CYAN EXHIBIT 1001
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`US005527533A
`
`United States Patent
`
`[19]
`
`[11] Patent Number:
`
`5,527,533
`
`T50 et al.
`
`[45] Date of Patent:
`
`Jun. 18, 1996
`
`[54] METHOD OF RETARDH‘IG AND
`AMELIORATING CENTRAL NERVOUS
`SYSTEM AND EYE DAMAGE
`
`[75]
`
`Inventors: Mark O. M. Tso, Northbrook;
`Tim-Tak Lam, Skokie, both of 111.
`
`[73] Assignee: Board of Trustees of the University of
`Illinois, Urbana, Ill.
`
`[21] Appl. No.2 330,194
`
`[22]
`
`Filed:
`
`Oct. 27, 1994
`
`Int. Cl.6 ..................................... A61K 31/00
`[51]
`[52] US. Cl. .......................... 424/422; 424/427; 514/912;
`514/913; 514/914; 514/915; 514/929
`[58] Field of Search ............................ 424/59, 60, 78.04,
`424/427; 128/645; 514/912, 913, 914, 915,
`929; 568/378
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,920,834
`5,243,094
`
`............................... 424/59
`11/1975 Klaui et a1.
`9/1993 Borg ........................................ 568/822
`
`FOREIGN PATENT DOCUMENTS
`
`467795
`
`_
`1/1992 Eumpean Pat. Off.
`OTHER PUBLICATIONS
`
`al., Current Eye Research,
`et
`Li
`pp.133—144.
`Johnson et al., Applied. Envim. Micro, vol. 35(6),]978 pp.
`1155—1159.
`
`10(2),1991,
`
`vol.
`
`Anon., “Bio & High Technology Announcement Itaro”, Itaro
`Refrigerated Food Co., Ltd.
`Anon., “Natural Astaxanthin & Krill Lecithin”, Itaro Refrig-
`erated Food Co., Ltd.
`DiMascio, P. et al., “Carotenoids, Tocopherols and Thiols as
`Biological Singlet Molecular Oxygen Quenchers”, Bio—
`chemical Society Transactions, 18, pp. 1054—1056 (1990).
`Hiramitsu, T. et al., “Preventative Effect of Antioxidants on
`Lipid Peroxidation in the Retina”, Ophthalmic Res, 23, pp.
`196—203 (1991).
`
`Johnson, E. A. et al., “Simple Method for the Isolation of
`Astaxanthin from the Basidomycetous Yeast Phaflia
`rhodozyma”, App. Environ. Microbiol.,
`35(6),
`pp.
`1155—1159 (1978).
`Kirschfeld, K., “Carotenoid Pigments: Their Possible Role
`in Pro tecting Against Photooxidation in Eyes and Photore—
`ceptor Cells”, Proc. R. Soc. Land, B216, pp. 71—85 (1982).
`Krinsky, N. I. et al., “Interaction of Oxygen and Oxy—radi-
`cals With Carotenoids”, J. Natl. Cancer Inst., 69(1), pp.
`205—210 (1982).
`Kurashige, M. et al., “Inhibition of Oxidative Injury of
`Biological Membranes by Astaxanthin”, Physiol. Chem.
`Phys. and Med. NMR, 22 pp. 27—38 (1990).
`Latscha, T., “Carotenoids—Carotenoids in Animal Nutri-
`tion”, I-loffinann—LaRoche Ltd, Basel, Switzerland.
`Li, Z. et al., “Desfem'oxime Ameliorated Retinal Photic
`Injury in Albino Rats”, CurrentEye Res, 10(2), pp. 133—144
`(1991).
`Mathews—Roth, M., “Porphyrin Photosensitization and
`Carotenoid Protection in Mice; In Vitro and In Vivo Stud-
`ies”, Photochemistry and Photobiology, 40(1), pp. 63—67
`(1984).
`Mathews—Roth, M., “Carotenoids and Cancer Prevention-
`—Experimental and Epidemiological Studies”, Pure and
`Appl. Chem., 57(5), pp. 717—722 (1985).
`Mathews—Roth, M., “Recent Progress in the Medical Appli-
`cations of Carotenoids”, Pure and Appl. Chem., 63(1), pp.
`147—156 (1991).
`
`(List continued on next page.)
`
`Primary Examiner—Thurman K. Page
`Assistant Examiner—Pamela S. Webber
`
`Attorney, Agent, or Firm—Marshall, O’Toole, Gerstein,
`Murray & Borun
`
`[57]
`
`ABSTRACT
`
`A method of retarding and ameliorating eye diseases and
`injuries is disclosed. The method comprises administering
`astaxanthin in a therapeutically—effective amount to prevent,
`retard or treat eye and central nervous system diseases or
`injuries, such as age-related macular degeneration and other
`central nervous system degenerative diseases, photic injury,
`ischemic diseases, and inflammatory diseases.
`
`27 Claims, 4 Drawing Sheets
`
`60
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`30
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`20
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`10
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`ThinlmessofONL(um)
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`C] Control
`- Treated
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`N
`
`Ave
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`

`

`5,527,533
`Page 2
`
`OTHER PUBLICATIONS
`
`Michon, J. J. et al., “A Comparative Study of Methods of
`Photoreceptor Morphometry”, Invest. Ophthalmol. Vis. Sci.,
`32, pp. 280—284 (1991).
`Miki, W., “Biological Functions and Activities of Animal
`Carotenoids”, Pure and Appl. Chem, 63(1), pp. 141—146
`(1991).
`Schalch, W., “Carotenoids in the Retina—A Review of Their
`Possible Role in Preventing or Limiting Damage Caused by
`Light and Oxygen”, Free Radicals andAging, I. Emerit et al.
`(ed.), Birkhauser Verlag, pp. 280—298 (1992).
`
`T30, M.O.M.. “Experiments on Visual Cells by Nature and
`Man: In Search of Treatment for Photoreceptor Degenera—
`tion”,
`Investigative Ophthalmology and Visual Science,
`30(12), pp. 2421—2454 (Dec. 1989).
`
`Tso, M.O.M., “Pathogenetic Factors of Aging Mascular
`Degeneration",Ophthalmology,92(5), pp. 628—635 (1985.
`
`Yu, D. et al., “Amelioration of Retinal Photic Injury by
`Beta—Carotene”, ARVO Abstracts Invest. Ophthalmol. Vis.
`Sci, 28 (Suppl.), p. 7, (1987).
`
`

`

`US. Patent
`
`Jun. 18, 1996
`
`Sheet 1 of 4
`
`5,527,533
`
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`US. Patent
`
`Jun. 18, 1996
`
`Sheet 2 of 4
`
`5,527,533
`
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`
`

`

`US. Patent
`
`Jun. 18, 1996
`
`Sheet 3 of 4
`
`5,527,533
`
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`US. Patent
`
`Jun. 18, 1996
`
`Sheet 4 of 4
`
`5,527,533
`
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`5,527,533
`
`1
`METHOD OF RETARDING AND
`AMELIORATING CENTRAL NERVOUS
`SYSTEM AND EYE DAMAGE
`
`FIELD OF THE INVENTION
`
`The present invention relates to a method of retarding and
`ameliorating central nervous system and eye diseases. More
`particularly, the present invention is directed to methods of
`treating central nervous system and eye insult resulting from
`disease or injury, such as age-related macular degeneration,
`photic injury, photoreceptor cell or ganglion cell damage,
`traumatic
`injury,
`ischemic insultarelated diseases and
`inflammatory diseases. The method comprises administering
`a therapeutically-efiective amount of astaxanthin to an indi-
`vidual, either orally, topically or parenterally, to ameliorate
`damage caused by the disease or injury or to retard the
`progress of a degenerative disease.
`
`BACKGROUND OF THE INVENTION
`
`Many diseases and injuries of the central nervous system
`presently are not treatable. These diseases and injuries are
`untreatable because, unlike many peripheral organs that can
`be removed in whole or in part, or transplanted, the central
`nervous system has very limited regeneration capability and
`cannot be totally excised without death. The eye is an
`extension of the brain, and therefore a part of the central
`nervous system. Accordingly, in the case'of an eye injury or
`disease, i.e., a retinal injury or disease, the diseases are often
`without treatment and the eye cannot be transplanted. Eye
`diseases and injuries that presently are untreatable include
`retinal photic injury, retinal ischemia—induced eye injury,
`age—related macular degeneration, and other eye diseases
`and injuries that are induced by free radical species.
`It has been hypothesized that a major cause of these
`untreatable central nervous system diseases and injuries is
`the generation and presence of singlet oxygen and other free
`radical species. Singlet oxygen and free radical species can
`be generated by a combination of light and oxygen, or
`during reperfusion after an ischemic insult.
`The eye is subjected to continuous light exposure because
`the primary purpose of the eye is light perception. Therefore,
`some untreatable diseases and injuries to the eye result from
`the continuous exposure of the eye to light, coupled with the
`highly—oxygenated environment in the eye.
`The process of light perception is initiated in the photo-
`receptor cells. The photoreceptor cells are a constituent of
`the outer neuronal layer of the retina, which is a component
`of the central nervous system. The photoreceptor cells are
`well sheltered in the center of the eye, and are protected
`structurally by the sclera, nourished by the highly-vascular-
`ized uvea and safeguarded by the blood-retinal barrier of the
`retinal pigment epithelium.
`The primary function of the photoreceptor cells is to
`convert light into a physio-chemical signal (transduction)
`and to transmit this signal to the other neurons (transmis-
`sion). During the transduction and transmission processes,
`the metabolic activities of these neurons are changed dra—
`matically. Even though the photoreceptor cells are securely
`protected in the interior of the eye, these cells are readily
`accessible to light because their primary function is light
`detection. Excessive light energy reaching the retina can
`cause damage to these neurons, either directly or indirectly,
`by overwhelming the metabolic systems of these cells.
`
`2
`The combination of continuous and/or excessive exposure
`to light, and the relatively high concentration of oxygen in
`the eye, generates singlet oxygen and other free radical
`species. Singlet oxygen and free radical species also can be
`generated by enzymatic processes independent from light
`exposure. The free radical species and singlet oxygen are
`reactive entities that can oxidize polyunsaturated fatty acids.
`The retina contains the highest concentration of polyunsatu-
`rated fatty acids of any tissue in the human body, and
`peroxidation of the polyunsaturated fatty acids in cell mem-
`branes of the retina by hydroxyl radicals (OH) or superoxide
`(02) radicals can propagate additional free radical species.
`These free radical species can lead to functional impairment
`of the cell membranes and cause temporary or permanent
`damage to retinal
`tissue. It has been theorized that the
`generation of singlet oxygen and free radical species there-
`fore underlies the pathogenesis of light-induced retinopathy
`and post—ischernic reflow injury (i.e., free radical generation
`during reperfusion after an ischemic insult). In addition, a
`deficiency in removing these species can also contribute to
`various diseases of the eye and the central nervous system.
`A number of natural mechanisms protect the photorecep~
`tor cells from light injury. For example, the ocular media,
`including the cornea, aqueous, lens, and vitreous, filter most
`of the light in the ultraviolet region. However, after cataract
`extraction or other surgical intervention, some of these
`protective barriers are removed or disturbed, whereby the
`photoreceptor cells are more susceptible to damage by
`radiant energy. The photoreceptor cells also possess other
`forms of protection from photic injury, for example,
`the
`presence of antioxidant compounds to counteract the free
`radical species generated by light. As will be demonstrated
`hereafter, antioxidants, which quench and/or scavenge sin-
`glet oxygen, hydrogen peroxide, superoxide and radical
`species, help minimize injury to the photoreceptor cells. In
`addition, the human eye has an excessive number of pho-
`toreceptor cells such that only destruction of a significant
`number of photoreceptor cells adversely affects visual func-
`tion.
`
`Even though several protective mechanisms are present in
`the eye, a leading cause of blindness in the United States is
`age-related photoreceptor degeneration. Clinically, photore-‘
`ceptor degeneration, as seen in age-related macular degen-
`eration, is causally related to excessive exposure to blue
`light. The causes of age—related macular degeneration, which
`is characterized by a loss of photoreceptor neurons resulting
`in decreased vision, are being investigated. Epidemiological
`studies indicate that age-related photoreceptor degeneration,
`or age-related macular degeneration, is caused by several
`factors including age, sex, family history, color of the iris,
`nutritional deficiency, immunologic disorders, cardiovascu-
`lar and respiratory diseases and preexisting eye diseases.
`Advancing age is the most significant factor. Recently, it has
`been demonstrated that aging eyes have a decreased amount
`of carotenoids. Clinical and laboratory studies indicate that
`photic injury is a cause of age-related macular degeneration
`because of the cumulative efiect of repeated mild photic
`insult which leads to a gradual loss of photoreceptor cells.
`Age-related macular degeneration is an irreversible blind-
`ing disease of the retina. Unlike cataract wherein vision can
`be restored by replacing the diseased lens, age—related macu-
`lar degeneration cannot be treated by replacing the diseased
`retina because the retina is a component of the central
`nervous system. Therefore, because no treatment for this
`disease exists once the photoreceptors are destroyed, pre-
`vention is the only way to confront age-related macular
`degeneration. Presently, prevention of age—related macular
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`5,527,533
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`3
`degeneration resides in limiting or preventing light and
`oxygen-induced (i.e., free radical-induced) damage to the
`retina because the retina is the only organ that is continu-
`ously exposed to high levels of light in a highly—oxygenated
`environment.
`
`In addition to photic injury, eye injury and disease can
`result from singlet oxygen and free radical species generated
`during reperfusion after an ischemic insult. Ischemic insult
`to retinal ganglion cells and to neurons of the inner layers of
`retina causes loss of vision. Loss of vision accompanies
`diabetic retinopathy, retinal arterial occlusion, retinal venous
`occlusion and glaucoma, each of which ischemically insults
`the eye, i.e., deprives the eye of oxygen and nutrition.
`The damage to the retinal ganglion cells has been attrib~
`uted to ischemia, and subsequent reperfusion during which
`free radicals are generated. During reperfusion, the reoxy-
`genation of tissue after an ischemic insult results in a
`relatively high concentration of oxygen flowing through the
`efiected tissue, which contributes to free radical formation.
`Ischemic insult and reperfusion accompanied by free radical
`generation also is a major contributor to central nervous
`system damage, such as damage caused by a stroke.
`The pathogenesis of photic injury, of age-related macular
`degeneration, of ischemia/reperfusion damage, of traumatic
`injury and of inflammations of the eye and central nervous
`system have been attributed to singlet oxygen and free
`radical generation, and subsequent free radical-initiated
`reactions. Investigators therefore studied the role of antioxi-
`dants in preventing or ameliorating these diseases and
`injuries of the central nervous system in general, and the eye
`in particular.
`For example, ascorbate was investigated as an agent to
`treat retinal photic injury. Ascorbate is a reducing agent
`which is present in the retina in a high concentration. Studies
`indicated that ascorbate in the retina can act as an antioxi-
`dant and is oxidized by free radical species generated during
`excessive light exposure.
`Administration of ascorbate reduced the loss of rhodopsin
`after photic exposure,
`thereby suggesting that ascorbate
`offered protection against retinal photic injury. A decrease in
`rhodopsin levels is an indicator of photic eye injury. The
`protective eifect of ascorbate is dose-dependent, and ascor-
`bate was effective when administered before light exposure.
`Morphometn'c studies of the photoreceptor nuclei remaining
`in the retina after light exposure showed that rats given
`ascorbate supplements had substantially less retinal damage.
`Morphologically,
`rats with ascorbate supplements also
`showed better preservation of retinal pigment epithelium.
`The above studies led to the hypothesis that ascorbate
`mitigates retinal photic injury because of its antioxidant
`properties, which are attributed to its redox properties.
`Ascorbate is a scavenger of superoxide radicals and hydroxy
`radicals and also quenches singlet oxygen and reduces
`hydrogen peroxide, all of which are formed in retinal photic
`injury. This hypothesis accounts for the presence of high
`levels of naturally-occurring ascorbate in a normal retina.
`Therefore, antioxidants which inhibit free radical forma-
`tion, or which quench singlet oxygen and scavenge for free
`radical species, can decrease lipid peroxidation and amelio-
`rate photic injury and ischemic/reperfusion injury in the
`central nervous system, and particularly in the retina. Anti—
`oxidants originally were investigated because they are
`known constituents of human tissue. However, antioxidants
`that are not naturally occurring in human tissue also were
`tested. In particular, in addition to ascorbate, antioxidants
`such as 2,6-di—tert-butylphenol,
`'y-oryzanol, oc-tocopherol,
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`mannitol, reduced glutathione, and various carotenoids have
`been studied for an ability to quench singlet oxygen and
`scavenge free radical species. These and other antioxidants
`are effective quenchers and scavengers for singlet oxygen
`and free radicals. In particular, the carotenoids, as a class of
`compounds, are very effective singlet oxygen quenchers and
`free radical scavengers. However, individual carotenoids
`differ in their ability to quench singlet oxygen and scavenge
`for free radical species.
`The carotenoids are naturally~occuning compounds that
`have antioxidant properties. The carotenoids are common
`compounds manufactured by plants or animals, and contrib—
`ute greatly to the coloring of plants and animals. A number
`of animals, including mammals, are unable to synthesize
`carotenoids de novo and accordingly rely upon diet to
`provide carotenoid requirements. Mammals also have a
`limited ability to modify carotenoids. A mammal can con-
`vert B-carotene to vitamin A, but most other carotenoids are
`deposited in mammalian tissue in unchanged form.
`With respect to humans, about ten carotenoids are found
`in human semm. The major carotenoids in human serum are
`B-carotene, oc-carotene, cryptoxanthin, lycopene and lutein.
`Small amounts of zeaxanthin, phytofluene, and phytoene are
`found in human organs. However, of the ten carotenoids
`found in human serum, only two, zeaxanthin and lutein, are
`found in the human retina. Zeaxanthin is the predominant
`carotenoid in the macula and is concentrated in the cone
`cells in the center of the retina, i.e., the macula. Lutein is
`located in the peripheral retina in the rod cells. Therefore,
`the eye preferentially assimilates zeaxanthin over lutein in
`the macula and also selectively concentrates zeaxanthin,
`which is a more efiective singlet oxygen scavenger than
`lutein, in the center of the eye. It has been theorized that
`zeaxanthin and lutein are concentrated in the retina because
`of their ability to quench singlet oxygen and scavenge free
`radicals, and thereby limit or prevent photic damage to the
`retina.
`
`Therefore only two of the about ten carotenoids present in
`human semm are found in the retina. Beta-carotene and
`lycopene, the two most abundant carotenoids in human
`serum, either have not been detected or have been detected
`only in minor amounts in the retina. Beta-carotene is rela-
`tively inaccessible to the retina because B-carotene is unable
`to cross the blood-retinal brain barrier of the retinal pigment
`epithelium effectively. As will be explained in detail here-
`inafter, small amounts of B-carotene have been found to
`cross the blood-retinal brain barrier.
`
`It also is known that another carotenoid, canthaxanthin,
`can cross the blood-retinal brain barrier and reach the retina.
`Canthaxanthin, like all carotenoids, is a pigment and can
`discolor the skin. Canthaxanthin provides a skin color that
`approximates a suntan, and accordingly has been used by
`humans to generate an artificial suntan. However, an unde-
`sirable side effect in individuals that ingested canthaxanthin
`at high doses for an extended time was the formation of
`crystalline canthaxanthin deposits in the inner layers of the
`retina. Therefore,
`the blood-retinal brain barrier of the
`retinal pigment epithelium permits only particular caro-
`tenoids to enter the retina. The carotenoids other than
`zeaxanthin and lutein that do enter the retina cause adverse
`
`eifects, such as the formation of crystalline deposits by
`canthaxanthin, which may take several years to dissolve.
`Canthaxanthin in the retina also caused a decreased adap-
`tation to the dark.
`
`Investigators have unsuccessfully sought an antioxidant
`to counteract the adverse affects of singlet oxygen and free
`
`

`

`5,527,533
`
`5
`
`radical species on the central nervous system in general and
`the eye in particular. The investigators have studied the
`antioxidant capabilities of several compounds,
`including
`various carotenoids. Even though the carotenoids are strong
`antioxidants,
`investigators have failed to find particular
`carotenoids among the 600 naturally—occurring carotenoids
`that effectively quench singlet oxygen and scavenge for free
`radical species, that are capable of crossing the blood-retinal
`brain barrier,
`that do not exhibit the adverse affects of
`canthaxanthin after crossing the blood-retinal brain barrier,
`and that ameliorate central nervous system or eye disease or
`injury and/or retard the progression of a degenerative dis-
`ease of the central nervous system or eye.
`Various publications are directed to eye diseases and
`injuries, such as age-related macular degeneration, causes of
`the damage resulting from the diseases or injuries, and
`attempts to prevent or treat such diseases and injuries. The
`publications, which discuss various antioxidants, including
`the carotenoids and other antioxidants like a—tocopherol,
`include:
`
`M. O. M. Tso, “Experiments on Visual Cells by Nature
`and Man: In Search of Treatment for Photoreceptor Degen—
`eration”, Investigative Ophthalmology and Visual Science,
`3002), pp. 2421—2454 (December, 1989);
`W. Schalch, “Carotenoids in the Retina—A Review of
`Their Possible Role in Preventing or Limiting Damage
`Caused by Light and Oxygen”, Free Radicals and Aging, I.
`Emerit et al. (ed), Birkhauser Verlag, pp. 280—298 (1992);
`M. O. M. Tso, “Pathogenetic Factors of Aging Macular
`Degeneration”, Ophthalmology, 92(5), pp. 628—635 (1985);
`M. Mathews-Roth, “Recent Progress in the Medical
`Applications of Carotenoids”, Pure and Appl. Chem., 63(1),
`pp. 147—156 (1991);
`W. Mild, “Biological Functions and Activities of Animal
`Carotenoids”, Pure and Appl. Chem, 63(1), pp. 141—146
`(1991);
`M. Mathews-Roth, “Carotenoids and Cancer Prevention-
`Experimental and Epidemiological Studies”, Pure and Appl.
`Chem, 57(5), pp. 717—722 (1985);
`M. Mathews-Roth, “Porphyrin Photosensitization and
`Carotenoid Protection in Mice; In Vitro and In Vivo Stud-
`ies”, Photochemistry and Photobiology, 40(1), pp. 63—67
`(1984);
`P. DiMascio et al., “Carotenoids, Tocopherols and Thiols
`as Biological Singlet Molecular Oxygen Quenchers”, Bio—
`chemical Society Transactions, 18, pp. 1054—1056 (1990);
`T. Hiramitsu et al., “Preventative Efl°ect of Antioxidants
`on Lipid Peroxidation in the Retina”, Ophthalmic Res, 23,
`pp. 196—203 (1991);
`K. Kirschfeld, “Carotenoid Pigments: Their Possible Role
`in Protecting Against Photooxidation in Eyes and Photore-
`ceptor Cells”, Proc. R. Soc. Lona'., B216, pp. 71—85 (1982);
`D. Yu et al., “Amelioration of Retinal Photic Injury by
`Beta-Carotene”, ARVO Abstracts Invest. Ophthalmol. Vis.
`Sci, 28 (Suppl), p. 7, (1987);
`M. Kurashige et al., “Inhibition of Oxidative Injury of
`Biological Membranes by Astaxanthin”, Physiol. Chem.
`Phys. and Med. NMR, 22, pp. 27—38 (1990); and
`N. I. Krinsky et al., “Interaction of Oxygen and Oxy-
`radicals With Carotenoids”, J. Natl. Cancer Inst, 69(1), pp.
`205—210 (1982).
`In general, the above—identified publications support the
`hypothesis that singlet oxygen and free radical species are
`significant contributors to central nervous system, and par~
`ticularly eye, injury and disease. For example, it has reported
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`that consumption of an antioxidant, such as ascorbic acid
`(Vitamin C), a-tocopherol (Vitamin E) or fi-carotene, can
`decrease the prevalence of age—related macular degenera-
`tion.
`
`The above-identified publications also demonstrated that
`several carotenoids, including astaxanthin, are strong anti-
`oxidants compared to B—carotene, ascorbic acid and other
`widely used antioxidants. The publications also relate that
`(1) only particular carotenoids selectively cross the blood-
`retinal brain barrier, and that (2) certain carotenoids other
`than zeaxanthin and lutein that cross the blood-retinal brain
`barrier cause adverse aifects.
`
`In general, the above—identified publications teach that
`astaxanthin is a more efiective antioxidant than carotenoids
`such as zeaxanthin, lutein, tunaxanthin, canthaxanthin and
`[i—carotene, and than Ot-tocopherol. For example, the in Vitro
`and in vivo studies disclosed in the Kurashige et al. publi-
`cation with respect to astaxanthin demonstrated that the
`mean efiective concentration of astaxanthin which inhibits
`lipid peroxidation was 500 times lower than that of a-toco-
`pherol. Similarly,
`the Miki publication discloses that, in
`Vitro, astaxanthin exhibits a strong quenching effect against
`singlet oxygen and a strong scavenging effect against free
`radical species.
`Investigators have theorized that free radical-mediated
`reactions are involved in the pathogenesis of degenerative
`diseases, such as Alzheimer’s disease, Parkinson’s disease
`and age—related macular degeneration, ischemic/reperfusion
`damage and traumatic injury of the brain, spinal cord and
`retina, as well as in inflammatory processes of the body, and
`particularly the eye. This theory has been advanced by
`investigators examining the eifectiveness of various antioxi—
`dants in ameliorating these diseases. For example, methyl-
`prednisolone, which at high doses is a strong lipid peroxi-
`dation inhibitor, has been recommended clinically for use in
`traumatic injury of the spinal cord.
`To date, investigative efiorts have been directed to pre-
`venting diseases and injury because the resulting free radi—
`cal-induced damage is not effectively treatable. Therefore, a
`need exists for a method not only to prevent or retard, but
`also to ameliorate, degenerative and traumatic diseases and
`injuries to the central nervous system, and particularly the
`eye. The present invention is directed to such methods.
`
`SUMMARY OF THE INVENTION
`
`The present invention is directed to methods of treating
`individuals suifering from central nervous system injury or
`disease. More particularly, the present invention is directed
`to methods of treating individuals suffering from an eye
`injury or disease, and to methods of retarding a degenerative
`disease of the eye.
`The method comprises administering a therapeutically-
`effective amount of astaxanthin to an individual to retard a
`degenerative disease, or to ameliorate damage to the central
`nervous system caused by a disease or an injury. In particu-
`lar, the method comprises administering a therapeutically-
`effective amount of astaxanthin to an individual to benefi-
`ciate the vision of an individual suffering from eye damage
`caused by disease or injury. The astaxanthin can be admin-
`istered parenterally, orally or topically.
`The method is used to treat free radical-induced eye
`damage, light-induced eye damage, photoreceptor cell darn-
`age, ganglion cell damage, damage to neurons of inner
`retinal layers, and age-related macular degeneration. The
`present method also ameliorates neuronal damage to the
`
`

`

`5,527,533
`
`7
`
`retina, wherein the neuronal damage is a result of photic
`injury, or ischemic, inflammatory or degenerative insult.
`One aspect of the present invention is to administer about
`5 to about 500 milligrams (mg) of astaxanthin, and prefer-
`ably about 10 to about 200 mg, per kilogram (kg) of body
`weight per dose,
`to retard a degenerative disease of the
`central nervous system or the eye, or to ameliorate damage
`resulting from an injury or a disease of the central nervous
`system or the eye.
`Another aspect of the present invention is to provide a
`method of treating an inflammatory disease of the eye by
`administering a therapeuticallyreffective amount of astax—
`anthin to an individual.
`
`Another aspect of the present invention is to treat diseases
`and injuries to the central nervous system by administering
`a therapeutically-effective amount of astaxanthin to an indi-
`vidual. The method is used to treat diseases and injuries
`effecting the brain, eye and spinal cord, such as injury
`caused by spinal cord trauma or by a stroke or neurodegen-
`erative diseases.
`
`These and other novel features and aspects of the present
`invention will become apparent from the following detailed
`description of the preferred embodiments taken in conjunc—
`tion with the figures.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a bar graph showing the thickness of the ONL
`(outer nuclear layer) of the retina in micrometers (urn) after
`light-induced receptor degeneration, and comparing the reti-
`nas of control animals to the retinas of animals treated with
`astaxanthin given parenterally both for the retina as a whole
`(average) and for each of the four quadrants of the retina;
`FIG. 2 is a bar graph showing the thickness of the inner
`retinal layer (IRL) in micrometers comparing the retinas of
`control animals to the retinas of animals after ischemic insult
`treated with astaxanthin;
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`FIG. 3 is a plot of rhodopsin level vs. time after photic
`injury comparing the retinas of animals treated orally with
`astaxanthin to the retinas of control animals; and
`
`4O
`
`FIG. 4 is a plot of ONL thickness vs. time after photic
`injury comparing the retinas of animals treated orally with
`astaxanthin to the retinas of control animals.
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`
`Certain diseases and injuries to the central nervous sys-
`tem, and particularly the eyes, presently are untreatable.
`Neuronal degeneration of the central nervous system or the
`eye can result from photic injury, ischemic or intraocular
`pressure-related insult, e.g., an occlusion or a stroke, or from
`a trauma, e.g., trauma to the spinal cord. The damage also
`can result from injury to the photoreceptor cells,
`to the
`ganglion cells in the retina of the eye or to neurons in the
`inner retinal layers of the eye, or from age-related macular
`degeneration.
`It has been theorized that the damage from such diseases
`and injuries can be attributed to increased generation, or
`decreased removal, of singlet oxygen and free radical spe—
`cies. Therefore, because antioxidants are known to quench
`singlet oxygen and to scavenge for free radical species, and
`because antioxidants are known to exist in humans, inves—
`tigators have sought effective antioxidants to scavenge free
`radicals, and thereby reduce the free radical-induced damage
`
`45
`
`50
`
`55
`
`60
`
`6S
`
`8
`caused to the central nervous system, and especially to the
`eye, by disease and injury.
`Numerous antioxidants have been investigated, such as
`for example, ascorbic acid, ot-tocopherol and B-carotene.
`Some investigators have focused on the carotenoids, e.g.,
`[ii—carotene, because over 600 carotenoids are known, are
`naturally occurring (and therefore are abundant), and are
`strong antioxidants. Also, with respect to the eyes,
`two
`carotenoids, zeaxanthin, and lutein, which are strong anti-
`oxidants, are found in the photoreceptor cells of the retina.
`However, although human plasma includes about ten caro-
`tenoids, only these two carotenoids are able to effectively
`cross the blood—retinal brain barrier and concentrate in the
`macula of the eye. Beta-carotene, the most abundant human
`plasma carotenoid, has a very limited ability to cross the
`blood—retinal brain barrier.
`
`The ability of a carotenoid to pass the blood-retinal brain
`barrier is important because carotenoids are not synthesized
`by the human body. The only source of carotenoids for
`humans is dietary intake. Furthermore, humans have a very
`limited ability to modify carotenoids. Therefore, the caro-
`tenoids accumulate in various organs in the ingested form.
`Accordingly, if a particular carotenoid is unable to cross the
`blood-retinal brain barrier, the carotenoid cannot accumulate
`in the retina and serve as an antioxidant.
`
`Furthermore, carotenoids that are not normal constituents
`of human plasma, but have an ability to cross the blood-
`retinal brain barrier, have demonstrated adverse affects on
`the retina. Canthaxanthin which is intentionally ingested to
`provide an artificial suntan has accumulated in the retina in
`the form of crystals and has temporarily afiected eye adap—
`tation to the dark. In addition, B-Carotene has a limited
`ability to cross the blood-retinal brain barrier.
`Therefore, even though the carotenoids are known as
`strong antioxidants and are present in abundant supply, the
`carotenoids have not been used for the treatment of central
`
`nervous system damage, or eye damage, caused by disease
`or injury. The carotenoids investigated to dat