`high intraocular pressure) depletes available free radical scavengers, thereby enabling
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`peroxidation of lipids in the photoreceptor membranes, which unleashes a free radical
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`barrage and resultant damage, injury, and (if vitamin A deficiency ensues) disease.
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`45.
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`Vitamin A deficiency inherently produces the same types of retinal damage and injury that
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`the experiments in the ‘533 patent produced. Grangaud et al. (in Ex. 1014) and Massonet et
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`al. (in Ex. 1010) administered to vitamin A-deficient rats astaxanthin to prevent, and to treat,
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`one type of eye damage, injury, and disease (xerophthalmia) caused by vitamin A deficiency
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`or by lack of antioxidant, but necessarily (inherently) treated other types of eye damage
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`and injury caused by vitamin A deficiency or by lack of antioxidant, including the type of
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`damage and injury that the experiments in the ‘533 patent produced.
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`46.
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`The preceding discussion of biochemical, histological, and pathological mechanisms of free
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`radical-induced retinal degeneration was confirmed in other animal models before the
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`Critical Date.
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`I quote from these other studies:
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`Berson, Eliot, “Nutrition And Retinal Degenerations: Vitamin A, Taurine, Omithine, and
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`Phytanic Acid,” Retina: Fall 1982 - Volume 2 - Issue 4 — pp 236-255. (Ex. 1028)
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`Bersonp.240, left col., top. (emphasis added)
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`“In contrast, rats raised on a vitamin A-
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`free diet supplemented with retinoic acid show changes in the outer segments at about
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`two months (Fig. 6B) and loss of outer segments, inner segments, and about half the
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`photoreceptor nuclei at about six months (Fig. 6C). At ten months (Fig. 6D) the
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`photoreceptors have disappeared except for one row of nuclei.
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`In fact, reversal of
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`fill’lCthl’l and structure can be achieved with refeeding vitamin A in early stages.
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`Figure 7 illustrates the retina of a control rat (A), that of a vitamin A-deficient rat at
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`six months with loss of outer segments and half the photoreceptors (B), and the retina
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`of a rat depleted for about six months and then given vitamin A for 16 days (C). No
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`increase in the thickness of the outer nuclear layer occurs (Fig. 7C compared with
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`Cyan.|PR.One and Cyan.|PR.Two
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`Fig. 7B), but new outer segments (Fig. 7C) with normal length and width regenerate
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`within 16 days.”
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`L. Carter-Dawson, T. Kuwabara, P.J. O'Brien and JG. Bieri: Structural and Biochemical
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`Changes in Vitamin A-Deficient Rat Retinas. Invest. Ophthalmol. Vis. Sci. 18: 437-446,
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`1979.
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`(EX. 1030)
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`See Fig. 2 showing reduction of rhodopsin levels, and Figs. 7 and 8 showing reduced
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`thickness of ONL in vitamin A-deficient rats, and pages 444-445 discussing recovery
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`of normal rhodopsin levels and ONL by administration of vitamin A.
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`Hayes, K.C., “Retinal degeneration in monkeys induced by deficiencies of vitamin E or A,”
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`Invest. Ophthalmol. Vis. SciJuly 1974 vol. 13 no. 7 499-510. (Emphasis added).
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`(EX.
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`1027)
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`Fig. 1 caption. “On the other hand, both peripheral retina (C) and macula (D) in the
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`vitamin-A deficient monkey have degenerated outer segments, the latter appearing
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`much worse than the former. Thinning of the ONL has also occurred in the
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`macula.”
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`p. 505 bottom, It. col. In a vitamin A deficient monkey, “The ONL was reduced in
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`thickness and contained degenerating nuclei corresponding to the degree of OS
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`[outer segment] degeneration (Fig. 1).”
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`p. 508 bottom to left col. top of page 509. “protracted vitamin A depletion in adult
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`monkeys produced classical signs of deficiency including xerophthalmia and
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`keratomalacia. Rupture of the cornea resulted in destructive panophthalmitis in one
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`monkey.
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`Both rods and cones appeared damaged in the macula and in the
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`surrounding retina of the more advanced lesion. Degeneration of the ONL and
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`numerous lipid-laden lysosomes in the pigment epithelium were the only other
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`changes observed.”
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`Kurashige, M. et al., "Inhibition of Oxidative Injury of Biological Membranes by
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`Astaxanthin", Physiol. Chem. Phys. and Med