(19) TZZ _ _T

(11) EP 2 874 612 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A23L 17/00 (2016.01) A61K 9/48 (2006.01) 07.09.2016 Bulletin 2016/36 A61K 45/06 (2006.01) A61K 8/00 (2006.01)

(21) Application number: 13740461.2 (86) International application number: PCT/US2013/050240 (22) Date of filing: 12.07.2013 (87) International publication number: WO 2014/014766 (23.01.2014 Gazette 2014/04)

(54) KRILL OIL AND REACTED ASTAXANTHIN COMPOSITION AND ASSOCIATED METHOD KRILLÖL UND REAGIERTE ASTAXANTHIN-ZUSAMMENSETZUNG SOWIE ZUGEHÖRIGES VERFAHREN HUILE DE KRILL ET COMPOSITION D’ASTAXANTHINE AYANT SUBI DES RÉACTIONS ET MÉTHODE ASSOCIÉE

(84) Designated Contracting States: (74) Representative: Ferreccio, Rinaldo AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Botti & Ferrari S.r.l. GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Via Cappellini, 11 PL PT RO RS SE SI SK SM TR 20124 Milano (IT)

(30) Priority: 19.07.2012 US 201213553025 (56) References cited: 09.07.2013 US 201313937547 WO-A2-2006/116755 US-A1- 2011 021 465 US-A1- 2011 237 548 US-A1- 2011 268 811 (43) Date of publication of application: 27.05.2015 Bulletin 2015/22 • "Neptune krill Oil’s Unique Properties", INTERNET CITATION, 30 September 2011 (73) Proprietor: U.S. Nutraceuticals LLC dba Valensa (2011-09-30), pages 1-3, XP002660404, Retrieved International from the Internet: Eustis, FL 32726 (US) URL:http://www.nowfoods.com/Products/Produ ctFAQs/081008/htm [retrieved on 2011-09-30] (72) Inventors: • BARBARA ANAN KOGAN: "Krill oil a potential • MINATELLI, John ocular anti-inflammatory", INTERNET CITATION, Eustis, Florida 32726 (US) 1 June 2006 (2006-06-01), pages 1-2, • HILL, Stephen XP002603663, Retrieved from the Internet: Ocala, Florida 34471 (US) URL:http://s129638273.onlinehome.us/pharma • MOERCK, Rudi nexmd/Files/2006-06-01-PrimaryCareOptometr Sanford, Florida 32771 (US) yNewsKrillOil.pdf [retrieved on 2010-06-10]

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 874 612 B1

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Description reaching the retina can cause damage to these neurons, either directly or indirectly, by overwhelming the meta- Related Application(s) bolic systems of these cells. [0008] The combination of continuous and/or exces- [0001] This application claims the benefit of U.S. non- 5 sive exposure to light, and the relatively high concentra- provisional application Serial No. 13/937,547 filed July tion of oxygen in the eye, generates singlet oxygen and 9,2013, and claims the benefit of U.S. non-provisional other free radical species. Singlet oxygen and free radical application Serial No. 13/553,025 filed July 19, 2012. species also can be generated by enzymatic processes independent from light exposure. The free radical spe- Field of the Invention 10 cies and singlet oxygen are reactive entities that can ox- idize polyunsaturated fatty acids. The retina contains the [0002] The present invention relates to a medicine de- highest concentration of polyunsaturated fatty acids of livery system comprising krill oil and mixed carotenoids. any tissue in the human body, and per-oxidation of the polyunsaturated fatty acids in cell membranes of the ret- 15 Background of the Invention ina by hydroxyl radicals (OH) or superoxide (O 2) radicals can propagate additional free radical species. These free [0003] The eye is an extension of the brain, and there- radical species can lead to functional impairment of the fore a part of the central nervous system. Accordingly, in cell membranes and cause temporary or permanent the case of an eye injury or disease, i.e., a retinal injury damage to retinal tissue. It has been theorized that the or disease, the diseases are often without treatment and 20 generation of singlet oxygen and free radical species the eye cannot be transplanted. Eye diseases and inju- therefore underlies the pathogenesis of light-induced ries that presently are untreatable include retinal photic retinopathy and post-ischemic reflow injury. In addition, injury, retinal ischemia-induced eye injury, age-related a deficiency in removing these reactive free radical spe- macular degeneration, and other eye diseases and inju- cies can also contribute to various diseases of the eye. ries that are induced by singlet oxygen and other free 25 [0009] A number of natural mechanisms protect the radical species. photoreceptor cells from light injury. For example, the [0004] It has been hypothesized that a major cause of ocular media, including the cornea, aqueous, lens, and these untreatable retinal and other eye diseases and in- vitreous, filter most of the light in the ultraviolet region. juries is the generation and presence of singlet oxygen However, after cataract extraction or other surgical inter- and other free radical species. Singlet oxygen and free 30 vention, some of these protective barriers are removed radical species can be generated by a combination of or disturbed, whereby the photoreceptor cells are more light, oxygen, other reactive oxygen species like hydro- susceptible to damage by radiant energy. The photore- gen peroxide, superoxide or during reperfusion after an ceptor cells also possess other forms of protection from ischemic insult resulting in highly reactive NOx release. photic injury, for example, the presence of antioxidant [0005] The eye is subjected to continuous light expo- 35 compounds to counteract the free radical species gen- sure because the primary purpose of the eye is light per- erated by light. As will be demonstrated hereafter, anti- ception. Therefore, some untreatable diseases and inju- oxidants, which quench and/or scavenge singlet oxygen, ries to the eye result from the continuous exposure of the hydrogen peroxide, superoxide and radical species, min- eye to light, coupled with the highly-oxygenated environ- imize injury to the photoreceptor cells. The most impor- ment in the eye. 40 tant area of the retina where such protection is necessary [0006] The process of light perception is initiated in the is the fovea or central region of the macula. Even though photoreceptor cells. The photoreceptor cells are a con- several protective mechanisms are present in the eye, a stituent of the outer neuronal layer of the retina, which is leading cause of blindness in the United States is age- a component of the central nervous system. The pho- related photoreceptor degeneration. Clinically, photore- toreceptor cells are well sheltered in the center of the 45 ceptor degeneration, as seen in age-related macular de- eye, and are protected structurally by the sclera, nour- generation, is causally related to excessive exposure to ished by the highly-vascularized uvea and safeguarded high energy UVA and UVB ultraviolet light. The causes by the blood-retinal barrier of the retinal pigmented epi- of age-related macular degeneration, which is character- thelium. ized by a loss of photoreceptor neurons resulting in de- [0007] The primary function of the photoreceptor cells 50 creased vision, are still being investigated. Epidemiolog- is to convert light into a physio-chemical signal (trans- ical studies indicate that age-related photoreceptor de- duction) and to transmit this signal to the other neurons generation, or age-related macular degeneration, is re- (transmission). During the transduction and transmission lated to several factors including age, sex, family history, processes, the metabolic activities of these neurons are color of the iris, nutritional deficiency, immunologic dis- changed dramatically. Even though the photoreceptor 55 orders, cardiovascular and respiratory diseases and pre- cells are securely protected in the interior of the eye, existing eye diseases. Advancing age is the most signif- these cells are readily accessible to light because their icant factor. Recently, it has been demonstrated that ag- primary function is light detection. Excessive light energy ing eyes have a decreased amount of carotenoids de-

2 3 EP 2 874 612 B1 4 posited on the retina. Clinical and laboratory studies in- [0016] The above studies led to the hypothesis that dicate that photic injury is at least one cause of age-re- ascorbate mitigates retinal photic injury because of its lated macular degeneration because of the cumulative antioxidant properties, which are attributed to its redox effect of repeated mild photic insult which leads to a grad- properties. Ascorbate is a scavenger of superoxide rad- ual loss of photoreceptor cells. 5 icals and hydroxy radicals and also quenches singlet ox- [0010] Age-related macular degeneration is an irre- ygen, all of which are formed during retinal photic injury. versible blinding disease of the retina. Unlike cataracts This hypothesis accounts for the presence of high levels which can be restored by replacing the diseased lens, of naturally-occurring ascorbate in a normal retina. age-related macular degeneration cannot be treated by [0017] Therefore, antioxidants which inhibit free radi- replacingthe diseased retina because theretina is acom- 10 cal formation, or which quench singlet oxygen and scav- ponent of the central nervous system. Therefore, be- enge free radical species, can decrease lipid per-oxida- cause no treatment for this disease exists once the pho- tion and ameliorate photic injury and ischemic/reper- toreceptors are destroyed, prevention is the only way to fusion injury in the retina. Antioxidants originally were address age-related macular degeneration. Presently, investigated because they are known constituents of hu- prevention of age-related macular degeneration resides 15 man tissue. However, antioxidants that are not naturally in limiting or preventing light and oxygen-induced (i.e., occurring in human tissue were also tested. In particular, free radical-induced) damage to the retina because the in addition to ascorbate, antioxidants such as 2,6-di-tert- retina is the only organ that is continuously exposed to butylphenol, gamma-oryzanol, alpha-tocopherol, manni- high levels of light in a highly-oxygenated environment. tol, reduced glutathione, and various carotenoids, includ- [0011] In addition to photic injury, eye injury and dis- 20 ing lutein, zeaxanthin and astaxanthin have been studied ease can result from singlet oxygen and free radical spe- for an ability to comparatively quench singlet oxygen and cies generated during reperfusion after an ischemic in- scavenge free radical species in vitro. These and other sult. Ischemic insult to retinal ganglion cells and to neu- antioxidants have been shown in vitro to be effective rons of the inner layers of retina causes loss of vision. quenchers and scavengers for singlet oxygen and free Loss of vision accompanies diabetic retinopathy, retinal 25 radicals. In particular, the carotenoids, as a class of com- arterial occlusion, retinal venous occlusion and glauco- pounds, are very effective singlet oxygen quenchers and ma, each of which insults the eye depriving the eye of free radical scavengers. However, individual carotenoids oxygen and nutrition via ischemic insult. differ in their ability to quench singlet oxygen and scav- [0012] The damage to the retinal ganglion cells has enge for free radical species. been attributed to ischemia, and subsequent reperfusion 30 [0018] The carotenoids are naturally-occurring com- during which free radicals are generated. pounds that haveantioxidant properties. The carotenoids [0013] The pathogenesis of photic injury, of age-relat- are common compounds manufactured by plants, and ed macular degeneration, of ischemia/reperfusion dam- contribute greatly to the coloring of plants and some an- age, of traumatic injury and of inflammations of the eye imals. A number of animals, including mammals, are un- have been attributed to singlet oxygen and free radical 35 able to synthesize carotenoids de novo and accordingly generation, and subsequent free radical-initiated reac- rely upon diet to provide carotenoid requirements. Mam- tions. Investigators therefore studied the role of antioxi- mals also have a limited ability to modify carotenoids. A dants in preventing or ameliorating these diseases and mammal can convert beta-carotene to vitamin A, but injuries of the central nervous system in general, and the most other carotenoids are deposited in mammalian tis- eye in particular. 40 sue in unchanged form. [0014] For example, ascorbate was investigated as an [0019] With respect to humans, about ten carotenoids agent to treat retinal photic injury. Ascorbate is a reducing are found in human serum. The major carotenoids in hu- agent which is present in the retina in a high concentra- man serum are beta-carotene, alpha-carotene, cryptox- tion. Studies indicated that ascorbate in the retina can anthin, lycopene and lutein. Small amounts of zeaxan- act as an antioxidant and is oxidized by free radical spe- 45 thin, phytofluene, and phytoene are found in human or- cies generated during excessive light exposure. gans. However, of the ten carotenoids found in human [0015] Administration of ascorbate reduced the loss of serum, only two, trans- and/or meso-zeaxanthin and rhodopsin after photic exposure, thereby suggesting that lutein, have been found in the human retina. Zeaxanthin ascorbate offered protection against retinal photic injury. is the predominant carotenoid in the central macula or A decrease in rhodopsin levels is an indicator of photic 50 foveal region and is concentrated in the cone cells in the eye injury. The protective effect of ascorbate is dose- center of the retina, i.e., the fovea. Lutein is predominant- dependent, and ascorbate was effective when adminis- ly located in the peripheral retina in the rod cells. There- tered before light exposure. Morphometric studies of the fore, the eye preferentially assimilates zeaxanthin over photoreceptor nuclei remaining in the retina after light lutein in the central macula which is a more effective sin- exposure showed that rats given ascorbate supplements 55 glet oxygen scavenger than lutein. It has been theorized had substantially less retinal damage. Morphologically, that zeaxanthin and lutein are concentrated in the retina rats with ascorbate supplements also showed better because of their ability to quench singlet oxygen and preservation of retinal pigmented epithelium. scavenge free radicals, and thereby limit or prevent phot-

3 5 EP 2 874 612 B1 6 ic damage to the retina. age Caused by Light and Oxygen," Free Radicals [0020] Therefore only two of the about ten carotenoids and Aging, I. Emerit et al. (ed.), Birkhauser Verlag, present in human serum are found in the retina. Beta- pp. 280-298 (1992); carotene and lycopene, the two most abundant carote- M. O. M. Tso, "Pathogenetic Factors of Aging Mac- noids in human serum, either have not been detected or 5 ular Degeneration," Ophthalmology, 92(5), pp. have been detected only in minor amounts in the retina. 628-635 (1985); Beta-carotene is relatively inaccessible to the retina be- M. Mathews-Roth, "Recent Progress in the Medical cause beta-carotene is unable to cross the blood-retinal Applications of Carotenoids," Pure and Appl. Chem., brain barrier of the retinal pigmented epithelium effec- 63(1), pp. 147-156 (1991); tively. It also is known that another carotenoid, canthax- 10 W. Miki, "Biological Functions and Activities of Ani- anthin, can cross the blood-retinal brain barrier and reach mal Carotenoids," Pure and Appl. Chem., 63(1), pp. the retina. Canthaxanthin, like all carotenoids, is a pig- 141-146 (1991); ment and can discolor the skin. Canthaxanthin provides M. Mathews-Roth, "Carotenoids and Cancer Pre- a skin color that approximates a suntan, and accordingly vention-Experimental and Epidemiological Studies," has beenused byhumans to generate anartificial suntan. 15 Pure and Appl. Chem., 57(5), pp. 717-722 (1985); However, an undesirable side effect in individuals that M. Mathews-Roth, "Porphyrin Photosensitization ingested canthaxanthin at high doses for an extended and Carotenoid Protection in Mice; In Vitro and In time was the formation of crystalline canthaxanthin de- Vivo Studies," Photochemistry and Photobiology, posits in the inner layers of the retina. Therefore, the 40(1), pp. 63-67 (1984); blood-retinal brain barrier of the retinal pigmented epi- 20 P. DiMascio et al., "Carotenoids, Tocopherols and thelium permits only particular carotenoids to enter the Thiols as Biological Singlet Molecular Oxygen retina. The carotenoids other than zeaxanthin and lutein Quenchers," Biochemical Society Transactions, 18, that do enter the retina cause adverse effects, such as pp. 1054-1056 (1990); the formation of crystalline deposits by canthaxanthin, T. Hiramitsu et al., "Preventative Effect of Antioxi- which may take several years to dissolve. Canthaxanthin 25 dants on Lipid Peroxidation in the Retina," Ophthal- in the retina also caused a decreased adaptation to the mic Res., 23, pp. 196-203 (1991); dark. D. Yu et al., "Amelioration of Retinal Photic Injury by [0021] Investigators have unsuccessfully sought addi- Beta-Carotene," ARVO Abstracts Invest. Ophthal- tional antioxidants to further counteract the adverse af- mol. Vis. Sci., 28 (Suppl.), p. 7, (1987); fects of singlet oxygen and free radical species on in the 30 M. Kurashige et al., "Inhibition of Oxidative Injury of eye. The investigators have studied the antioxidant ca- Biological Membranes by Astaxanthin," Physiol. pabilities of several compounds, including various caro- Chem. Phys. and Med. NMR, 22, pp. 27-38 (1990); tenoids. Even though the carotenoids are strong antioxi- and dants, investigators have failed to find particular carote- N. I. Krinsky et al., "Interaction of Oxygen and Oxy- noids among the 600 naturally-occurring carotenoids that 35 radicals With Carotenoids," J. Natl. Cancer Inst., effectively quench singlet oxygen and scavenge for free 69(1), pp. 205-210 (1982). radical species, that are capable of crossing the blood- Anon., "Bio & High Technology Announcement retinal brain barrier, that do not exhibit the adverse affects Itaro," Itaro Refrigerated Food Co., Ltd. of canthaxanthin after crossing the blood-retinal brain Anon., "Natural Astaxanthin & Krill Lecithin," Itaro barrier, and that ameliorate eye disease or injury and/or 40 Refrigerated Food Co., Ltd. retard the progression of a degenerative disease of the Johnson, E. A. et al., "Simple Method for the Isolation eye and are more potent anti-oxidants than either lutein of Astaxanthin from the Basidomycetous Yeast or zeaxanthin. Phaffia rhodozyma," App. Environ. Microbiol., 35(6), [0022] Many scientific papers are directed to eye dis- pp. 1155-1159 (1978). eases and injuries, such as age-related macular degen- 45 Kirschfeld, K., "Carotenoid Pigments: Their Possible eration, causes of the damage resulting from the diseas- Role in Protecting Against Photooxidation in Eyes es or injuries, and attempts to prevent or treat such dis- andPhotoreceptor Cells," Proc. R. Soc. Lond.,B216, eases and injuries. The publications, which discuss var- pp. 71-85 (1982). ious antioxidants, including the carotenoids and other Latscha, T., "Carotenoids-Carotenoids in Animal antioxidants like alpha-tocopherol, include: 50 Nutrition," Hoffmann-LaRoche Ltd., Basel, Switzer- land. M.O. M.Tso, "Experiments on Visual Cells by Nature Li, Z. et al., "Desferrioxime Ameliorated Retinal Phot- and Man: In Search of Treatment for Photoreceptor ic Injury in Albino Rats," Current Eye Res., 10(2), pp. Degeneration," Investigative Ophthalmology and 133-144(1991). Visual Science, 30(12), pp. 2421-2454 (December, 55 Mathews-Roth, M., "Porphyrin Photosensitization 1989); and Carotenoid Protection in Mice; In Vitro and In W. Schalch, "Carotenoids in the Retina--A Review Vivo Studies," Photochemistry and Photobiology, of Their Possible Role in Preventing or Limiting Dam- 40(1), pp. 63-67 (1984).

4 7 EP 2 874 612 B1 8

Michon, J. J. et al., "A Comparative Study of Methods containing phospholipid bound and triglyceride bound of Photoreceptor Morphometry," Invest. Ophthalmol. EPA and DHA in which said krill oil contains at least 30% Vis. Sci., 32, pp. 280-284 (1991). total phospholipids. The composition includes 50 to 1000 Tso, M.O.M., "Pathogenetic Factors of Aging Mas- mg of krill oil, 0.5 to 8 mg of astaxanthin, 2 to 15 mg of cular Degeneration,"Ophthalmology, 92(5), pp.5 lutein and 0.2 to 12 mg of trans-zeaxanthin. Additional 628-635 (1985). relevant prior art includes US 2011/021465. Yu, D. et al., "Amelioration of Retinal Photic Injury [0028] Unexpectedly, it has been found that the addi- by Beta-Carotene," ARVO Abstracts Invest. Oph- tion of carotenoids and especially astaxanthin to krill oil thalmol. Vis. Sci., 28 (Suppl.), p. 7, (1987). results in an apparent chemical reaction between the two 10 components with the possible trans-esterification occur- [0023] In general, the above-identified publications ring between the krill oil fatty acid esters and partially support the hypothesis that singlet oxygen and free rad- esterified carotenoids and creating a new compound. ical speciesare significantcontributors to central nervous Therefore, a delivery mechanism is beneficial for the system, and particularly eye injury and disease. For ex- composition to prevent the disappearance of caroten- ample, it has reported that consumption of an antioxidant, 15 oids. such as ascorbic acid (Vitamin C), alpha-tocopherol (Vi- tamin E) or beta-carotene (which is converted in vivo to Summary of the Invention lutein), can decrease the prevalence of age-related mac- ular degeneration. [0029] In one aspect, the present invention relates to [0024] The above-identified publications also demon- 20 a medicine delivery system according to claim 1. Pre- strated that several carotenoids, including astaxanthin, ferred features of the medicine delivery system according are strong antioxidants compared to beta-carotene, to the invention are defined in claims 2 to 6. In another ascorbic acid and other widely used antioxidants in vitro. aspect, the present invention relates to a dietary supple- The publications also relate that (1) only particular caro- ment delivery system for use in the enhancement of car- tenoids selectively cross the blood-retinal brain barrier, 25 diovascular health according to claim 7. The krill oil and and that (2) certain carotenoids other than zeaxanthin astaxanthin may be reacted together after ingesting an and lutein that cross the blood-retinal brain barrier cause outer capsule containing the krill oil and an inner capsule adverse affects. containing the astaxanthin. [0025] In general, the above-identified publications [0030] The medicine delivery system includes an inner teachthat astaxanthin isa more effective antioxidant than 30 capsule containing carotenoids and an outer capsule in carotenoids such as zeaxanthin, lutein, tunaxanthin, can- which the inner capsule is contained within the outer cap- thaxanthin, beta-carotene, and alpha-tocopherol in vitro. sule and the outer capsule containing a therapeutically For example, the in vitro and in vivo studies disclosed in effective amount of krill oil. The carotenoids comprise at the Kurashige et al. publication with respect to astaxan- least S, S’-astaxanthin derived from Haematococcus plu- thin demonstrated that the mean effective concentration 35 vialis, and one or more of lutein and/or trans-zeaxanthin of astaxanthin which inhibits lipid peroxidation was 500 or meso-zeaxanthin. The medicine delivery system also times lower than that of alpha-tocopherol. Similarly, the includes 0.5 to 8 mg of astaxanthin, 2 to 15 mg of lutein Miki publication discloses that, in vitro, astaxanthin ex- and 0.2 to 12 mg of trans-zeaxanthin contained within hibits a strong quenching effect against singlet oxygen the inner capsule. In a specific example, the medicine and a strong scavenging effect against free radical spe- 40 delivery system includes about 4 mg of astaxanthin, cies. about 10 mg of lutein and about 1.2 mg of trans-zeaxan- [0026] This free radical theory of retinal damage has thin contained within the inner capsule. been advanced by investigators examining the effective- ness of various antioxidants in ameliorating these dis- Brief Description of the Drawings eases. 45 [0027] To date, investigative efforts have been direct- [0031] Other objects, features and advantages of the ed to preventing diseases and injury because the result- present invention will become apparent from the detailed ing free radical-induced damage is not effectively treat- description of the invention which follows, when consid- able. Therefore, a need exists for a method not only to ered in light of the accompanying drawings in which: prevent or retard, but also to ameliorate, degenerative 50 and traumatic diseases and injuries to the central nerv- FIG. 1 is an example of a medicine delivery system ous system, and particularly the eye. The copending ‘396 that includes an inner capsule containing caroten- parent application discloses a therapeutically effective oids and an outer capsule containing krill oil in ac- amount of a synergistic multi-ingredient composition of cordance with a non-limiting example. mixed carotenoids comprising at least S, S’-astaxanthin 55 FIG. 2 is chart showing stability data of various for- derived from Haematococcus pluvialis, and one or more mulation blends of the composition of carotenoids of lutein and/or trans-zeaxanthin or meso-zeaxanthin ad- and krill oil. mixed with a therapeutically effective amount of krill oil FIGS. 3 and 4 show stability analytical charts for var-

5 9 EP 2 874 612 B1 10

ious formulation blends of the composition of caro- AMDdevelopment and progression have been examined tenoids and krill oil at respective 20 degrees C and in several studies including AREDSI, a NEI-sponsored 50 degrees C. study, the LAST, TOZAL and CARMIS studies for exam- FIG. 5 is a chart showing a summary of changes by ple. AREDS was a multi-center study of the natural his- ultraviolet radiation of the astaxanthin in the compo- 5 tory of AMD and cataract. AREDS included a controlled sition. randomized clinical trial designed to evaluate the effect FIG. 6 is another chart showing astaxanthin stability of pharmacological doses of zinc and/or a formulation for the composition at 20 degrees C and 50 degrees containing nutrients with antioxidant properties (vitamin C. C, vitamin E, and β-carotene) on the rate of progression FIG. 7 are chemical formula of various carotenoids 10 to advanced AMD and on visual acuity outcomes. The and showing the differences among them. use of the combination of antioxidants and zinc reduced the risk of development of advanced AMD in participants Detailed Description of the Preferred Embodiments who had at least a moderate risk of developing AMD by about 25%. The overall risk of moderate vision loss [ ≥15 [0032] The present invention will now be described 15 letters on the Early Treatment Diabetic Retinopathy more fully hereinafter. This invention may, however, be Study (ETDRS) chart] was reduced by 19% at 5 years. embodied in many different forms and should not be con- [0038] Of approximately 600 carotenoids identified in strued as limited to the embodiments set forth herein. nature in the human diet, and 20 in human serum, only Rather, these embodiments are provided so that this dis- two forms of dietary xanthophylls, lutein and zeaxanthin, closure will be thorough and complete, and will fully con- 20 arepresent in humanmacular pigment. Luteinrepresents vey the scope of the invention to those skilled in the art. approximately 36% of all retinal carotenoids; zeaxanthin [0033] The leading cause of visual loss among elderly and meso-zeaxanthin each represent about 18%. persons is dry or atropic AMD, which has an increasingly [0039] Thenatural tissue distribution, biochemical, and important social and economic impact in the United biophysical characteristics of lutein provide a reasonable States. As the size of the elderly population increases in 25 basis for speculating that this nutrient acts in biological this country, AMD will become a more prevalent cause systems as: (1) an important structural molecule within of blindness than both diabetic retinopathy and glaucoma cell membranes; (2) a short-wavelength light filter; (3) a combined. Although laser treatment has been shown to modulator of intra- and extracellular reduction-oxidation reduce the risk of extensive macular scarring from the (redox) balance; and (4) a modulator in signal transduc- "wet" or neovascular form of the disease, there are cur- 30 tion pathways. Lutein and zeaxanthin were considered rently no effective treatments for the vast majority of pa- for inclusion in the AREDS formulation; however, at the tients with wet AMD. time of AREDS’ initiation, neither carotenoid was readily [0034] The Eye Diseases Prevalence Research Group available for manufacturing in a research formulation. (EDPRG) attributes AMD as the major cause of blindness [0040] The evidence base suggests that macular xan- among elderly people of European ancestry. Among35 thophylls in combination with omega-3 LCPUFAs from white persons, AMD is believed to account for more than fish oil may act as modifiable factors capable of modu- 50% of all blinding conditions. lating processes implicated in existing AMD pathogene- [0035] The EDPRG estimates that approximately 1.2 sis and progression and is the basis for the on-going US million residents of the US are living with neovascular Government sponsored AREDS II study. Intake of these AMD and 970,000 are living with geographic atrophy, 40 compounds may also show merit as a well-tolerated pre- while 3.6 million are living with bilateral large drusen. In ventive intervention. Biochemical and biophysical prop- the next 20 years these values are expected to increase erties of these compounds demonstrate a capacity to by 50% with projected demographic shifts. modulate factors and processes that activate and are [0036] Age-related developmental changes in retinal activated by exposures associated with aging. These ex- morphology and energy metabolism, as well as cumula- 45 posures include developmental changes associated with tive effects of environmental exposures may render the aging, chronic light exposure, alterations in energy me- neural and vascular retina and retinal pigment epithelium tabolism, and cellular signaling pathways. more susceptible to damage in late adulthood. Along with these metabolic and structural changes and exposures, Dry Eye Syndrome the aging eye also experiences a reduction in the potency 50 of endogenous and exogenous defense systems. Phar- [0041] According to C Stephen Foster, MD, FACS, macological and surgical treatment options are of limited FACR, FAAO, Clinical Professor of Ophthalmology, Har- scope and efficacy currently. They are costly and may vard Medical School; Consulting Staff, Department of result in complications as severe as end-stage disease. Ophthalmology, Massachusetts Eye and Ear Infirmary; The likelihood of vision loss among persons with neo- 55 Founder and President, Ocular Immunology and Uveitis vascular AMD can be reduced with anti-VEGF treatment, Foundation, Massachusetts Eye Research and Surgery photodynamic therapy, and laser photocoagulation. Institution et al’ dry eye is a very common disorder af- [0037] Nutrient-based preventative treatments for fecting a significant percentage (approximately 10-30%)

6 11 EP 2 874 612 B1 12 of the population, especially those older than 40 years. led by a neural reflex arc, with afferent nerves (trigeminal [0042] In the United States, an estimated 3.23 million sensory fibers) in the cornea and the conjunctiva passing women and 1.68 million men, a total of 4.91 million peo- to the pons (superior salivary nucleus), from which effer- ple, aged 50 years and older are affected. ent fibers pass, in the nervus intermedius, to the ptery- [0043] Dry eye is a multi-factorial disease of the tears 5 gopalatine ganglion and postganglionic sympathetic and and the ocular surface that results in symptoms of dis- parasympathetic nerves terminating in the lacrimal comfort, visual disturbance, and tear film instability with glands. potential damage to the ocular surface. Dry eye is ac- [0051] Keratoconjunctivitis sicca (KCS) is the name companied by increased osmolarity of the tear film and given to this ocular surface disorder. KCS is subdivided inflammation of the ocular surface. 10 into Sjogren syndrome (SS) associated KCS and non- [0044] The tear layer covers the normal ocular surface. SS associated KCS. Patients with aqueous tear deficien- Generally, it is accepted that the tear film is made up of cy have SS if they have associated xerostomia and/or 3 intertwined layers, as follows: connective tissue disease. Patients with primary SS have [0045] 1) A superficial thin lipid layer (0.11 mm) is pro- evidence of a systemic autoimmune disease as mani- duced by the meibomian glands, and its principal function 15 festedby the presence ofserum auto-antibodiesand very is to retard tear evaporation and to assist in uniform tear severe aqueous tear deficiency and ocular surface dis- spreading. ease. These patients, mostly women, do not have a sep- [0046] 2) A middle thick aqueous layer (7 mm) is pro- arate, identifiable connective tissue disease. Subsets of duced by the main lacrimal glands (reflex tearing), as patients with primary SS lack evidence of systemic im- well as the accessory lacrimal glands of Krause and20 mune dysfunction, but they have similar clinical ocular Wolfring (basic tearing). presentation. Secondary SS is defined as KCS associ- [0047] 3) An innermost hydrophilic mucin layer ated with a diagnosable connective tissue disease, most (0.02-0.05 mm) is produced by both the conjunctiva gob- commonly rheumatoid arthritis but also SLE and system- let cells and the ocular surface epithelium and associates ic sclerosis. itself with the ocular surface via its loose attachments to 25 [0052] Non-SS KCS is mostly found in postmenopau- the glycocalyx of the microplicae of the epithelium. It is sal women, in women who are pregnant, in women who the hydrophilic quality of the mucin that allows the aque- are taking oral contraceptives, or in women who are on ous to spread over the corneal epithelium. hormone replacement therapy (especially estrogen only [0048] The lipid layer produced by the meibomian pills). The common denominator here is a decrease in glands acts as a surfactant, as well as an aqueous barrier 30 androgens, either from reduced ovarian function in the (retarding evaporation of the underlying aqueous layer), postmenopausal female or from increased levels of the and provides a smooth optical surface. It may also act sex hormone binding globulin in pregnancy and birth con- as a barrier against foreign particles and may also have trol pill use. Androgens are believed to be trophic for the some antimicrobial properties. The glands are holocrine lacrimal and meibomian glands. They also exert potent in nature, and so the secretions contain both polar lipids 35 anti-inflammatoryactivity through theproduction of trans- (aqueous-lipid interface) and nonpolar lipids (air-tear in- forming growth factor beta (TGF-beta), suppressing lym- terface) as well as proteinaceous material. All of these phocytic infiltration. are held together by ionic bonds, hydrogen bonds, and [0053] Lipocalins (previously known as tear-specific van der Waals forces. The secretions are subject to neu- prealbumin), which are present in the mucous layer, are ronal (parasympathetic, sympathetic,and sensory sourc- 40 inducible lipid-binding proteins produced by the lacrimal es), hormonal (androgen and estrogen receptors), and glands that lower the surface tension of normal tears. vascular regulation. Evaporative loss is predominantly This provides stability to the tear film and also explains due to meibomian gland dysfunction (MGD). the increase in surface tension that is seen in dry eye [0049] The aqueous component is produced by the lac- syndromes characterized by lacrimal gland deficiency. rimal glands. This component includes about 60 different 45 Lipocalin deficiency can lead to the precipitation in the proteins, electrolytes, and water. Lysozyme is the most tear film, forming the characteristic mucous strands seen abundant (20-40% of total protein) and also the most in patients with dry eye symptomatology. alkaline protein present in tears. It is a glycolytic enzyme [0054] The glycocalyx of the corneal epithelium con- that is capable of breaking down bacterial cell walls. tains the transmembrane mucins (glycosylated glycopro- Lactoferrin has antibacterial and antioxidant functions, 50 teins present in the glycocalyx) MUC1, MUC4, and and the epidermal growth factor (EGF) plays a role in MUC16. These membrane mucins interact with soluble, maintaining the normal ocular surface and in promoting secreted, gel-forming mucins produced by the goblet corneal wound healing. Albumin, transferrin, immu- cells (MUC5AC) and also with others like MUC2. The noglobulin A (IgA), immunoglobulin M (IgM), and immu- lacrimal gland also secretes MUC7 into the tear film. noglobulin G (IgG) are also present. 55 [0055] These soluble mucins move about freely in the [0050] Aqueous tear deficiency (ATD) is the most com- tear film (a process facilitated by blinking and electrostat- mon cause of dry eye, and it is due to insufficient tear ic repulsion from the negatively charged transmembrane production. The secretion of the lacrimal gland is control- mucins), functioning as clean-up proteins (picking up dirt,

7 13 EP 2 874 612 B1 14 debris, and pathogens), holding fluids because of their disease also have increased viscosity of meibum, de- hydrophilic nature, and harboring defense molecules creased tear break-up time, and increased tear film de- produced by the lacrimal gland. Transmembrane mucins bris, all indicative of a deficient or abnormal tear film. prevent pathogen adherence (and entrance) and provide [0060] It is known that in various tissues pro-inflamma- a smooth lubricatingsurface, allowing lidepithelia to glide 5 tory cytokines may cause cellular destruction. For exam- over corneal epithelia with minimal friction during blinking ple including interleukin 1 (IL-1), interleukin 6 (IL-6), in- and other eye movements. Recently, it has been sug- terleukin 8 (IL-8), TGF-beta, TNF-alpha, and RANTES, gested that the mucins are mixed throughout the aque- are altered in patients with KCS. IL-1 beta and TNF-al- ous layer of tears (owing to their hydrophilic nature) and, pha, which are present in the tears of patients with KCS, being soluble, move freely within this layer. 10 cause the release of opioids that bind to opioid receptors [0056] Mucin deficiency (caused by damage to the on neural membranes and inhibit neurotransmitter re- goblet cells or the epithelial glycocalyx), as seen in Ste- lease through NF-K β production. IL-2 also binds to the vens-Johnson syndrome or after a chemical burn, leads delta opioid receptor and inhibits cAMP production and to poor wetting of the corneal surface with subsequent neuronal function. This loss of neuronal function dimin- desiccation and epithelial damage, even in the presence 15 ishes normal neuronal tone, leading to sensory isolation of adequate aqueous tear production. of the lacrimal gland and eventual atrophy. [0061] Pro-inflammatory neurotransmitters, such as Pathophysiology substance Pand calcitoningene related peptide (CGRP), are released, which recruit and activate local lym- [0057] A genetic predisposition in SS associated KCS 20 phocytes. Substance P also acts via the NF-AT and NF- exists as evident by the high prevalence of human leu- K β signaling pathway leading to ICAM-1 and VCAM-1 kocyte antigen B8 (HLA-B8) haplotype in these patients. expression, adhesions molecules that promote lym- This condition leads to a chronic inflammatory state, with phocyte homing and chemotaxis to sites of inflammation. the production of auto-antibodies, including antinuclear Cyclosporin A is an NK-1 and NK-2 receptor inhibitor that antibody (ANA), rheumatoid factor, fodrin (a cytoskeletal 25 can down-regulate these signaling molecules and is a protein), the muscarinic M3 receptor, or SS-specific an- novel addition to the therapeutic armamentarium for dry tibodies (eg, anti-RO [SS-A], anti-LA [SS-B]), inflamma- eye, being used to treat both aqueous tear deficiency tory cytokine release, and focal lymphocytic infiltration and meibomian gland dysfunction. It has been shown to (ie, mainly CD4+ T cells but also B cells) of the lacrimal improve the goblet cell counts and to reduce the numbers and salivary gland, with glandular degeneration and in- 30 of inflammatory cells and cytokines in the conjunctiva. duction of apoptosis in the conjunctiva and lacrimal [0062] These pro-inflammatory cytokines, in addition glands. This results in dysfunction of the lacrimal gland, to inhibiting neural function, may also convert androgens with reduced tear production, and loss of response to into estrogens, resulting in meibomian gland dysfunction, nerve stimulation and less reflex tearing. Active T lym- as discussed above. An increased rate of apoptosis is phocytic infiltrate in the conjunctiva also has been report- 35 also seen in conjunctival and lacrimal acinar cells, per- ed in non-SS associated KCS. haps due to the cytokine cascade. Elevated levels of tis- [0058] Both androgen and estrogen receptors are lo- sue-degrading enzymes called matrix metalloproteinas- cated in the lacrimal and meibomian glands. SS is more es (MMPs) are also present in the epithelial cells. common in postmenopausal women. At menopause, a [0063] Mucin synthesizing genes, designated MUC1- decrease in circulating sex hormones (ie, estrogen, an- 40 MUC17, representing both transmembrane and goblet- drogen) occurs, possibly affecting the functional and se- cell secreted, soluble mucins, have been isolated, and cretory aspect of the lacrimal gland. Forty years ago, in- their role in hydration and stability of the tear film are itial interest in this area centered on estrogen and/or pro- being investigated in patients with dry eye syndrome. gesterone deficiency to explain the link between KCS Particularly significant is MUC5AC, expressed by strati- and menopause. However, recent research has focused 45 fied squamous cells of the conjunctiva and whose prod- on androgens, specifically testosterone, and/or metabo- uct is the predominant component of the mucous layer lized androgens. of tears. A defect in this and other mucin genes may be [0059] It has been shown that in meibomian gland dys- a factor in dry eye syndrome development. In addition to function, a deficiency in androgens results in loss of the dry eye, other conditions, such as ocular cicatricial pem- lipidlayer, specifically triglycerides, cholesterol, monoun- 50 phigoid, Stevens-Johnson syndrome, and vitamin A de- saturated essential fatty acids (eg, oleic acid), and polar ficiency, which lead to drying or keratinization of the oc- lipids (eg, phosphatidylethanolamine, sphingomyelin). ular epithelium, eventually lead to goblet cell loss. Both The loss of polar lipids (present at the aqueous-tear in- classes of mucins are decreased in these diseases, and, terface) exacerbates the evaporative tear loss, and the on a molecular level, mucin gene expression, translation, decrease in unsaturated fatty acids raises the melting 55 and posttranslational processing are altered. point of meibum, leading to thicker, more viscous secre- [0064] Normal production of tear proteins, such as lys- tions that obstruct ductules and cause stagnation of se- ozyme, lactoferrin, lipocalin, and phospholipase A2, is cretions. Patients on anti-androgenic therapy for prostate decreased in KCS.

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[0065] It is clear from the above discussion that com- Stern ME, Gao J, Siemasko KF, et al. The role of the mon causes of dry eye syndromes may be ameliorated lacrimal functional unit in the pathophysiology of dry by treatment with anti-inflammatory agents such as top- eye. Exp Eye Res. Mar 2004; 78(3):409-16. ical corticosteroids, topical cyclosporine A and/or topi- [Medline]. cal/systemic omega-3 fatty acids. 5 Tatlipinar S, Akpek EK. Topical ciclosporin in the treatment of ocular surface disorders. Br J Ophthal- Dry Eye References mol. Oct 2005; 89(10):1363-7. [Medline]. Yoon KC, Heo H, Im SK, et al. Comparison of autol- [0066] ogous serum and umbilical cord serum eye drops 10 for dry eye syndrome. Am J Ophthalmol. Jul 2007; Basic teachings regarding dry eye can be found in 144(1):86-92. [Medline]. the following references: Zoukhri D. Effect of inflammation on lacrimal gland Dry Eye Workshop (DEWS) Committee. 2007 Re- function. Exp Eye Res. May 2006;82(5):885-98. port of the Dry Eye Workshop (DEWS). Ocul Surf. [Medline]. April 2007;5(2):65-204. 15 Behrens A, Doyle JJ, Stern L, et al. Dysfunctional Associated AMD References tear syndrome: a Delphi approach to treatment rec- ommendations. Cornea. Sep 2006;25(8):900-7. [0067] Associated AMD teachings can be found in the [Medline]. following references: Abelson MB. Dry eye, today and tomorrow. Review 20 in Ophthalmology. 2000;11:132-34. 1) Macular Photocoagulation Study Group: Argon American Academy of Ophthalmology. External dis- Laser Photocoagulation for senile macular degener- ease and cornea. In: Section Seven: Basic & Clinical ation: Results of a randomized clinical trial. Arch Science Course. American Academy of Ophthalmol- Ophthalmol, 1982; 100:912-918. ogy; 2007-2008. 25 2) TAP study group. Photodynamic therapy of sub- BarabinoS, Rolando M, CamicioneP, et al. Systemic foveal choroidal neovascularization in age-related linoleic and gamma-linolenic acid therapy in dry eye macular degeneration with verteporfm. One-year re- syndrome with an inflammatory component. Cornea. sults of 2 randomized clinical trials - TAP report 1. Mar 2003;22(2):97-101. [Medline]. Arch Ophthalmol, 1999; 117:1329-1345. Bron AJ, Tiffany JM, Gouveia SM, et al. Functional 30 3) Gragoudas ES, Adamis AP, Cunningham ET, et aspects of the tear film lipid layer. Exp Eye Res. Mar al. Pegaptanib for Neovascular Age-Related Macu- 2004;78(3):347-60. [Medline]. lar Degeneration. NEJM, 2004; 351:2805-2816. Geerling G, Maclennan S, Hartwig D. Autologous 4) Michels S, Rosenfeld PJ. Treatment of neovas- serum eye drops for ocular surface disorders. Br J cular age-related macular degeneration with Ranibi- Ophthalmol. Nov 2004;88(11):1467-74. [Medline]. 35 zumab/Lucentis. Klin Monatsbl Augenheilkd, 2005; Gilbard JP. Dry eye disorders. In: Albert DM, Jako- 222(6):480-484. biec FA, eds. Principles and Practice of Ophthalmol- 5)Kini MM, Leibowitz HM,Colton T, et al. Prevalence ogy. Vol 2. WB Saunders Co; 2000:982-1000. of senile cataract, diabetic retinopathy, senile mac- Karadayi K, Ciftci F, Akin T, et al. Increase in central ular degeneration, and open-angle glaucoma in the corneal thickness in dry and normal eyes with appli- 40 Framingham Eye Study. Am J Ophthalmol, 1978; cation of artificial tears: a new diagnostic and follow- 85:28-34. up criterion for dry eye. Ophthalmic Physiol Opt. Nov 6) Smiddy WE, Fine SL. Prognosis of patients with 2005;25(6):485-91. [Medline]. bilateral macular drusen. Ophthalmol, 1984; McCulley JP, Shine WE. The lipid layer of tears: de- 91:271-277. pendent on meibomian gland function. Exp Eye Res. 45 7) Friedman DS, O’Colmain BJ, Munoz B, et al. (Eye Mar 2004;78(3):361-5. [Medline]. Disease Prevalence Research Group.) Prevalence Murube J, Nemeth J, Hoh H, et al. The triple classi- of age-related macular degeneration in the United fication of dry eye for practical clinical use. Eur J States. Arch Ophthalmol, 2004; 122:564-572. Ophthalmol. Nov-Dec 2005;15(6):660-7. [Medline]. 8) Age-Related Eye Disease Study Research Group. Ohashi Y, Dogru M, Tsubota K. Laboratory findings 50 A randomized, placebo-controlled, clinical trial of in tear fluid analysis. Clin Chim Acta. Jul 15 2006; high-dose supplementation with vitamins C and E, 369(1):17-28. [Medline]. beta-carotene, and zinc for age-related macular de- Perry HD, Donnenfeld ED. Dry eye diagnosis and generation and vision loss: AREDS Report No. 8. management in 2004. Curr Opin Ophthalmol. Aug Arch Ophthalmol, 2001; 119:1417-36. 2004; 15(4):299-304. [Medline]. 55 9) Dietary Preference Intakes for Vitamin C, Vitamin Pflugfelder SC. Advances in the diagnosis and man- E, Selenium, and Carotenoids. Washington, DC: agement of keratoconjunctivitis sicca. Curr Opin Academy Press; 2000. Ophthalmol. Aug 1998; 9(4):50-3. [Medline]. 10) Khachik F, Spangler CJ, Smith JC, Jr., Canfield

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69) Hu et al. Types of dietary fat and risk of coronary AMD in humans, was not based on clinical trials per- heart disease: A critical review. J. Am. Coll. Nutrition formed on human subjects but instead on a different 2001:20:5-19. mammalian species, namely in rats. 70) Kris-Etherton et al. Curr. Atheroscler. Report [0072] Therefore, there remains no conclusive evi- 2008; 10: 503-509. 5 dence that astaxanthin alone can prevent or ameliorate 71) Breslow J. N-3 fatty acids and cardiovasualr dis- dry AMD in man since no human study has ever been ease Am. J. Clin. Nutrition 2006; 83(suppl):1477S- performed using astaxanthin supplementation alone, nor 82S. has any human study shown that astaxanthin actually 72) Leaf et al. Prevention of sudden death by omega- deposits anywhere in the human retina, the first required 3 polyunsaturated fatty acids. Pharmacol. Therapy 10 step to retinal protection by this powerful carotenoid. 2003; 98: 355-77. 73) Maki et al. Krill oil supplementation increases Potential Roles of Polyunsaturated Fatty Acids plasma concentrations of eicosapentaenoic and do- cosahexaenoic acids in overweight and obese men in Eye Physiology and women. Nutrition Research 29; (2009): 609-15 . 15 74) Massrieh, W. Health benefits of omega-3 fatty [0073] An inverse relationship of dietary omega-3 acids from Neptune Krill oil. Lipid Technology, May LCPUFA intake with advanced AMD has been reported 2008, Vol 20, No. 5. in six studies examining the issue. For prevalent disease, 75) See: http://emedicine.medscape.com/arti- the magnitude of odds ratios for highest versus lowest cle/1210417-overview. 20 omega-3 LCPUFA intake ranged from 0.4 to 0.9. [0074] Among these studies, the one containing the [0068] Five of six studies examining the association of largest number of subjects with neovascular or "wet" dietary lutein/zeaxanthin intake with advanced AMD AMD yielded a significantly lower likelihood of having the have yielded inverse relationships that are statistically disease among participants reporting the highest con- significant. The magnitude of odds ratios in these studies 25 sumption of omega-3. ranged from 0.1 to 0.7. Both sets of findings are germane [0075] The scientific literature is replete with the certain in guiding applied clinical research on prevention and human benefits of triacylglyceride bound EPA and DHA treatment of retinal disease, since: (1) tissue concentra- found in fish oil and fish oil concentrates and more re- tions of DHA, lutein, and zeaxanthin per unit area are cently the potential utility of phospholipid bound EPA and substantially higher in the retina than elsewhere in the 30 DHA found in krill oil derived from Euphasia superba or body; and (2) retinal tissue status of these compounds Antarctic krill. is modifiable and dependent upon intake. [0076] The cardiovascular benefits as well as the anti- [0069] The AREDS II study protocol (concluded its sci- inflammatory benefits of such fish and krill oils 66-67, and entific rational by stating: "There is a compelling need to in particular triacylglyceride bound EPA and DHA derived implement a clinical trial on nutrients that are both con- 35 from fish oils as well as algae derived triacylglyceride centrated in the retina and implicated in modulation of bound DHA are well known 68-73 Such algae derived DHA pathogenic factors and processes of AMD." is used in large part as a supplement in infant formulas [0070] It has been well established that lutein and to ensure brain health in the developing fetus and in in- trans-zeaxanthin are present in human retinal tissue and fants. that they function to protect the eye from photo induced 40 [0077] LCPUFAs affect factors and processes impli- injury. The CARMIS study, which included a mixture of cated in the pathogenesis of vascular and neural retinal lutein, trans-zeaxanthin and astaxanthin, is the only clin- disease.13 Evidence characterizing structural and func- ical trial which reported the use of astaxanthin. Unfortu- tional properties of LCPUFAs indicates that these nutri- nately, there have been no reports of the use of astax- ents may operate both as: (1) essential factors in the anthin alone in any human clinical trial for the prevention 45 visual-sensory process, and (2) protective agents or amelioration of dry AMD. The CARMIS study failed to against retinal disease. determine if supplementation with astaxanthin alone is a [0078] Docosahexaenoic Acid (DHA) is the major key determinate of the positive outcomes of the study or structural lipid of retinal photoreceptor outer segment that astaxanthin deposited on retinal epithelial cells. One membranes.14-15 Tissue DHA status affects retinal cell possible interpretation of the CARMIS study is that lutein 50 signaling mechanisms involved in phototransduc- and zeaxanthin alone provided the observed benefits of tion.16-17 Tissue DHA insufficiency is associated with the formulation employed, or in another interpretation conditions characterized by alterations in retinal func- that astaxanthin in combination with lutein and zeaxan- tion,18-20 and functional deficits have been ameliorated thin provided the observed benefits. However, in no pos- with DHA supplementation in some cases. 21 Biophysical sible interpretation can one conclude unequivocally that 55 and biochemical properties of DHA may affect photore- astaxanthin alone prevents or ameliorates dry AMD. ceptor function by altering membrane permeability, flu- [0071] In addition, the work of Tso, though claiming idity,thickness, and lipid phaseproperties. 22-23DHA may utility of astaxanthin for prevention or amelioration of dry operate in signaling cascades to enhance activation of

12 23 EP 2 874 612 B1 24 membrane-bound retinal proteins. 16-17,24 DHA may also tention deficit hyperactivity disorder.74 be involved in rhodopsin degeneration.25 [0084] However one mustuse caution when evaluating [0079] DHA and Eicosapentaenoic Acid (EPA) may such information since all krill oil clinical trials to date serveas protectiveagents because of their effect on gene havebeen conducted using krill oil that contains amixture expression,26-29 retinal cell differentiation,30-32 and sur- 5 of triacylglyceride bound and phospholipid bound EPA vival.30-34 DHA activates a number of nuclear hormone and DHA. In addition, such krill oils usually contain ap- receptors that operate as transcription factors for mole- proximately 30-40% weight-weight phospholipid bound cules that modulate redox-sensitive and proinflammatory fatty acids, principally in the form of saturated phosphati- genes; these include the peroxisome proliferator-activat- dylcholines which themselves are important cellular ed receptor-α (PPAR-α)27 and the retinoid X receptor 10 membrane components. (RXR).26 In the case of PPAR- α, this action is thought to [0085] Thus, it is difficult at the present time, to distin- prevent endothelial cell dysfunction and vascular remod- guish which form of EPA and DHA present in krill oil is eling through inhibition of vascular smooth muscle cell useful as reported in the references cited above as well proliferation, inducible nitric oxide synthase production, as the clinical trials described therein. It is also well known interleukin(IL)-1 induced cyclooxygenase (COX)-2 pro- 15 that phospholipids in general act as excellent emulsifiers duction, and thrombin-induced endothelin-1 produc- and are known to improve the stability of emulsions and tion.35 the bio-availability of many active ingredients. Phosphol- [0080] Research on model systems demonstrates that ipids also play an important role in the production of mi- omega-3 LCPUFAs also have the capacity to affect pro- celle based drug delivery systems containing active in- duction and activation of angiogenic growth factors, 36-38 20 gredients with vastly improved bio-availability. Therefore arachidonic acid-based proangiogenic eicosanoids, 39-43 it remains undetermined what the clinical value of krill oil, and matrix metalloproteinases involved in vascular re- either alone or in combination with carotenoids, is in the modeling.44 prevention or amelioration of eye related diseases such [0081] EPA depresses vascular endothelial growth as AMD, cataracts or dry eye syndromes. factor (VEGF) -specific tyrosine kinase receptor activa- 25 tion and expression.36,45 VEGF plays an essential role Cataracts in induction of endothelial cell migration and proliferation, microvascular permeability, endothelial cell release of [0086] A cataract is an opacity, or clouding, of the lens metalloproteinases and interstitial collagenases, and en- of the eye. The prevalence of cataracts increases dra- dothelial cell tube formation. 46 The mechanism of VEGF 30 matically with age. It typically occurs in the following way. receptor down-regulation is believed to occur at the ty- The lens is an elliptical structure that sits behind the pupil rosine kinase nuclear factor-kappa B (NFkB) site be- and is normally transparent. The function of the lens is cause EPA treatment causes suppression of NFkB acti- to focus light rays into images on the retina (the light- vation. NFkB is a nuclear transcription factor that up-reg- sensitive tissue at the back of the eye). ulates COX-2 expression, intracellular adhesion mole- 35 [0087] In young people, the lens is elastic and changes cule (ICAM), thrombin, and nitric oxide synthase. All four shape easily, allowing the eyes to focus clearly on both factors are associated with vascular instability. 35 COX-2 near and distant objects. As people reach their mid-40s, drives conversion of arachidonic acid to a number of an- biochemical changes occur in the proteins within the lens, giogenic and pro-inflammatory eicosanoids. causing them to harden and lose elasticity. This causes [0082] Although the mechanistic benefits of dietary 40 a number of vision problems. For example, loss of elas- supplementation with EPA and DHA polyunstaruated fat- ticity causes presbyopia, or far-sightedness, requiring ty acids in triacylglyceride form are well know, it remains reading glasses in almost everyone as they age. speculative that such triacylglyceride bound EPA and [0088] In some people, the proteins in the lens, notably DHA can improve vision. Such hypothesis is now under those called alpha crystallins, may also clump together, exploration under the National Eye Institute’s 5-year45 forming cloudy (opaque) areas called cataracts. They AREDS II study. usually develop slowly over several years and are related to aging. In some cases, depending on the cause of the Krill Oil cataracts, loss of vision progresses rapidly. Depending on how dense they are and where they are located, cat- [0083] Nowhere does the literature teach that phos- 50 aracts can block the passage of light through the lens pholipid bound EPA and DHA derived from Antarctic krill and interfere with the formation of images on the retina, imparts any benefit in ameliorating eye related diseases causing vision to become cloudy. such as AMD and/or syndromes such as dry eye syn- [0089] Nuclear cataracts form in the nucleus (the inner drome, although more recent research indicates that krill core) of the lens. This is the most common variety of oil extracts containing some phospholipid bound EPA 55 cataract associated with the aging process. Cortical cat- and DHA may be useful in the treatment of hyperlipi- aracts form in the cortex (the outer section of the lens). demia, joint disease as manifested in osteoarthritis Posterior subcapsular cataracts form toward the back of and/or rheumatoid arthritis, blood sugar control and at- a cellophane-like capsule that surrounds the lens. They

13 25 EP 2 874 612 B1 26 are more frequent in people with diabetes, who are over- plays a significant role in cataract development. Dark weight, or those taking steroids. Although the causes of colored (green, red, purple, and yellow) fruits and vege- cataract formation remain largely unknown, researchers tables usually have high levels of important plant chem- have been focusing on particles called oxygen-free rad- icals (phytochemicals) and may be associated with a low- icals as a major factor in the development of cataracts. 5 er risk for cataracts. They cause harm in the following way: [0094] In analyzing nutrients, researchers have fo- [0090] Oxygen-free radicals (also called oxidants) are cused on antioxidants and carotenids. Studies have not molecules produced by natural chemical processes in demonstrated that antioxidant vitamin supplements the body. Toxins, smoking, ultraviolet radiation, infec- (such as vitamins C and E) help prevent cataracts. Lutein tions, and many other factors can create reactions that 10 and zeaxanthin are the two carotenids that have been produce excessive amounts of these oxygen-free radi- most studied for cataract prevention. They are xantho- cals. When oxidants are overproduced, these chemical phylis compounds, which are a particular type of carote- reactions can be very harmful to nearly any type of cell nid. Lutein and zeaxanthin are found in the lenses of the in the body. At times these reactions can even affect ge- eyes. Some evidence indicates that xanthophyll-rich netic material in cells. 15 foods (such as dark green leafy vegetables) may help [0091] Cataract formation is one of many destructive retard the aging process in the eye and protect against changes that can occur with overproduction of oxidants, cataracts. However, there is not enough evidence to sug- possibly in concert with deficiencies of an important pro- gest that taking supplements with these carotenoids low- tective anti-oxidant called glutathione. Glutathione oc- ers the risk of cataract formation. Since little is known curs in high levels in the eye and helps clean up these 20 about the exact mechanism for formation of cataracts, it free radicals. One theory is that in the aging eye, barriers is not surprising that there are no known drugs or dietary develop that prevent glutathione and other protective supplements including the carotenoids that prevent cat- antioxidants from reaching the nucleus in the lens, thus aract formation there remains a need to find a suitable making it vulnerable to oxidation. Sunlight consists of ul- preventative treatment to prevent or ameliorate further traviolet (referred to as UVA or UVB) radiation, which 25 cataract formation. Since no drugs can reverse nor pre- penetrates the layers of the skin. Both UVA and UVB vent cataract formation, the only current treatment suit- have destructive properties that can promote cataracts. able for advanced cataract in humans is lens replace- The eyes are protected from the sun by eyelids and the ment surgery. structure of the face (overhanging brows, prominent cheekbones, and the nose). Long-term exposure to sun- 30 Cataract References light, however, can overcome these defenses. [0092] UVB radiation produces the shorter wave- [0095] length, and primarily affects the outer skin layers. It is the primary cause of sunburn. It is also the UV radiation pri- Basic teachings regarding cataracts can be found in marily responsible for cataracts. Long-term exposure to 35 the following references: even low levels of UVB radiation can eventually cause Allen D. Cataract. BMJ Clinical Evidence. Web pub- changes in the lens, including pigment changes, which lication date: 01 April 2007 (based on October 2006 contribute to cataract development. (UVB also appears search). Accessed July 1, 2008. to play a role in macular degeneration, an age-related American Academy of Ophthalmology. Cataract in disorder of the retina.) UVA radiation is composed of40 the Adult Eye, Preferred Practice Pattern. San Fran- longer wavelengths. They penetrate more deeply and cisco: American Academy of Ophthalmology, 2006. efficiently into the inner skin layers and are responsible Accessed July 1, 2008. for tanning. The main damaging effect of UVA appears Awasthi N, Guo S, Wagner BJ. Posterior capsular to be the promotion of the release of oxidants. Cataracts opacification: a problem reduced but not yet eradi- are common side effects of total body radiation treat-45 cated. Arch Ophthalmol. 2009 Apr;127(4):555-62. ments, which are administered for certain cancers. This Bell CM, Hatch WV, Fischer HD, Cernat G, Paterson observation indicates that ionizing radiation, which pro- JM, Gruneir A, et al. Association between tamsulosin duces large numbers of free radicals dramatically accel- and serious ophthalmic adverse events in older men erates cataract formation. following cataract surgery. JAMA. 2009 May [0093] Glaucoma and its treatments, including certain 50 20;301(19):1991-6 drugs (notably miotics) and filtering surgery, pose a high Clinical Trial of Nutritional Supplements and Age- risk for cataracts. The glaucoma drugs posing a particular Related Cataract Study Group, Maraini G, Sperduto risk for cataracts including demecarium (Humorsol), iso- RD, Ferris F, Clemons TE, Rosmini F, et al. A ran- flurophate (Floropryl), and echothiophate (Phospholine). domized, double-masked, placebo-controlled clini- Uveitis is chronic inflammation in the eye, which is often 55 cal trial of multivitamin supplementation for age-re- caused by an autoimmune disease or response. Often lated lens opacities. Clinical trial of nutritional sup- the cause is unknown. It is a rare condition that carries plements and age-related cataract report no. 3. Oph- a high risk for cataracts. It is not clear whether nutrition thalmology. 2008 Apr;115(4):599-607.e1.

14 27 EP 2 874 612 B1 28

Fernandez MM, Afshari NA. Nutrition and the pre- least S, S’-astaxanthin derived from Haematococcus plu- vention of cataracts. Curr Opin Ophthalmol. 2008 vialis, and one or more of lutein and/or trans-zeaxanthin Jan;19(1):66-70. or meso-zeaxanthin, and, in an inner capsule, a thera- Friedman AH. Tamsulosin and the intraoperative peutically effective amount of krill oil containing phos- floppy iris syndrome. JAMA. 2009 5 pholipid May bound and triglyceride bound EPA and DHA. 20;301(19):2044-5. Interestingly, none of the carotenes tested to date, and Guercio JR, Martyn LJ. Congenital malformations of most of the xanthophylls tested to date do not pass the eye and orbit. Otolaryngol Clin North Am. 2007 through the blood brain barrier with a few notable excep- Feb;40(1):113-40, vii. tions. These exceptions include lutein, trans-zeaxanthin, Long V, Chen S, Hatt S. Surgical interventions for 10 canthaxanthin and astaxanthin. bilateral congenital cataract. Cochrane Database [0100] Human serum typically contains about ten car- Syst Rev. 2006 Jul 19;3:CD003171. otenoids. The major carotenoids in human serum include Moeller SM, Voland R, Tinker L, Blodi BA, Klein ML, beta-carotene, alpha-carotene, cryptoxanthin, lycopene Gehrs KM, et al. Associations between age-related andlutein. Small amounts of zeaxanthin, phytoflueneand nuclear cataract and lutein and zeaxanthin in the diet 15 phytoene are also found in human organs. However, of and serum in the Carotenoids in the Age-Related all of these carotenoids, only zeaxanthin and lutein are Eye Disease Study, an Ancillary Study of the Wom- found in the human retina. In addition to certain carote- en’s Health Initiative. Arch Ophthalmol. 2008 noids, the retina also has the highest concentration of Mar;126(3):354-64. polyunsaturated fatty acids of any tissue in the human Olitsky SE, Hug D, and Smith LP. Abnormalities of 20 body. These polyunsaturated fatty acids are highly sus- the lens. In: Kliegman RM, Behrman RE, Jenson HB, ceptible to free radial and singlet oxygen induced decom- StantonBF, eds. Nelson Textbookof Pediatrics. 18th position. Therefore there is an absolute need to protect ed. St. Louis, MO: WB Saunders; 2007; chap 627. these polyunsaturated fatty acids, which make up a por- Wishart MS, Dagres E. Seven-year follow-up of com- tion of the cellular membrane bi-layer, from photo in- bined cataract extraction and viscocanalostomy. J 25 duced free radical or singlet oxygen degradation. Cataract Refract Surg. 2006 Dec;32(12):2043-9. [0101] It has been theorized that zeaxanthin and lutein are concentrated in the retina because of their ability to [0096] The ability of a carotenoid to pass the blood- quench singlet oxygen and to scavenge free radicals, retinal brain barrier is important because carotenoids are because they pass the blood and eye brain barriers and not synthesized by the human body. The only source of 30 are required in the oxygen rich environment of the retina carotenoids for humans is dietary intake. Furthermore, to prevent light mediated free radical damage to the ret- humans have a very limited ability to modify carotenoids. ina. Therefore, the carotenoids accumulate in various organs [0102] In fact, zeaxanthin is the predominant caroten- in the ingested form. Accordingly, if a particular carote- oid found in the central portion of the retina and more noid is unable to cross the blood-retinal brain barrier, the 35 specifically is located in concentration in the retinal cones carotenoid cannot accumulate in the retina and serve as located in the central area of the retina (ie. the macula). an antioxidant. Lutein, on the other hand, is located in the peripheral [0097] Furthermore, some carotenoids that are not area of the retina in the rod cells. Therefore, the eye pref- normal constituents of human plasma, but have the ability erentially accumulates zeaxanthin over lutein in the crit- to cross the blood-retinal brain barrier, have demonstrat- 40 ical central macular retinal area, (zeaxanthin interesting- ed adverse affects on the retina. Canthaxanthin which is ly, is a much more effective singlet oxygen scavenger intentionally ingested to provide an artificial suntan has than lutein), where the greatest level of light impinges. accumulated in the retina in the form of crystals and has [0103] Biochemists have determined the exact, yet temporarily affected eye adaptation to the dark. In addi- complicated, mechanism for light sensory response in tion, beta-carotene has a very limited ability to cross the 45 the eye. It involves a key protein called rhodopsin whose blood-retinal brain barrier. structure includes a bound polyunsaturated compound [0098] Therefore, even though the carotenoids are called retinal (retinal is structurally related to vitamin A). known as strong antioxidants and are present in abun- When light enters the eye, cis-retinal isomerizes to all its dant supply, the carotenoids have not been used for the all-trans isomer, causing disassociation of itself from its treatment of central nervous system damage, or eye50 protein carrier. The disassociation triggers a complicated damage, caused by disease or injury. The carotenoids cascade leading to nerve based transmission of elec- investigated to date either could not effectively cross the trons to the brain via the optic nerve. All of this "photo- blood-retinal barrier (i.e., beta-carotene) or adversely af- chemistry" takes a mere 200 femtoseconds to occur mak- fected the eye (i.e., canthaxanthin). ing it one of the fastest biochemical to electron transfor- [0099] In accordance with an important feature, the 55 mations known. medicine delivery system comprises, in an outer capsule, [0104] Chemists have learned that the retina is highly a therapeutically effective amount of a synergistic multi- susceptible to polymerization by localized free radicals ingredient composition of mixed carotenoids including at and highly reactive singlet oxygen. Because the retina is

15 29 EP 2 874 612 B1 30 a strong absorber of light and because the retina is highly can become pro-oxidants under certain conditions, how- vascularized and thus rich in dissolved oxygen, nature ever because astaxanthin is the most potent of all caro- has provided zeaxanthin as the key retinal carotenoid for tenoids, Beutner et al. showed that astaxanthin can never protection of the central foveal region of the retina from be nor has it ever exhibited any pro-oxidant activity unlike light induced damage at that point in the center of the 5 the zeaxanthin found in the human eye. Since humans retina where the most significant light impingement oc- already have an abundant source of lutein and trans- curs. zeaxanthin in their diets from many vegetable sources [0105] Clinical studies in man indicate that photic injury and are already present in the human eye, it appears that is a cause of age related macular degeneration because astaxanthin with its unique qualifying properties, unlike of the cumulative effect of repeated photic insult leading 10 lutein or trans-zeaxanthin, may be the eye healthcare to the gradual loss of photoreceptor cells. supplement of choice. With astaxanthin’s extraordinarily [0106] There have been many clinical trials designed potent antioxidant properties, its ability to cross the blood to support the supplementation of the diet with lutein, brain/eye barrier and concentrate in the retinal macula however, as of 2007, there appears to be no unequivocal in other mammalian species, without the side effects evidence that lutein supplementation is necessary in eye 15 seen with canthaxanthin, and in light of Tso’s contribu- healthcare despite its wide acceptance as a supplement. tions, astaxanthin, in a convenient dietary supplement This may simply imply that supplementation with extra presentation, may emerge as the pre-eminent new in- lutein is not necessary since it is a readily available xan- gredient addition to lutein and/or zeaxanthin eye health- thophyll in many vegetables. More recently trans-zeax- care supplementation for the management of eye related anthin and meso-zeaxanthin have also entered the mar- 20 oxidative stress and thus the prevention and mitigation ketplace as an eye healthcare supplements which indeed of degenerative diseases of the eye such as age related makes sense. However, is there yet a better carotenoid macular degeneration (ARMD)and cataract formation if meeting all the requirements associated withastaxanthin deposition can be experimentally confirmed eye/blood/brain barrier transport, accumulation in the in human retinal tissue. macula and capable of long term use? The answer is 25 [0110] In addition, Tso found that light induced dam- found in the xanthophyll astaxanthin. age, photo-receptor cell damage, ganglion cell damage [0107] Dr. Mark Tso, at the Univ. of Ill, has demonstrat- and damage to neurons of the inner retinal layers can be ed that astaxanthin is one such naturally occurring anti- prevented or ameliorated by the use of astaxanthin in- oxidant meeting all of these critical criteria in rats. Astax- cluding neuronal damage from ischemic, photic, inflam- anthin is the carotenoid xanthophyll responsible for the 30 matory and degenerative insult in rats. Tso’s patent red color in salmon, lobster, krill, crab, other shell fish claims the use of astaxanthin across a wide range of eye and in the micro algae Haematoccous pluvialis. The latter diseases including age related macular degeneration, di- source has made astaxanthin readily available worldwide abetic neuropathy, cystoid macular edema, central reti- for such uses. US 5,527,533 was issued to the Univ. of nal arterial and veneous occlusion, glaucoma and inflam- Ill. describing the use of astaxanthin more fully in eye 35 matory eye diseases such as retinitis, uveitis, iritis, kera- related diseases. titis and scleritis, all disease states common to eye insult [0108] In addition, astaxanthin is a much more power- by oxidative species such as free radicals however this ful antioxidant than canthoaxanthin, beta-carotene, ze- work was never confirmed in humans. axanthin,lutein and alpha-tocopherol. Shimidzu etal. dis- [0111] Oral administration of astaxanthin confirms that covered that astaxanthin is 550 times more potent than 40 it is at least transported into human blood stream, how- alpha-tocopherol, 27.5 times more potent than lutein and ever, its deposition in human retinal tissue has never 11 times more potent that beta-carotene in quenching been confirmed. singlet oxygen. In addition, Bagchi discovered that nat- [0112] Astaxanthin is the major pigment of certain mi- ural astaxanthin is 14 times more potent than alpha-to- cro algae and crustaceans. Astaxanthin is a lipid-soluble copherol, 54 times more potent that beta-carotene and 45 pigment primarily used for pigmenting cultured fish, like 65 times more potent that ascorbic acid (Vitamin C) in salmon, which must ingest astaxanthin to yield consum- scavenging oxygen free radicals. Thus, though there are er-acceptable pink-colored salmon muscle. Astaxanthin dramatic differences in the potency of astaxanthin when also is an antioxidant which is about 100 to about 1000 comparing the quenching of singlet oxygen and the scav- times more effective than alpha-tocopherol. enging of oxygen free radicals, it is clear that astaxanthin 50 [0113] The prime source of commercial S,S’-astaxan- compares very favorably to zeaxanthin and lutein, the thin is micro algae, and, to a very small extent, is found two carotenoids that are found naturally in the retina. in krill oil derived from Euphasia superba (Antarctic Krill). [0109] There is one more aspect of carotenoids, name- Astaxanthin also is available synthetically, however syn- ly that some carotenoids can act as pro-oxidants. This is thetic astaxanthin may not be safe for use in humans important since a carotenoid with pro-oxidant capability 55 since it contains 3 known enantiomers including R,R’, R, actually causes oxidation to occur in the body when high S’ and S,S’ which are not easily nor economically sepa- concentrations are present in tissue. Martin, et al. rated two of which have unknown human safety data. showed that beta-carotene, lycopene and zeaxanthin The preferred naturally-occurring S,S’-astaxanthin can

16 31 EP 2 874 612 B1 32 be used in the medicine delivery system of the present able to address not only the eye diseases known to be invention. ameliorated by carotenoids but also the inflammatory dis- [0114] As previously stated, the retinal pigment epithe- eases of the eye (including but not limited to dry eye lium protects the retina by providing a blood-retinal brain syndrome) associated with the known anti-inflammatory barrier. The barrier excludes plasma constituents that 5 activity of omega-3’s. Just because phospholipid bound are potentially harmful to the retina. As also previously EPA and DHA are active alone as an anti-hyperlipidemic stated, the blood-retinal brain barrier only permits lutein or as a reducer of pain in arthritic joints does not insure and zeaxanthin to enter the retina, and excludes other that such phospholipids from any source such as krill oil carotenoids present in human serum, including beta-car- would prove effective for the treatment of inflammatory otene which is the most abundant carotenoid in human 10 conditions of the eye. The AREDS2 study cited above is serum. Astaxanthin is not a naturally-occurring constitu- an example. This study was designed to determine if trig- ent in the retina. Therefore, the presence of a physiolog- lyceride bound omega-3 fatty acids would be effective in ically significant amount of astaxanthin in the retina of amelioration of inflammatory eye diseases. rats may illustrate the ability of astaxanthin to readily [0118] As noted before, the astaxanthin disappears cross the blood-retinal brain barrier into the retina of hu- 15 and is converted to a new product in the presence of the mans. krill oil. Other carotenoids can possibly have a similar effect. This is an unexpected result such that the addition "Reacted" Carotenoids and Delivery System of some carotenoids and primarily astaxanthin to krill oil results in the chemical reaction. The design of the cap- [0115] The analytical analysis of the product blend of 20 sule in a capsule prevents initial mixing. krill oil and carotenoids, including astaxanthin during test- [0119] In accordance with a non-limiting example, FIG. ing, gave conflicting recovery data for the added astax- 1 illustrates a medicine or drug delivery system at 10 that anthin when evaluated using HPLC (High-Performance includes a central capsule 12 containing carotenoids 13 Liquid Chromatography, also known as HighPressure whilean outer capsule14 contains krill oil 15 and contains Liquid Chromatography) analysis. It was determined that 25 the inner capsule as illustrated. This capsule within the over time, astaxanthin apparently, and rapidly, chemical- capsule prevents the disappearance of the carotenoids ly combined in what appears to be an unknown way with and the inability of standard HPLC (high-performance one or more krill oil components to produce a compound liquidchromatography) methods toquantitatively recover that is not detectable using the HPLC methodology used the carotenoids, especially with astaxanthin. Both cap- to quantify the amount of astaxanthin in such initially30 sules 12,14 could be formed of gelatin or similar material blended product. As a result, a "capsule in a capsule" and formed to prevent mixing of carotenoids and krill oil was designed to prevent such loss of astaxanthin to the until after capsule ingestion. krill oil in order to retain label claims. This is an unexpect- [0120] When added alone or in combination with other ed observation/result with great benefits. Further, it is carotenoids, astaxanthin HPLC based recovery quickly believed that astaxanthin when mixed with pure fish oil 35 abates much to the surprise of those skilled in the art. containing essentially all triacylglycerides bound fatty ac- There is possible trans-esterification occurring between ids, does not result in quantitative loss of astaxanthin to krilloil fatty acid esters and partiallyesterified carotenoids fish oil components as observed in krill oils. One skilled that creates a new compound. It is possible that the com- in the art would not have anticipated the instability of pound could be EPA transesterified onto astaxanthin. astaxanthin and other carotenoids in such added mix- 40 Thus, to deliver the label amount of each carotenoid in tures. a single dose, the krill oil and the carotenoids are not [0116] Testing has determined that astaxanthin added allowed to mix because of the incompatibility issue. FIG. to krill oil results in an unstable product with respect to 1 shows the technical solution using the capsule within recoverable astaxanthin by HPLC analysis. A chemical a capsule in accordance with a non-limiting example. reaction most likely takes place between krill oil and at 45 [0121] FIG. 1 shows the medicine delivery system 10 least astaxanthin in such mixtures. Nevertheless, the that includes the inner capsule 12 containing mixed car- astaxanthin does not seem to be destroyed in such mix- otenoids in one example comprising at least S, S’-astax- tures since UV based analysis indicates that the astax- anthin derived from Haematococcus pluvialis and one or anthin moiety is still present without structural loss be- more of lutein and/or trans-zeaxanthin or meso-zeaxan- cause it is still able to absorb UV at the appropriate quan- 50 thin. The outer capsule 14 contains a therapeutically ef- tifying absorption wavelength of astaxanthin at essential- fective amount of krill oil comprising phospholipid bound ly 100% quantification of the amount of astaxanthin add- and triglyceride bound EPA and DHA and the krill oil con- ed to such blended product. Had UV alone been used to tains at least 30% total phospholipids. determine product stability the decrease in recoverable [0122] In an example, 50-1,000 mg of krill oil can be astaxanthin over time would not have been observed. 55 contained in the outer capsule 14. In another example, The test results, tables and charts of FIGS. 2-6 show the the mixed carotenoids can include 0.5 to 8 mg of astax- decrease in recoverable astaxanthin over time, anthin, 2 to 15 mg of lutein, and 0.2 to 12 mg of trans- [0117] The carotenoids admixed with krill oil should be zeaxanthin within the inner capsule. About 4 mg of astax-

17 33 EP 2 874 612 B1 34 anthin, about 10 of lutein and about 1.2 mg of trans-ze- ipids but other experiments can be conducted with other axanthin is contained within the inner capsule in one ex- chemical species to determine the scope of this reaction. ample. The use of this medicine delivery system 10 pro- Further determination can be made whether all keto-al- vides a new composition after ingestion in vivo that in- cohols (e.g., astaxanthin) react, and alcohols, amines, cludes the therapeutically effective amount of synergistic 5 etc. A full strength eye care composition product included multi-ingredient composition of krill oil containing phos- 4 mg of astaxanthin, 10 mg lutein and 1.2 mg zeaxanthin pholipid bound and triglyceride bound EPA and DHA in in an inner capsule with krill oil in the outer capsule to whichthe krilloil contains at least 30%total phospholipids prevent comingling of the carotenoids with krill oil and and mixed carotenoids comprising at least S, S’-astax- preserve label claims in the event of an HPLC analysis anthin derived from Haematococcus pluvialis that has 10 of a simple blended product. reacted with the krill oil, and one or more of lutein and/or [0126] FIG. 3 is another chart showing a stability ana- trans-zeaxanthin or meso-zeaxanthin. This allows a lytical report for the astaxanthin, lutein and zeaxanthin method of treating an individual by administering the cap- with percent change from day 0 to day 12 at 20 degrees sule that includes the therapeutically effective amount of C. This is compared to FIG. 2 charts that show for day a synergistic multi-ingredient composition of krill oil and 15 13. The charts in FIG. 2 show the Aker Biomarine sample carotenoids and reacting carotenoids in vivo with the krill while the charts in FIG. 3 show use for an eye formula oil after ingestion. as disclosed and also FlexPro MD™ such as disclosed [0123] FIG. 2 is a chart showing data of astaxanthin at in commonly assigned U.S. patent application Serial No. 13 days post formulation reacting with krill oil, but essen- 12/840,372 and issued as U.S. Patent No. 8,481,072 as tially neither lutein nor zeaxanthin. Astaxanthin is a keto- 20 used for joint care. In the charts, A corresponds to Aker alcohol while lutein and zeaxanthin are alcohols and this Biomarine krill oil and E corresponds to enzymotec krill can possibly make some difference. It should also be oil. The FlexPro MD™ product as disclosed in the ’072 understood that the krill oil and "reacted" carotenoids, patent is a composition that alleviates joint pain and may such as astaxanthin, and possibly zeaxanthin and lutein, be used to reduce symptoms of osteoarthritis and/or lose their ability to be recovered over time from the mix- 25 rheumatoid arthritis and is formed as a krill oil and/or ture but can still be quantitated by ultraviolet (UV) anal- marine oil in combination with astaxanthin and low mo- ysis. For example, the chromophores of the carotenoids lecular weight polymers of hyaluronic acid or sodium hy- are not destroyed when the reaction takes place over aluronate (hyaluronan) in an oral dosage form. It can be time and can be used to develop a number of products produced as a dietary supplement composition or phar- useful for cardiovascular applications, joint health care 30 maceutical. applications, eye care and other diseases. [0127] In one example, the composition as a reaction [0124] Referring now to the chart in FIG. 2, there are product may be used to enhance cardiovascular health shown various samples and explanation. Sample 3/1 is by administering atherapeutic amount ofthe composition a mixture of astaxanthin in krill oil only and showing a comprising krill oil reacted with the astaxanthin. In one 23% reduction in HPLC recoverable astaxanthin by day 35 example, the astaxanthin comprises S,S’-astaxanthin 13 while retaining total carotenoid concentration as derived from Haematococcus pluvialis. The krill oil com- measured by UV analysis. Similar results were obtained prises phospholipid bound and triglyceride bound EPA in the presence of lutein, however the "loss" of recover- and DHA and the krill oil comprises 30% total phosphol- able lutein was minimal while astaxanthin provided the ipids. It is possible to administer 50 to 1,000 mg of krill same loss as the 3/1 sample. Sample 5/1 is mixed car- 40 oil in one example. It is also possible to administer one otenoids. In this example, there is essentially no loss of or more of lutein and/or trans-zeaxanthin or meso-zeax- HPLC recoverable astaxanthin while there is minimal anthin. In one example, 0.5 to 8 mg of astaxanthin, 2 to loss of lutein and zeaxanthin (probably within experimen- 15 mg of lutein, and 0.2 to 12 mg of trans-zeaxanthin are tal error). Recoveries of lutein and zeaxanthin in the pres- administered. ence of krill oil were within experimental error. Recover- 45 [0128] The composition may be administered as a di- ies of these same carotenoids without krill oil, however, etary supplement composition, as a food product, and in were less than expected because lutein and zeaxanthin another example, as a cosmetic applied to the skin. As are not very soluble in astaxanthin oleoresin. described above, the krill oil and astaxanthin may be re- [0125] Admixture of astaxanthin derived from hp bio- acted when an outer capsule containing the krill oil is mass extraction with krill oil above normal levels shows 50 ingested, and the inner capsule contains the astaxanthin. a continued loss of recoverable astaxanthin over day 1 [0129] Such use of the composition as a reaction prod- concentrations while the total carotenoid concentration uct may also be used to improve blood lipid profiles and as measured by UV analysis remains essentially un- reduce low density lipoprotein (LDL) oxidation in humans changed, indicating that a chemical reaction is taking such as disclosed in commonly assigned U.S. patent ap- place when relatively high concentrations of astaxanthin 55 plication serial no. 13/093,201 and published as U.S. Pat- are added to krill oil. Further testing with LC/MS/MS can ent Publication No. 2011/0268811. determine the resulting chemical species being formed. [0130] As noted in the ’201 application, krill oil is sup- It is believed this "reaction" is unique to krill oil phosphol- plemented with astaxanthin to improve a formulated

18 35 EP 2 874 612 B1 36 product utility. In one study, 4 mg of astaxanthin per day this burp-back feature of fish oils, it has been found that for two weeks resulted in a 26% reduction of LDL cho- approximately 50% of all consumers who try fish oil never lesterol oxidation. 4 mg of astaxanthin for eight weeks buy it again. resulted in a 21 % decrease in C-reactive protein scores. [0135] Astaxanthin has an excellent safety record. A 3.6 mg of astaxanthin per day for two weeks demonstrat- 5 conducted study obtained the results as follows: ed that astaxanthin protects LDL cholesterol against in- duced in vitro oxidation. Oral LD 50: 600 mg/kg (rats); [0131] Astaxanthin is also known to reduce C-Reactive NOAEL: 465 mg/kg (rats); or Protein (C-RP) blood levels in vivo. For example, in hu- Serum Pharmacokinetics: Stewart et al. 2008 man subjects with high risk levels of C-RP three months 10 of astaxanthin treatment resulted in 43% drop in the pa- 1) T1/2: 16 hours; tient population’s serum C-RP levels a drop which is be- 2) Tmax: 8 hours; low the unacceptable cardiovascular event risk level. 3) Cmax: 65 mg/L. Astaxanthin is so powerful that it has been shown to ne- gate the pro-oxidant activity of Vioxx in vitro, a COX-2 15 [0136] At eight weeks of supplementation at 6 mg per inhibitor belonging to the NSAIDS drug class which is day, there was no negative effect in healthy adults. Spiller known to cause cellular membrane lipid per-oxidation et al. 2003. leading to heart attacks and strokes. For this reason Vi- [0137] As set forth in the ’201 application, astaxanthin oxx was subsequently removed from the US market by has three prime sources, e.g., 3 mg astaxanthin per 240 the FDA. Astaxanthin is also absorbedin vitro by lens 20 g serving of non-farmed raised salmon or a 1% to 12% epithelial cells where it suppresses UVB induced lipid astaxanthin oleoresin or 1.5-2.5% beadlet derived from per-oxidative mediated cell damage at umol/L concen- microalgae. Literature references pertinent to the above trations. Reduction of C-Reactive protein (CRP), reduc- discussion can be found in Lee et al., Molecules and Cells tion of LDL oxidation and an increase in the omega-3 16(1): 97-105, 2003; Ohgami et al., Investigative Oph- index in vivo would presumably all be important positive 25 thalmology and Visual Science 44(6): 2694-2701, 2003; contributors to cardiovascular health since each are well Spiller et al., J. of the American College of Nutrition 21(5): know biomarkers for cardiovascular health risk. These October 2002; and Fry et al., University of Memphis, Hu- results have been shown in: man Performance Laboratories, 2001 and 2004, Reports 1 and 2. 1) Lee et al., Molecules and Cells, 16(1):97-105;30 [0138] The krill oil in one example is derived from Eu- 2003; phasia spp., comprising Eicosapentaenoic (EPA) and 2) Olzgami et al., Investigative Ophthalmology and Docosahexaenoic (DHA) fatty acids in the form of tria- Visual Science 44(6):2694-2701, 2003; cylglycerides and phospholipids, although not less than 3) Spiller et al., J. of the Amer. College of Nutrition, 1% EPA and 5% DHA has been found advantageous. In 21(5): October 2002; and 35 another example, the krill oil includes at least 15% EPA 4) Harris, Pharmacol. Res. 2007 March; 55(3) and 9% DHA, of which not less than 45% are in the form 217-223. of phospholipids, and in one example, greater than 50% and in another example, 10% EPA and 5% DHA of which [0132] In one embodiment, 300-500 mg of krill oil and not less than 40% are in the form of phospholipids. 2 mg astaxanthin is used and can form the reaction prod- 40 [0139] FIG. 4 is a chart showing results for 50 degrees uct as disclosed above. C. FIG. 5 is a chart showing summary of changes by UV [0133] As noted before, krill oil is typically produced as described above. FIG. 6 is another chart showing re- from Antarctic krill (euphausia superba), which is a zoo- sults with a day 26 added with the eye formulation as plankton (base of food chain). It is one of the most abun- described. dant marine biomass of about 500 million tons according 45 [0140] FIG. 7 shows the different chemical formula for to some estimates. Antarctic krill breeds in the pure un- different carotenoids. This indicates the differences and contaminated deep sea waters. It is a non-exploited ma- why krill oil can react more readily with one type of car- rine biomass and the catch per year is less than or equal otenoid and not others. to about 0.02% according to some estimates. [0134] It is believed that Krill oil based phospholipid 50 bound EPA and DHA uptake into cellular membranes is Claims far more efficient than triacylglyercide bound EPA and DHA since liver conversion of triacylglycerides is itself 1. A medicine delivery system comprising: inefficient and because phospholipid bound EPA and DHA can be transported into the blood stream via the 55 an outer capsule containing a therapeutically ef- lympathic system, thus, avoiding liver breakdown. In ad- fective amount of krill oil comprising phospholi- dition, krill oil consumption does not produce the burp- pid bound and triglyceride bound EPA and DHA back observed with fish oil based products. Because of and the krill oil comprising at least 30% total

19 37 EP 2 874 612 B1 38

phospholipids; and Patentansprüche an inner capsule within the outer capsule and containing mixed carotenoids comprising at 1. Ein Abgabesystem für Arzneimittel umfassend: least S, S’-astaxanthin derived from Haemato- coccus pluvialis, and one or more of lutein and/or 5 eine äußere Kapsel enthaltend eine therapeu- trans-zeaxanthin or meso-zeaxanthin, wherein tisch effektive Menge von Krillöl umfassend the outer and inner capsules are formed to pre- Phospholipid-gebundene und Triglycerid-ge- vent mixing of the carotenoids and krill oil until bundene EPS und DHS und das Krillöl umfas- after capsule ingestion. send wenigstens 30 % Gesamtphospholipide; 10 und 2. The medicine delivery system according to Claim 1, eine innere Kapsel innerhalb der äußeren Kap- further comprising 50 to 1000 mg of krill oil contained sel und enthaltend gemischte Carotenoide um- in the outer capsule. fassend wenigstens S,S’-Astaxanthin stam- mend von Haematococcus pluvialis, und ein 3. The medicine delivery system according to Claim 1, 15 oder mehrere von Lutein und/oder trans-Zea- further comprising 0.5 to 8 mg of astaxanthin, 2 to xanthin oder meso-Zeaxanthin, wobei die äuße- 15 mg of lutein and 0.2 to 12 mg of trans-zeaxanthin ren und inneren Kapseln so gestaltet sind, dass contained within the inner capsule. die Vermischung der Carotenoide und des Krill- öls bis nach der Einnahme der Kapsel unterbun- 4. The medicine delivery system according to Claim 3, 20 den wird. further comprising about 4 mg of astaxanthin, about 10 mg of lutein and about 1.2 mg of trans-zeaxanthin 2. Das Abgabesystem für Arzneimittel gemäß An- contained within the inner capsule. spruch 1, weiter umfassend 50 bis 1.000 mg Krillöl enthalten in der äußeren Kapsel. 5. The medicine delivery system according to claim 4, 25 for use in the eye care. 3. Das Abgabesystem für Arzneimittel gemäß An- spruch 1, weiter umfassend 0,5 bis 8 mg Astaxan- 6. The medicine delivery system according to claim 1, thin, 2 bis 15 mg Lutein und 0,2 bis 12 mg trans- wherein the medicine delivery system is formulated Zeaxanthin enthalten in der inneren Kapsel. to deliver as a dietary supplement composition30 formed from the krill oil and mixed carotenoids. 4. Das Abgabesystem für Arzneimittel gemäß An- spruch 3, weiter umfassend ca. 4 mg Astaxanthin, 7. A dietary supplement delivery system comprising: ca. 10 mg Lutein und ca. 1,2 mg trans-Zeaxanthin enthalten in der inneren Kapsel. an outer capsule containing a therapeutically ef- 35 fective amount of krill oil comprising phospholi- 5. Das Abgabesystem für Arzneimittel gemäß An- pid bound and triglyceride bound EPA and DHA spruch 4 für die Verwendung in der Augenpflege. and the krill oil comprising at least 30% total phospholipids; and 6. Das Abgabesystem für Arzneimittel gemäß An- an inner capsule within the outer capsule and 40 spruch 1, wobei das Abgabesystem für Arzneimittel containing mixed carotenoids comprising at für die Abgabe als Nahrungsergänzungszuberei- least S, S’-astaxanthin derived from Haemato- tung, gebildet aus dem Krillöl und den gemischten coccus pluvialis, and one or more of lutein and/or Carotenoiden formuliert ist. trans-zeaxanthin or meso-zeaxanthin, wherein the outer and inner capsules are formed to pre- 45 7. Ein Abgabesystem für Nahrungsergänzungsmittel vent mixing of the carotenoids and krill oil until umfassend: after capsule ingestion, for use in the enhance- ment of cardiovascular health. eine äußere Kapsel enthaltend eine therapeu- tisch effektive Menge von Krillöl umfassend 8. The dietary supplement delivery system for the use 50 Phospholipid-gebundene und Triglycerid-ge- according to claim 7, wherein krill oil is contained in bundene EPS und DHS und das Krillöl umfas- the outer capsule in an amount of 50 to 1000 mg. send wenigstens 30 % Gesamtphospholipide; und 9. The dietary supplement delivery system for the use eine innere Kapsel innerhalb der äußeren Kap- according to claim 7, wherein the inner capsule con- 55 sel und enthaltend gemischte Carotenoide um- tains 0.5 to 8 mg of astaxanthin, 2 to 15 mg of lutein fassend wenigstens S,S’-Astaxanthin stam- and 0.2 to 12 mg of trans-zeaxanthin. mend von Haematococcus pluvialis, und ein oder mehrere von Lutein und/oder trans-Zea-

20 39 EP 2 874 612 B1 40

xanthin oder meso-Zeaxanthin, wobei die äuße- 6. Système d’administration de médicament selon la ren und inneren Kapseln so gestaltet sind, dass revendication 1, dans lequel le système d’adminis- die Vermischung der Carotenoide und des Krill- tration de médicament est formulé pour être admi- öls bis nach der Einnahme der Kapsel unterbun- nistré comme une composition de complément ali- den wird, für die Verwendung in der Verbesse- 5 mentaire formée à partir de l’huile de krill et de ca- rung der kardiovaskulären Gesundheit. roténoïdes mixtes.

8. Das Abgabesystem für Nahrungsergänzungsmittel 7. Système d’administration de complément alimen- zur Verwendung gemäß Anspruch 7, wobei das Krill- taire comprenant : öl in der äußeren Kapsel in einer Menge von 50 bis 10 1.000 mg enthalten ist. une capsule externe contenant une quantité thé- rapeutiquement efficace d’huile de krill compre- 9. Das Abgabesystem für Nahrungsergänzungsmittel nant de l’acide EPA et du DHA liés aux phos- für die Verwendung gemäß Anspruch 7, wobei die pholipides et aux triglycérides et l’huile de krill innere Kapsel 0,5 bis 8 mg Astaxanthin, 2 bis 15 mg 15 comprenant au moins 30 % de phospholipides Lutein und 0,2 bis 12 mg trans-Zeaxanthin enthält. totaux ; et une capsule interne à l’intérieur de la capsule externe et contenant des caroténoïdes mixtes Revendications comprenant au moins la S, S’-astaxanthine dé- 20 rivée d’Haematococcus pluvialis, et une ou plu- 1. Système d’administration de médicament sieurs de la lutéine et/ou de la trans-zéaxanthine comprenant : ou de la méso-zéaxanthine, dans lequel les cap- sules externe et interne sont formées afin que une capsuleexterne contenant une quantité thé- les caroténoïdes et l’huile de krill ne se mélan- rapeutiquement efficace d’huile de krill compre- 25 gent qu’après ingestion de la capsule, pour une nant de l’acide EPA et du DHA liés aux phos- utilisation dans l’amélioration de la santé cardio- pholipides et aux triglycérides et l’huile de krill vasculaire. comprenant au moins 30 % de phospholipides totaux ; et 8. Système d’administration de complément alimen- une capsule interne à l’intérieur de la capsule 30 taire pour l’utilisation selon la revendication 7, dans externe et contenant des caroténoïdes mixtes lequel l’huile de krill est contenue dans la capsule comprenant au moins la S, S’-astaxanthine dé- externe à raison de 50 à 1000 mg. rivée d’Haematococcus pluvialis, et une ou plu- sieurs de la lutéine et/ou de la trans-zéaxanthine 9. Système d’administration de complément alimen- ou de la méso-zéaxanthine, dans lequel les cap- 35 taire pour l’utilisation selon la revendication 7, dans sules externe et interne sont formées afin que lequel la capsule interne contient 0,5 à 8 mg d’as- les caroténoïdes et l’huile de krill ne se mélan- taxanthine, 2 à 15 mg de lutéine et 0,2 à 12 mg de gent qu’après ingestion de la capsule. trans-zéaxanthine.

2. Système d’administration de médicament selon la 40 revendication 1, comprenant en outre 50 à 1000 mg d’huile de krill dans la capsule externe.

3. Système d’administration de médicament selon la revendication 1, comprenant en outre 0,5 à 8 mg 45 d’astaxanthine, 2 à 15 mg de lutéine et 0,2 à 12 mg de trans-zéaxanthine à l’intérieur de la capsule in- terne.

4. Système d’administration de médicament selon la 50 revendication 3, comprenant en outre environ 4 mg d’astaxanthine, environ 10 mg de lutéine et environ 1,2 mg de trans-zéaxanthine à l’intérieur de la cap- sule interne. 55 5. Système d’administration de médicament selon la revendication 4, pour une utilisation dans les soins oculaires.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 13937547 A [0001] • US 840372 A [0126] • US 13553025 A [0001] • US 8481072 B [0126] • US 2011021465 A [0027] • US 093201 A [0129] • US 5533527 A, Tso [0067] • US 20110268811 A [0129] • US 5527533 A [0107]

Non-patent literature cited in the description

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