US 200901 24574A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0124574 A1 LOckW00d et al. (43) Pub. Date: May 14, 2009

(54) CAROTENOIDANALOGS AND DERIVATIVES A63L/365 (2006.01) FOR THE PREVENTION OF PLATELET A6II 3/66 (2006.01) AGGREGATION A613/60 (2006.01) A 6LX 3L/727 (2006.01) (76) Inventors: Samuel F. Lockwood, Lake A63L/366 (2006.01) Linden, MI (US); R. Preston A63L/4365 (2006.01) Mason, Manchester, MA (US) (52) U.S. Cl...... 514/56; 514/547: 514/483; 514/479; Correspondence Address: 514/473; 514/136; 514/163; 514/460; 514/301 MEYERTONS, HOOD, KIVLIN, KOWERT & (57) ABSTRACT GOETZEL, P.C. P.O. BOX 398 The presently described embodiments are directed to compo AUSTIN, TX 78767-0398 (US) sitions that include one or more carotenoid analogs or deriva tives for use in the treatment of a disorder associated with (21) Appl. No.: 12/079,253 platelet aggregation. Certain embodiments provide for the use of said carotenoid analogs or derivatives in preparing (22) Filed: Mar. 24, 2008 compositions suitable for use in Such treatments. Further embodiments provide for pharmaceutical compositions that Related U.S. Application Data include one or more carotenoid analogs or derivatives in (60) Provisional application No. 60/919,637, filed on Mar. R with St. E. still Ein or 23, 2007, provisional application No. 60/948,787, medicaments suitable Ior une treatment of a disorder associ filed on Jul. 10, 2007. ated with platelet aggregation. Yet farther embodiments pro s vide for methods of treating a disorder associated with plate O O let aggregation that include administering to a Subiect who Publication Classification E. from Such treatment Nitti . (51) Int. Cl. tions suitable for inhibiting platelet aggregation in a subject A6 IK 3L/225 (2006.01) undergoing said treatments, and that include carotenoidana A6IP 7/02 (2006.01) logs or derivatives, optionally in combination with one or A6 IK3I/27 (2006.01) more additional antiplatelet agents. Patent Application Publication May 14, 2009 Sheet 1 of 21 US 2009/O124574 A1

FIG. 1A

FIG. 1B

FIG 1C

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nano Sensor (tip 200 um)

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A 25

20

15

O

Control Astaxanthin Clopidogrel AstaXanthin -- Clopidogrel

3 O O

2 5 O

2 O O

150

100

50

Astaxanthin Clopidogrel AStaxanthin -- Clopidogrel

FIG. 3 Patent Application Publication May 14, 2009 Sheet 3 of 21 US 2009/O124574 A1

009Z 09ZZ 000Z 09/,?. 009|| 097|| 000|| 09/ 009 09% uOle Juego UOO ON Patent Application Publication May 14, 2009 Sheet 4 of 21 US 2009/0124574 A1

7OO

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Control Astaxanth n Clopidogrel Astaxanthin -- Clopidogrel

FIG. 5

Control Astaxanth r Clopidogrel Astaxanthin r Clopidogrel FIG. 6 Patent Application Publication May 14, 2009 Sheet 5 of 21 US 2009/0124574 A1

CN LO

Control Astaxanthin Clopidogrel Astaxanthin -- Clopidogrel FIG. 7

Patent Application Publication May 14, 2009 Sheet 8 of 21 US 2009/O124574 A1

2500 5 c 2OOO O. N

w 1500 X O Zas 1 OOO 8 500 o d Control Astaxanthin Aspirin Simvastatin

500

400

200

1OO

Control AStaxanthin Aspirin Simvastatin

FIG. 12 Patent Application Publication May 14, 2009 Sheet 9 of 21 US 2009/0124574 A1

500

400

300

200

1OO

Control Aspirin Aspirin Aspirin Aspirin Aspirin -- -- H H AstaX AstaX AS tax Astax 0.1 uM 1.0 uM 5.0 uM 10.0 uM

FIG. 13

4500 4000 3500 3000 2500 2000 1500 1 OOO 500

Control Aspirin Aspirin Aspirin Aspirin Aspirin Ha H Astax Astax AstaX AStax 0.1 uM 1.0 uM 5.0 uM 10.0 M

FIG. 14 Patent Application Publication May 14, 2009 Sheet 10 of 21 US 2009/O124574 A1

375 T 300

225 -

- 150 -

Control Simva Simva Simva Simva Sinva s ------Astax Astax As tax AstaX 0.1 puM 1.0 uM 5.0 puM 10.0 uV

FIG. 15

3500

3OOO

2500 2000

1500

1000

500

Control Simva Simva Simva Simva Simva ------Astax AstaX Astax Astax 0.1 uM 1.0 uM 5.0 uM 10.0 uM FIG. 16 Patent Application Publication May 14, 2009 Sheet 11 of 21 US 2009/0124574 A1

14

1 2

1 O

6

Control Aspirin Aspirin Aspirin Aspirin Aspirin H ------Astax AStax AStax AStax 0.1 uv 1.0 uM 5.0 uM 10.0 uM

FIG. 17

Control Simva Simva Simva Simva Simva ------AstaX AStax AStax Astax 0.1 uM 1.0 uM 5.0 uM 10.0 uM

FIG. 18 Patent Application Publication May 14, 2009 Sheet 12 of 21 US 2009/O124574 A1

Control Astaxanthin Aspirin Simvastatin

FIG. 19 Patent Application Publication May 14, 2009 Sheet 13 of 21 US 2009/0124574 A1

Oz'91-'

Patent Application Publication May 14, 2009 Sheet 14 of 21 US 2009/0124574 A1

Astaxanthin in Rat Plasma. 4 Hours. After Ingestion of Astaxanthin 50 40 30 20 10 O -10

Astaxanthin in Rat Plasma: 4 Hours. After ingestion of ADS

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Astaxanthin in Rat Plasma. 4 Hours. After Ingestion of ADL

FIG 21 Patent Application Publication May 14, 2009 Sheet 15 of 21 US 2009/0124574 A1

Astaxanthin in Rat Plasma: 8 Hours. After Ingestion of Astaxanthin 50 40 30

Astaxanthin in Rat Plasma. 8 Hours. After ingestion of ADS 50 40

an ag 30

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O - C O a.

Astaxanthin in Rat Plasma. 8 Hours. After Ingestion of ADL 50 40 30

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FIG 21 (Continued) Patent Application Publication May 14, 2009 Sheet 16 of 21 US 2009/O124574 A1

ZOZZOy?dXO–Kpn?SXAd Patent Application Publication May 14, 2009 Sheet 17 of 21 US 2009/O124574 A1 Patent Application Publication May 14, 2009 Sheet 18 of 21 US 2009/0124574 A1

m Patent Application Publication May 14, 2009 Sheet 19 of 21 US 2009/O124574 A1

Patent Application Publication May 14, 2009 Sheet 20 of 21 US 2009/O124574 A1

90710yIdxo–Ápn?SXd Patent Application Publication May 14, 2009 Sheet 21 of 21 US 2009/O124574 A1

ZOGLOMdXO–Ápn}SXAd US 2009/O124574 A1 May 14, 2009

CAROTENOID ANALOGS AND DERVATIVES granular contents of platelets Supply additional adhesion mol FOR THE PREVENTION OF PLATELET ecules, growth factors, coagulation enzymes and other spe AGGREGATION cialized molecules instrumental in the process of thrombus formation and the initiation of the healing process. PRIORITY CLAIM 0008. In addition to coronary artery disease/myocardial 0001. This application claims the benefit of priority under infarction, cerebrovascular disease and peripheral vascular 35 U.S.C. 119 (d)(e) to Provisional Patent Application Ser. disease, diseases and disorders associated with inappropriate No. 60/919,637, entitled “CAROTENOIDANALOGS AND platelet activity and arterial thrombosis also include, for DERIVATIVES FOR THE INHIBITION OF AGGREGA example, stable and unstable angina, transient ischemic TION' filed Mar. 23, 2007 and to Provisional Patent Appli attacks, placental insufficiency, unwanted thromboses Subse cation Ser. No. 60/948,787, entitled “CAROTENOIDANA quent to Surgical procedures (e.g., aortocoronary bypass Sur LOGS AND DERIVATIVES FOR THE PREVENTION OF gery, angioplasty and stent placement, and heart Valve PLATELET AGGREGATION filed Jul. 10, 2007. The replacement), or thromboses Subsequent to atrial fibrillation. above-cited applications are commonly assigned with the Inhibitors of platelet activity can provide therapeutic and present invention, and the entire contents thereof are incor preventive benefits for each of these diseases or disorders. It porated by reference as though fully set forth herein. is also possible that inappropriate platelet activation plays a role in venous thrombosis, such that platelet inhibitors can be BACKGROUND OF THE INVENTION useful for the treatment or prevention of disorders associated with such thromboses. 0002 1. Field of the Invention 0009. A connection is emerging between platelet activa 0003. The present invention generally relates to the field of tion and inflammation, particularly allergic inflammation medicinal chemistry. More specifically, the present invention (e.g., in asthma) and inflammation at the sites of atheroscle relates to the use of synthetic carotenoid analogs, derivatives rotic damage. Therefore, compounds that inhibit platelet acti and compositions made using same for the treatment and vation can also be useful in the treatment or prevention of prevention of platelet aggregation and thrombus formation in disorders involving inflammation. a subject. 0010. There are a number of agents presently available 0004 2. Description of the Relevant Art that target platelet function. For example, aspirin is a rela 0005 Platelet accumulation at sites of vascular injury is a tively weak platelet inhibitor. However, aspirin can cause dynamic process that mediates formation of both the primary life-threatening allergic reactions in sensitive individuals. hemostatic plug and pathologic thrombus formation. The 0011. Another platelet inhibiting agent is ticlopidine mechanisms by which platelet surface proteins direct platelet (TICLIDTM, Roche Pharmaceuticals). Because it requires the recruitment to thrombi under flow conditions have been stud production of active metabolites to be effective, the effect of ied in detail. In addition to directing initial platelet adhesion, ticlopidine is delayed 24-48 hours. The drug can also cause cell-surface receptor interactions activate intracellular signal thrombotic thrombocytopenic purpura, a life-threatening ing. Intracellular signaling stimulates the release of thrombo condition, as well as nausea, abdominal pain, dyspepsia, diar genic Substances from platelet granules. Signaling also medi rhea and skin rash. ates activation of the platelet integrin CfB (gpIb-IIIa) that (0012 Clodiprogel (PLAVIXTM, Bristol-Meyers Squibb/ facilitates firm adhesion of platelets to thrombi. Sanofi Pharmaceuticals) is another platelet inhibitor that 0006 Arterial thrombosis mediates tissue infarction in requires the generation of active metabolites for its therapeu coronary artery disease, cerebrovascular disease, and periph tic efficacy. Therefore, clodiprogel also has a delay of 24-48 eral vascular disease, and, thus, is the single most common hours for its effect. Clodiprogel can also cause thrombotic cause of morbidity and mortality in the United States. Plate thrombocytopenia purpura as well as agranulocytopenia, lets are key mediators of arterial thrombosis. Thus, the iden both life-threatening conditions. The drug has also been asso tification of compounds that inhibit platelet function is of ciated with rash, edema, hypertension, hypercholesterolemia, great importance to medicine. nausea, abdominal pain, dyspepsia, diarrhea, urinary tract 0007 Platelets form the body's primary means of hemo infections, liver enzyme elevations and arthralgia. Stasis and, as Such, have developed an elaborate mechanism of Surveying the vasculature for defects in endothelial integ 0013 The platelet inhibitory agents Abiximab and c7E3 rity. This mechanism involves the ability to respond to sub Fab (REOPRO ABCIXIMABTM, manufacturer Centocor endothelial matrices, shear forces, neighboring platelets, the B.V., distributor Eli Lilly and Co.) are only available in a adrenal axis, as well as soluble proteinacious, nucleotide, and parenteral form. The drugs can cause severe thrombocytope lipid signals. Despite this plethora of physiologic activators, nia. Both have a very long half-life and, therefore, complicate the platelet has only a small repertoire of major functional Surgery that is sometimes required in the setting of life-threat outputs. Upon activation, platelets change shape, aggregate, ening arterial occlusion (e.g., emergent cardiac Surgery in the and secrete their granular contents. The process of platelet setting of a myocardial infarction). activation involves the expression of activities not shared by (0014 Tirofiban (AGGRASTATTM, Merck and Co., Inc.) is functionally merit resting platelets, including, for example, another platelet inhibitory agent that is only available in a ATP release, serotonin release, lysosomal release, alpha gran parenteral form. Tirofiban can cause thrombocytopenia, coro ule release, dense granule release, and cell Surface expression nary artery dissection, bradycardia and edema, as well as of markers of activated platelets (including, but not limited to dizziness and Vasovagal reactions. CD9, gplb, gplIb, gpIIa, CDIa-IIa, P-selectin, PECAM-1, (0.015 Eptifibatide (INTEGRILINTM, COR Therapeutics, activated gpIb/IIIa, and vitronectin receptor). In addition, Inc., Key Pharmaceuticals Inc.) is another platelet inhibitory platelet activation results in the aggregation of platelets with agent that is only available for parenteral administration. It each other and with non-platelet Surrounding cells. The can cause hypotension. US 2009/O124574 A1 May 14, 2009

0016. There is only limited clinical experience with the shape, chemical reactivity, and light-absorbing properties of oral anti-GPIIbIIIa agents lamifiban, sibrafiban, orofiban and carotenoids. Additionally, isomerism around C=C double Xemilofiban. Similarly, clinical experience is limited with the bonds yields distinctly different molecular structures that phosphodiesterase inhibitors cilostazol, trapidil and trifusal. may be isolated as separate compounds (known as Z ('cis' There is more clinical experience with the phosphodiesterase and E (“trans'), or geometric, isomers). Of the more than 750 inhibitor dipyridamole, but its activity is so weak that it is not described carotenoids, an even greater number of the theo frequently used. retically possible mono-Z and poly-Z isomers are sometimes 0017. There is a need in the art for additional platelet encountered in nature. The presence of a Z double bond inhibitory agents for the treatment and prevention of diseases creates greater steric hindrance between nearby hydrogen or disorders characterized by platelet activation and throm bosis. There is a need in the area of cardiovascular and cere atoms and/or methyl groups, so that Z isomers are generally brovascular therapeutics for an agent that can be used in the less stable thermodynamically, and more chemically reactive, prevention and treatment of thrombi, with minimal side than the corresponding all-E form. The all-E configuration is effects, such as unwanted prolongation of bleeding, while an extended, linear, and rigid molecule. Z-isomers are, by preventing or treating target thrombi. contrast, not simple, linear molecules (the so-called “bent chain' isomers). The presence of any Z in the polyene chain Antioxidant Properties of Carotenoids creates a bent-chain molecule. The tendency of Z-isomers to crystallize or aggregate is much less than all-E, and Z isomers 0.018 Carotenoids are a group of natural pigments pro are more readily solubilized, absorbed, and transported in duced principally by plants, yeast, and microalgae. The fam vivo than their all-E counterparts. This has important impli ily of related compounds now numbers greater than 750 described members, exclusive of Z and E isomers. Humans cations for enteral (e.g., oral) and parenteral (e.g., intrave and other animals cannot synthesize carotenoids de novo and nous, intra-arterial, intramuscular, and Subcutaneous) dosing must obtain them from their diet. All carotenoids share com in mammals. mon chemical features, such as a polyisoprenoid structure, a 0021 Carotenoids with chiral centers may exist either as long polyene chain forming the chromophore, and near sym the R (rectus) or S (sinister) configurations. As an example, metry around the central double bond. Tail-to-tail linkage of astaxanthin (with 2 chiral centers at the 3 and 3' carbons) may two Cogeranyl-geranyl diphosphate molecules produces the exist as 4 possible stereoisomers: 3S, 3'S:3R,3'S and 3S, 3R parent Cao carbon skeleton. Carotenoids without oxygenated (identical meso forms); or 3R,3R. The relative proportions of functional groups are called "carotenes”, reflecting their each of the stereoisomers may vary by natural source. For hydrocarbon nature; oxygenated carotenes are known as example, Haematococcus pluvialis microalgal meal is 99% “xanthophylls.” “Parent carotenoids may generally refer to 3S, 3'S astaxanthin, and is likely the predominant human those natural compounds utilized as starting scaffold for evolutionary source of astaxanthin. Krill (3R,3R) and yeast structural carotenoid analog synthesis. Carotenoid deriva Sources yield different stereoisomer compositions than the tives may be derived from a naturally occurring carotenoid. microalgal Source. Synthetic astaxanthin, produced by large Naturally occurring carotenoids may include lycopene, lyco manufacturers such as Hoffmann-LaRoche AG, Buckton phyll, lycoxanthin, astaxanthin, beta-carotene, lutein, Zeax Scott (USA), or BASF AG, are provided as defined geometric anthin, and/or canthaxanthin to name a few. isomer mixtures of a 1:2:1 stereoisomer mixture 3S, 3'S, 3R, 0019 Cyclization at one or both ends of the molecule 3'S, 3R.3S (meso); 3R, 3R of non-esterified, free astaxan yields 7 identified end groups (illustrative structures shown in thin. Natural source astaxanthin from Salmonid fish is pre FIG. 1). Examples of uses of carotenoid derivatives and ana dominantly a single stereoisomer (3S.3'S), but does contain a logs are illustrated in U.S. patent application Ser. No. 10/793, mixture of geometric isomers. Astaxanthin from the natural 671 filed on Mar. 4, 2004, entitled “CAROTENOID ETHER Source Haematococcus pluvialis may contain nearly 50% Z ANALOGS ORDERIVATIVES FOR THE INHIBITION isomers. As stated above, the Z conformational change may AND AMELIORATION OF DISEASE by Lockwood et al. lead to a higher steric interference between the two parts of published on Jan. 13, 2005, as Publication No. US-2005 the carotenoid molecule, rendering it less stable, more reac 0009758 and PCT International Application Number PCT/ tive, and more Susceptible to reactivity at low oxygen ten US2003/023706 filed on Jul. 29, 2003, entitled “STRUC sions. In Such a situation, in relation to the all-E form, the Z TURAL CAROTENOID ANALOGS FOR THE forms: (1) may be degraded first; (2) may better Suppress the INHIBITION AND AMELIORATION OF DISEASE by attack of cells by reactive oxygen species such as Superoxide Lockwood et al. (International Publication Number WO anion; and (3) may preferentially slow the formation of radi 2004/011423 A2, published on Feb. 5, 2004) both of which cals. Overall, the Z forms may initially be thermodynamically are incorporated by reference as though fully set forth herein. favored to protect the lipophilic portions of the cell and the 0020 Documented carotenoid functions in nature include cell membrane from destruction. It is important to note, how light harvesting, photoprotection, and protective and sex ever, that the all-E form of astaxanthin, unlike B-carotene, related coloration in microscopic organisms, mammals, and retains significant oral bioavailability as well as antioxidant birds, respectively. A relatively recent observation has been capacity in the form of its dihydroxy- and diketo-substitu the protective role of carotenoids againstage-related diseases tions on the B-ionone rings, and has been demonstrated to in humans as part of a complex antioxidant network within have increased efficacy over 3-carotene in most studies. The cells. This role is dictated by the close relationship between all-E form of astaxanthin has also been postulated to have the the physicochemical properties of individual carotenoids and most membrane-stabilizing effect on cells in vivo. Therefore, their in vivo functions in organisms. The long system of it is likely that the all-E form of astaxanthin in natural and alternating double and single bonds in the central part of the synthetic mixtures of stereoisomers is also extremely impor molecule (delocalizing the L-orbital electrons over the entire tant in antioxidant mechanisms, and may be the form most length of the polyene chain) confers the distinctive molecular Suitable for particular pharmaceutical preparations. US 2009/O124574 A1 May 14, 2009

0022. The antioxidant mechanism(s) of carotenoids, and and the progression to cirrhosis by affecting the activation of in particular astaxanthin, includes singlet oxygen quenching, hepatic stellate cells early in the fibrogenetic pathway. Reduc direct radical scavenging, and lipid peroxidation chain-break tion in the level of ROS by the administration of a potent ing. The polyene chain of the carotenoid absorbs the excited antioxidant can therefore be crucial in the prevention of the energy of singlet oxygen, effectively stabilizing the energy activation of both HSC and Kupffer cells. This protective transfer by delocalization along the chain, and dissipates the antioxidant effect appears to be spread across the range of energy to the local environment as heat. Transfer of energy potential therapeutic antioxidants, including water-soluble from triplet-state chlorophyll (in plants) or other porphyrins (e.g., vitamin C, glutathione, resveratrol) and lipophilic (e.g., and proto-porphyrins (in mammals) to carotenoids occurs Vitamin E, B-carotene, astaxanthin) agents. Therefore, a co much more readily than the alternative energy transfer to antioxidant derivative strategy in which water-soluble and oxygen to form the highly reactive and destructive singlet lipophilic agents are combined synthetically is a particularly oxygen ("O.). Carotenoids may also accept the excitation useful embodiment. energy from singlet oxygen if any should be formed in situ, 0029. Vitamin E is generally considered the reference and again dissipate the energy as heat to the local environ antioxidant. When compared with vitamin E, carotenoids are ment. This singlet oxygen quenching ability has significant more efficient in quenching singlet oxygen in homogeneous implications in cardiac ischemia, macular degeneration, por organic solvents and in liposome systems. They are better phyria, and other disease states in which production of singlet chain-breaking antioxidants as well in liposomal systems. oxygen has damaging effects. In the physical quenching They have demonstrated increased efficacy and potency in mechanism, the carotenoid molecule may be regenerated vivo. They are particularly effective at low oxygen tension, (most frequently), or be lost. Carotenoids are also excellent and in low concentration, making them extremely effective chain-breaking antioxidants, a mechanism important in agents in disease conditions in which ischemia is an impor inhibiting the peroxidation of lipids. Astaxanthin can donate tant part of the tissue injury and pathology. These carotenoids a hydrogen (H) to the unstable polyunsaturated fatty acid also have a natural tropism for the liver after oral administra (PUFA) radical, stopping the chain reaction. Peroxyl radicals tion. Therefore, therapeutic administration of carotenoids may also, by addition to the polyene chain of carotenoids, be should provide a greater benefit in limiting fibrosis than vita the proximate cause for lipid peroxide chain termination. The min E. appropriate dose of astaxanthin has been shown to completely 0030 Problems related to the use of naturally occurring Suppress the peroxyl radical chain reaction in liposome sys carotenoids and some structural carotenoid analogs include: tems. Astaxanthin shares with vitamin Ethis dual antioxidant (1) the complex isomeric mixtures, including non-carotenoid defense system of singlet oxygen quenching and direct radi contaminants, provided in natural and synthetic sources lead cal scavenging, and in most instances (and particularly at low ing to costly increases in safety and efficacy tests required by oxygen tension in vivo) is Superior to Vitamin E as a radical such agencies as the FDA; (2) limited bioavailability upon Scavenger and physical quencher of singlet oxygen. administration to a subject; and (3) the differential induction 0023 Carotenoids, and in particular astaxanthin, are of cytochrome P450 enzymes (this family of enzymes exhib potent direct radical scavengers and singlet oxygen quench its species-specific differences which must be taken into ers and possess all the desirable qualities of Such therapeutic account when extrapolating animal work to human studies). agents for inhibition or amelioration of ischemia-reperfusion (I/R) injury. Synthesis of novel carotenoid derivatives with SUMMARY OF THE INVENTION “soft-drug properties (i.e. activity in the derivatized form), 0031. This present application provides methods and com with physiologically relevant, cleavable linkages to pro-moi positions suitable for use in preventing or treating diseases or eties, can generate significant levels of free carotenoids in conditions associated with platelet aggregation; such diseases both plasma and Solid organs. This is critically important, for including but not limited to venous thrombosis, thrombophle in mammals, diesters of carotenoids generate the non-esteri bitis, arterial embolism, coronary and cerebral arterial throm fied or “free” parent carotenoid, and may be viewed as elegant bosis, unstable angina, myocardial infarction, stroke, cerebral synthetic and novel delivery vehicles with improved proper embolism, kidney embolisms and pulmonary embolisms. ties for delivery of free carotenoid to the systemic circulation The method is also directed to a method of preventing, treat and ultimately to target tissue. In the case of non-esterified, ing or reducing the incidence of thrombosis, thrombotic free astaxanthin, this is a particularly useful embodiment events, embolic events or pathological conditions associated (characteristics specific to non-esterified, free astaxanthin with such events, where the thrombosis, thrombotic event or below): embolic event occurs during or after Surgery. 0024 Lipid soluble in natural form; may be modified to 0032. In some embodiments, uses of carotenoids, caro become more water soluble tenoid analogs or derivatives, including pharmaceutically (0025 Molecular weight of 597 Daltons size.<600 dal acceptable salts thereof, may include the uses thereof in the tons (Da) readily crosses the blood brain barrier, or formulation of pharmaceutical compositions Suitable for the BBB treatment of a disorder associated with platelet aggregation in 0026 Long polyene chain characteristic of carotenoids a Subject. effective in singlet oxygen quenching and lipid peroxi 0033. In some embodiments, pharmaceutical composi dation chain breaking tions suitable for use in the treatment of a disorder associated 0027 No pro-vitamin A activity in mamnimals (elimi with plateletaggregation in a Subject may include one or more nating concerns of hypervitaminosis A and retinoid tox carotenoid analogs or derivatives in an amount Sufficient to at icity in humans). least partially decrease the risk of thrombotic events occur 0028. The administration of antioxidants that are potent ring in a subject. singlet oxygen quenchers and direct radical scavengers, par 0034. In an embodiment, pharmaceutical compositions ticularly of superoxide anion, should limit hepatic fibrosis formulated for use in the treatment of a disorder associated US 2009/O124574 A1 May 14, 2009

with platelet aggregation may include one or more carotenoid able cellular and/or biochemical processes that occur in dis analogs or derivatives in an amount Sufficient to affect one or orders associated with platelet aggregation. more biochemical pathways associated with platelet activa 0039. In some of the foregoing embodiments, analogs or tion, including but not limited to increasing the bioavailability derivatives of carotenoids may be at least partially water of NO to platelets, increasing the production and secretion of soluble. “Water-soluble' structural carotenoid analogs or NO by platelets, and or reducing sources of oxidative stress derivatives are those analogs or derivatives that may be for and peroxynitrite formation. mulated in aqueous solution, either alone or with one or more 0035. In some embodiments, pharmaceutical composi excipients. Water-soluble carotenoid analogs or derivatives tions formulated for use in the treatment of a disorder asso may include those compounds and synthetic derivatives that ciated with plateletaggregation are provided that may include form molecular self-assemblies, and may be more properly one or more carotenoid analogs orderivatives in combination with one or more additional compositions or medicaments termed “water dispersible' carotenoid analogs or derivatives. used in the treatment of platelet disorders, including but not Water-soluble and/or “water-dispersible' carotenoid analogs limited to one or more additional agents that inhibit platelet or derivatives may be preferred in some embodiments. aggregation. A composition in accordance with Such an 0040 Water-soluble carotenoid analogs or derivatives embodiment may include, for example, one or more caro may have a water solubility of greater than about 1 mg/mL in tenoid analogs orderivatives in combination with one or more Some embodiments. In certain embodiments, water-soluble additional anti-platelet agents. carotenoid analogs or derivatives may have a water Solubility 0036. In some embodiments, methods are provided for of greater than about 5 mg/ml-10 mg/mL. In certain embodi treating a disorder associated with platelet aggregation in a ments, water-soluble carotenoid analogs or derivatives may Subject. Such methods may include administering to an indi have a water solubility of greater than about 20 mg/mL. In vidual who would benefit from such treatment a therapeuti certain embodiments, water-soluble carotenoid analogs or cally effective amount of a pharmaceutical composition that derivatives may have a water solubility of greater than about includes one or more carotenoid analogs orderivatives. Meth 25 mg/mL. In some embodiments, water-soluble carotenoid ods are also provided for the treatment a disorder associated analogs or derivatives may have a water solubility of greater with platelet aggregation, such methods comprising admin than about 50 mg/mL. istering to an individual having need for Such treatment a 0041. In some embodiments, water-soluble analogs or therapeutically effective amount of a pharmaceutical compo derivatives of carotenoids may be administered to a subject sition that includes one or more carotenoid analogs or deriva tives and also administering to Such an individual atherapeu alone or in combination with additional carotenoids or struc tically effective amount of one or more additional anti tural analogs or derivatives thereof. In some embodiments, platelet agents. water-soluble analogs or derivatives of carotenoids may be administered to a subject alone or in combination with other 0037. In certain embodiments, the carotenoid analogs or derivatives may be administered to a subject concurrently antioxidants. with one or more additional compositions or medicaments 0042. The uses, methods and compositions contemplated used in the treatment of a disorder associated with platelet herein include the use of one or more carotenoid analogs or aggregation, including but not limited to one or more addi derivatives. In some embodiments, a carotenoid analogs or tional anti-platelet agents. In an embodiment, the one or more derivatives may have the structure: additional compositions or medicaments may be adminis tered to the Subject either as a co-formulation, or as separate pharmaceutical and/or nutraceutical formulation adminis tered as part of a co-therapy regimen. In one such embodi ment, carotenoid analogs or derivatives may be administered to the Subject undergoing such treatment prior to the com mencement of drug therapy with the one or more additional compositions or medicaments used in the treatment of a dis order associated with platelet aggregation. In another I0043 where each R is independently hydrogen or methyl, embodiment, carotenoids analogs or derivatives may be and where R' and Rare each independently: administered to the Subject following the commencement of drug therapy with the one or more additional compositions or medicaments used in the treatment of a disorder associated with platelet aggregation. 0038 Administration of the carotenoid analogs or deriva tives to a Subject in accordance with the preceding embodi ments may be provided to a subject with the intention of at least partially inhibiting and/or influencing some of the nega tive or undesirable cellular and/or biochemical processes that occur in pathologies associated with platelet aggregation. Administering one or more carotenoid analogs or derivatives by one skilled in the art as provided for herein including consideration of the pharmacokinetics and pharmacodynam ics of therapeutic drug delivery is expected to reduce and/or ameliorate at least a portion of the of the negative or undesir

US 2009/O124574 A1 May 14, 2009 -continued tr.-continued

—(C(O). CH-NHMe, C(O) NHMe-CH CO.R. and pharmaceutically acceptable salts of any of these com pounds, where each R is independently H, alkyl, aryl, benzyl, Group IA metal, or co-antioxidant. 0049. In some embodiments, carotenoid analogs or derivatives suitable for use with the present compositions, methods and uses may have the structure O H O. N. Yso --~~ R2 I0050 where each R is independently hydrogen or methyl, and wherein each R' and Rare independently:

O H O N Srson

US 2009/O124574 A1 May 14, 2009

-continued -continued OMe O ONn P-O O O, O o ------MeO OMe

pharmaceutically acceptable salts of any of these compounds, where each R is independently H, alkyl, aryl, benzyl, Group IA metal, or co-antioxidant. 0058. In some embodiments, a composition may include one or more carotenoids, carotenoid analogs, carotenoid derivatives, and pharmaceutically acceptable derivatives of carotenoids, carotenoid analogs, and carotenoid derivatives O having the general structure: H O N rol

Each R may be independently hydrogen or methyl. Each R' and R may be independently:

O H O NSro

US 2009/O124574 A1 May 14, 2009 12

wherein R is independently hydrogen, OH, methyl, -continued —CHOH, or —OR; wherein at least one R group in the R4 R4 R4 carotenoid analog orderivative may be —OR; wherein each R is independently: alkyl: aryl;-alkyl-N(R);-aryl-N(R): R R -alkyl-N-(R7); -aryl-N-(R7); -alkyl-COR: -aryl-COR: -alkyl-CO.; -aryl-CO: C(O)-alkyl-N(R); —C(O)- aryl-N(R): C(O)-alkyl-N-(R): C(O)-aryl-N-(R): R4 R4 O R4 —C(O)-alkyl-COR: —C(O)-aryl-COR: C(O)-alkyl R4 R4 R4 R4 CO: C(O)-aryl-CO: C(O)–(NR)-alkyl-N(R): —C(O)–(NR)-aryl-N(R7);-C(O)–(NR7)-alkyl-N-(R7) R O : C(O)–(NR)-aryl-N-(R): C(O)–(NR)-alkyl R4 4 s R4 , COR: –C(O)–(NR)-aryl-COR: –C(O)–(R)-alkyl R CO: C(O)–(NR7)-aryl-CO: C(O)–(NR7)-alkyl-N O R4 O R4 R4 (R)-alkyl-N(R): C(O) OR: -P(O)(OR); S(O) R4 R4 R4 R4 (OR)2; —C(O)—Co-C. Saturated hydrocarbon): —C(O)—C-C, monounsaturated hydrocarbon; R O —C(O)—C-C, a peptide; a carbohydrate; a nucleoside R4 , or reside; or a co-antioxidant; where R is hydrogen, alkyl, or aryl; where R is hydrogen, alkyl, aryl, benzyl or a co-anti O R4 O R4 oxidant; and where R is hydrogen, alkyl, aryl, -P(O)(OR) , -S(O)(OR), an amino acid, a peptide, a carbohydrate, a I0064 wherein R is independently hydrogen, OH, nucleoside, or a co-antioxidant. methyl, -CHOH, or —OR; wherein at least one R group 0.066 Each co-antioxidant may be independently Vitamin in the carotenoidanalog orderivative may be—OR; wherein C. Vitamin C analogs, Vitamin C derivatives, Vitamin E, each R is independently: alkyl: aryl; -alkyl-N(R);-aryl-N Vitamin E analogs, Vitamin E derivatives, flavonoids, fla (R) -alkyl-N-(R); -aryl-N-(R); -alkyl-COR: -aryl vonoid derivatives, or flavonoid analogs. Flavonoids include, CO.R., -alkyl-CO.; -aryl-CO.; —C(O)-alkyl-N(R): but are not limited to, quercetin, Xanthohumol, isoxanthohu —C(O)-aryl-N(R); —C(O)-alkyl-N-(R): C(O)-aryl mol, or genistein. Selection of the co-antioxidant should not N"(R); –C(O)-alkyl-COR: –C(O)-aryl-COR: be seen as limiting for the therapeutic application of the —C(O)-alkyl-CO: C(O)-aryl-CO: C(O) (NR)- current invention. alkyl-N(R): C(O)–(NR)-aryl-N(R): C(O)— 0067. In some embodiments, pharmaceutical composi (NR)-alkyl-N-(R7): —C(O)–(NR)-aryl-N-(R): tions are provided that may include one or more carotenoids —C(O)–(NR)-alkyl-COR: - C(O)–(NR)-aryl (“a co-formulation' strategy), or synthetic derivatives orana COR: C(O)–(NR)-alkyl-CO: C(O)–(NR)-aryl logs thereof, in combination with one or more additional CO: —C(O)–(NR)-alkyl-N(R)-alkyl-N(R7): compositions or medicaments used in the treatment of plate C(O) OR: P(O)(OR), S(O)(OR): C(O) let disorders. Certain embodiments may be further directed to C-C saturated hydrocarbon; —C(O)—C-C monoun pharmaceutical compositions that include combinations of saturated hydrocarbon; —C(O)—C-C polyunsaturated two or more carotenoids or synthetic analogs or derivatives hydrocarbon; a peptide; a carbohydrate; a nucleoside reside; thereof. or a co-antioxidant; where R is hydrogen, alkyl, or aryl; 0068. In some embodiments, separate pharmaceutical where R is hydrogen, alkyl, aryl, benzylor a co-antioxidant; compositions are provided, Such that the one or more addi and where R is hydrogen, alkyl, aryl, -P(O)(OR), S(O) tional compositions or medicaments used in the treatment of (OR)2, an amino acid, a peptide, a carbohydrate, a nucleo a disorder associated with platelet aggregation is/are deliv side, or a co-antioxidant. ered separately from the carotenoid, or synthetic derivatives 0065. In some embodiments, a composition may include or analogs thereof (sometimes referred to in the art as a one or more carotenoids, carotenoid analogs, carotenoid “co-administration' strategy). The pharmaceutical composi derivatives, and pharmaceutically acceptable derivatives of tions may be adapted to be administered orally, or by one or carotenoids, carotenoid analogs, and carotenoid derivatives more parenteral routes of administration. In an embodiment, having the general structure: the pharmaceutical composition may be adapted Such that at

US 2009/O124574 A1 May 14, 2009

least a portion of the dosage of the carotenoid or synthetic 0080 FIG. 10 shows the tongue bleeding time (sec) before derivative or analog thereof is delivered prior to, during, or and after occlusive thrombus formation in the LCA and sub after at least a portion of the one or more additional compo sequent treatment with DDA (10, 30 or 50 mg/kg) or 0.9% sitions or medicaments used in the treatment of a disorder NaCl solution (0 mg/kg. DDA) followed immediately by rt associated with platelet aggregation is/are delivered to the PA; Subject. I0081 FIG. 11 shows the comparative effects of astaxan 0069 Embodiments directed to pharmaceutical composi thin, aspirin and simvastatin (all at 1.0 LM) on platelet-de tions may further include appropriate vehicles for delivery of rived nitric oxide release following stimulation with IP3 (1.0 said pharmaceutical composition to a desired site of action uM); (i.e., the site a subject's body where the biological effect of the I0082 FIG. 12 shows the comparative effects of astaxan pharmaceutical composition is most desired). Pharmaceuti thin, aspirin and simvastatin (all at 1.0 LM) on platelet-de cal compositions including carotenoids or analogs that may rived peroxynitrite release following stimulation with IP3 be administered orally or intravenously may be particularly (1.0 uM); advantageous for and Suited to embodiments described I0083 FIG. 13 shows the effects of aspirin (1.0 uM) on herein. In yet a further embodiment, an injectable pharma platelet peroxynitrite release in the absence and presence of ceutical composition may be prepared. increasing levels of astaxanthin following stimulation with IP3 (1.0 uM); BRIEF DESCRIPTION OF THE DRAWINGS I0084 FIG. 14 shows the effects of aspirin (1.0 uM) on 0070. The above brief description as well as further platelet nitric oxide release in the absence and presence of objects, features and advantages of the methods and appara increasing levels of astaxanthin following stimulation with tus of the present invention will be more fully appreciated by IP3 (1.0 uM); reference to the following detailed description of presently I0085 FIG. 15 shows the effects of simvastatin (1.0 uM) on preferred but nonetheless illustrative embodiments in accor platelet peroxynitrite release in the absence and presence of dance with the present invention when taken in conjunction increasing levels of astaxanthin following stimulation with with the accompanying drawings. IP3 (1.0 uM); 0071 FIG. 1 shows several examples of the structures of I0086 FIG.16 shows the effects of simvastatin (1.0 uM) on various synthetic carotenoid derivatives or analogs that may platelet nitric oxide release in the absence and presence of be used according to some embodiments. (A) disuccinate increasing levels of astaxanthin following stimulation with divitamin Castaxanthin; (B) disodium disuccinic acid ester IP3 (1.0 uM); astaxanthin salt; (C) dilysinate astaxanthin ester salt; I0087 FIG. 17 shows the effects of aspirin (1.0 uM) on 0072 FIG. 2 is a schematic diagram of a nitric oxide platelet nitric oxide/peroxynitrite release ratio in the absence nanosensor placed in close proximity to the Surface of a single and presence of increasing levels of astaxanthin following vascular platelet; stimulation with IP3 (1.0 uM); 0073 FIG.3 shows the effects of nitric oxide release from I0088 FIG. 18 shows the effects of simvastatin (1.0 uM) on vascular platelets following incubation with homochiral platelet nitric oxide/peroxynitrite release ratio in the absence astaxanthin and clopidogrel, either alone or in combination; and presence of increasing levels of astaxanthin following 0074 FIG. 4 shows representative amperograms of plate stimulation with IP3 (1.0 uM); let-derived nitric oxide release following incubation with 0089 FIG. 19 shows the effects of astaxanthin, simvasta 3S.3'S-Astaxanthin alone and in combination with clopi tin, and aspirin on platelet nitric oxide?peroxynitrite release dogrel; ratio following stimulation with IP3 (1.0 LM): 0075 FIG. 5. shows the effects on calcium ionophore 0090 FIG. 20 shows a table summarizing the experiments (Call)-stimulated total nitric oxide release from human run to determine the metabolism of carotenoid derivatives in umbilical vein endothelial cells following incubation with various animals; homochiral astaxanthin and clopidogrel, either alone or in 0091 FIG.21 shows chromatograms of rat plasma 4 and 8 combination; hours after the administration of carotenoid derivatives; 0076 FIG. 6 shows the effects on calcium ionophore 0092 FIG. 22 shows pharmacokinetic data determined (Call)-stimulated total peroxynitrite release from human after the administration of carotenoid derivatives to rats: umbilical vein endothelial cells following incubation with 0093 FIG. 23 shows pharmacokinetic data determined homochiral astaxanthin and clopidogrel, either alone or in after the administration of ADL carotenoid derivatives to combination; dogs: 0077 FIG. 7 shows the effects on calcium ionophore 0094 FIG. 24 shows accumulation of carotenoid in vari (Call)-stimulated total nitric oxide and peroxynitrite release ous tissues after the administration of carotenoid derivatives (expressed as a ratio) from human umbilical vein endothelial to dogs; cells following incubation with homochiral astaxanthin and 0.095 FIG. 25 shows a graph of the blood plasma concen clopidogrel, either alone or in combination; tration of carotenoid determined after the administration of 0078 FIG. 8 shows the percent platelet aggregation carotenoid derivatives to dogs; responses to ADP(A) and AA (B) before and after occlusive 0096 FIG. 26 shows pharmacokinetic data determined thrombus formation in the LCA and subsequent treatment after the administration of ADG and ADSacarotenoid deriva with DDA (10, 30 or 50 mg/kg) or 0.9% NaCl solution (0 tives to dogs; and mg/kg. DDA) followed immediately by rt-PA: 0097 FIG. 27 shows pharmacokinetic data determined 0079 FIG. 9 shows thrombus weights after occlusive after the administration of carotenoid derivatives to monkeys. thrombus formation in the LCA and subsequent treatment 0098. While the invention is susceptible to various modi with DDA (10, 30 or 50 mg/kg) or 0.9% NaCl solution fol fications and alternative forms, specific embodiments thereof lowed immediately by rt-PA; are shown by way of example in the drawings and will herein US 2009/O124574 A1 May 14, 2009

be described in detail. It should be understood, however, that carotenoid derivative may also generally refer to chemical the drawing and detailed description thereto are not intended compounds or compositions that are synthetically derived to limit the invention to the particular form disclosed, but on from non-carotenoid based parent compounds; however, the contrary, the intention is to cover all modifications, which ultimately substantially resemble a carotenoid derived equivalents and alternatives falling within the spirit and scope analog. Non-limiting examples of carotenoid analogs and of the present invention as defined by the appended claims. derivatives that may be used according to some of the embodiments described herein are depicted schematically in DETAILED DESCRIPTION OF THE FIG 1. EMBODIMENTS 0104. As used herein, the term “organ', when used in reference to a part of the body of an animal or of a human Definitions generally refers to the collection of cells, tissues, connective 0099. It is to be understood that the present invention is not tissues, fluids and structures that are part of a structure in an limited to particular devices or biological systems, which animal or a human that is capable of performing some spe may, of course, vary. It is also to be understood that, as used cialized physiological function. Groups of organs constitute in this specification and the appended claims, the singular one or more specialized body systems. The specialized func forms “a”, “an', and “the include singular and plural refer tion performed by an organ is typically essential to the life or ents unless the content clearly dictates otherwise. Thus, for to the overall well-being of the animal or human. Non-limit example, reference to “a linker' includes one or more linkers. ing examples of body organs include the heart, lungs, kidney, It is to be yet further understood that any terminology used ureter, urinary bladder, adrenal glands, pituitary gland, skin, herein is for the purpose of describing particular embodi prostate, uterus, reproductive organs (e.g., genitalia and ments only, and is not intended to be limiting. accessory organs), liver, gall-bladder, brain, spinal cord, 0100. The terms used throughout this specification gener stomach, intestine, appendix, pancreas, lymph nodes, breast, ally have their ordinary meanings in the art, within the context salivary glands, lacrimal glands, eyes, spleen, thymus, bone of the invention, and in the specific context where each term marrow. Non-limiting examples of body systems include the is used. Certainterms are discussed below, or elsewhere in the respiratory, circulatory, cardiovascular, lymphatic, immune, specification, to provide additional guidance to the practitio musculoskeletal, nervous, digestive, endocrine, exocrine, ner in describing the general embodiments of the invention, as hepato-biliary, reproductive, and urinary systems. In animals, well as how to make and use them. It will be readily appre the organs are generally made up of several tissues, one of ciated that the same thing can be said in more than one way. which usually predominates, and determines the principal Consequently, alternative language and synonyms may be function of the organ. used for any one or more of the terms discussed herein, nor is 0105. As used herein, the term “tissue', when used in any special significance to be placed upon whether or not a reference to a part of a body or of an organ, generally refers to term is elaborated or discussed in greater detail herein. Syn an aggregation or collection of morphologically similar cells onyms for certain terms are provided. A recital of one or more and associated accessory and Support cells and intercellular synonyms does not exclude the use of other synonyms. The matter, including extracellular matrix material, vascular Sup use of examples anywhere in this specification, including ply, and fluids, acting together to perform specific functions in examples of any terms discussed herein, is illustrative only, the body. There are generally four basic types of tissue in and in no way limits the scope and meaning of the invention animals and humans including muscle, nerve, epithelial, and or of any exemplified term. connective tissues. 0101. As used herein the term "carotenoid analogs and 0106. As used herein the terms “reducing.” “inhibiting derivatives' may be generally defined as biologically active and “ameliorating, when used in the context of modulating a structural analogs and derivatives of carotenoids. Typical ana pathological or disease state, generally refers to the preven logs include molecules which demonstrate equivalent or tion and/or reduction of at least a portion of the negative improved biologically useful and relevant function, but which consequences of the disease state. When used in the context of differ structurally from the parent (i.e., naturally occurring) a biochemical event or pathway, the term generally refers to a compounds. Parent carotenoids are selected from the more net reduction in the magnitude or activity of said pathway. than 600 naturally-occurring carotenoids described in the 0107 As used herein, the phrase “increase the bioavail literature, and their stereo- and geometric isomers. Such ana ability of nitric oxide (NO) generally refers to a physiologi logs may include, but are not limited to, esters, ethers, car cal event, stimulus or treatment that produces a net increase in bonates, amides, carbamates, phosphate esters and ethers, the amount of biologically active NO available to affect Sulfates, glycoside ethers, with or without spacers (linkers). physiological responses dependent thereon. In the context of 0102. As used herein, the term “xanthophyll carotenoid' the present application, the phrase, unless other wise speci generally refers to a naturally occurring or synthetic 40-car fied, is generally meant in reference to net amount of biologi bonpolyene chain with a carotenoid structure that contains at cally active NO that is available to affect platelet function. least one oxygen-containing functional group. The chain may The bioactive NO described in the context of the instant include terminal cyclic end groups. Exemplary, though non application is not limited to any one particular source or limiting, Xanthophyll carotenoids include astaxanthin, Zeax sources. Nevertheless, it will be readily appreciated by the anthin, lutein, echinenone, lycophyll, canthaxanthin, and the skilled practitioner that the majority of bioactive NO avail like. Non-limiting examples of carotenoids that are not Xan able to platelets is typically derived from vascular endothe thophyll carotenoids include B-carotene and lycopene. lium as well as from the platelets themselves. Generally, NO 0103) As used herein, terms such as "carotenoid analog has a net inhibitory effect on platelet aggregation. and “carotenoid derivative' generally refer to chemical com 0108. As used herein, the term “platelet' generally refers pounds or compositions derived from a naturally occurring or to the cell fragments circulating in the blood that are involved synthetic carotenoid. Terms such as carotenoid analog and in the cellular mechanisms of primary hemostasis leading to US 2009/O124574 A1 May 14, 2009

the formation of blood clots. Dysfunction or low levels of 0111 Various drugs are known to inhibit platelet function. platelets predisposes to bleeding, while high levels, although Non-limiting examples of platlet inhibitor drugs include: usually asymptomatic, may increase the risk of thrombosis. Aspirin, which irreversibly inhibits cyclooxygenase-1 and Platelets are produced in the bone marrow and are the frag blocks the formation of TXA to platelets; clopidogrel, which mented products of megakaryocytes. Platelets are anuclear is an antiplatelet drug that irreversibly inhibits ADP recep and discoid cell fragments measuring 1.5-3.0 um in diameter. tors, non-steroidal anti-inflammatory drugs (NSAIDs), The body has a very limited reserve of platelets, so they can be which inhibit prostaglandin synthesis, Abciximab, another rapidly depleted. They contain RNA, mitochondria, a canali antiplatelet drug, which blocks fibrinogen receptors; and qui cular system, and several different types of granules; lysos nidine, a calcium channel blocker. omes (containing acid hydrolases), dense bodies (containing 0112. As used herein, phrases such as “one or more addi ADP, ATP serotonin and calcium) and alpha granules (con tional medicaments or compositions Suitable for the treat taining fibrinogen, factor V. vitronectin, thrombospondin and ment of a disorder associated with platelet aggregation in a von Willebrand factor), the contents of which are released Subject, or more simply, “one or more additional composi upon activation of the platelet. Platelets are activated when tions or medicaments.” generally refer to a pharmaceutical brought into contact with collagen (which is exposed when composition that contains at least one pharmaceutically the endothelial blood vessel lining is damaged), thrombin active compound that is used for the treatment of platelet (primarily through PAR-1), ADP receptors expressed on aggregation disorders, but which is distinct from the caro white blood cells or the endothelial cells of the blood vessels, tenoid analogs or derivatives that form the basis of the present a negatively charged surface (e.g., glass), or several other disclosure. Typically one or more additional medicaments or activating factors. Once activated, they release a number of compositions suitable for the treatment of a disorder associ different coagulation factors and platelet activating factors. ated with platelet aggregation in a Subject as presently described may include one or more antiplateletagents. Exem Platelet activation further results in the transport of negatively plary though non-limiting antiplatelet agents Suitable for use charged phospholipids to the platelet Surface. These phospho in the presently described embodiments include: anticoagu lipids provide a catalytic surface (with the charge provided by lants; statins; ADP receptor inhibitors (e.g., thienopyridines); phosphatidylserine and phosphatidylethanolamine) for the thrombin inhibitors; factor Xa inhibitors; agonists of puriner tenase and prothrombinase complexes. The platelets adhere gic receptors; antagonists of CD40 or CD40 ligand (CD40L) to each other via adhesion receptors or integrins, and to the or compounds that disrupt (e.g., reduce) the interaction of endothelial cells in the wall of the blood vessel forming a CD40 and CD40L: eicosanoid related targets (e.g., COX haemostatic plug in conjunction with fibrin. The high con inhibitors, PGE1 agonists, PG synthase inhibitors, TX syn centration of myosin and actin filaments to platelets are thase inhibitors, and TXA antagonists); and glycoprotein stimulated to contract during aggregation, further reinforcing IIb/IIIa antagonists. Exemplary anticoaglulants Suitable for the plug. The most abundant platelet adhesion receptor is use in accordance with the presently disclosed treatment glycoprotein (GP) IIb/IIIa; this is a calcium-dependent recep methods include, e.g., aspirin, warfarin, a combination of tor for fibrinogen, fibronectin, vitronectin, thrombospondin aspirin and warfarin. Exemplary non-fractionated or fraction and von Willebrand factor (VWF). Other receptors include ated heparins may include low molecular weight heparins, GPIb-V-IX complex (vWF) and GPVI (collagen). Such as ardeparin, certoparin, dalteparin, enoxaparin, nadro 0109) A variety of molecules are know to function as parin, reviparin, or tinaZaparin, and those compounds platelet activators. Examples of platelet activating molecules described in WO97/35592. Exemplary statins suitable foruse include: collagen, which is exposed when endothelial blood in accordance with the presently disclosed treatment methods vessel lining is damaged and binds to its receptors GPVI and include, e.g., atorvastatin, fluvastatin, lovastatin, pravastatin, C-B; von Willebrand factor (VWF), which circulates in the or simvastatin. Exemplary ADP receptor inhibitors suitable blood and binds to its receptor GPIb-IX-V; thrombin, prima for use in accordance with the presently disclosed treatment rily through cleavage of the extracellular domain of PAR1 and methods include, e.g., clopidogrel, ditazole, pirozadil, Sar PAR4; thromboxane A (TXA), which binds to its receptor, pogrelate, and ticlopidine. Exemplary thrombin inhibitors TP-R; ADP through action on its two cell surface receptors, suitable for use in accordance with the presently disclosed P2Y and P2Y, adrenaline through its receptor (alpha 2): treatment methods include, e.g., argatroban, dermatan, serotonin, through its receptor (5HT-2c); human neutrophil desirudin, efegatran, inogatran, lepirudin, melagatran, elastase (HNE), which cleaves the C-B integrin on the mesoglycan, PEG-r-hirudin, and reviparin. Factor Xa inhibi platelet surface; and P-selectin, which is exposed on the sur tors include, for example, danaparoid, fondaparinux, and tifa face of activated platelets and which binds PSGL-1 on endot cogin. Exemplary purinergic receptor agonists suitable for helial cells and white blood cells. use in accordance with the presently disclosed treatment 0110. Likewise, a variety of endogenous molecules are methods include, e.g., adenosine and adenosine analogs, e.g., know to function as platelet inhibitors. Examples of endog 2-(N-pyrazolyl) derivatives of adenosine (e.g., CVT 3146), enous molecules known to inhibit platelet function include: and 2-propynylcyclohexyl-5'-N-ethylcarboxamido deriva prostacyclin, which opposes the actions of most if not all tives of adenosine (e.g., ATL-146e (4-(3-6-amino-9-(5-eth platelet agonists by increasing intracellular cAMP levels: ylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-pu adenosine, which signals through its receptor (A2 receptor) rin-2-yl)-prop-2-ynyl)-cyclohexanecarboxylic acid methyl on the surface of platelets and increases intracellular cAMP ester), and ATL-193. Exemplary antagonists of CD40 or levels; nitric oxide (NO), which is released by vascular endot CD40 ligand (CD40L) or compounds that disrupt (e.g., helial cells and activated platelets, increases cGMP levels in reduce) the interaction of CD40 and CD40L include mono platelets by directly activating the enzyme guanylyl cyclase in clonal antibodies (e.g., Antora and IDEC 131 and see US platelets; clotting factors II, IX, X, XI, XII; and nucleotidases, 2002/0031512 and WO01/34649), free CD40, and antisense such as CD39 ecto-ADPase, which hydrolyze ADP. nucleic acids. Exemplary eicosanoid related targets include US 2009/O124574 A1 May 14, 2009

alprostadil, beraprost, carbasalate, cloricromene, epoprost, tional, the following can result: vasoconstriction (e.g., coro etersalate, iloprost, indobufin, indometacin farnesil, nary vasospasm, elevated systemic vascular resistance, limaprost, OZagrel, pamicogrel, picotamide, ramatroban, ter hypertension); platelet aggregation and adhesion leading to bogrel, and triflusal. Exemplary glycoprotein IIb/IIIa antago thrombosis; upregulation of leukocyte and endothelial adhe nists for use in the methods and compositions of the invention sion molecules leading to enhanced inflammation; vascular include Xemilofiban, abciximab, cromafiban, elarofiban, Stenosis, or restenosis as occurs following balloon angio orbofiban, roxifiban, sibrafiban, RPR 1098.91, eptifibatide, plasty and stent placement, increased inflammation and tissue and tirofiban. damage mediated by reactive oxygen species such as Super 0113. The term “nitric oxide' (NO), as used herein, gen oxide anion and hydroxyl radical. erally refers to a chemical compound that is a key biological 0118. As used herein, the phrase “disorder associated with messenger, playing a role in a variety of biological processes. platelet aggregation' generally refers to medical disorders or Nitric oxide (NO) is produced by vascular endothelium and complications characterized by unwanted or excessive plate Smooth muscle, cardiac muscle, and many other cell types. let activation and or aggregation. Examples of such disorders The substrate for NO is L-arginine that is transported into the or conditions include, though are not limited to, thrombosis, cell. When acted upon by nitric oxide synthase (NOS), NO primary arterial thrombotic complications of atherosclerotic and citrulline are formed. There are two general forms of disease, thrombotic complications of interventions ofathero NOS: constitutive and inducible. NO is continuously pro Sclerotic disease, thrombotic complications of Surgical or duced by constitutive NO synthase (cNOS). The cMOS found mechanical damage, mechanically-induced platelet activa in endothelial cells is also referred as eNOS, ecNOS, or Type tion, shunt occlusion, thrombosis secondary to vascular dam III NOS. The activity of cMOS is modulated by calcium that age and inflammation, indications with a diffuse thrombotic/ is released from Subsarcolemmal storage sites in response to platelet consumption component, venous thrombosis, the binding of certain ligands to their receptors. Substances coronary arterial thrombosis, pathological effects of athero Such as acetylcholine, bradykinin, histamine, insulin, and Sclerosis and arteriosclerosis, platelet aggregation and clot substance P stimulate NO production by this mechanism. formation in blood and blood products during storage, Another important mechanism regulating the release of NO is chronic or acute states of hyper-aggregability, reocclusion of shearing forces acting on the luminal Surface of vascular an artery or vein following fibrinolytic therapy, platelet adhe endothelium. By this mechanism, increased flow velocity sion associated with extracorporeal circulation, thrombotic stimulates calcium release and increased cnOS activity. The complications associated with thrombolytic therapy, throm inducible form of NOS (iNOS, or Type II NOS) is not cal botic complications associated with coronary and other cium-dependent, but instead is stimulated by the actions of angioplasty, or thrombotic complications associated with cytokines (e.g., tumor necrosis factor, interleukins) and bac coronary artery bypass procedures. Additional medical or terial endotoxins (e.g., lipopolysaccharide). Induction of Surgical procedures that may cause unwanted platelet aggre iNOS occurs over several hours and results in NO production gation include, for example, cardiac interventional proce that may be more than a 1,000-fold greater than that produced dures, tissue or organ transplantation and angioplastic proce by cnOS. This is an important mechanism in the pathogen dures. These procedures include, without limitation, esis of inflammation. percutaneous transluminal coronary angioplasty with or 0114 Nitric oxide serves many important functions in the without placement of an intracoronary Stent, cardiac bypass cardiovascular system, including but not limited to: Vasodi Surgery, hemodialysis, extra-corporeal circulation associated lation (ligand mediated and flow dependent); inhibition of with a Surgical procedure, intracranial angioplasty, and vasoconstrictor influences (e.g., inhibits angiotensin II and angioplasty on peripheral arteries. Additional medical condi sympathetic vasoconstriction); inhibition of platelet adhesion tions that are amendable to treatment in accordance with the to the vascular endothelium (anti-thrombotic); inhibition of presently described methods an compositions include, with leukocyte adhesion to vascular endothelium (anti-inflamma out limitation, sickle cell anemia crisis, heparin-induced tory); antiproliferative (e.g., inhibits Smooth muscle hyper thrombotic thrombocytopenia (HITT), idiopathic thrombotic plasia following vascular injury); and Scavenging Superoxide thrombocytopenia (ITTP), stroke, atherosclerosis, angiogen anion (anti-inflammatory). esis, thrombosis, thromboembolic conditions such as deep 0115 The mechanism of many of these actions of NO venous thrombosis, pulmonary embolism or thrombophlebi involves the formation of c(GMP. When NO is formed by an tis, disseminated intravascular coagulation or thromboembo endothelial cell, for example, it readily diffuses out of the cell lic syndromes associated with cancer, sepsis, or obstetrical and into adjacent Smooth muscle cells where it binds to and complications, peripheral arterial occlusive disease, acute stimulates the enzyme guanylyl cyclase to c(GMP. Increased coronary syndromes such as unstable angina and myocardial cGMP activates a kinase that subsequently leads to the inhi infarction, diabetes, or tissue damage caused by phospholi bition of calcium influx into the smooth muscle cell, and pases A (PLA). decreased calcium-calmodulin stimulation of myosin light 0119) The phrase “thrombolytic therapy” or “thromboly chain kinase (MLCK). This in turn decreases the phosphory sis” refers to a medical treatment or intervention that is per lation of myosin light chains, thereby decreasing Smooth formed to promote the pharmacologic dissolution of a clot or muscle tension development and causing vasodilation. a thrombus in a blood vessel. Thrombolytic therapy works by 0116. The anti-platelet aggregatory effects of NO are also stimulating fibrinolysis by plasmin through infusion of ana related to the increase incCMP Drugs that inhibit the break logs of tissue plasminogen activator, the protein that normally down of c(GMP (inhibitors of c(3MP-dependent phosphodi activates plasmin. Formation of blood clots lies at the basis of esterase such as sildenafil) potentiate the effects of NO-me a number of serious diseases (see below). By breaking down diated actions on the target cell. the clot, the disease process can be arrested, or the complica 0117. When NO production is impaired as occurs when tions reduced. While other anticoagulants (such as heparin) the vascular endothelium becomes damaged or dysfunc decrease the “growth of a clot, thrombolytic agents actively US 2009/O124574 A1 May 14, 2009

reduce the size of the clot. Disorders for which thrombolytic by contacting the free base form with a sufficient amount of therapy may be indicated include disorders involving venous the desired acid to produce the salt in the conventional man thromboses such as, e.g., Deep venous thrombosis (with or ner. The free base form may be regenerated by contacting the without pulmonary embolism; together classified as venous salt form with a base, and isolating the free base in the con thromboembolism/VTE), Portal vein thrombosis, Renal vein ventional manner. The free base forms differ from their thrombosis, hepatic vein thrombosis (Budd-Chiari syn respective salt forms somewhat in certain physical properties drome), Paget-Schroetter disease (upper extremity vein), such as solubility in polar solvents, but otherwise the salts are Thoracic outlet syndrome, and disorders involving arterial equivalent to their respective free base for purposes of the thromboses Such as, e.g., stroke (either thrombotic or embo present invention. Pharmaceutically acceptable base addition lic), myocardial infarction (usually coronary thrombosis due salts are formed with metals or amines, such as alkali and to rupture of an atherosclerotic plaque), and thoracic outlet alkaline earth metal hydroxides, or of organic amines. syndrome (may precipitate arterial thrombosis as well as Examples of metals used as cations are sodium, potassium, venous). magnesium, calcium, and the like. Examples of Suitable 0120. Thrombolytic agents work by activating the enzyme amines are N,N'-dibenzylethylenediamine, chloroprocaine, plasminogen, which clears the cross-linked fibrin mesh (the choline, diethanolamine, ethylenediamine, N-methylglu backbone of a clot). This makes the clotsoluble and subject to camine, and procaine; see, for example, Berge et al., Supra. further proteolysis by other enzymes, and restores blood flow 1977. The base addition salts of acidic compounds are pre over occluded blood vessels. Thrombolysis requires the use pared by contacting the free acid form with a sufficient of thrombolytic drugs, which are either derived from Strep amount of the desired base to produce the salt in the conven tomyces spp. or (more recently) the effect of recombinant tional manner. The free acid form may be regenerated by technology, where human activators of plasminogen (e.g. contacting the salt form with an acid and isolating the free tissue plasminogen activator, tRA) are manufactured by bac acidina conventional manner. The free acid forms differ from teria. Some commonly used thrombolytics include streptoki their respective salt forms somewhat in certain physical prop nase, urokinase, alteplase (recombinant tissue plasminogen erties such as solubility in polar solvents, but otherwise the activator or rtPA), reteplase, and tenecteplase. salts are equivalent to their respective free acid for purposes 0121 The phrase “pharmaceutically acceptable' is of the present invention. Lists of suitable salts are found in employed herein to refer to those compounds, materials, Remington's Pharmaceutical Sciences, 17th ed., Mack Pub compositions, and/or dosage forms which are, within the lishing Company, Easton, Pa., 1985, p. 1418, the disclosure of scope of sound medical judgment, suitable for use in contact which is hereby incorporated by reference. with the tissues of human beings and animals without exces I0123. The phrase “combination therapy” (or "co sive toxicity, irritation, allergic response, or other problem or therapy'), as used herein embraces the administration of one complication, commensurate with a reasonable benefit/risk or more carotenoid analogs or derivatives, and of one or more ratio. additional agents suitable for the treatment of a disorder asso 0122. As used herein, “pharmaceutically acceptable salts' ciated with plateletaggregation, as part of a specific treatment refer to derivatives of the disclosed compounds wherein the regimen intended to provide a beneficial effect from the co parent compound is modified by making acid or base salts action of these therapeutic agents. The beneficial effect of the thereof. Examples of pharmaceutically acceptable salts combination includes, but is not limited to, pharmacokinetic include, but are not limited to, mineral or organic acid salts of or pharmacodynamic co-action resulting from the combina basic residues such as amines; alkali or organic salts of acidic tion of therapeutic agents. Administration of these therapeu residues such as carboxylic acids; and the like. The pharma tic agents in combination typically is carried out over a ceutically acceptable salts include the conventional non-toxic defined time period (usually minutes, hours, days or weeks salts or the quaternary ammonium salts of the parent com depending upon the combination selected). The term is pound formed, for example, from non-toxic inorganic or intended to embrace administration of these therapeutic organic acids. Pharmaceutically acceptable acid addition agents in a sequential manner, that is, wherein each therapeu salts of the compounds of the invention include salts derived tic agent is administered at a different time, as well as admin forminorganic acids such as hydrochloric, nitric, phosphoric, istration of these therapeutic agents, or at least two of the Sulfuric, hydrobromic, hydroiodic, phosphorus, and the like, therapeutic agents, in a Substantially simultaneous manner. as well as the salts derived from organic acids, such as ali Substantially simultaneous administration can be accom phatic mono- and dicarboxylic acids, phenyl-substituted plished, for example, by administering to the Subject a phar alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, maceutical preparation having a fixed ratio of each therapeu aromatic acids, aliphatic and aromatic Sulfonic acids, etc. tic agent or in multiple preparations for each of the Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, therapeutic agents. Sequential or Substantially simultaneous bisulfite, nitrate, phosphate, monohydrogenphosphate, dihy administration of each therapeutic agent can be effected by drogenphosphate, metaphosphate, pyrophosphate, chloride, any appropriate route including, but not limited to, oral bromide, iodide, acetate, propionate, caprylate, isobutyrate, routes, intravenous routes, intramuscular routes, and direct oxalate, malonate. Succinate, Suberate, sebacate, fumarate, absorption through mucous membrane tissues. The therapeu maleate, mandelate, benzoate, chlorobenzoate, methylben tic agents can be administered by the same route or by differ Zoate, dinitrobenzoate, phthalate, benzenesulfonate, toluene ent routes. For example, a first therapeutic agent of the com Sulfonate, phenylacetate, citrate, lactate, maleate, tartrate, bination selected may be administered by intravenous methanesulfonate, and the like. Also contemplated are the injection while the other therapeutic agents of the combina salts of amino acids such as arginate, gluconate, galactur tion may be administered orally. Alternatively, for example, onate, and the like; see, for example, Berge et al., “Pharma all therapeutic agents may be administered orally or all thera ceutical Salts. J. of Pharmaceutical Science, 1977; 66:1 19. peutic agents may be administered by intravenous injection. The acid addition salts of the basic compounds are prepared The sequence in which the therapeutic agents are adminis US 2009/O124574 A1 May 14, 2009

tered is not narrowly critical. “Combination therapy' also can combinations thereof. Included in a pharmaceutical prepara embrace the administration of the therapeutic agents as tion may be one or more carriers, preservatives, flavorings, described above in further combination with other biologi excipients, coatings, stabilizers, binders, solvents and/or aux cally active ingredients (such as, but not limited to, additional iliaries that are, typically, pharmacologically inert. It will be pharmacologic agents) and non-drug therapies (such as, but readily appreciated by an ordinary practitioner of the art that, not limited to, Surgery or radiation treatment). included within the meaning of the term are pharmaceutically 0124. As used herein, the term “inhibitor, when used in acceptable salts of compounds. It will further be appreciated the context of receptor inhibitor, such as in “receptor antago by an ordinary practitioner of the art that the term also encom nist, generally refers to a molecule that binds to a specific passes those pharmaceutical compositions that contain an receptor and decrease their the activity of that receptor by at admixture of two or more pharmacologically active com least 5%, at least 15%, at least 30%, at least 50% or at least pounds. Such compounds being administered, for example, as 75%. Many drug molecules are receptor antagonists so their a combination therapy. discovery and improvement is an active area of research in I0127. As used herein the terms “subject' generally refers biochemistry and pharmacology. A medicinal receptor to a mammal, and in particular to a human. antagonist is often judged by its specificity (its lack of binding 0128. The terms “in need of treatment,” “in need thereof.” to other proteins) and its potency (its dissociation constant, “who would benefit from such treatment or the like when which indicates the concentration needed to inhibit the used in the context of a subject being administered a pharma enzyme). A high specificity and potency ensure that a drug cologically active composition, generally refers to a judg will have few side effects and thus low toxicity. ment made by an appropriate healthcare provider that an 0125. As used herein the terms “administration.” “admin individual or animal requires or will benefit from a specified istering or the like, when used in the context of providing a treatment or medical intervention. Such judgments may be pharmaceutical or nutraceutical composition to a Subject gen made based on a variety of factors that are in the realm of erally refers to providing to the Subject one or more pharma expertise of healthcare providers, but include knowledge that ceutical, “over-the-counter (OTC) or nutraceutical compo the individual or animal is ill, will be ill, or is at risk of sitions in combination with an appropriate delivery vehicle by becoming ill, as the result of a condition that may be amelio any means such that the administered compound achieves one rated or treated with the specified medical intervention. or more of the intended biological effects for which the com I0129. The phrases “therapeutically effective amount” and pound was administered. By way of non-limiting example, a “effective amount” are synonymous unless otherwise indi composition may be administered parenteral, subcutaneous, cated, and mean an amount of a compound of the present intravenous, intracoronary, rectal, intramuscular, intra-peri invention that is Sufficient to improve the condition, disease, toneal, transdermal, or buccal routes of delivery. Alterna or disorder being treated. Determination of a therapeutically tively, or concurrently, administration may be by the oral effective amount, as well as other factors related to effective route. The dosage administered will be dependent upon the administration of a compound of the present invention to a age, health, weight, and/or disease state of the recipient, kind patient in need of treatment, including dosage forms, routes of concurrent treatment, ifany, frequency of treatment, and/or of administration, and frequency of dosing, may depend upon the nature of the effect desired. The dosage of pharmacologi the particulars of the condition that is encountered, including cally active compound that is administered will be dependent the patient and condition being treated, the severity of the upon multiple factors, such as the age, health, weight, and/or condition in a particular patient, the particular compound disease state of the recipient, concurrent treatments, if any, being employed, the particular route of administration being the frequency of treatment, and/or the nature and magnitude employed, the frequency of dosing, and the particular formu of the biological effect that is desired. lation being employed. Determination of a therapeutically 0126. As used herein, terms such as “pharmaceutical com effective treatment regimen for a patient is within the level of position.” “pharmaceutical formulation.” “pharmaceutical ordinary skill in the medical or veterinarian arts. In clinical preparation, or the like, generally refer to formulations that use, an effective amount may be the amount that is recom are adapted to deliver a prescribed dosage of one or more mended by the U.S. Food and Drug Administration, or an pharmacologically active compounds to a cell, a group of equivalent foreign agency. The amount of active ingredient cells, an organ or tissue, an animal or a human. Methods of that can be combined with the carrier materials to produce a incorporating pharmacologically active compounds into single dosage form varies depending upon the mammalian pharmaceutical preparations are widely known in the art. The host treated and the particular mode of administration. determination of an appropriate prescribed dosage of a phar 0.130 By “prophylactically effective amount” is meant an macologically active compound to include in a pharmaceuti amount of a pharmaceutical composition that will Substan cal composition in order to achieve a desired biological out tially prevent, delay or reduce the risk of occurrence of the come is within the skill level of an ordinary practitioner of the biological or physiological event in a cell, a tissue, a system, art. A pharmaceutical composition may be provided as Sus animal or human that is being sought by a researcher, Veteri tained-release or timed-release formulations. Such formula narian, physician or other caregiver. tions may release abolus of a compound from the formulation I0131 The term “pharmacologically inert,” as used herein, at a desired time, or may ensure a relatively constant amount generally refers to a compound, additive, binder, vehicle, and of the compound present in the dosage is released over a given the like, that is Substantially free of any pharmacologic or period of time. Terms such as “sustained release.” “controlled “drug-like activity. release,” or “timed release' and the like are widely used in the 0.132. A “pharmaceutically or nutraceutically acceptable pharmaceutical arts and are readily understood by a practi formulation as used herein, generally refers to a non-toxic tioner of ordinary skill in the art. Pharmaceutical preparations formulation containing a predetermined dosage of a pharma may be prepared as Solids, semi-solids, gels, hydrogels, liq ceutical and/or nutraceutical composition, wherein the dos uids, Solutions, Suspensions, emulsions, aerosols, powders, or age of the pharmaceutical and/or nutraceutical composition is US 2009/O124574 A1 May 14, 2009 adequate to achieve a desired biological outcome. The mean generation in vascular endothelial cells. Thus, agents that ing of the term may generally include an appropriate delivery directly stimulate NO release may have important therapeutic vehicle that is suitable for properly delivering the pharmaceu advantages in the prevention and treatment of cardiovascular tical composition in order to achieve the desired biological disease. OutCOme. 0.138 Astaxanthin, a naturally-occurring hydrophobic 0133. As used herein the term “antioxidant may be gen C40 carotenoid, is a potent physical quencher of singlet oxy erally defined as any of various Substances (as beta-carotene, gen and chain-breaking antioxidant with favorable physico vitamin C, and C-tocopherol) that inhibit oxidation or reac chemical properties. Applicant has recently demonstrated tions promoted by Reactive Oxygen Species (ROS) and other distinct benefits for homochiral (3S,3'S)-astaxanthin in the radical and non-radical species. reduction of the pro-oxidant stress induced by the presence of 0134. As used herein the term “co-antioxidant may be rofecoxib (VIOXXTM) in model cellular membranes (see U.S. generally defined as an antioxidant that is used and that acts in patent application Ser. No. 1 1/417.30, which is commonly combination with another antioxidant (e.g., two antioxidants owned with the instant application and hereby fully incorpo that are chemically and/or functionally coupled, or two anti rated by reference). Applicant has also recently provided oxidants that are combined and function with each another in evidence Suggesting that non-esterified astaxanthin acts as a a pharmaceutical preparation). The effects of co-antioxidants selective inhibitor of the coMP-hydrolyzing enzyme PDE5A may be additive (i.e., the anti-oxidative potential of one or by binding to the active site of the enzyme. more anti-oxidants acting additively is approximately the 0.139. When derivatized according to retrometabolic drug Sum of the oxidative potential of each component anti-oxi design principles, novel carotenoid esters retain the ability to dant) or synergistic (i.e., the anti-oxidative potential of one or form the carotenoid radical cation, nearly identical to the more anti-oxidants acting synergistically may be greater than parent astaxanthin compound. The disodium disuccinate the Sum of the oxidative potential of each component anti diesters and the tetrahydrochloride dilysinate esters retain oxidant). and improve the potent Superoxide anion scavenging ability 0135 Compounds described herein embrace isomers mix of astaxanthin itself. The antioxidant properties carotenoids, tures, racemic, optically active, and optically inactive Stere including synthetic analogs and derivatives thereof, may be oisomers and compounds. exploited in platelet activation, if levels of NO could be enhanced in the presence of therapeutic concentrations of Platelet Aggregation: these potent compounds. Moreover, this activity may be 0.136 Platelet activation occurs in three stages: (1) expo increased further still in the presence of other platelet inhibi Sure to a platelet agonist; (2) generation of second messen tors (e.g., antiplatelet agents). gers; and (3) initiation of a response cascade that includes 0140. Thienopyridines, such as clopidogrel, effectively cytoskeletal rearrangements, shape change, storage granule interfere with platelet activation by selectively and irrevers release, and ultimately, platelet aggregation. Many factors ibly blocking a subunit of the adenosine diphosphate (ADP) can initiate platelet activation including arachidonic acid receptor (P2Y receptor). This provides a potent antiplatelet (AA), collagen, plasma protein, thrombin, hormones and effect that is additive to the inhibition of the thromboxane A2 products of platelet metabolism, including adenosine diphos pathway by aspirin. Thus, the combination of clopidogrel phate (ADP) and thromboxane A2. In normal (physiologic) with agents that have complementary benefits on platelet platelets, the initial stimulus by platelet activators is followed function is a standard, and clinically relevant, approach to by the release of nitric oxide (NO), an inhibitor of platelet reducing atherothrombotic risk. aggregation. Dysfunctional platelets, by contrast, generate 0.141. In accordance with the above, and without being nitroxidative stress (increased levels of peroxynitrite instead limited to any one particular theory or mechanism of action, of normal NO levels)—which is followed by platelet aggre it is Applicant's belief that carotenoids and synthetic analogs gation and adhesion to the endothelium of the vasculature. and derivatives thereof, such as, e.g., homochiral (3S.3'S)- Therefore, the capability of platelets to maintain a normal astaxanthin, may increase NO bioavailability in vascular balance between NO and oxidative stress is critical for the platelets by reducing sources of oxidative stress and peroX maintenance of normal vascular homeostasis. Such function ynitrite formation. It is also Applicant's belief that caro is essential for preventing thrombotic events that are associ tenoids and synthetic analogs and derivatives thereof may ated with stroke, heart attack, and peripheral arterial throm potentiate the bioactivity of NO in platelets, at least in part by bosis. inhibiting PDE5A and prolonging the amplitude and duration 0.137 In addition to platelets, the vascular endothelium of c(GMP signaling in platelets. It is Applicant's belief further modulates vascular tone and platelet function through the still, that the effects of astaxanthin may be enhanced by the release of NO, a potent vasodilator that regulates regional presence of antiplatelet agents such as clopidogrel, due to its blood flow. A reduction in NO bioavailability contributes to complementary physicochemical and pharmacologic proper elevated vascular resistance and loss of sensitivity to stimuli ties. of vasodilation, hallmark features of hypertension. Reversal 0.142 Homochiral astaxanthin and clopidogrel were of these pathological effects with antioxidants validates the tested alone, as well as in combination, to determine the main mechanistic underpinnings of nitric oxide bioavailability in effects of each compound on NO bioavailability and the the vasculature. Beyond vasodilation, NO has well character potential interaction of the 2 complementary agents. The ized vascular benefits, including inhibition of smooth muscle combination of homochiral astaxanthin and clopidogrel cell proliferation and migration, and reduction in the adhesion enhanced the ability of vascular platelets to release NO in a and transmigration of leukocytes. In patients at higher risk for highly synergistic fashion, with both the kinetics and magni cardiovascular disease and its clinical consequences, such as tude of NO release improved by the combination of drugs. African Americans, there is evidence for reduced NO-medi The current results indicate a novel approach to reducing ated vasodilation associated with increased Superoxide anion atherothrombotic risk with clopidogrel that extends beyond US 2009/O124574 A1 May 14, 2009 20

ADP inhibition, and to Applicant's knowledge, represents the Such as deep venous thrombosis, pulmonary embolism or first demonstration of the utility of astaxanthin in favorable thrombophlebitis, disseminated intravascular coagulation or modulation of platelet activation. thromboembolic syndromes associated with cancer, sepsis, 0143. It is an object of the present application to provide a or obstetrical complications, peripheral arterial occlusive dis method of preventing or treating disorders or conditions asso ease, acute coronary syndromes Such as unstable angina and ciated with platelet aggregation; Such diseases include myocardial infarction, diabetes, or tissue damage caused by venous thrombosis, thrombophlebitis, arterial embolism, phospholipases A (PLA). coronary and cerebral arterial thrombosis, unstable angina, 0145. In one embodiment, uses of carotenoid analogs or myocardial infarction, stroke, cerebral embolism, kidney derivatives, including pharmaceutically acceptable salts embolisms and pulmonary embolisms. The method is also thereof, in applications directed to preventing or treating dis directed to a method of preventing, treating or reducing the orders or conditions associated with platelet aggregation may incidence of thrombosis, thrombotic events, embolic events include the preparation of a pharmaceutical composition Suit orpathological conditions associated with Such events, where able for Such treatment. The pharmaceutical compositions the thrombosis, thrombotic event or embolic event occurs may typically include one or more carotenoid analogs or during or after Surgery. derivatives in an amount Sufficient to increase the biological 0144. Thus, it is an object of the present application to availability of nitric oxide in a subject administered such a provide a method of preventing or treating disorders or con composition. ditions associated with platelet aggregation; such diseases 0146 In one embodiment, uses of carotenoid analogs or include venous thrombosis, thrombophlebitis, arterial embo derivatives, including pharmaceutically acceptable salts lism, coronary and cerebral arterial thrombosis, unstable thereof, in applications directed to preventing or treating dis angina, myocardial infarction, stroke, cerebral embolism, orders or conditions associated with platelet aggregation in a kidney embolisms and pulmonary embolisms. The method is Subject may include administering to a subject having need also directed to a method of preventing, treating or reducing for such treatment a therapeutically effective amount of a the incidence of thrombosis, thrombotic events, embolic pharmaceutically acceptable composition comprising a caro events or pathological conditions associated with Such events, tenoid analog or derivative. where the thrombosis, thrombotic event or embolic event 0.147. In an embodiment, uses of carotenoid analogs or occurs during or after Surgery. It is a further object of the derivatives, including pharmaceutically acceptable salts present application to provide pharmaceutical compositions thereof, in applications directed to preventing or treating dis suitable for use in the treatment methods described herein. In orders or conditions associated with platelet aggregation in a Some embodiments, specific disorders or conditions associ Subject in a Subject may include the preparation of pharma ated with platelet aggregation that are amenable to treatment ceutical compositions for use with additional pharmaceutical in accordance with the present disclosure may include, compositions which, when co-administered, act synergisti though are not limited to, thrombosis, primary arterial throm cally to prevent or treat disorders or conditions associated botic complications of atherosclerotic disease, thrombotic with platelet aggregation in a subject. complications of interventions of atherosclerotic disease, 0.148. In one embodiment, uses of carotenoid analogs or thrombotic complications of Surgical or mechanical damage, derivatives, including pharmaceutically acceptable salts mechanically-induced platelet activation, shunt occlusion, thereof, in applications directed to preventing or treating dis thrombosis secondary to vascular damage and inflammation, orders or conditions associated with platelet aggregation in a indications with a diffuse thrombotic/platelet consumption Subject may include the preparation of pharmaceutical com component, venous thrombosis, coronary arterial thrombo positions having at least one carotenoid analog or derivative, sis, pathological effects of atherosclerosis and arteriosclero in addition to at least one additional composition or medica sis, platelet aggregation and clot formation in blood and blood ment Suitable for use as preventing or treating disorders or products during storage, chronic or acute states of hyper conditions associated with platelet aggregation in a Subject, aggregability, reocclusion of an artery or vein following including but not limited to one or more antiplatelet agents. fibrinolytic therapy, platelet adhesion associated with extra Exemplary though non-limiting antiplatelet agents Suitable corporeal circulation, thrombotic complications associated for use in the presently described embodiments include: anti with thrombolytic therapy, thrombotic complications associ coagulants; statins; ADP receptor inhibitors (e.g., thienopy ated with coronary and otherangioplasty, or thrombotic com ridines); thrombin inhibitors; factor Xa inhibitors; agonists of plications associated with coronary artery bypass procedures. purinergic receptors; antagonists of CD40 or CD40 ligand Additional medical or Surgical procedures that may cause (CD40L) or compounds that disrupt (e.g., reduce) the inter unwanted platelet aggregation include, for example, cardiac action of CD40 and CD40L, eicosanoid related targets (e.g., interventional procedures, tissue or organ transplantation and COX inhibitors, PGE1 agonists, PG synthase inhibitors, TX angioplastic procedures. These procedures include, without synthase inhibitors, and TXA2 antagonists); and glycopro limitation, percutaneous transluminal coronary angioplasty tein IIb/IIIa antagonists. Exemplary anticoaglulants Suitable with or without placement of an intracoronary stent, cardiac for use in accordance with the presently disclosed treatment bypass Surgery, hemodialysis, extra-corporeal circulation methods include, e.g., aspirin, warfarin, a combination of associated with a Surgical procedure, intracranial angioplasty, aspirin and warfarin. Exemplary non-fractionated or fraction and angioplasty on peripheral arteries. Additional medical ated heparins may include low molecular weight heparins, conditions that are amendable to treatment in accordance Such as ardeparin, certoparin, dalteparin, enoxaparin, nadro with the presently described methods and compositions parin, reviparin, or tinaZaparin, and those compounds include, without limitation, sickle cell anemia crisis, heparin described in WO97/35592. Exemplary statins suitable foruse induced thrombotic thrombocytopenia (HITT), idiopathic in accordance with the presently disclosed treatment methods thrombotic thrombocytopenia (ITTP), stroke, atherosclero include, e.g., atorvastatin, fluvastatin, lovastatin, pravastatin, sis, angiogenesis, thrombosis, thromboembolic conditions or simvastatin. Exemplary ADP receptor inhibitors suitable US 2009/O124574 A1 May 14, 2009

for use in accordance with the presently disclosed treatment absorption, metabolism, pharmacokinetics; etc in accordance methods include, e.g., clopidogrel, ditazole, pirozadil, sar with standard pharmaceutical practice. pogrelate, and ticlopidine. Exemplary thrombin inhibitors suitable for use in accordance with the presently disclosed Combination Therapy and Treatments: treatment methods include, e.g., argatroban, dennatan, 0152. As described above, the presently contemplated desirudin, efegatran, inogatran, lepirudin, melagatran, treatment methods and compositions are not limited solely to mesoglycan, PEG-r-hirudin, and reviparin. Factor Xa inhibi the administration of a formulation containing synthetic caro tors include, for example, danaparoid, fondaparinux, and tifa tenoid analogs and derivatives as the Sole medically active cogin. Exemplary fractionated heparins include low molecu component. On the contrary, equally contemplated are com lar weight heparins, such as ardeparin, certoparin, dalteparin, positions and methods in which the Subject carotenoid ana enoxaparin, nadroparin, reviparin, or tinazaparin. Exemplary logs or derivatives may be administered in conjunction with purinergic receptor agonists Suitable for use in accordance one or more additional compositions or medicaments Suitable with the presently disclosed treatment methods include, e.g., for use in preventing or treating disorders or conditions asso adenosine and adenosine analogs, e.g., 2-(N-pyrazolyl) ciated with platelet aggregation. Typically, Such agents will derivatives of adenosine (e.g., CVT 3146), and 2-propynyl cyclohexyl-5'-N-ethylcarboxamido derivatives of adenosine include at least one antiplatelet agent. (e.g., ATL-146e (4-(3-6-amino-9-(5-ethylcarbamoyl-3,4-di 0153. To use the present invention in combination with the hydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl)-prop-2- administration of a one or more additional compositions or medicaments suitable for use in preventing or treating disor ynyl)-cyclohexanecarboxylic acid methyl ester), and ATL ders or conditions associated with platelet aggregation, one 193. Exemplary antagonists of CD40 or CD40 ligand may simply administer to a Subject a carotenoid analog or (CD40L) or compounds that disrupt (e.g., reduce) the inter derivative composition in combination with the second com action of CD40 and CD40L include monoclonal antibodies position (i.e., an antiplatelet agent) in a manner effective to (e.g., Antora and IDEC 131 and see US 2002/0031512 and result in their combined or synergistic actions within the WOO1/34649), free CD40, and antisense nucleic acids. subject. These agents would, therefore, be provided in an Exemplary eicosanoid related targets include alprostadil, amount effective and for a period of time effective to result in beraprost, carbasalate, cloricromene, epoprost, etersalate, their combined presence within the cardiovascular system of iloprost, indobufin, indometacin farnesil, limaprost, oZagrel, the Subject. To achieve this goal, the carotenoid analog or pamicogrel, picotamide, ramatroban, terbogrel, and triflusal. derivative composition and one or more additional composi Exemplary glycoprotein IIb/IIIa antagonists for use in the tions or medicaments may be administered to the subject methods and compositions of the invention include simultaneously, either in a single composition, or as two Xemilofiban, abciximab, cromafiban, elarofiban, orbofiban, distinct compositions using different administration routes. roxifiban, sibrafiban, RPR 109891, eptifibatide, and tirofiban. 0154. In embodiments where the one or more additional 0149. In an embodiment, carotenoids or synthetic deriva compositions or medicaments and carotenoid analog or tives or analogs thereof may be administered to a subject derivative composition are administered separately to the concurrently with at least one additional composition or Subject, one would generally ensure that a significant period medicament Suitable for use as preventing or treating disor of time did not expire between the time of each delivery, such ders or conditions associated with platelet aggregation. In an that the one or more additional compositions or medicaments embodiment, carotenoids or synthetic derivatives or analogs and carotenoid analog or derivative composition would still thereof may be administered to a subject prior to the com be able to exert an advantageously combined effect on the mencement of drug therapy with the one or more composi cardiovascular system. In Such instances, it is contemplated tions or medicaments Suitable for use as preventing or treating that one would contact platelets with both agents within about disorders or conditions associated with platelet aggregation. 5 minutes to about one week of each other and, more prefer In an embodiment, carotenoids or synthetic derivatives or ably, within about 12-72 hours of each other, with a delay time analogs thereof may be administered to a Subject following of only about 24–48 hours being most preferred. In some the commencement of therapy with the one or more addi situations, it may be desirable to extend the time period for tional compositions or medicaments suitable for use as pre treatment significantly, where several days (2, 3, 4, 5, 6 or 7) venting or treating disorders or conditions associated with or even several weeks (1,2,3,4, 5, 6, 7 or 8) lapse between the platelet aggregation. respective administrations. It also is conceivable that more 0150. The carotenoids or synthetic derivatives or analogs than one administration of either the carotenoid analog or thereof may be provided in a single pharmaceutical prepara derivative or the one or more additional compositions or tion together with at least one additional composition or medicaments will be desired. To achieve optimal inhibition of medicament Suitable for use as preventing or treating disor platelet aggregation, both agents may be delivered in a com ders or conditions associated with platelet aggregation. Alter bined amount effective to inhibit platelet aggregation, irre natively, the carotenoids or synthetic derivatives or analogs spective of the times for administration. thereof may be provided to a subject in a pharmaceutical 0155 Salts encompassed within the term “pharmaceuti preparation that is distinct from that which includes the one or cally acceptable salts' refer to non-toxic salts of the com more additional compositions or medicaments suitable for pounds which are generally prepared by reacting a free base use as preventing or treating disorders or conditions associ with a suitable organic or inorganic acid or by reacting the ated with platelet aggregation. acid with a suitable organic or inorganic base. Particular 0151. The suitability of any particular composition or mention may be made of the pharmaceutically acceptable medicament for use as treatment for a disorder associated inorganic and organic acids customarily used in pharmacy. with platelet aggregation can be readily determined by evalu Those suitable are in particular water-soluble and water-in ation of its potency and selectivity using methods known to soluble acid addition salts with acids such as, for example, those skilled in the art, followed by evaluation of its toxicity, hydrochloric acid, hydrobromic acid, phosphoric add, nitric US 2009/O124574 A1 May 14, 2009 22 acid, Sulfuric acid, acetic acid, citric acid, D-gluconic acid, tional composition suitable for the treatment of a disorder benzoic acid, 2-(4-hydroxybenzoyl)-benzoic acid, butyric associated with platelet aggregation are also contemplated acid, Sulfosalicylic acid, maleic acid, lauric acid, malic acid, herein. Such kits will generally contain, in Suitable container fumaric acid, Succinic acid, oxalic acid, tartaric add, embonic means, a pharmaceutically acceptable formulation of at least add, Stearic add, toluenesulfonic acid, methanesulfonic acid or 1-hydroxy-2-naphthoic add, the adds being employed in one carotenoid analog or derivative compound. The kits also salt preparation—depending on whether it is a mono- or may contain other pharmaceutically acceptable formulations, polybasic acid and depending on which salt is desired in an Such as those containing components to target the carotenoid equimolar quantitative ratio or one differing therefrom. analog or derivative compounds to distinct regions of a 0156. As examples of salts with bases are mentioned the patient where treatment is needed, or any one or more of a lithium, Sodium, potassium, calcium, aluminum, magnesium, range of drugs which may workin concert with the carotenoid titanium, ammonium; meglumine or guanidinium salts, here, analog or derivative compounds, for example, antiplatelet too, the bases being employed in salt preparation in an agents as described above. equimolar quantitative ratio or one differing therefrom. 0157. It is understood that the active compounds and their 0.161 The kits may have a single container means that pharmaceutically acceptable salts mentioned can also be contains the carotenoid analog or derivative compounds, with present, for example, in the form of their pharmaceutically or without any additional compositions or medicaments, or acceptable solvates, in particular in the form of their hydrates. they may have distinct container means for each desired com 0158. The pharmaceutical preparation may be adminis position. When the components of the kit are provided in one tered orally, in the form of a tablet, a capsule, an emulsion, a or more liquid solutions, the liquid solution is an aqueous liquid, or the like. Alternatively, the pharmaceutical prepara Solution, with a sterile aqueous solution being particularly tion may be administered via a parenteral route. A more preferred. However, the components of the kit may be pro detailed description of the types of pharmaceutical prepara vided as dried powder(s). When reagents or components are tions that are suitable for some embodiments is described in provided as a dry powder, the powder can be reconstituted by detail below. Some embodiments may be particularly suited the addition of a suitable solvent. It is envisioned that the timed or Sustained release pharmaceutical preparations, in Solvent also may be provided in another container means. The which the preparation is adapted to deliver a known dosage of container means of the kit will generally include at least one carotenoids or synthetic derivatives or analogs thereof at or vial, test tube, flask, bottle, Syringe or other container means, over a predetermined time. In an embodiment, a pharmaceu into which the medically active agent(s), and any other tical preparation may be a 'soft drug, in that the compound desired agent, may be placed and, preferably, Suitably ali is active in the derivatized state, and may yield a known quoted. Where additional components are included, the kit carotenoid after metabolic conversion in vivo. In an embodi will also generally contain a second vial or other container ment, a pharmaceutical preparation may be adapted to one into which these are placed, enabling the administration of drug, or a portion thereof, before delivering the second drug. separated designed doses. The kits also may comprise a sec For example, a pharmaceutical preparation may be adapted in ond/third container means for containing a sterile, pharma Such a way that at least a portion of the Xanthophyll caro ceutically acceptable buffer or other diluent. tenoid or structural analog or derivative thereof is released 0162 The kits also may contain a means by which to into the body of a subject before the additional compositions administer the pharmaceutical compositions to an animal or or medicaments are released. patient, e.g., one or more needles or syringes, or even an eye 0159. One or more of the additional compositions or medi dropper, pipette, or other Such like apparatus, from which the caments suitable for the treatment of a disorder associated formulation may be injected into the animal or applied to a with platelet aggregation for the uses presently contemplated diseased area of the body. The kits of the present invention may be formulated as a separate pharmaceutical composition will also typically include a means for containing the vials, or to be administered in conjunction with the Subject carotenoid Such like, and other component, in close confinement for analogs or derivatives as part of a therapeutic regimen, or may commercial sale, such as, e.g., injection or blow-molded plas be formulated in a single preparation together with the one or tic containers into which the desired vials and other apparatus more carotenoid analogs or derivatives. Such a composition are placed and retained. may be administered orally, parenterally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, Carotenoids and the Preparation and Use Thereof adjuvants, and vehicles as desired. Topical administration 0163. In some embodiments, a composition may include may involve the use of transdermal administration Such as one or more carotenoid analogs or derivatives, optionally in transdermal patches or iontophoresis devices. The term combination with one or more additional compositions or parenteral generally embraces non-oral routes of administra medicaments suitable for the treatment of a disorder associ tion, including but not limited to, Subcutaneous injections, ated with platelet aggregation. Carotenoid analogs and intravenous, intramuscular, intrasternal injection, or infusion derivative suitable for use in accordance with the may include techniques. Formulation of drugs is discussed in, for carotenoids having the general structure: example, Hoover, John E., Remington's Pharmaceutical Sci ences, Mack Publishing Co., Easton, Pa. 1975. Another dis cussion of drug formulations can be found in Liberman, H. A. and Lachman, L., Eds. Pharmaceutical Dosage Forms, Mar cel Decker, New York, N.Y., 1980. Therapeutic Kits: 0160 Therapeutic kits comprising the carotenoid analogs or derivatives, either alone or in combination with an addi

US 2009/O124574 A1 May 14, 2009 25

-continued -continued OMe O oal O O O | N s ---, O o O

MeO OMe O O O N--H

O O O n-n-so O O H O O N-1S-1N |-OMe, NH2.: NH2: o1 P\ O OMe O

O -- NH OH: pharmaceutically acceptable salts of any of these compounds, r n- where each R is independently H, alkyl, aryl, benzyl, Group O O IA metal, or co-antioxidant. O 0170 In some embodiments, carotenoid analogs or derivatives suitable for use with the present compositions, O NH2: methods and uses may have the structure NH2 O H O N OH --- R2 O NH2: NH2 (0171 where each Risindependently hydrogen or methyl, "S OH: and wherein each R' and Rare independently:

O O

O NH2: O NH2 O H O N OH

O NH2:

US 2009/O124574 A1 May 14, 2009 27

Alpha-tocopherol is used to designate when R'=R=CHs. 0182. In some embodiments, decreased antioxidant prop Beta-tocopherol is used to designate when R'=CH and erties associated with shorter polyene chains may be over R’—H. Gamma-tocopherol is used to designate when R'=H come by increasing the dosage administered to a Subject or and R=CH. Delta-tocopherol is used to designate when patient. R—R—H. 0183 In some embodiments, a chemical compound 0177. The second category of Vitamin E may include including a carotenoid derivative or analog may have the tocotrienols having a general structure general structure:

Each R'' may be independently hydrogen or methyl. Rand Alpha-tocotrienol is used to designate when R'=R=CHs. R" may be independently H, an acyclic alkene with one or Beta-tocotrienol is used to designate when R'=CH and more Substituents, or a cyclic ring including one or more R’—H. Gamma-tocotrienol is used to designate when R'=H Substituents. y may be 5 to 12. In some embodiments, y may and R=CH. Delta-tocotrienol is used to designate when be 3 to 15. In certain embodiments, the maximum value of y R—R—H. may only be limited by the ultimate size of the chemical 0.178 Quercetin, a flavonoid, has the structure compound, particularly as it relates to the size of the chemical compound and the potential interference with the chemical compound's biological availability as discussed herein. In OH Some embodiments, Substituents may be at least partially hydrophilic. These carotenoid derivatives may be included in OH. a pharmaceutical composition. 0184. In some embodiments, a method for treating a dis HO O order associated with platelet aggregation in a Subject may include administering to a subject who would benefit from Such treatment a therapeutically effective amount of a phar OH maceutically acceptable composition comprising a caro tenoid analog or derivative having the structure OH O

In some embodiments, one or more co-antioxidants may be coupled to a carotenoid or carotenoid derivative or analog. Derivatives of one or more carotenoid analogues may be formed by coupling one or more free hydroxy groups of the co-antioxidant to a portion of the carotenoid. 0179 Derivatives or analogs may be derived from any known carotenoid (naturally or synthetically derived). Spe (0185 where each Risindependently hydrogen or methyl, cific examples of naturally occurring carotenoids which com and where R' and Rare each independently: pounds described herein may be derived from include for

example zeaxanthin, lutein, lycophyll, astaxanthin, and lyco pene. 0180. In some embodiments, carotenoid analogs or derivatives may have increased water solubility and/or water dispersibility relative to some or all known naturally occur ring carotenoids. Contradictory to previous research, improved results are obtained with derivatized carotenoids relative to the base carotenoid, wherein the base carotenoid is R5 N derivatized with substituents including hydrophilic substitu ents and/or co-antioxidants. 0181. In some embodiments, the carotenoid derivatives R5 may include compounds having a structure including a poly ene chain (i.e., backbone of the molecule). The polyene chain R5 may include between about 5 and about 15unsaturated bonds. In certain embodiments, the polyene chain may include R N s between about 7 and about 12 unsaturated bonds. In some embodiments a carotenoid derivative may include 7 or more conjugated double bonds to achieve acceptable antioxidant R5 properties. US 2009/O124574 A1 May 14, 2009 28

-continued -continued R5 R3 R3 R3 2 O R3 R3 R3 R3

RO 0186 where each R is independently hydrogen, —CH, —OH, -CH-OH or -OR wherein at least one R group in s the carotenoid analog or derivative is —OR; wherein each 2^ R is independently: H; alkyl: aryl; -alkyl-N(R); -aryl-N (R); -alkyl-N-(R); -aryl-N'(R); -alkyl-CO.R., -aryl CO.R., -alkyl-CO.; -aryl-CO: C(O)-alkyl-N(R): —C(O)-aryl-N(R); —C(O)-alkyl-N-(R): C(O)-aryl RO N"(R); –C(O)-alkyl-COR: –C(O)-aryl-COR: O —C(O)-alkyl-CO.; —C(O)-aryl-CO.; C(O)–(NR)-

S

R 5 hydrocarbon; —C(O)—C-C monounsaturated hydro carbon; —C(O)—C-C polyunsaturated hydrocarbon; a O peptide; a carbohydrate; a nucleoside reside; or a co-antioxi dant; where R is hydrogen, alkyl, or aryl; where R is hydro gen, alkyl, aryl, benzyl or a co-antioxidant; and where R is hydrogen, alkyl, aryl, -P(O)(OR), S(O)(OR), an amino acid, a peptide, a carbohydrate, a nucleoside, or a co-antioxidant. O R6 0187. In some embodiments, a method for treating a dis order associated with platelet aggregation in a Subject may s include administering to the Subject an effective amount of a N pharmaceutically acceptable formulation including a syn thetic analog or derivative of a carotenoid where each —OR R5 group may independently be:

3 R3 R3

O OH

OH OH

OH US 2009/O124574 A1 May 14, 2009 29

-continued -continued OH O NH2: O

O H O N r OH O NH2:

pharmaceutically acceptable salts of any of these compounds, where each R is independently H, alkyl, aryl, benzyl, Group IA metal, or co-antioxidant. 0188 Each co-antioxidant may be independently Vitamin C. Vitamin C analogs, Vitamin C derivatives, Vitamin E, Vitamin E analogs, Vitamin E derivatives, flavonoids, fla vonoid derivatives, or flavonoid analogs. Flavonoids include, O but are not limited to, quercetin, Xanthohumol, isoxanthohu H O N mol, or genistein. Selection of the co-antioxidant should not be seen as limiting for the therapeutic application of the rol current invention. 0189 In some embodiments, a method for treating a dis order associated with platelet aggregation in a Subject may include administering to a subject who would benefit from Such treatment a therapeutically effective amount of a phar maceutically acceptable composition comprising a caro tenoid analog or derivative. The carotenoid analog or deriva tive of the carotenoid may have the structure

US 2009/O124574 A1 May 14, 2009

-continued -continued

O NH2: O NH2 O H O N Yso O NH2:

O O H n P-OM O O, O N >, | N OH: O O NH2 O MeO OMe

O O s ------O

O O O

o1 | N -OMe, O lu-Ns OMe

pharmaceutically acceptable salts of any of these compounds, where each R is independently H, alkyl, aryl, benzyl, Group IA metal, or co-antioxidant. (0193 When R is an amino acid derivative or a peptide, coupling of the amino acid or the peptide is accomplished O through an ester linkage or a carbamate linkage. Specifically, H O N an ester linked amino acid group —OR has the general You Structures:

O O -- NH2 NH O O R 14 R 14 US 2009/O124574 A1 May 14, 2009 32

0194 Depending on if the free form of the salt form is desired. A carbamate linked amino acid group —OR will -continued have the general structure:

O "S -- "S H O COR O CO O N

O YorR 14 When R is an amino acid derivative or a peptide, coupling of O the amino acid or the peptide is accomplished through an H amide linkage. The amide linkage may be formed between O N the terminal carboxylic acid group of the linker attached to the r O Xanthophyll carotene and the amine of the amino acid or O R 14 peptide. (0197) When R is a carbohydrate, R includes, but is not limited to the following side chains: 0.195 Depending on if the free form of the salt form is (0198 –CH (CHOH), COH: -CH (CHOH)— desired. For both ester linked and carbamate linked amino CHO: acids, the group R' represents an amino acid side chain. (0199 CH (CHOH), CHOH, CH (CHOH) Specifically, R' can be: 0196) —H (glycine); —CH (alanine); —CH(CH)— CH (valine); —CH-CH(CH)—CH (leucine); —CH OH (CH)—CH2—CH (isoleucine); —CH-Ph (phenylala OH nine); —CH2—CH2—S CH (methionine); —CH2—OH O (serine); —CH(CH)—OH (threonine); —CH SH (cys teine); —CH-Ph-OH (tyrosine); —CH2—C(O) NH (as pargine); —CH, CH, C(O) NH (glutamine); OH, —CH COH (aspartic acid); —CH2—CH COH OR 10 (glutamic acid); —CH2—CH2—CH2—CH2—NH (lysine); —CH, CH, CH, NH (ornithine); where R' is hydrogen or CH, CH, CH NH CONH) NH (arginine);

OH

OH O O OH

OH: (histidine); and OH HO R13 where R' is hydrogen or -OH. (0200. When R is a nucleoside, R may have the structure:

/ O R12 N H HO R13 (tryptophan). Amino acid side chains can be in the neutral form (as depicted above) or in a salt form. When R' repre 0201 where R' is a purine or pyrimidine base, and R' is sents the side chain from the amino acid proline, the following hydrogen or —OH. compounds result: (0202) When R is —C(O)—C-C saturated hydrocar bon), the substituent, R, is derived from coupling of a satu rated fatty acid with the carotenoid parent structure. Examples of saturated fatty acids include, but are not limited to: hexanoic acid (caproic acid); octanoic acid (caprylic acid); decanoic acid (capric acid); dodecanoic acid (lauric acid); tridecanoic acid; tetradecanoic acid (myristic acid); pentade ----'o canoic acid; hexadecanoic acid (palmitic acid); heptade canoic acid (margaric acid); octadecanoic acid (Stearic acid); US 2009/O124574 A1 May 14, 2009 33 eicosanoic acid (arachidic acid); docosanoic acid (behenic C-parinaric acid); and all trans-octadeca-9,11,13,15-trienoic acid); tricosanoic acid; and tetracosanoic acid (lignoceric acid (B-parinaric acid). Examples of omega-6 polyunsatu acid). rated fatty acids include, but are not limited to:9,12-octadeca dienoic acid (linoleic acid); 6.9,12-octadecatrienoic acid (0203) When R is C(O)—C-C monounsaturated (gamma-linolenic acid); 11,14-eicosadienoic acid (eicosadi hydrocarbon), the substituent, R, is derived from coupling of enoic acid); 8,11,14-eicosatrienoic acid (homo-gamma-lino a monounsaturated fatty acid with the carotenoid parent lenic acid); 5,8,11,14-eicosatetraenoic acid (arachidonic structure. Examples of monounsaturated fatty acids include, acid); 13,16-docosadienoic acid (docosadienoic acid); 7.10, but are not limited to:9-tetradecenoic acid (myristoleic acid); 13,16-docosatetraenoic acid (adrenic acid); 4,7,10,13,16 9-hexadecenoic acid (palmitoleic acid); 11-octadecenoic acid docosapentaenoic acid (docosapentaenoic acid); 8E.10E, (vaccenic acid); 9-octadenoic acid (oleic acid); 11-eicosenoic 12Z-octadecatrienoic acid (calendic acid); 1 OE,12Z acid; 13-docosenoic acid (erucic acid); 15-tetracosanoic acid octadeca-9,1'-dienoic acid; 8E,10E,12Z-octadecatrienoic (nervonic acid): 9-trans-hexadecenoic acid (palmitelaidic acid (C-calendic acid); 8E.10E, 12E-octadecatrienoic acid acid): 9-trans-octadecenoic acid (elaidic acid); 8-eicosaenoic (B-calendic acid); 8E.10Z,12E-octadecatrienoic acid (jacaric acid; and 5-eicosaenoic acid. acid); and 5Z.8Z.10E, 12E.14Z-eicosanoic acid (bosseopen 0204. When R is C(O)—C-C polyunsaturated taenoic acid). Examples of conjugated polyunsaturated fatty hydrocarbon), the substituent, R, is derived from coupling of acids include, but are not limited to: 9Z, 11E-octadeca-9,11 a polyunsaturated fatty acid with the carotenoid parent struc dienoic acid (rumenic acid); 10E,12Z-octadeca-9,11-dienoic ture. Examples of polyunsaturated fatty acids include, but are acid; 8E,10E, 12Z-octadecatrienoic acid (C-calendic acid); not limited to omega-3 polyunsaturated fatty acids, omega-6 8E.10E, 12E-octadecatrienoic acid (B-calendic acid); polyunsaturated fatty acids; and conjugated polyunsaturated 8E.10Z,12E-octadecatrienoic acid (jacaric acid); 9E, 11E, fatty acids. Examples of omega-3 polyunsaturated fatty acids 13Z-octadeca-9,11,13-trienoic acid (C-eleostearic acid); 9E, include, but are not limited to: 9,12,15-octadecatrienoic acid 11E. 13E-octadeca-9,11,13-trienoic acid (B-eleostearic (alpha-linolenic acid); 6.9,12,15-octadecatetraenoic acid acid); 9Z, 11Z,13E-octadeca-9,11,13-trienoic acid (catalpic (Stearidonic acid); 11.14, 17-eicosatrienoic acid (eicosa acid); 9E, 11Z,13E-octadeca-9,11,13-trienoic acid (punicic trienoic acid (ETA)); 8,11,14, 17-eicsoatetraenoic acid (eic acid); 9E, 11Z., 15E-octadeca-9,11,15-trienoic acid (rume Soatetraenoic acid); 5,8,11,14, 17-eicosapentaenoic acid lenic acid); 9E, 11Z., 13Z., 15E-octadeca-9,11,13,15-trienoic (eicosapentaenoic acid (EPA)); 7,10,13,16,19-docosapen acid (C-parinaric acid); all trans-octadeca-9,11,13,15 taenoic acid (docosapentaenoic acid (DPA)); 4,7,10,13,16, trienoic acid (B-parinaric acid); and 5Z.8Z.10E, 12E.14Z 19-docosahexaenoic acid (docosahexaenoic acid (DHA)); eicosanoic acid (bosseopentaenoic acid). 6.9,12,15, 18.21-tetracosahexaenoic acid (nisinic acid); 0205 Specific examples of carotenoid derivatives include, 9E,11Z,15E-octadeca-9,11,15-trienoic acid (rumelenic but are not limited to, the following compounds and pharma acid); 9E, 11Z,13Z,15E-octadeca-9,11,13,15-trienoic acid ceutically acceptable salts of these compounds:

O O --- OH. O S1S1s1S1s1s1s1s1s O is-- O O O

O N~~~ O S1S 1S 1 N1sn 1 n 1s 1S 1 N. O -----, O

S-1S-1s-1S-1s-1s-1s-1s-1s

US 2009/O124574 A1 May 14, 2009 36

-continued O No.O N S. O NH2 HN O N N N1N1 N1 n1 N. HO ls N O H O O

NH2 O N1-S-1s-1S HO us N O H O O H O N r OH N1N1N1 N1N O NH2

0206. Further details regarding the use and synthesis of CHIRAL DIHYDROXY KETONE INTERMEDIATES carotenoid derivatives and analogs suitable for use in the USEFUL FOR THE CHIRAL SYNTHESIS OF CARO presently described embodiments may be found in the fol TENOIDS”; U.S. Patent Application Publication No. 2006/ lowing U.S. patent documents: U.S. Pat. No. 7,145,025 OO88904 entitled METHODS FOR THE SYNTHESIS OF entitled STRUCTURAL CAROTENOIDANALOGS FOR ASTAXANTHIN'; U.S. Patent Application Publication No. THE INHIBITION AND AMELIORATION OF DISEASE 2006/0270590 entitled REDUCTION IN COMPLEMENT issued Dec. 5, 2006: U.S. Patent Application Publication No. ACTIVATION AND INFLAMMATION DURING TISSUE 2005/O113372 entitled “CAROTENOID ANALOGS OR INJURY BY CAROTENOIDS, CAROTENOID ANA DERIVATIVES FOR THE INHIBITION AND AMELIO LOGS, ORDERIVATIVES THEREOF: U.S. Patent Appli RATION OF DISEASE: U.S. Patent Application Publica cation Publication No. 2006/0270.589 entitled “CARO tion No. 2005/0075337, entitled “PHARMACEUTICAL TENOIDS, CAROTENOID ANALOGS, OR COMPOSITIONS INCLUDING CAROTENOID ANA CAROTENOID DERIVATIVES FOR THE STABILIZA LOGS ORDERIVATIVES FOR THE INHIBITION AND TION OR IMPROVEMENT OF VISUAL ACUITY”; U.S. AMELIORATION OF DISEASE: U.S. Patent Application Patent Application Publication No. 2007/0015735 entitled Publication No. 2005/026.1254 entitled “CAROTENOID “WATER-DISPERSIBLE CAROTENOIDS, INCLUDING ANALOGS ORDERIVATIVES FOR THE INHIBITION ANALOGS AND DERIVATIVES”; U.S. Patent Application AND AMELIORATION OF INFLAMMATION”; U.S. Publication No. 2006/0276372 entitled “CAROTENOIDS, Patent Application Publication No. 2006/0058269 entitled CAROTENOIDANALOGS, ORCAROTENOIDDERIVA “CAROTENOIDANALOGS ORDERIVATIVES FORTHE TIVES FOR THE TREATMENT OF PROLIFERATIVE INHIBITION AND AMELIORATION OF INFLAMMA DISORDERS”; U.S. application Ser. No. 1 1/417.307 entitled TION”; U.S. Patent Application Publication No. 2006/ USE OF CAROTENOIDS AND/OR CAROTENOID O1785.38 entitled METHODS FOR THE SYNTHESIS OF DERIVATIVES/ANALOGS FOR REDUCTIONAINHIBI CHIRAL DIHYDROXY INTERMEDIATES USEFUL FOR TION OF CERTAIN NEGATIVE EFFECTS OF COX THE CHIRAL SYNTHESIS OF CAROTENOIDS”; U.S. INHIBITORS: U.S. Ser. No. 60/691,518 entitled “METH Patent Application Publication No. 2006/0183947 entitled ODS FOR SYNTHESIS OF CAROTENOIDS, INCLUD METHODS FOR THE SYNTHESIS OF ASTAXAN ING ANALOGS, DERIVATIVES, AND SYNTHETIC AND THIN'; U.S. Patent Application Publication No. 2006/ BIOLOGICAL INTERMEDIATES”; U.S. Patent Applica O15515O entitled METHODS FOR THE SYNTHESIS OF tion Publication No. 2006/0293545 entitled “METHODS LUTEIN'; U.S. Patent Application Publication No. 2006/ FOR SYNTHESIS OF CAROTENOIDS, INCLUDING OO88905 entitled METHODS FOR THE SYNTHESIS OF ANALOGS, DERIVATIVES, AND SYNTHETIC AND ZEAXANTHIN”; U.S. Patent Application Publication No. BIOLOGICAL INTERMEDIATES”; U.S. application Ser. 2006/O1673 19 entitled METHODS FOR THE SYNTHE No. 1 1/636,401 entitled “STRUCTURAL CAROTENOID SIS OF UNSATURATED KETONE INTERMEDIATES ANALOGS ORDERIVATIVES FOR THE MODULATION USEFUL FOR THE SYNTHESIS OF CAROTENOIDS”; OF SYSTEMIC AND/ORTARGET ORGAN REDOX STA U.S. Patent Application Publication No. 2006/0183.185 TUS: and U.S. Ser. No. 1 1/699,924 entitled “SYNTHESIS entitled METHODS FOR THE SYNTHESIS OF ASTAX OF CAROTENOIDANALOGS ORDERIVATIVES WITH ANTHIN'; U.S. Patent Application Publication No. 2006/ IMPROVED ANTIOXIDANT CHARACTERISTICS, all O111580 entitled METHODS FOR THE SYNTHESIS OF of which are commonly owned with the present application US 2009/O124574 A1 May 14, 2009 37 and which are hereby expressly incorporated by reference in 0212. Although the above structure, and subsequent struc their entirety as though fully set forth herein. tures, depict alkenes in the E configuration this should not be 0207 Water-soluble carotenoid analogs or derivatives seen as limiting. Compounds discussed herein may include may have a water solubility of greater than about 1 mg/mL in embodiments where alkenes are in the Z configuration or Some embodiments. In certain embodiments, water-soluble include alkenes in a combination of Z and E configurations carotenoid analogs or derivatives may have a water Solubility within the same molecule. The compounds depicted herein of greater than about 5 mg/mL. In certain embodiments, may naturally convert between the Z and E configuration water-soluble carotenoid analogs or derivatives may have a and/or exist in equilibrium between the two configurations. water solubility of greater than about 10 mg/mL. In certain embodiments, water-soluble carotenoid analogs or deriva 0213 Compounds described herein embrace isomers mix tives may have a water solubility of greater than about 20 tures, racemic, optically active, and optically inactive Stere mg/mL. In some embodiments, water-soluble carotenoid oisomers and compounds. Carotenoid analogs or derivatives analogs or derivatives may have a water Solubility of greater may have increased water solubility and/or water dispersibil than about 50 mg/mL. ity relative to some or all known naturally occurring caro 0208 Naturally occurring carotenoids such as xantho tenoids. In some embodiments, one or more co-antioxidants phyll carotenoids of the C40 series, which includes commer may be coupled to a carotenoid or carotenoid derivative or cially important compounds such as lutein, Zeaxanthin, and analog. astaxanthin, have poor aqueous solubility in the native state. 0214. In some embodiments, carotenoid analogs or Varying the chemical structure(s) of the esterified moieties derivatives may be employed in 'self-formulating aqueous may vastly increase the aqueous solubility and/or dispersibil Solutions, in which the compounds spontaneously self-as ity of derivatized carotenoids. semble into macromolecular complexes. These complexes 0209. In some embodiments, highly water-dispersible Cao may provide stable formulations in terms of shelf life. The carotenoid derivatives may include natural source RRR same formulations may be parenterally administered, upon lutein (Be-carotene-3,3'-diol) derivatives. Derivatives may which the spontaneous self-assembly is overcome by inter be synthesized by esterification with inorganic phosphate and actions with serum and/or tissue components in vivo. Succinic acid, respectively, and Subsequently converted to the 0215. Some specific embodiments may include phos Sodium salts. Deep orange, evenly colored aqueous Suspen phate. Succinate, co-antioxidant (e.g., Vitamin C, Vitamin C sions were obtained after addition of these derivatives to analogs, Vitamin C derivatives, Vitamin E. Vitamin E analogs, USP-purified water. Aqueous dispersibility of the disuccinate Vitamin E derivatives, or flavonoids), or combinations Sodium salt of natural lutein was 2.85 mg/mL, the diphos thereof derivatives or analogs of carotenoids. Flavonoids may phate salt demonstrated a >10-fold increase in dispersibility include, for example, quercetin, Xanthohumol, isoxanthohu at 29.27 mg/mL. Aqueous Suspensions may be obtained with mol, or genistein. Derivatives or analogs may be derived from out the addition of heat, detergents, co-solvents, or other any known carotenoid (naturally or synthetically derived). additives. Specific examples of naturally occurring carotenoids which 0210. The direct aqueous superoxide scavenging abilities compounds described herein may be derived from include for of these derivatives were subsequently evaluated by electron example zeaxanthin, lutein, lycophyll, astaxanthin, and lyco paramagnetic resonance (EPR) spectroscopy in a well-char pene. acterized in vitro isolated human neutrophil assay. The 0216. The synthesis of water-soluble and/or water-dis derivatives may be potent (millimolar concentration) and persible carotenoids (e.g., Cao) analogs or derivatives—as nearly identical aqueous-phase Scavengers, demonstrating potential parenteral agents for clinical applications may dose-dependent Suppression of the Superoxide anion signal improve the injectability of these compounds as therapeutic (as detected by spin-trap adducts of DEPMPO) in the milli agents, a result perhaps not achievable through other formu molar range. Evidence of card-pack aggregation was lation methods. The methodology may be extended to caro obtained for the diphosphate derivative with UV-Vis spec tenoids with fewer than 40 carbon atoms in the molecular troscopy (discussed herein), whereas limited card-pack and/ skeleton and differing ionic character. The methodology may or head-to-tail aggregation was noted for the disuccinate be extended to carotenoids with greater than 40 carbon atoms derivative. These lutein-based soft drugs may find utility in in the molecular skeleton. The methodology may be extended those commercial and clinical applications for which aque to non-symmetric carotenoids. The aqueous dispersibility of ous-phase singlet oxygen quenching and direct radical scav these compounds allows proof-of-concept studies in model enging may be required. systems (e.g., cell culture), where the high lipophilicity of 0211. The absolute size of a carotenoid derivative (in 3 these compounds previously limited their bioavailability and dimensions) is important when considering its use in biologi hence proper evaluation of efficacy. Esterification or etheri cal and/or medicinal applications. Some of the largest natu fication may be useful to increase oral bioavailability, a for rally occurring carotenoids are no greater than about Cso. tuitous side effect of the esterification process, which can This is probably due to size limits imposed on molecules increase solubility in gastric mixed micelles. These com requiring incorporation into and/or interaction with cellular pounds, upon introduction to the mammalian GI tract, are membranes. Cellular membranes may be particularly rapidly and effectively cleaved to the parent, non-esterified co-evolved with molecules of a length of approximately 30 compounds, and enter the systemic circulation in that manner nm. In some embodiments, carotenoid derivatives may be and form (see, e.g., FIG. 2). The effect of the intact ester greater than or less than about 30 nm in size. In certain and/or ether compound on the therapeutic endpoint of interest embodiments, carotenoid derivatives may be able to change can be obtained with parenteral administration of the com conformation and/or otherwise assume an appropriate shape, pound(s). The net overall effect is an improvement in poten which effectively enables the carotenoid derivative to effi tial clinical utility for the lipophilic carotenoid compounds as ciently interact with a cellular membrane. therapeutic agents. US 2009/O124574 A1 May 14, 2009 38

0217. In one embodiment, a subject may be administered form for retrometabolic syntheses: (1) synthetic handles (hy a pharmaceutical composition comprising a carotenoid ana droxyl groups) for conjugation, and (2) an excellent safety log or derivative. The analog or derivative may be broken profile for the parent compound. As stated above, lutein is down according to the following reaction: available commercially from multiple sources in bulk as pri

N1S 1s 1N 1S 1S 1s 1N1)

Cs2H64Na2O20P2 Mol. Wit.: 1116.98

O

OH

S1N1,N1S 1S 1S 1S 1s 1N

HO

C40H52O4 MOI. Wit.: 596.84

O | HO-P/NoH HO HOP MOI. Wit.: 98.00

C6HsO6 MOI. Wit.: 176.12

0218. In some embodiments, the principles of retrometa marily the RRR-stereoisomer, the primary isomer in the bolic drug design may be utilized to produce novel soft drugs human diet and human retinal tissue. from the asymmetric parent carotenoid scaffold (e.g., RRR 0219. In some embodiments, carotenoid analogs or lutein (Be-carotene-3,3'-diol)). For example, lutein scaffold derivatives may have increased water solubility and/or water for derivatization was obtained commercially as purified dispersibility relative to some or all known naturally occur natural plant source material, and was primarily the RRR ring carotenoids. Stereoisomer (one of 8 potential stereoisomers). Lutein 0220. In some embodiments, the carotenoid derivatives (Scheme 1) possesses key characteristics—similar to starting may include compounds having a structure including a poly material astaxanthin which make it an ideal starting plat ene chain (i.e., backbone of the molecule). The polyene chain US 2009/O124574 A1 May 14, 2009 39 may include between about 5 and about 15unsaturated bonds. tially increases the water solubility of the naturally occurring In certain embodiments, the polyene chain may include carotenoid. The carotenoid derivatives may retain and/or between about 7 and about 12 unsaturated bonds. In some improve the antioxidant properties of the parent carotenoid. embodiments a carotenoid derivative may include 7 or more The carotenoid derivatives may retain the non-toxic proper conjugated double bonds to achieve acceptable antioxidant ties of the parent carotenoid. The carotenoid derivatives may properties. have increased bioavailability, relative to the parent caro 0221. In some embodiments, decreased antioxidant prop tenoid, upon administration to a Subject. The parent caro erties associated with shorter polyene chains may be over tenoid may be naturally occurring. come by increasing the dosage administered to a subject or patient. 0227 Other embodiments may include the administering a composition comprised of the Synergistic combination of 0222 Some embodiments may include solutions or phar maceutical preparations of carotenoids and/or carotenoid more than one Xanthophyll carotenoid or structural caro derivatives combined with co-antioxidants, in particular vita tenoid analog or derivative or synthetic intermediate to a min C and/or vitamin Canalogs or derivatives. Pharmaceuti Subject Such that disease severity and/or complications asso cal preparations may include about a 2:1 ratio of vitamin C to ciated with a disorder associated with platelet aggregation are carotenoid respectively. thereby at least partially reduced, inhibited and/or amelio 0223. In some embodiments, co-antioxidants (e.g., vita rated. The composition may be a “racemic” (i.e. mixture of min C) may increase solubility of the chemical compound. In the potential Stereoisomeric forms) mixture of carotenoid certain embodiments, co-antioxidants (e.g., vitamin C) may derivatives. Included as well are pharmaceutical composi decrease toxicity associated with at least Some carotenoid tions comprised of structural analogs or derivatives or syn analogs or derivatives. In certain embodiments, co-antioxi thetic intermediates of carotenoids in combination with a dants (e.g., vitamin C) may increase the potency of the chemi pharmaceutically acceptable carrier. In one embodiment, a cal compound synergistically. Co-antioxidants may be pharmaceutically acceptable carrier may be serum albumin. coupled (e.g., a covalent bond) to the carotenoid derivative. In one embodiment, structural analogs or derivatives or Syn Co-antioxidants may be included as a part of a pharmaceuti thetic intermediates of carotenoids may be complexed with cally acceptable formulation. human serum protein Such as, for example, human serum 0224. As used herein terms such as “structural carotenoid albumin (i.e., HSA) in a solvent. In an embodiment, HSA may analogs or derivatives' may be generally defined as caro act as a pharmaceutically acceptable carrier. tenoids and the biologically active structural analogs or 0228. In some embodiments, a single stereoisomer of a derivatives thereof. “Derivative' in the context of this appli cation is generally defined as a chemical Substance derived structural analog or derivative or synthetic intermediate of from another substance either directly or by modification or carotenoids may be administered to a human Subject in order partial Substitution. “Analog in the context of this applica to ameliorate a pathological condition. Administering a tion is generally defined as a compound that resembles single stereoisomer of a particular compound (e.g., as part of another in structure but is not necessarily an isomer. Typical a pharmaceutical composition) to a human Subject may be analogs or derivatives include molecules which demonstrate advantageous (e.g., increasing the potency of the pharmaceu equivalent or improved biologically useful and relevant func tical composition). Administering a single stereoisomer may tion, but which differ structurally from the parent compounds. be advantageous due to the fact that only one isomer of Parent carotenoids are selected from the more than 700 natu potentially many may be biologically active enough to have rally occurring carotenoids described in the literature, and the desired effect. their stereo- and geometric isomers. Such analogs or deriva 0229. In some embodiments, compounds described herein tives may include, but are not limited to, esters, ethers, car may be administered in the form of nutraceuticals. “Nutra bonates, amides, carbamates, phosphate esters and ethers, ceuticals' as used herein, generally refers to dietary Supple Sulfates, glycoside ethers, with or without spacers (linkers). ments, foods, or medical foods that: 1. possess health benefits 0225. As used herein the terms “the synergistic combina generally defined as reducing the risk of a disease or health tion of more than one carotenoid or structural analog or condition, including the management of a disease or health derivative or synthetic intermediate of carotenoids' may be condition or the improvement of health; and 2. are safe for generally defined as any composition including one Xantho human consumption in Such quantity, and with Such fre phyll carotenoid or a structural carotenoid analog or deriva quency, as required to realize such properties. Generally a tive or synthetic intermediate combined with one or more different xanthophyll carotenoids or structural carotenoid nutraceutical is any substance that is a food or a part of a food analogs or derivatives or synthetic intermediates or co-anti and provides medical or health benefits, including the preven oxidants, either as derivatives or in Solutions and/or formula tion and treatment of disease. Such products may range from tions. isolated nutrients, dietary Supplements and specific diets to 0226 Certain embodiments may include administering a genetically engineered designer foods, herbal products, and carotenoid or a structural carotenoid analogs or derivatives or processed foods such as cereals, Soups and beverages. It is synthetic intermediates alone or in combination to a subject important to note that this definition applies to all categories Such that disease severity and/or complications associated of food and parts of food, ranging from dietary Supplements with a disorder associated with platelet aggregation are such as folic acid, used for the prevention of spina bifida, to thereby at least partially reduced, inhibited and/or amelio chicken Soup, taken to lessen the discomfort of the common rated. The xanthophyll carotenoid or a structural carotenoid cold. This definition also includes a bio-engineered designer analogs or derivatives or synthetic intermediates may be Vegetable food, rich in antioxidant ingredients, and a stimu water-soluble and/or water dispersible derivatives. The caro lant functional food or pharmafood. Within the context of the tenoid derivatives may include any Substituent that Substan description herein where the composition, use and/or delivery US 2009/O124574 A1 May 14, 2009 40 of pharmaceuticals are described nutraceuticals may also be cal, the compounds may be administered as part of a pharma composed, used, and/or delivered in a similar manner where ceutical preparation containing Suitable pharmaceutically appropriate. acceptable carriers, preservatives, excipients and auxiliaries which facilitate processing of the structural carotenoid analog Dosage and Administration or derivative or synthetic intermediates which may be used 0230. The carotenoids, carotenoid derivative or analog pharmaceutically. The preparations, particularly those prepa may be administered at a dosage level up to conventional rations which may be administered orally and which may be dosage levels for Such derivatives or analogs, but will typi used for the preferred type of administration, such as tablets, cally be less than about 2 gm per day. Suitable dosage levels softgels, lozenges, dragees, and capsules, and also prepara may depend upon the overall systemic effect of the chosen tions which may be administered rectally, such as Supposito Xanthophyll carotenoids, carotenoid derivatives or analogs, ries, as well as Suitable Solutions for administration by injec but typically suitable levels will be about 0.001 to 50 mg/kg tion or orally or by inhalation of aerosolized preparations, body weight of the patient per day, from about 0.005 to 30 may be prepared in dose ranges that provide similar bioavail mg/kg per day, or from about 0.05 to 10 mg/kg per day. The ability as described above, together with the excipient. While compound may be administered on a regimen of up to 6 times individual needs may vary, determination of the optimal per day, between about 1 to 4 times per day, or once per day. ranges of effective amounts of each component is within the 0231. In the case where an oral composition is employed, skill of the art. a Suitable dosage range is, e.g. from about 0.01 mg to about 0236. In some embodiments in which one or more addi 100 mg of a xanthophyll carotenoid, carotenoid derivative or tional medicaments or compositions Suitable for the treat analog per kg of body weight per day, preferably from about ment of a disorder associated with platelet aggregation in a 0.1 mg to about 10 mg per kg and for cytoprotective use from Subject are administered in conjunction with a carotenoid, a 0.1 mg to about 100 mg of a Xanthophyll carotenoid, caro carotenoid derivative or a carotenoid analog, the carotenoid, tenoid derivative or analog per kg of body weight per day. carotenoid derivative or analog may be administered sepa 0232. It will be understood that the dosage of the thera rately in separate dosage forms or together in a single unit peutic agents will vary with the nature and the severity of the dosage form. Where separate dosage formulations are used, condition to be treated, and with the particular therapeutic the Xanthophylls carotenoid, carotenoid derivative or analog agents chosen. The dosage will also vary according to the age, and one or more additional medicaments or compositions weight, physical condition and response of the individual may be administered at Substantially the same time, i.e., con patient. The selection of the appropriate dosage for the indi currently, or at separately staggered times, i.e., sequentially, vidual patient is within the skills of a clinician. and in any order. In certain embodiments the Xanthophyll 0233. In some embodiments, compositions may include carotenoid, carotenoid derivative or analog the one or more all compositions of 1.0 gram or less of a particular structural additional medicaments or compositions may be co-admin carotenoid analog, in combination with 1.0 gram or less of istered concurrently on a once-a-day (QD) dosing schedule: one or more other structural carotenoid analogs orderivatives however, varying dosing schedules, such as the Xanthophyll or synthetic intermediates and/or co-antioxidants, in an carotenoid, carotenoid derivative or analog once per day and amount which is effective to achieve its intended purpose. the one or more additional medicaments or compositions While individual subject needs vary, determination of opti once, twice or more times per day, or the one or more addi mal ranges of effective amounts of each component is with tional medicaments or compositions once per day and the the skill of the art. Typically, a structural carotenoid analog or carotenoid derivative or analog once, twice or more times per derivative or synthetic intermediates may be administered to day, is also encompassed herein. According to certain appli mammals, in particular humans, orally at a dose of 5 to 100 cation(s) of the present embodiments, a single oral dosage mg per day referenced to the body weight of the mammal or formulation comprising the carotenoid derivative or analog human being treated for a particular disease. Typically, a and the one or more additional medicaments or compositions structural carotenoid analog or derivative or synthetic inter may be preferred. In other embodiments, it may be desirable mediate may be administered to mammals, in particular to administer the carotenoid derivative or analog separately humans, parenterally at a dose of between 5 to 1000 mg per from the one or more additional medicaments or composi day referenced to the body weight of the mammal or human tions. A single dosage formulation will provide convenience being treated for a particular disease. In other embodiments, for the patient. about 100 mg of a structural carotenoid analog or derivative 0237. The one or more additional medicaments or compo or synthetic intermediate is either orally or parenterally sitions suitable for the treatment of a disorder associated with administered to treat or prevent disease. platelet aggregation in a Subject may be administered at a 0234. The unit oral dose may comprise from about 0.25 dosage level up to conventional dosage levels for Such com mg to about 1.0 gram, or about 5 to 25 mg. of a structural pounds. Suitable dosage levels will depend upon the effect carotenoid analog. The unit parenteral dose may include from and the pharmacological porterties of the chosen additional about 25 mg to 1.0 gram, or between 25 mg and 500 mg. of a medicaments or compositions, but typically Suitable levels structural carotenoid analog. The unit intracoronary dose may will be between about 0.001 to 50 mg/kg body weight of the include from about 25 mg to 1.0 gram, or between 25 mg and patient per day, between about 0.005 to 30 mg/kg per day, or 100 mg. of a structural carotenoid analog. The unit doses may between about 0.05 to 10 mg/kg per day. In some embodi be administered one or more times daily, on alternate days, in ments, the compound may be administered on a regimen of up loading dose or bolus form, or titrated in a parenteral Solution to 6 times per day, from 1 to 4 times per day, or once per day. to commonly accepted or novel biochemical Surrogate mark 0238. In the case where an oral composition is employed, er(s) or clinical endpoints as is with the skill of the art. an exemplary dosage range is, e.g. from about 0.01 mg to 0235. In addition to administering a structural carotenoid about 100 mg of each additional medicament or composition analog orderivative or synthetic intermediate as a raw chemi per kg of body weight per day, or from about 0.1 mg to about US 2009/O124574 A1 May 14, 2009

10 mg per kg of each additional medicament or composition inhibiting platelet aggregation. Suitable GPIIb/IIIa receptor per kg of body weight per day. antagonists are disclosed in WO 99/45913, being herewith 0239 Dosage levels of additional compositions or medi incorporated by reference. Suitable GP IIb/IIIa receptor caments suitable for the treatment of disorders associated antagonists may be selected from Xemilofiban, abciximab, with platelet aggregation may be on the order of about 0.1 mg cromafiban, elarofiban, orbofiban, roxifiban, sibrafiban, RPR to about 10,000 mg of the active ingredient compound are 1098.91, eptifibatide, tirofiban, DMP 754, and fradafiban. useful in the treatment of the above conditions, with preferred 0246 Oral dosages of GP IIb/IIIa receptor antagonists levels of about 1.0 mg to about 1,000 mg. The amount of when used for the indicated effects may typically range active ingredient that may be combined with other anticancer between about 0.001 mg per kg of body weight per day agents to produce a single dosage form will vary depending (mg/kg/day) to about 50 mg/kg/day and preferably 0.005 upon the host treated and the particular mode of administra mg/kg/day -20 mg/kg/day, most preferably 0.01 mg/kg/day tion. 10 mg/kg/day. Suitable oral tablets and capsules contain 0240. It is understood, however, that a specific dose level between 0.01 mg and 5 g, preferably between 0.1 mg and 2 g, for any particular patient will depend upon a variety of factors most preferably between 0.5 mg and 1 mg, for example, 0.5 including the activity of the specific compound employed, the mg, 1 mg, 5 mg, 10 mg, 150 mg, 250 mg or 500 mg of GP age, body weight, general health, sex, diet, time of adminis IIb/IIIa receptor antagonists. Oral administration may be in tration, rate of excretion, drug combination, and the severity one or divided doses of two, three or four times daily. In an of the particular disease being treated and form of adminis embodiment, a single daily dose may be preferred. tration. 0247 Intravenously, the most preferred doses for GP IIb/ 0241 Treatment dosages generally may be titrated to opti IIIa receptorantagonists will range from about 0.5ug to about mize safety and efficacy. Typically, dosage-effect relation 5 mg/kg/minute during a constant rate infusion, to achieve a ships from invitro initially can provide useful guidance on the plasma level concentration during the period of time of proper doses for patient administration. Studies in animal administration of between 0.1 ng/ml and 1 ug/ml. models also generally may be used for guidance regarding 0248 Clopidogrel may be administered orally in a daily effective dosages for treatment of cancers in accordance with dosage of about 25 mg to 500 mg, preferably from 75 mg to the present invention. In terms of treatment protocols, it 375 mg, and most preferably from 75 mg to 150 mg. For should be appreciated that the dosage to be administered will example, a formulation or dosage unit may contain 25 mg, 50 depend on several factors, including the particular agent that mg, 75 mg, 150 mg, 250 mg or 500 mg of clopidogrel. Oral is administered, the route administered, the condition of the administration may be in one or divided doses of two, three or particular patient, etc. Generally speaking, one will desire to four times daily. In an embodiment, a single daily dose may administer an amount of the compound that is effective to be preferred. achieve a serum level commensurate with the concentrations found to be effective in vitro. Thus, where a compound is 0249 Ticlopidine can be administered orally in a daily found to demonstrate in vitro activity at, e.g., 10uM, one will dosage of about 50 mg to 1000 mg, preferably from 100 mg to desire to administer an amount of the drug that is effective to 750 mg, and most preferably from 200 mg to 500 mg. For provide about a 10 uM concentration in vivo. Determination example, a formulation or dosage unit may contain 50 mg. of these parameters is well within the skill of the art. 100 mg, 200 mg, 250 mg or 500 mg of ticlopidine. Oral 0242. These considerations, as well as effective formula administration may be in one or divided doses of two, three or tions and administration procedures are well known in the art four times daily. In an embodiment, a single daily dose may and are described in standard textbooks. The following dos be preferred. ages, which are provided by way of illustrative example, may 0250 Cilostazol can be administered orally in a daily dos serve as general guidance in the determination of Suitable age of about 50 mg to 500 mg, preferably from 100 mg to 300 dosage ranges for certain of the additional antiplatelet agent mg, and most preferably from 150 mg to 250 mg. For contemplated for use herein. It will of course be readily example, a formulation or dosage unit may contain 50 mg. appreciated by the skilled practitioner that alternative dosages 100 mg, 200 mg, 250 mg or 500 mg of cilostazol. Oral may be employed without departing from the spirit and scope administration may be in one or divided doses of two, three or of the presently described embodiments. four times daily. In an embodiment, a single daily dose may 0243 Antiplatelet agents suitable for use in the present be preferred. invention may include glycoprotein (GP) IIb/IIIa receptor 0251 Dipyridamole can be administered orally in a daily antagonists, clopidogrel, ticlopidine, dipyridamole, cilosta dosage of about 25 mg to 500 mg, preferably 75 mg to 375 Zol and ASA. mg, most preferred 75 mg to 150 mg. For long-term treatment 0244. Herein, with respect to all aspects of the invention, it is of advantage to administer repeated doses such as a dose the term “antiplatelet agent is intended to encompass all of 25 mg dipyridamole retard or any other instant release pharmaceutically acceptable salts, esters and Solvate forms, formulation three or four times a day. For example, a formu including hydrates, of compounds which have platelet aggre lation or dosage unit may contain 25 mg, 50 mg, 75 mg, 150 gation inhibitory activity as well as pro-drug forms. Those mg, 250 mg or 500 mg of dipyridamole. Oral administration compounds having one or more chiral centers may occur as may be in one or divided doses of two, three or four times racemates, racemic mixtures and as individual diastereomers daily. In an embodiment, a single daily dose may be preferred. or enantiomers with all such isomeric forms and mixtures 0252 For parenteral administration dipyridamole could thereof being included within the scope of the present disclo be given in a dosage of 0.5 to 5 mg/kg body weight, preferably Sure. Any crystalline form of antiplatelet agent forming poly 1 to 3.5 mg/kg body weight, during 24 hours as slow i.v. morphs are also intended to be included. infusion. 0245 GPIIb/IIIa receptor antagonists inhibit the binding 0253 Oral dosage amounts of ASA for the indicated of fibrinogen to the IIb/IIIa platelet receptor site, thereby effects generally range from about 10 mg to about 325 mg per US 2009/O124574 A1 May 14, 2009 42 day. For example, a formulation or dosage unit may contain described herein be administered parenterally. In yet other 10 mg, 20 mg, 50 mg, 75 mg, 80 mg, 100 mg, 150 mg, 250 mg embodiments, it may be advantageous that the compositions or 325 mg of ASA. described herein be administered locally, at the site of tissue 0254 Standard heparin therapy which may be combined injury. with the method of treatment according to the invention com 0259. The compositions may include those compositions prises the administration of low-molecular-weight heparin Suitable for oral, rectal, topical, parenteral (including Subcu (LMWH), unfractionated heparin (UFH), hirudin, hirulog, taneous, intramuscular, and intravenous), ocular (oph argatroban, melagatran, lepirudin orbivalirudin, for instance thalmic), pulmonary (aerosol inhalation), or nasal adminis subcutaneous LMWH (e.g. nadroparin 87 IU/kg BID or tration, although the most Suitable route in any given case will enoxaparin 1 mg/kg, BID) or iv UFH given as an initial bonus depend on the nature and severity of the conditions being of 5000 IU followed by continuous infusion at 1000 IU per treated and on the nature of the active ingredient. They may be hour for 7 days. The activated partial-thromboplastin time conveniently presented in unit dosage form and prepared by (APTT) can be used to assess the degree of anticoagulation in any of the methods well known in the art of pharmacy. patients receiving iv UFH. Patients can be tested every 12 0260 For administration by inhalation, the drugs used in hours on the first day and every 24 hours subsequently. The the present invention are conveniently delivered in the form of APTT should be maintained in the range of 45 to 87 seconds anaerosol spray presentation from pressurized packs or nebu (normal value, 30+/-5 seconds). lizers. The compounds may also be delivered as powders 0255 Viewed from a second aspect the present invention which may be formulated and the powder composition may provides a pharmaceutical composition comprising a thera be inhaled with the aid of an insufflation powder inhaler peutically or prophylactically effective amount of a caro device. tenoid analog or derivative and a therapeutically or prophy 0261 Suitable topical formulations for use in the present lactically effective amount of an antiplatelet agent for use in embodiments may include transdermal devices, aerosols, treating a disorder associated with platelet aggregation. The creams, ointments, lotions, dusting powders, and the like. instant pharmaceutical combinations may include a single 0262. In practical use, drugs used can be combined as the pharmaceutical dosage formulation containing both a caro active ingredient in intimate admixture with a pharmaceutical tenoid analog or derivative and the antiplatelet agent in a carrier according to conventional pharmaceutical compound pharmaceutical dosage formulation for simultaneous use, as ing techniques. The carrier may take a wide variety of forms well as a combined preparation or a kit of parts comprising a depending on the form of preparation desired for administra carotenoid analog or derivative in a pharmaceutical dosage tion, e.g., oral or parenteral (including intravenous). In pre formulation and the antiplatelet agent in a separate pharma paring the compositions for oral dosage form, any of the usual ceutical dosage formulation for simultaneous, separate or pharmaceutical media may be employed, such as, for sequential use. example, water, glycols, oils, alcohols, flavoring agents, pre 0256 For instance, the kit of parts embodiment may bean servatives, coloring agents and the like in the case of oral oral dosage formulation of a carotenoid analog or derivative liquid preparations, such as, for example, Suspensions, elixirs and an oral dosage formulation of ASA. The packaging for the and solutions; or carriers such as starches, Sugars, microcrys kit may be designed and manufactured in a variety of ways. A talline cellulose, diluents, granulating agents, lubricants, preferred example is a blister package containing rows of a binders, disintegrating agents and the like in the case of oral carotenoid analog or derivative tablet and an ASA tablet side Solid preparations such as, for example, powders, capsules by side on the same blister card, each of the two tablets in its and tablets, with the solid oral preparations being preferred own blister bubble, with calendar or similar type markings on over the liquid preparations. Because of their ease of admin the card that convey to the user that one “pair of tablets is to istration, tablets and capsules represent the most advanta be ingested per day. geous oral dosage unit form in which case Solid pharmaceu 0257 General guidance in determining effective dose tical carriers are obviously employed. If desired, tablets may ranges for pharmacologically active compounds and compo be coated by standard aqueous or nonaqueous techniques. sitions for use in the presently described embodiments may 0263. The pharmaceutical preparations may be manufac be found, for example, in the publications of the International tured in a manner which is itself known to one skilled in the Conference on Harmonisation and in REMINGTON'S art, for example, by means of conventional mixing, granulat PHARMACEUTICAL SCIENCES, 8' Edition Ed. Bertram ing, dragee-making, softgel encapsulation, dissolving, G. Katzung, chapters 27 and 28, pp. 484-528 (Mack Publish extracting, or lyophilizing processes. Thus, pharmaceutical ing Company 1990) and yet further in BASIC & CLINICAL preparations for oral use may be obtained by combining the PHARMACOLOGY, chapters 5 and 66, (Lange Medical active compounds with Solid and semi-solid excipients and Books/McGraw-Hill, New York, 2001). Suitable preservatives, and/or co-antioxidants. Optionally, the resulting mixture may be ground and processed. The resulting Pharmaceutical Compositions mixture of granules may be used, after adding Suitable aux 0258 Any suitable route of administration may be iliaries, if desired or necessary, to obtain tablets, softgels, employed for providing a patient with an effective dosage of loZenges, capsules, or dragee cores. drugs of the present invention. For example, oral, rectal, 0264 Suitable excipients may be fillers such as saccha topical, parenteral, ocular, intracranial, pulmonary, nasal, and rides (e.g., lactose, Sucrose, or mannose). Sugar alcohols (e.g., the like may be employed. Dosage forms may include tablets, mannitol or Sorbitol), cellulose preparations and/or calcium troches, dispersions, Suspensions, solutions, capsules, phosphates (e.g., tricalcium phosphate or calcium hydrogen creams, ointments, aerosols, and the like. In certain embodi phosphate). In addition binders may be used such as starch ments, it may be advantageous that the compositions paste (e.g., maize or corn starch, wheat starch, rice starch, described herein be administered orally. In other embodi potato starch, gelatin, tragacanth, methyl cellulose, hydrox ments, it may be advantageous that the compositions ypropylmethyl-cellulose, Sodium carboxymethylcellulose, US 2009/O124574 A1 May 14, 2009

and/or polyvinyl pyrrolidone). Disintegrating agents may be able lipophilic solvents, co-solvents (such as DMSO or etha added (e.g., the above-mentioned starches) as well as car nol), and/or vehicles including fatty oils, for example, rice boxymethyl-Starch, cross-linked polyvinyl pyrrolidone, agar, bran oil or peanut oil and/or palm oil, or synthetic fatty acid or alginic acid or a salt thereof (e.g., Sodium alginate). AuX esters, for example, ethyl oleate or triglycerides, may be used. iliaries are, above all, flow-regulating agents and lubricants Aqueous injection Suspensions may contain Substances that (e.g., silica, talc, Stearic acid or salts thereof. Such as magne increase the Viscosity of the Suspension including, for sium Stearate or calcium Stearate, and/or polyethylene glycol, example, Sodium carboxymethyl cellulose, Sorbitol, dextran, or PEG). Dragee cores are provided with suitable coatings, and/or cyclodextrins. Cyclodextrins (e.g., B-cyclodextrin) which, if desired, are resistant to gastric juices. Softgelatin may be used specifically to increase the water solubility for capsules ('softgels') are provided with Suitable coatings, parenteral injection of the structural carotenoid analog. Lipo which, typically, contain gelatin and/or Suitable edible dye(s). Somal formulations, in which mixtures of the structural caro Animal component-free and kosher gelatin capsules may be tenoid analog or derivative with, for example, egg yolk phos particularly suitable for the embodiments described herein photidylcholine (E-PC), may be made for injection. for wide availability of usage and consumption. For this pur Optionally, the Suspension may contain stabilizers, for pose, concentrated saccharide Solutions may be used, which example, antioxidants such as BHT, and/or preservatives, may optionally contain gum arabic, talc, polyvinyl pyrroli Such as benzyl alcohol. done, polyethylene glycol (PEG) and/or titanium dioxide, 0269. The compounds of this invention can be adminis lacquer Solutions and Suitable organic solvents or solvent tered in Such oral dosage forms as tablets, capsules (each of mixtures, including dimethylsulfoxide (DMSO), tetrahydro which includes sustained release or timed release formula furan (THF), acetone, ethanol, or other suitable solvents and tions), pills, powders, granules, elixirs, tinctures, Suspen co-solvents. In order to produce coatings resistant to gastric sions, syrups, and emulsions. They may also be administered juices, Solutions of Suitable cellulose preparations such as in intravenous (bolus or infusion), intraperitoneal, Subcuta acetylcellulose phthalate or hydroxypropylmethyl-cellulose neous, or intramuscular form, all using dosage forms well phthalate, may be used. Dye stuffs or pigments may be added known to those of ordinary skill in the pharmaceutical arts. to the tablets or dragee coatings or softgelatin capsules, for They can be administered alone, but generally will be admin example, for identification or in order to characterize combi istered with a pharmaceutical carrier selected on the basis of nations of active compound doses, or to disguise the capsule the chosen route of administration and standard pharmaceu contents for usage in clinical or other studies. tical practice. 0265. Other pharmaceutical preparations that may be used (0270. The dosage regimen for the compounds of the orally include push-fit capsules made of gelatin, as well as present invention will, of course, vary depending upon known soft, thermally sealed capsules made of gelatin and a plasti factors, such as the pharmacodynamic characteristics of the cizer Such as glycerol or Sorbitol. The push-fit capsules may particular agent and its mode and route of administration; the contain the active compounds in the form of granules that species, age, sex, health, medical condition, and weight of the may be mixed with fillers such as, for example, lactose, bind recipient; the nature and extent of the symptoms; the kind of ers such as starches, and/or lubricants such as talc or magne concurrent treatment; the frequency of treatment; the route of sium Stearate and, optionally, stabilizers and/or preservatives. administration, the renal and hepatic function of the patient, In soft capsules, the active compounds may be dissolved or and the effect desired. A physician or veterinarian may deter Suspended in Suitable liquids. Such as fatty oils such as rice mine and prescribe the effective amount of the drug required bran oil or peanut oil or palm oil, or liquid paraffin. In some to prevent, counter, or arrest the progress or the development embodiments, stabilizers and preservatives may be added. prostate cancer in a Subject. 0266. In some embodiments, pulmonary administration of 0271. By way of general guidance, the daily oral dosage of a pharmaceutical preparation may be desirable. Pulmonary each active ingredient, when used for the indicated effects, administration may include, for example, inhalation of aero will range between about 0.001 to 1000 mg/kg of body solized or nebulized liquid or solid particles of the pharma weight, between about 0.01 to 100 mg/kg of body weight per ceutically active component dispersed in and Surrounded by a day, or between about 1.0 to 20 mg/kg/day. Intravenously gaS. administered doses may range from about 1 to about 10 0267 Possible pharmaceutical preparations, which may mg/kg/minute during a constant rate infusion. Compounds of be used rectally, include, for example, Suppositories, which this invention may be administered in a single daily dose, or consist of a combination of the active compounds with a the total daily dosage may be administered in divided doses of Suppository base. Suitable Suppository bases are, for two, three, or four or more times daily. example, natural or synthetic triglycerides, or paraffin hydro 0272. The pharmaceutical compositions described herein carbons. In addition, it is also possible to use gelatin rectal may further be administered in intranasal form via topical use capsules that consist of a combination of the active com of Suitable intranasal vehicles, or via transdermal routes, pounds with a base. Possible base materials include, for using transdermal skin patches. When administered in the example, liquid triglycerides, polyethylene glycols, or paraf form of a transdermal delivery system, the dosage adminis fin hydrocarbons. tration will, of course, be continuous rather than intermittent 0268 Suitable formulations for parenteral administration throughout the dosage regimen. include, but are not limited to, aqueous solutions of the active 0273. The compounds are typically administered in compounds in water-soluble and/or water dispersible form, admixture with Suitable pharmaceutical diluents, excipients, for example, water-soluble salts, esters, carbonates, phos or carriers (collectively referred to herein as “pharmacologi phate esters or ethers, sulfates, glycoside ethers, together with cally inert carriers’) suitably selected with respect to the spacers and/or linkers. Suspensions of the active compounds intended form of administration, that is, oral tablets, capsules, as appropriate oily injection Suspensions may be adminis elixirs, syrups and the like, and consistent with conventional tered, particularly Suitable for intramuscular injection. Suit pharmaceutical practices. US 2009/O124574 A1 May 14, 2009 44

0274 For instance, for oral administration in the form of a tion preferably containa water-soluble salt of the active ingre tablet or capsule, the pharmacologically active component dient, Suitable stabilizing agents, and if necessary, buffer may be combined with an oral, non-toxic, pharmaceutically Substances. Antioxidizing agents such as Sodium bisulfite, acceptable, inert carrier Such as lactose, starch, Sucrose, glu Sodium Sulfite, orascorbic acid, either alone or combined, are cose, methyl cellulose, magnesium Stearate, dicalcium phos Suitable stabilizing agents. Also used are citric acid and its phate, calcium sulfate, mannitol, Sorbitol and the like; for oral salts and sodium EDTA. In addition, parenteral solutions can administration in liquid form, the oral drug components can contain preservatives, such as benzalkonium chloride, be combined with any oral, non-toxic, pharmaceutically methyl- or propyl-paraben, and chlorobutanol. acceptable inert carrier Such as ethanol, glycerol, water, and 0280 Suitable pharmaceutical carriers are described in the like. Moreover, when desired or necessary, suitable bind ers, lubricants, disintegrating agents, and coloring agents can Remington's Pharmaceutical Sciences, Mack Publishing also be incorporated into the mixture. Suitable binders Company, a standard reference text in this field. include starch, gelatin, natural Sugars such as glucose or 0281. In an embodiment, the pharmaceutical composi beta-lactose, corn Sweeteners, natural and synthetic gums tions can be administered locally to the site of neural injury Such as acacia, tragacanth, or sodium alginate, carboxymeth Such as by local injection, a minimally invasive drug delivery ylcellulose, polyethylene glycol, waxes, and the like. Lubri system, or during a Surgical procedure. Alternatively, in some cants used in these dosage forms include Sodium oleate, embodiments it may be preferable to administer the compo Sodium Stearate, magnesium Stearate, Sodium benzoate, sition(s) systemically (such as e.g., by oral or parenteral Sodium acetate, Sodium chloride, and the like. Disintegrators delivery) Such that the concentration of the pharmacologi include, without limitation, starch, methyl cellulose, agar, cally active agents is Sufficient to increase the bioavailability bentonite, Xanthan gum, and the like. of NO to platelets in the vasculature of the subject. 0275. The compounds of the present invention may also be 0282. In an embodiment, the active compounds may be administered in the form of liposome delivery systems, such administered to the patient systemically. The term systemic as as Small unilamellar vesicles, large unilamellar vesicles, and used herein includes Subcutaneous injection; intravenous, multilamellar vesicles. Liposomes can beformed from a vari intramuscular, intraestemal injection; infusion; inhalation, ety of phospholipids, such as cholesterol, Stearylamine, or transdermal administration, oral administration; and intra phosphatidylcholines. operative instillation. 0276 Compounds of the present invention may also be 0283. One systemic method involves an aerosol suspen coupled with soluble polymers as targetable drug carriers. sion of respirable particles comprising the active compound, Such polymers can include polyvinylpyrrolidone, pyran which the subject inhales. The active compound would be copolymer, polyhydroxypropylmethacrylamide-phenol, absorbed into the bloodstream via the lungs, and subse polyhydroxyethylaspartamidephenol, or polyethyleneoxide quently contact the lacrimal glands in a pharmaceutically polylysine substituted with palmitoyl residues. Furthermore, effective amount. The respirable particles may be liquid or the compounds of the present invention may be coupled to a Solid, with a particle size Sufficiently small to pass through the class of biodegradable polymers useful in achieving con mouth and larynx upon inhalation; in general, particles rang trolled release of a drug, for example, polylactic acid, polyg ing from about 1 to 10 microns, but more preferably 1-5 lycolic acid, copolymers of polylactic and polyglycolic acid, microns, in size are considered respirable. polyepsilon caprolactone, polyhydroxybutyric acid, poly 0284 Another method of systemically administering the orthoesters, polyacetals, polydihydropyrans, polycyanoacy active compounds involves administering a liquid/liquid Sus lates, and crosslinked or amphipathic block copolymers of pension in the form of eye drops or eye wash or nasal drops of hydrogels. a liquid formulation, or a nasal spray of respirable particles 0277 Dosage forms (pharmaceutical compositions) suit that the Subject inhales. Liquid pharmaceutical compositions able for administration may contain from about 1 milligram of the active compound for producing a nasal spray or nasal or to about 100 milligrams or more of active ingredient per eye drops may be prepared by combining the active com dosage unit. In these pharmaceutical compositions the active pound with a suitable vehicle. Such as Sterile pyrogen free ingredient will ordinarily be present in an amount of about water or sterile saline by techniques known to those skilled in 0.5-95% by weight based on the total weight of the compo the art. sition. 0285. The active compounds may also be systemically 0278 Gelatin capsules may contain the active ingredient administered through absorption by the skin using transder and powdered carriers, such as lactose, starch, cellulose mal patches or pads. The active compounds are absorbed into derivatives, magnesium Stearate, Stearic acid, and the like. the bloodstream through the skin. Plasma concentration of the Similar diluents can be used to make compressed tablets. active compounds can be controlled by using patches con Both tablets and capsules can be manufactured as Sustained taining different concentrations of active compounds. release products to provide for continuous release of medica 0286. Other methods of systemic administration of the tion over a period of hours. Compressed tablets can be Sugar active compound involves oral administration, in which phar coated or film coated to mask any unpleasant taste and protect maceutical compositions containing active compounds are in the tablet from the atmosphere, or enteric coated for selective the form of tablets, lozenges, aqueous or oily suspensions, disintegration in the gastrointestinal tract. Viscous gels, chewable gums, dispersible powders or gran 0279 Liquid dosage forms for oral administration can ules, emulsion, hard or soft capsules, or syrups or elixirs. contain coloring and flavoring to increase patient acceptance. Additional means of systemic administration of the active In general, water, a Suitable oil, Saline, aqueous dextrose compound to the Subject may involve a Suppository form of (glucose), and related Sugar Solutions and glycols such as the active compound. Such that a therapeutically effective propylene glycolor polyethylene glycols are Suitable carriers amount of the compound reaches the eyes via systemic for parenteral Solutions. Solutions for parenteral administra absorption and circulation. US 2009/O124574 A1 May 14, 2009

0287 Further means of systemic administration of the calcium phosphate or kaolin, or as Soft gelatin capsules active compound involve direct intra-operative instillation of wherein the active ingredient is mixed with water or an oil a gel, cream, or liquid Suspension form of a therapeutically medium, for example, peanut oil, liquid paraffin or olive oil. effective amount of the active compound. Formulation for oral use may also be presented as chewable 0288 For topical application, the solution containing the gums by embedding the active ingredient in gums so that the active compound may contain a physiologically compatible active ingredient is slowly released upon chewing. vehicle, as those skilled in the art can select, using conven 0292 For rectal administration, the compositions in the tional criteria. The vehicles may be selected from the known form of Suppositories can be prepared by mixing the active pharmaceutical vehicles which include, but are not limited to, ingredient with a suitable non-irritating excipient which is saline solution, water polyethers such as polyethylene glycol, Solidat ordinary temperatures but liquid at the rectal tempera polyvinyls such as polyvinyl alcohol and povidone, cellulose ture and will therefore melt in the rectum to release the com derivatives such as methylcellulose and hydroxypropyl meth pound. Such excipients include cocoa butter and polyethyl ylcellulose, petroleum derivatives Such as mineral oil and ene glycols. white petrolatum, animal fats Such as lanolin, polymers of acrylic acid Such as carboxypolymethylene gel, vegetable fats 0293. The pharmaceutical activity of carotenoid analogs Such as peanut oil and polysaccharides such as dextrans, and and derivatives as well as antiplatelet agents in accordance glycosaminoglycans such as sodium hyaluronate and salts with the objects of this invention may be assessed using, for example, any of the methods shown below. Other methods for Such as Sodium chloride and potassium chloride. assessing the pharmacological activity of the present formu 0289 For systemic administration such as injection and infusion, the pharmaceutical formulation is prepared in a lations are well within the skill level of the ordinary practi sterile medium. The active ingredient, depending on the tioner of the pharmaceutical arts. vehicle and concentration used, can either be suspended or 0294 The present invention provides that platelet aggre dissolved in the vehicle. Adjuvants such as local anaesthetics, gation be inhibited or reduced in vivo by administration of preservatives and buffering agents can also be dissolved in the various pharmacologically active agents, or combinations vehicle. The sterile injectable preparation may be a sterile thereof. The present invention describes the utility of various injectable solution or Suspension in a non-toxic acceptable carotenoid analogs and derivatives, various antiplatelet agent, diluent or solvent. Among the acceptable vehicles and Sol and various combinations of carotenoid analogs and deriva vents that may be employed are sterile water, saline solution, tive with antiplatelet agents, by addressing a plurality of or Ringer's solution. diseases under which atherapeutic modality is clinically ben 0290 For oral use, an aqueous suspension is prepared by eficial. addition of water to dispersible powders and granules with a 0295) The invention is illustrated further by the following dispersing or Wetting agent, Suspending agent one or more examples which are not to be construed as limiting the inven preservatives, and other excipients. Suspending agents tion in Scope or spirit to the specific procedures described in include, for example, sodium carboxymethylcellulose, meth them. ylcellulose and Sodium alginate. Dispersing or wetting agents 0296 Having now described the invention, the same will include naturally-occurring phosphatides, condensation be more readily understood through reference to the follow products of an allylene oxide with fatty acids, condensation ing example(s), which are provided by way of illustration, products of ethylene oxide with long chain aliphatic alcohols, and are not intended to be limiting of the present invention. condensation products of ethylene oxide with partial esters from fatty acids and a hexitol, and condensation products of Materials and Methods ethylene oxide with partial esters derived from fatty acids and hexitol anydrides. Preservatives include, for example, ethyl, Materials: and n-propyl p-hydroxybenzoate. Other excipients include 0297. Non-esterified, all-Eastaxanthin (3S,3'S-AST) was Sweetening agents (e.g., Sucrose, saccharin), flavoring agents synthesized by Synchem, Inc. (Des Plaines, Ill.) and used and coloring agents. Those skilled in the art will recognize the without further modification (>97% chiral purity by HPLC: many specific excipients and wetting agents encompassed by FIG. 1). Clopidogrel (2-(2-chlorphenyl)-2-(2,4,5,6,7,7a the general description above. hexahydrothieno3.2cpyridine-5-yl-acetic acid methylester 0291 For oral application, tablets are prepared by mixing hydrogen sulfate, 7S) was obtained and used without further the active compound with nontoxic pharmaceutically accept modification. Both compounds were introduced into the able excipients suitable for the manufacture of tablets. These model system using Tyrode-HEPES buffer. excipients may be, for example, inert diluents, such as cal cium carbonate, sodium carbonate, lactose, calcium phos phate or sodium phosphate; granulating and disintegrating Donors and Endothelial Cell Preparation: agents, for example, corn starch, or alginic acid; binding 0298 Human umbilical vein endothelial cells (HUVECs) agents, for example, starch, gelatin or acacia; and lubricating were isolated into primary cultures from white female donors agents, for example magnesium Stearate, Stearic acid or talc. by Clonetics (San Diego, Calif.), essentially as described The tablets may be uncoated or they may be coated by known (Mason et al. 2005). All donors were reported to be healthy techniques to delay disintegration and absorption in the gas and none had pregnancy or prenatal complications. All mea trointestinal tract and thereby provide a Sustained action over Surements were recorded in vitro using a sensitive porphy a longer period. For example, a time delay material Such as rinic probe, also as previously described. HUVEC cells were glyceryl monostearate or glyceryl distearate may be grown in Ham's F12K medium with 2 mM L-glutamine employed. Formulations for oral use may also be presented as adjusted to contain 1.5 g/L Sodium bicarbonate and Supple hard gelatin capsules wherein the active ingredient is mixed mented with 0.1 mg/mL heparin and 0.03-0.05 mg/mL endot with an inert Solid diluent, for example, calcium carbonate, helial cell growth supplement (ECGS) plus 10% fetal bovine US 2009/O124574 A1 May 14, 2009 46 serum. The HUVEC cells were kept in an atmosphere of cisely measure the NO concentration (with a detection limit elevated CO, concentration (5%). of about 10 M, a response time better than 100 us, and precision better than +7%). Animals and Platelet Isolation: Statistical Analysis: 0299 Wistar-Kyoto (WKY) rats were anaesthetized and (0303 Statistical analyses were performed with the NCSS blood was collected directly from the heart using a 10 mL statistical software package (NCSS 2001 and PASS 2002, syringe that contained 3.8% sodium citrate (9:1, v v'). The Kaysville, Utah). One-way analysis of variance (one-way blood was centrifuged at 200xg for 15 min at room tempera ANOVA) was followed by Student-Newman-Keuls multiple ture. The platelet rich plasma (PRP) was then removed by comparisons post-hoc test. All statistical tests were per aspiration. The washed platelets were finally suspended in formed at an O.-0.05. A sample size of six (6) was evaluated calcium (Ca")-free Tyrode-HEPES buffer to ascertain that for each treatment group. Data is reported as meanistandard NO was not generated by activation of the aggregating pro deviation (meantS.D.). cess in the presence of extracellular Ca". The number of platelets was counted and adjusted to 3.5x10 per mL with EXAMPLES Tyrode-HEPES buffer. The platelets were incubated in an Example 1 aggregometer with a NO nanosensor (working electrode) placed in the platelet Suspension. Separate versus Combined Effects of Homochiral Astaxanthin and Clopidogrel on Platelet-Dependent Measurement of NO: Nitric Oxide (NO) Release 0304. The effects of inositol triphosphate (IP) stimulation 0300. As shown in FIG. 2, measurement of NO was con on total NO release from vascular platelets following incuba ducted using an electrochemical nanosensor. All measure tion with either 5.0LM homochiralastaxanthin and/or 1.0LM ments of NO release were conducted in Hank's balanced salt clopidogrel are shown in FIG. 3 (panels A and B). The sepa solution (HBSS) at 37°C. The sensor operated with a three rate versus combined effects of these agents on NO release is electrode system: nanosensor (working electrode), Saturated reported in absolute terms FIG. 3A, as well as the percentage calomel electrode (reference electrode), and platinum wire (%) increase over mean control levels in FIG. 3B. (counter electrode, 0.5 mm diameter). The three electrodes 0305 Pre-treatment of vascular platelets with homochiral were connected to a potentiostat/galvanostat PAR273. The astaxanthin alone induced a pronounced mean increase in baseline was stabilized after ~20s. With a nanoinjector, the their capacity to generate bioavailable NO by almost two-fold test compounds were injected onto the Surface of the cells vs. controls (6.6+0.3 to 12.9+0.3 molecules NOx10/plate following solubilization in buffer. In experiments with mul let). Albeit to a lesser extent, clopidogrel also caused a tiple compounds, the compounds were injected simulta marked mean increase in platelet-derived NO release by more neously. The current (amps) proportional to the NO concen than 50% vs. controls (6.6+0.3 to 10.2+0.4 molecules tration was measured with the sensor, which operated in NOx10°/platelet). Significant main effects of monotherapy, amperometric mode at a constant potential of 0.63 V. Data then, were seen for each agent. were acquired with the use of custom Software. Amperograms 0306 Synergy is observed with after the administration of (current vs. time curves) were recorded with a Guniry FAS1 both compounds; the relative increase in NO produced by the Femtostat (Warminster, Pa.). combination exceeded that of astaxanthin alone and clopi 0301 The nanosensor for NO was calibrated using satu dogrel alone, Supporting a co-formulation strategy for the two rated solution (concentration 1.82 mM verified with the cou compounds. The absolute mean amounts of NO produced by lometric method). Linear calibration curves were constructed the combination exceeded that of homochiral astaxanthin for each sensor from 5x10 to 3x10 MNO before and after alone by more than 75%, and clopidogrel alone by more than two-fold (panel 3A). When standardized as a mean % measurements of cell activity. The data were presented as increase, the relative increase in NO produced by the combi meantS.D. for each of the triplicate measurements. The data nation exceeded that of homochiral astaxanthin alone by (calculation and plotting) were transferred to Microcal Origin more than 2-fold, and exceeded that of clopidogrel alone by Software (OriginLab Corp., Northampton, Mass.). more than 4-fold (panel 3B). The amperogram that describes 0302) The nanosensors (diameter 200-300 nm) were the relationship for NO release with time was also distinct for placed in the suspension of 3.5x10 platelets in 250 L of the combination, as evidenced by more Sustained production buffer A solution of inositol triphosphate (IP3), or a solution with time (34s), as compared to only astaxanthin (13s. FIG. of 3S,3'S-AST, clopidogrel, or a combination of 3S,3'S-AST 4). This resulted in a greater net production of NO. These & clopidogrel was added to the platelet Suspension with help findings indicate an important and statistically significant of a computer controlled nanoinjector. The final concentra synergistic benefit for homochiral astaxanthin and clopi tions measured were 5uM and 1 uM, respectively, for 3S.3'S- dogrel with respect to platelet-derived NO production that AST and clopidogrel in each experiment. These Suspensions may indicate complementary mechanisms of action on this were allowed to incubate with the platelets for 30 min prior to cellular process. the NO measurement readings being obtained. Current pro portional to NO concentration was continuously recorded in Example 2 time intervals of 40s. The NO concentration was calculated using current responses obtained from standard NO Solution 0307 Turning to FIG. 4, representative inositol triphos (prepared from 0.176 mM stock solution, saturated aqueous phate (IP)-stimulated amperograms of platelet-derived nitric NO Solution). Nanosensor approaches allowed us to sample a oxide (NO) release following 15 min incubation with 3.S.3'S- Small Volume of Solution with Suspended platelets and pre AST (5.0 LM) alone (black line) and in combination with US 2009/O124574 A1 May 14, 2009 47 clopidogrel (1.0 uM; blue line) are shown. More sustained IP3 (1.0 uM). Values are reported as meani.S.D. (N=5). *p-0. production over time (34s) is observed with the combination 001 and ip-0.05 versus control; Sp-0.001 versus aspirin when compared to the amperogram of 3S,3'S-AST alone (13 treatment (ANOVA Student-Newman-Keuls multiple com s), resulting in greater overall net production of NO with the parisons test; overall ANOVA: p-0.0001; F=52.475). combination. Example 8 Example 3 0313 Shown in FIG. 13 are the effects of aspirin (1.0 uM) 0308 FIG. 5 shows the effects on calcium ionophore on platelet peroxynitrite release in the absence and presence (Call)-stimulated total nitric oxide release (nM) from human of increasing levels of astaxanthin following stimulation with umbilical vein endothelial cells (HUVEC) following incuba IP3 (1.0 uM). Values are reported as meani.S.D. (N=5). *p-0. tion with homochiral astaxanthin (3S.3'S-AST: 5.0 LM) and 001 versus control and ip-0.001 versus aspirin alone clopidogrel (1.0 uM), either alone or in combination. Statis (ANOVA Student-Newman-Keuls multiple comparisons tically significant increases in NO release were seen after test; overall ANOVA: p-0.0001; F-21.905). Abbreviations: treatment with homochiral astaxanthin alone or in combina Astax Astaxanthin. tion with clopidogrel (p<0.001 versus controls and clopi dogrel-treated cells). Treatment with clopidogrel alone did Example 9 not significantly change NO release from HUVEC, and no 0314. Shown in FIG. 14 are the effects of aspirin (1.0 uM) additional significant increase was seen after combination on platelet nitric oxide release in the absence and presence of with homochiral astaxanthin. increasing levels of astaxanthin following stimulation with IP3 (1.0 uM). Values are reported as meani.S.D. (N=5). *p-0. Example 4 001 versus control and ip-0.001 versus aspirin alone 0309 FIG. 6 shows the effects on calcium ionophore (ANOVA Student-Newman-Keuls multiple comparisons (Call)-stimulated total peroxynitrite (OONO ) release (nM) test; overall ANOVA: p-0.0001; F-358.23). Abbreviations: from human umbilical vein endothelial cells (HUVEC) fol Astax Astaxanthin. lowing incubation with homochiralastaxanthin (3S,3'S-AST: 5.0 LM) and clopidogrel (1.0 LM), either alone or in combi Example 10 nation. Statistically significant decreases in peroxynitrite 0315 Shown in FIG. 15 are the effects of simvastatin (1.0 release were seen after treatment with homochiral astaxan uM) on platelet peroxynitrite release in the absence and pres thin (p<0.01 versus controls and p<0.001 versus clopi ence of increasing levels of astaxanthin following stimulation dogrel-treated cells). Treatment with clopidogrel alone did with IP3 (1.0 uM). Values are reported as meantS.D. (N=5). not significantly change peroxynitrite release from HUVEC, *p-0.05 and **p-0.001 versus control; p <0.05 and ip-0. and no additional significant decrease was observed after 001 versus simvastatin alone (ANOVA Student-Newman combination with homochiral astaxanthin. Keuls multiple comparisons test; overall ANOVA: p<0.0001; F=13.716). Abbreviations: Astax Astaxanthin; Example 5 Simva-Simvastatin. 0310 Shown in FIG. 7 are the effects on calcium iono phore (Call)-stimulated total nitric oxide (NO) and peroxyni Example 11 trite (OONO) release (expressed as a ratio) from human 0316 Shown in FIG.16 are the effects of simvastatin (1.0 umbilical vein endothelial cells (HUVEC) following incuba uM) on platelet nitric oxide release in the absence and pres tion with homochiral astaxanthin (3S.3'S-AST: 5.0 LM) and ence of increasing levels of astaxanthin following stimulation clopidogrel (1.0 uM), either alone or in combination. Statis with IP3 (1.0 uM). Values are reported as meantS.D. (N=5). tically significant increases in the NO/OONO release ratio *p-0.001 versus control and ip-0.001 versus simvastatin were seen after treatment with homochiralastaxanthin (p<0. alone (ANOVA Student-Newman-Keuls multiple compari 001 versus controls and clopidogrel-treated cells). Treatment sons test; overall ANOVA: p<0.0001; F=158.53). Abbrevia with clopidogrel alone did not significantly change the tions: Astax=Astaxanthin; Simva-Simvastatin. NO/OONO release from HUVEC, and no additional signifi cant increase in the release ratio was observed after combi Example 12 nation with homochiral astaxanthin. 0317 Shown in FIG. 17 are the effects of aspirin (1.0 uM) Example 6 on platelet nitric oxide/peroxynitrite release ratio in the absence and presence of increasing levels of astaxanthin fol 0311 Shown in FIG. 11 are the comparative effects of lowing stimulation with IP3 (1.0 LM). Values are reported as astaxanthin, aspirin and simvastatin (all at 1.0 LM) on plate meani.S.D. (N=5). *p-0.05 and **p-0.001 versus control; let-derived nitric oxide release following stimulation with IP3 p<0.01 and p-0.001 versus aspirin alone (ANOVA Stu (1.0 uM). Values are reported as mean+S.D. (N=5). *p-0.001 dent-Newman-Keuls multiple comparisons test; overall versus control or simvastatin: ip-0.01 versus aspirin treat ANOVA: p-0.0001; F=129.98). Abbreviations: ment (ANOVA Student-Newman-Keuls multiple compari Astax Astaxanthin. sons test; overall ANOVA: p-0.0001; F=70.756). Example 13 Example 7 0318. Shown in FIG. 18 are the effects of simvastatin (1.0 0312 Shown in FIG. 12 are the comparative effects of uM) on platelet nitric oxide?peroxynitrite release ratio in the astaxanthin, aspirin and simvastatin (all at 1.0 LM) on plate absence and presence of increasing levels of astaxanthin fol let-derived peroxynitrite release following stimulation with lowing stimulation with IP3 (1.0 LM). Values are reported as US 2009/O124574 A1 May 14, 2009 48 meani.S.D. (N=5). *p-0.05 and **p-0.001 versus control; therapy, thereby documenting for the first time a favorable p<0.001 versus simvastatin alone (ANOVA Student-New effect on nitric oxide bioavailability and a potential reduction man-Keuls multiple comparisons test; overall ANOVA: p<0. in oxidative stress invascular platelets forastaxanthin. Physi 0001; F-84.477). Abbreviations: Astax=Astaxanthin; ologic levels of astaxanthin in serum could likely be achieved Simva-Simvastatin. with a 200 to 250 mg dose of this agent in appropriate lipo philic vehicle, if dose-proportionality is achieved upon Example 14 stepped dosing of the non-esterified compound. Significantly higher doses of astaxanthin and its derivatives are well toler 0319. Shown in FIG. 19 are the comparative effects of ated in animals after both I.V. and oral administration. astaxanthin, aspirin and simvastatin (all at 1.0 LM) on plate 0323 Astaxanthin may intersect the nitric oxide-perox let-derived nitric oxide?peroxynitrite release ratio following ynitrite axis to platelets through modulation of Superoxide stimulation with IP3 (1.0 LM). Values are reported as anion bioavailability. Astaxanthin is a well-documented Scav meani.S.D. (N=5). *p-0.05 and ip-0.001 versus control; enger of biologically-produced Superoxide. As well, soft Sp-0.001 versus astaxanthin treatment (ANOVA Student drugs based on the astaxanthin Scaffold are capable of potent Newman-Keuls multiple comparisons test; overall ANOVA: Superoxide anion scavenging, even in the aqueous phase. The p-0.0001; F=40.020). additional anti-inflammatory effects of the parent compound, Interpretation of Results and its bioactive synthetic diesters, are the subject of recent reviews. Additional favorable anti-inflammatory effects of 0320. The beneficial cardiovascular effects of clopidogrel astaxanthin-based soft drugs cannot be ruled out in the setting in vivo have been well documented, particularly as they relate of platelet activation; direct Scavenging of peroxynitrite, as to platelet inhibition through blockade of one of the two described for chemically similar C40 carotenoids (albeit not adenosine diphosphate (ADP) receptors. ADP is released astaxanthin), is also possible. Lycopene is the only other C40 from platelet dense granules upon platelet activation by carotenoid with apparent activity on platelet aggregation, numerous agonists, and Subsequently amplifies platelet through mechanisms separate from the current mechanism thrombotic response regardless of the inciting stimulus. The identified in the current study. P2Y receptor on which clopidogrel is active is linked to G, 0324 Strikingly, the combination of homochiral astaxan it plays a special role in the amplification of platelet activation thin (at 5uM) with clopidogrel (at 1 uM) exhibited synergistic initiated by numerous other pathways. Such activation can improvement on NO bioavailability in the combination result in multiple responses that are critical to arterial throm treated platelets. The combination resulted in a mean % bosis and the inflammatory responses associated with it: (1) increase in NO release (normalized to levels in control, platelet aggregation; (2) dense and alpha granule secretion; untreated platelets) of approximately 4-fold. This was a sig and (3) procoagulant activity. Current therapeutic regimens nificant, non-additive improvement over the favorable using clopidogrel can sometimes yield variable and incom increases seen with each agent as monotherapy. The kinetics plete P2Y receptor blockade; more effective strategies to ofNO release were altered by the combination, resulting in an block P2Y receptor activation, as well as combination increased length of NO production by the platelets (from 13s therapies with aspirin to improve overall anti-platelet efficacy with homochiral astaxanthin alone, to 34 s with the combi offer the potential of improved clinical efficacy. Additional nation). The agents therefore appear to be operating on at least strategies to improve the overall cardiovascular effects of this partially independent mechanisms of action which are thienopyridine agent would include identification and exploi complementary in nature. Further investigations of these tation of novel mechanisms of action on the vascular platelet complementary mechanism(s) of action—alone and in com for clopidogrel as well as to identify novel combination bination—are therefore suggested by this seminal work. therapies based on compounds with complementary, and per 0325 In conclusion, the studies presented herein demon haps synergistic, action(s) on platelet activation. strate a potent and synergistic effect of carotenoids or caro 0321. In the present application, the main effects of homo tenoid derivatives (e.g., astaxanthin) and clopidogrel on chiral (3S,3'S)-astaxanthin and clopidogrel were tested on platelet-derived NO release. The novel platelet effects NO generation from isolated mammalian platelets using pre observed with this combination may offer additional protec viously-described nanosensor technology. The absolute gen tion against atherothrombotic events, already documented in eration of NO after treatment with each agent was compared humans for clopidogrel. AS astaxanthin appears to be safe for to baseline levels of NO generation in control (untreated) human consumption at recommended doses in humans, addi vascular platelets. Significant mean main effects on platelet tional studies of the anti-platelet effects of this agent in NO generation were observed for both agents. When normal ized to mean control levels in untreated platelets, clopidogrel humans may be warranted. at 1 uM increased the generation of NO by almost 50%— thereby identifying a secondary, and important, in vitro In Vivo Model of Reocclusion Following Fibrinolytic potential function of this cardiovascular agent on the platelet. Therapy: The mechanism of action of clopidogrel on NO generation is Drug Preparation: not completely understood at the present time, although ben eficial antioxidant and anti-inflammatory effects of this agent 0326 Disodium disuccinate astaxanthin (DDA: see FIG. have been recently suggested. Although in vivo metabolic 1B) was synthesized from commercially available crystalline activation of clopidogrel is believed to be necessary for effec astaxanthin (Buckton Scott, India) as previously described in tive anti-aggregating properties, in the current study, we Frey, et al. (“The efficient synthesis of disodium disuccinate found activity of the parent compound on NO bioavailability astaxanthin (Cardax).” Org Process Res Dev 8: 796-801, that appears to be separate from the ADP-receptor binding 2004.). The final product purity was 97% (as area under the function. This seminal report will require additional follow curve) by HPLC analysis. DDA was dissolved directly in up to document the mechanism of action described herein. sterile-filtered deionized water. The maximum aqueous dis 0322 Similarly, homochiralastaxanthin at 5uM increased persibility of DDA is approximately 10 mM, or 8.64 mg/ml. the generation of NO by almost 100% when used as mono Vehicle consisted of isotonic sterile 0.9% NaCl solution. US 2009/O124574 A1 May 14, 2009 49

Canine Thrombosis Model: sodium citrate as the anticoagulant (1:100 volume ratio of citrate to blood). A whole blood cell count was determined 0327. The experimental model used is a modification of with an H-2000 cell counter (Texas International Laborato that described by Hong, et al., (Thromb Res 117: 333-342, ries, Inc., Culver City, Calif.). Platelet-rich plasma (PRP), the 2006), Rebello, et al., (Arterioscler Thromb VascBiol 18: Supernatant present after centrifugation of whole blood at 954-960, 1998), and Rote, et al. (Cardiovasc Res 27: 500-507, 100xg for 10 min, was diluted with Platelet-poor plasma 1993). Healthy male or female purpose-bred beagle dogs (PPP) to achieve a platelet count of approximately 200,000/ (9-13 kg) were anesthetized with sodium pentobarbital (30 ml. PPP was prepared by centrifuging the remaining blood at mg/kg, i.v.), intubated and ventilated with room air at a tidal 1,500xg for 10 min and discarding the bottom cellular layer. volume of 30 ml/kg and a rate of 12 breaths per minute. The Ex vivo platelet aggregation was assessed by established left carotid artery (LCA) was exposed by blunt dissection spectrophotometric methods with the use of a four-channel using care not to injure the vessel. An external adjustable aggregometer (BioData PAP-4: BioData Corp., Horsham, stainless steel constrictor was shaped to fit each vessel and Pa.) by recording the increase in light transmission through a adjusted with a nylon Screw (2 mm in diameter) to reduce the stirred suspension of PRP maintained at 37° C. Aggregation pulsatile flow pattern by 50% without altering the mean was induced with ADP (20 uM) or AA (0.65 mM). A subag carotid artery blood flow rate. Vascular injury was accom gregatory concentration of epinephrine (550 nM) was used to plished with the application of a low anodal current to the prime the platelets before addition of the agonists to induce intimal surface of the vessel via an intravascular electrode platelet aggregation. Values for platelet aggregation are composed of a Teflon R insulated, silver-coated, copper wire. expressed as percentage of light transmission standardized to The intravascular electrode was connected to the positive pole PRP and PPP samples yielding 0 and 100% light transmis (anode) of a dual-channel Square wave generator (Grass S88 Sion, respectively. stimulator) and a Grass Constant Current Unit; model CCUIA (Grass Instrument Division, Astro-Med, Inc. West Warwick, R.I.). The cathode was attached at a remote subcu Bleeding Time Determinations: taneous site. The current (300 LA) applied to the intimal 0330 Tongue bleeding time (BT) was determined with the surface of the vessel was monitored continuously. The intra use of a SurgiCutTM (International Technidyne Corp., Edison, vascular electrode and mechanical constrictor were upstream N.J.) device that makes a uniform incision 5 mm long and 1 with respect to the flow probe. Blood flow in the carotid artery mm deep on the upper Surface of the tongue. The tongue was quantified using a graded patency scoring system of 0-3 lesion was blotted with filter paper every 20 sec until the as described previously (16), in which a score of 0 represents transfer of blood to the filter paper was no longer apparent. no flow (total occlusion), 1-2 represents varying degrees of The interval, from the time of the tongue incision until the reduced and cyclic patterns of flow and 3 represents non time that blood was no longer transferred to the filter paper, oscillatory or uninterrupted blood flow. was recorded as the tongue bleeding time. Experimental Design: Statistical Analysis: 0328 FIG. 8 illustrates the experimental protocol. 0331. The data are expressed as meant-SEM and were Twenty-eight dogs were randomized between four treatment analyzed by one-way analysis of variance for group compari groups receiving either DDA (10.30 or 50 mg/kg i.v. n=7 per sons followed by a Dunnett's post hoc t-test to determine the group) or 0.9% NaCl (n=7). Experimental thrombosis of the level of significance. The incidence of carotid artery reocclu left carotid artery (LCA) was induced to achieve total occlu sion was compared between the groups by chi-square test. sion of blood flow. Thirty minutes later, the animals received The change over time in the vessel blood flow patency score an intravenous infusion of DDA or an equivalent volume of was compared between two groups by two-way analysis of 0.9% NaCl solution for thirty minutes (flow rate depended on variance followed by Bonferroni post-tests to determine the dose of DDA). The drug treatment was followed by intra level of significance. Values are considered statistically dif carotid administration of rtPA (0.54 ug/kg bolus+3.26 ug/kg/h infusion) delivered immediately proximal to the ferent at p-0.05. occlusive thrombus. The infusion of rtPA was discontinued Example 15 10 min after establishing 20% of the original flow. The end point determinations consisted of: (1) time to thrombolysis, 0332 The hemodynamic variables of mean arterial blood (2) incidence of LCA reocclusion (secondary thrombosis), pressure and heart rate did not differ significantly throughout (3) vessel patency scores assessed by dynamic flow record the experimental protocol in any of the groups (Table 1). ings, and (4) thrombus weight. However, a small (<15%) transient decrease in arterial blood pressure was seen in 5 dogs receiving the highest dose of Ex Vivo Platelet Aggregation: DDA (50 mg/kg). The blood pressure returned to original 0329 Whole blood (10 ml) was drawn from a right femo values within 30 min and did not affect the outcome of the ral vein cannula into a plastic syringe containing 3.7% study (data not shown).

TABLE 1.

Thrombotic effects of the combined administration of DDA and rt-PA.

Platelet Platelet Time to Time to Count Count occlusion thrombolysis Incidence of (Baseline) (Post rt-PA) (min) (min) Reocclusion O mg/kg DDA 364 - 38 338. 36 86.1 6.7 1645.5 5/7 (0.9% NaCl) 10 mg/kg DDA 392 - 36 35237 843 - 10.2 20.3 6.7 6.7 US 2009/O124574 A1 May 14, 2009 50

TABLE 1-continued

Thrombotic effects of the combined administration of DDA and rt-PA.

Platelet Platelet Time to Time to Count Count occlusion thrombolysis Incidence of (Baseline) (Post rt-PA) (min) (min) Reocclusion 30 mg/kg DDA 349 36 316 32 74.9 14.2 20.6 - 7.9 3/7 50 mg/kg DDA 431 31 363 - 66 95.6 19.8 24.O 6.0 1.7%

Example 16 Ex vivo platelet aggregation responses to AA and ADP, how ever, were dose dependently attenuated by administration of Incidence of Left Carotid Artery Reocclusion DDA. Significant reductions in platelet aggregation were 0333. The LCA was subjected to electrolytic injury lead observed at 30 and 50 mg/kg doses. FIG. 8 shows the percent ing to the development of an occlusive thrombus as indicated platelet aggregation responses to ADP(A) and AA (B) before by the cessation of blood flow in the injured vessel. Thirty and after occlusive thrombus formation in the LCA and sub minutes later, either DDA (10.30 or 50 mg/kg) or 0.9% NaCl sequent treatment with DDA (10, 30 or 50 mg/kg) or 0.9% solution was administered followed by the intra-carotid infu NaCl solution (0 mg/kg. DDA) followed immediately by rt sion of rt-PA that was delivered immediately proximal to the PA. The data represent the mean of 7 experiments+SEM. (*) obstructive thrombus. The time to clot lysis was similar Indicates p-0.05 when compared with respective baseline between the DDA and 0.9% NaCl-treated animals (Table 1). values within each group by one-way ANOVA followed by However, the incidence of reocclusion of the LCA after suc Dunnets post test. (ii) Indicates p<0.05 when the same time cessful thrombolysis was dose dependently reduced by DDA point values are compared between DDA and 0.9% NaCl (0 (6/7, 3/7, 1/7 for 10, 30 and 50 mg/kg doses, respectively) mg/kg. DDA) treatment by unpaired t-test. Baseline (pretreat compared to the 0.9% NaCl-treated group (5/7) (Table 1). ment), black bars: Post drug treatment, white bars. Example 17 Example 19 Effect of DDA on Thrombus Weight Vessel Patency Score 0336 Intravenous administration of DDA dose depen 0334. Successful thrombolysis of the LCA was achieved dently reduced thrombus weight measured at the end of the with the local administration of rt-PA in both the 0.9% NaCl experimental protocol. DDA treatment resulted in a 43+4% and DDA treated animals. In the absence of an adjunctive and 42+5% (30 and 50 mg/kg doses, respectively) reduction anticoagulant, however, most recanalized vessels exhibit in thrombus weights compared to those dogs treated with poor quality of blood flow and progress toward reocclusion. 0.9% NaCl solution. Turning to FIG. 9, thrombus weights Table 2 summarizes the patency scores of the left carotid after occlusive thrombus formation in the LCA and subse arteries assessed at 30-min intervals after the administration quent treatment with DDA (10.30 or 50 mg/kg) or 0.9% NaCl of rt-PA. In animals receiving the intermediate and highest solution followed immediately by rt-PA are shown. The data doses of DDA (30 and 50 mg/kg), there was a progressive represent the mean of 7 experiments: SEM. * indicates p-0. improvement in the vessel patency score during the protocol. 05 when compared with 0.9% NaCl solution (0 mg/kg. DDA, The benefit of DDA was more apparent when compared to the control) by unpaired t-test. 0.9% NaCl-treated group. Although the time to thrombolysis in the LCA was not significantly different between the Example 20 groups, the vessel patency scores of and the incidence of Effects of DDA on Bleeding Time reocclusion were improved significantly in the DDA-treated 0337 Tongue template bleeding time was assessed using a animals. SurgiCut TM device. Bleeding times were similar at baseline

TABLE 2 Effect of DDA on arterial patency score, a determinant of the quality of blood flow. Time period (min) O-30 30-60 60-90 90-120 120-150 O mg/kg DDA 1.3 0.2 17 O.3 1.0 0.4 O.7 O.S. O.7 O.S (0.9% NaCl) 10 mg/kg DDA 1.4 + 0.2 1.3 + 0.4 1.1 + 0.3 1.1 + 0.4 0.4 + 0.4 0.4 + 0.4 30 mg/kg DDA 1.3 0.2 13 O-4 100.4 1.6 OS 1.1 - O.S 1.4 - O.S 50 mg/kg DDA 1.90.3 2.4 - 0.3 2.3 + 0.4 2.3 - 0.5* 2.3 - 0.4 2.3 O.S*

Example 18 and after drug treatment in all of the treatment groups. Addi tionally there were no differences in bleeding times observed Effects of DDA on Ex Vivo Platelet Aggregation between groups at baseline of after drug treatment. FIG. 10 0335. As illustrated in Table 1, whole blood platelet counts shows the Tongue bleeding time (sec) before and after occlu were unaffected by the administration of any dose of DDA. sive thrombus formation in the LCA and subsequent treat US 2009/O124574 A1 May 14, 2009

ment with DDA (10, 30 or 50 mg/kg) or 0.9% NaCl solution 0344 Metabolism of Carotenoid Derivatives (0 mg/kg. DDA) followed immediately by rt-PA. The data 0345 Pharmacokinetic data showing that the carotenoids represent the mean of 7 experiments+SEM. Baseline (pre derivatives described herein, when administered to animals, treatment), black bars: Post DDA treatment, white bars. results in the accumulation of the parent carotenoid in the 0338. As shown in FIG. 11, treatment of platelets with plasma has been determined. For example, we have deter astaxanthin or aspirin alone (1.0 LM) caused a pronounced mined that when astaxanthin derivatives are introduced into increase in the capacity of platelets to generate NO by more than 30%. The rapid and potent effects of astaxanthin indicate animals, theastaxanthin derivates are metabolized to produce a favorable effect on NOS coupling efficiency. The mecha free astaxanthin in the blood stream of the animal. nism for enhanced NOS activity with astaxanthin may be through preservation of NOS and its co-factors in a non Example 21 oxidized and fully functional state. 0339 Enhanced oxidative stress mechanisms cause the 0346. In one experiment, astaxanthin; all-trans 3S.3'S-as NOS enzymes to produce superoxide, the precursor of per taxanthin diester disuccinate disodium salt (hereinafter oxynitrite (ONOO ). With NOS uncoupling, the enzyme ADS); and all-trans 3S.3'S-astaxanthin diester dilysinate acts like peroxynitrite synthase, a stimulant of NF-kB activ tetrahydrochloride salt (hereinafter ADL) were adminis ity. Peroxynitrite is also known to oxidize NOS co-factors tered orally to separate rats as a lipid suspension. A single such as BH4 that leads to further NOS uncoupling. The com dose that included 500 mg/kg of the carotenoid/carotenoid bination of NOS uncoupling and co-factor depletion will derivative was administered to each rat. (See FIG. 20, PK ultimately lead to a disruption in the NO/ONOO cycle, thus Study # CXPK07201). Plasma from each of the rats was contributing to the pathogenesis of chronic diseases. Agents collected 4 hours after ingestion and 8 hours after ingestion that block or reverse NOS uncoupling processes would have and all samples were analyzed by high performance liquid a potential role in the treatment of chronic diseases. chromatography (“HPLC). HPLC analysis was used to 0340. In contrast to astaxanthin and aspirin, simvastatin detect the presence of free underivatized carotenoid (e.g., in had no effect on platelet-derived NO release. The absence of this example, astaxanthin) in the plasma. HPLC chromato activity with simvastatin may be due to the limited incubation grams were collected for each plasma sample taken. The period or a lack of an effect on platelet NOS expression. With HPLC chromatograms are presented in FIG. 21. As can be a longer exposure, simvastatin may be able to influence seen in the chromatograms, four hours after ingestion of genomic expression or stability of NOS RNA transcripts. astaxanthin, there was no significant amount of free astaxan 0341. In FIG. 12, we observed that astaxanthin and simv thin in the rat plasma collected. Eight hour after ingestion of astatin decreased ONOO– release while aspirin actually astaxanthin, there was no significant amount of free astaxan increased nitroxidative stress in a highly significant fashion. thin in the rat plasma collected. This indicates that very little The beneficial effect of astaxanthin is support for its potent (if any) astaxanthin is absorbed by the rats through oral dos and rapid scavenging activity that maintains NOS function. age. Four hours after ingestion of ADS and ADL shows a Remarkably, astaxanthin was able to reverse the effect of significant amount of free astaxanthin in the rat plasma col aspirin on ONOO– release in a dose-dependent manner lected. Eight hour after ingestion of ADS and ADL also shows (FIG. 13). The effect of simvastatin was unexpected but may a significant amount of free astaxanthin in the rat plasma indicate an antioxidant effect associated with its chemical collected. This indicates that carotenoid derivatives are composition, including conjugated ring structures that may absorbed and metabolized by the rats to produce underiva stabilize the unpaired electrons in various resonance struc tized carotenoid. tures. 0342. In the combination study, astaxanthin potentiated Example 22 (by 80%) the effect of NO release observed by aspirin alone (1.0 LM) and a 3-fold increase over untreated samples (FIG. 0347 In a another experiment, all-trans 3S.3'S-astaxan 14). Astaxanthin also increased NOS coupling efficiency (ra thin diester diglycinate dihydrochloride salt (hereinafter tio of NO/ONOO ) by approximately three-fold over con ADG'); all-trans 3S,3'S-astaxanthin diester disarcosinate trol levels as well as aspirin alone (FIG. 16). In fact, aspirin dihydrochloride salt (hereinafter ADSa'); and ADL were separately failed to produce a favorable effect on administered orally to separate rats as a aqueous Suspension NOf ONOO ratio. The favorable effect of astaxanthin on containing 0.5% carboxymethylcellulose. A single dose that NO release with aspirin was dose-dependent and attributed, included 40 mg/kg of the carotenoid derivative was adminis in part, to its ability to attenuate or reverse aspirin-induced tered to each rat. (See FIG. 20, PK Study # CXPK07202). For nitroxidative stress. Astaxanthin also enhanced the effect of each carotenoid derivative given to the rats in this example, simvastatin on platelet-derived NO release in a dose-depen the amount of all astaxanthin isomers, the amount of trans dent fashion. The separate effects of these compounds on the astaxanthin isomers, and the amount of cis astaxanthin iso NO/ONOO– ratio are reviewed in FIG.17 and highlight the mers in the plasma was determined at predefined time inter benefits of astaxanthin. vals for 3 days (72 hours). Cmax (the peak plasma concen 0343 Astaxanthin enhanced platelet-derived NO release tration of astaxanthin, trans astaxanthin and cisastaxanthin); while reducing nitroxidative stress by maintaining NOS in a Tmax (the time it took for the plasma concentration to reach functional state. The effect of astaxanthin was potentiated in Cmax); AUC (the area under the concentration curve); and the presence of aspirin which separately had adverse effects T (elimination half life) was determined for each sample on nitroxidative stress. These findings indicate a new and is presented in FIG.22. As shown in FIG.22, oral dosages approach to reducing atherothrombotic risk with astaxanthin of ADG, ADSa, and ADL were absorbed by the rats and in a manner that complements other anti-platelet therapy, metabolized to produce various Cmax concentrations of Such as aspirin. astaxanthin. This indicates that carotenoid derivatives ADG, US 2009/O124574 A1 May 14, 2009 52

ADSa, and ADL are absorbed and metabolized by the rats to ADL are absorbed and metabolized by the dogs to produce produce underivatized carotenoid (in this example, astaxan underivatized carotenoid (in this example, astaxanthin). thin). Example 26 Example 23 0351. In another experiment, all-trans 3S.3' S-astaxanthin diester di-beta-alanine dihydrochloride salt (hereinafter 0348. In another experiment, ADG; ADSa; and ADL were “ADA'); ADG. ADL: ADSa; and ADS were administered administered to separate rats intravenously as an aqueous orally to separate non-naive dogs as a aqueous Suspension Solution. A single dose that included 5 mg/kg of the caro containing 0.5% carboxymethylcellulose. A single dose that tenoid derivative was administered to each rat. (See FIG. 20. included 20 mg/kg of the carotenoid derivative was adminis PK Study # CXPK07202). For each carotenoid derivative tered to each dog. (See FIG. 20, PK Study # CXPK07405). given to the rats in this example, the amount of all astaxanthin For each carotenoid derivative given to the dogs in this example, the amount of all astaxanthin isomers was deter isomers, the amount of trans astaxanthin isomers, and the mined at predefined time intervals for 3 days (72 hours). amount of cis astaxanthin isomers in the plasma was deter Cmax was determined for each sample and a graph represent mined at predefined time intervals for 3 days (72 hours). ing the obtained data is presented in FIG. 25. As shown in Cmax; Tmax; AUC.; and T1/2 was determined for each FIG. 25, oral dosages of ADA, ADG, ADL, ADSa, and ADS sample and is presented in FIG. 22. As shown in FIG. 22. were absorbed by the dogs and metabolized to produce vari intravenous dosages of ADG, ADSa, and ADL were absorbed ous Cmax concentrations of astaxanthin. This indicates that by the rats and metabolized to produce various Cmax con carotenoid derivatives ADA, ADG, ADL, ADSa, and ADS are centrations of astaxanthin. This indicates that carotenoid absorbed and metabolized by the dogs to produce underiva derivatives ADG, ADSa, and ADL are absorbed and metabo tized carotenoid (in this example, astaxanthin). lized by the rats to produce underivatized carotenoid (in this example, astaxanthin). Example 27 0352. In another experiment, ADG or ADSa was admin Example 24 istered to non-naive beagle dogs orally (as an aqueous Sus pension containing 0.5% carboxymethylcellulose). Oral 0349. In another experiment, ADL was administered to doses included either 50 mg/kg of the carotenoid derivative or non-naive beagle dogs orally (as an aqueous Suspension con 100 mg/kg of the carotenoid derivative. (See FIG. 20, PK taining 0.5% carboxymethylcellulose) and intravenously as Study # CXPK07406). For each carotenoid derivative given an aqueous solution. Oral doses that included 10 mg/kg of the to the dogs in this example, the amount of all astaxanthin carotenoid derivative, 100 mg/kg of the carotenoid derivative, isomers, the amount of trans astaxanthin isomers, and the and 500 mg/kg of the carotenoid derivative were administered amount of cis astaxanthin isomers in the plasma was deter to each dog. The intravenous dosage was 5 mg/kg. (See FIG. mined at predefined time intervals for 3 days (72 hours). 20, PK Study # CXPK07402). For each carotenoid derivative Cmax, Tmax; AUC.; and T was determined for each sample given to the dogs in this example, the amount of all astaxan and is presented in FIG. 26. As shown in FIG. 26, oral dosages thin isomers, the amount of trans astaxanthin isomers, and the of ADG or ADSa were absorbed by the dogs and metabolized amount of cis astaxanthin isomers in the plasma was deter to produce various Cmax concentrations of astaxanthin. This mined at predefined time intervals for 3 days (72 hours). indicates that carotenoid derivatives ADL are absorbed and Cmax, Tmax; AUC.; and T was determined for each sample metabolized by the dogs to produce underivatized carotenoid and is presented in FIG. 23. As shown in FIG. 23, intravenous (in this example, astaxanthin). and oral dosages of ADL were absorbed by the dogs and metabolized to produce various Cmax concentrations of Example 28 astaxanthin. This indicates that carotenoid derivatives ADL 0353. In another experiment, ADSa was administered to are absorbed and metabolized by the dogs to produce non-naive beagle monkeys orally (as an aqueous Suspension underivatized carotenoid (in this example, astaxanthin). containing 0.5% carboxymethylcellulose) and intravenously as an aqueous solution. Oral doses that included 10 mg/kg of Example 25 the carotenoid derivative, 300 mg/kg of the carotenoid deriva tive, and 500 mg/kg of the carotenoid derivative were admin 0350. In another experiment, ADL was administered to istered to each monkey. The intravenous dosage was 5 mg/kg. naive beagle dogs orally (as an aqueous suspension contain (See FIG. 20, PK Study # CXPK07502). For each carotenoid ing 0.5% carboxymethylcellulose) twice a day for six days. derivative given to the monkeys in this example, the amount Each oral dose included 75 mg/kg of the carotenoid deriva of all astaxanthin isomers, the amount of trans astaxanthin tive. (See FIG. 20, PK Study # CXPK07404). For each caro isomers, and the amount of cis astaxanthin isomers in the tenoid derivative given to the dogs in this example, the plasma was determined at predefined time intervals for 3 days amount of all astaxanthin isomers, the amount of trans astax (72 hours). Cmax; Tmax: AUC; and T was determined for anthin isomers, and the amount of cisastaxanthin isomers in each sample and is presented in FIG. 27. As shown in FIG. 27. the plasma was determined at predefined time intervals for 6 intravenous and oral dosages of ADSa were absorbed by the days. Cmax; Tmax: AUC.; and T was determined for each monkeys and metabolized to produce various Cmax concen sample and is presented in FIG. 24. As shown in FIG. 24, oral trations of astaxanthin. This indicates that carotenoid deriva dosages of ADL were absorbed by the dogs and metabolized tives ADL are absorbed and metabolized by the monkeys to to produce various Cmax concentrations of astaxanthin over produce underivatized carotenoid (in this example, astaxan the six-day period. This indicates that carotenoid derivatives thin). US 2009/O124574 A1 May 14, 2009 53

0354 We have shown through these, and other unreported experiments, that carotenoid ester derivatives are readily metabolized by a variety of animals. Metabolism of caro tenoid derivatives produces biologically significant amounts of the parent carotenoid in the blood stream of the animal. 0355. In this patent, certain U.S. patents, U.S. patent appli cations, and other materials (e.g., articles) have been incor porated by reference. The text of such U.S. patents, U.S. patent applications, and other materials is, however, only R incorporated by reference to the extent that no conflict exists N between Such text and the other statements and drawings set forth herein. In the event of such conflict, then any such conflicting text in Such incorporated by reference U.S. pat- R5 ents, U.S. patent applications, and other materials is specifi R5 cally not incorporated by reference in this patent. 0356. Further modifications and alternative embodiments of various aspects of the invention may be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner R5 ch of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as the presently preferred embodiments. Elements and 2 materials may be substituted for those illustrated and 2 described herein, parts and processes may be reversed, and certain features of the invention may be utilized indepen dently, all as would be apparent to one skilled in the art after RO having the benefit of this description to the invention. Changes may be made in the elements described herein with out departing from the spirit and scope of the invention as described in the following claims. In addition, it is to be 2^ understood that features described herein independently may, in certain embodiments, be combined. 1. A method of treating a disorder associated with platelet aggregation in a subject comprising administering to a Sub O ject who would benefit from such treatment a pharmaceuti- Or cally acceptable composition comprising a therapeutically R N effective amount of a carotenoid analog or derivative. 2-11. (canceled) 12. A pharmaceutical composition comprising: R5 a therapeutically effective amount of a one or more caro tenoid analogs or derivatives; and R5 atherapeutically effective amount of at least one additional O non-carotenoid medicament or composition Suitable for \ the treatment of a disorder associated with platelet aggregation. 13. The composition of claim 12, wherein the amount of the carotenoid analog or derivative is sufficient to at least partially increase the bioavailability of nitric oxide (NO) in the subject. 14. The composition of claim 12, wherein the carotenoid analog or derivative has the structure

where each R is independently hydrogen or methyl, and where R' and Rare each independently:

US 2009/O124574 A1 May 14, 2009 55

16. The composition of claim 12, wherein the carotenoid analog or derivative has the structure -continued O

OR6 where each R is independently hydrogen or methyl, and where each R' and Rare independently: where R is hydrogen or methyl; where each R is indepen dently hydrogen, OH, or -OR wherein at least one R group is —OR; wherein each R is independently: alkyl: aryl; -alkyl-N(R); -aryl-N(R); -alkyl-CO, H: -aryl-COH: -O-C(O) R; -P(O)(OR); S(O) (OR); an amino acid; a peptide, a carbohydrate; —C(O)-(CH2), CO.R. a nucleoside reside, or a co antioxidant; where R is hydrogen, alkyl, or aryl; wherein R is hydrogen, alkyl, aryl, benzyl or a con antioxidant; where R is hydrogen; alkyl: aryl; P(O) (OR); S(O)(OR); an amino acid; a peptide, a car bohydrate; a nucleoside, or a co-antioxidant; and where n is 1 to 9. 17. The composition of claim 12, wherein the carotenoid analog or derivative has the structure

---~~ R2

where each R' and Rare independently:

US 2009/O124574 A1 May 14, 2009 56

18. The composition of claim 12, where each —OR is -continued independently

2

HO

2 O O OH HO O O O X, O, O S. R HO OH OH R5 O O O O, O S. O s HO OH OH

O

O

O O

MeO OMe

OR6 O

O s where each R is independently hydrogen, OH, or O —OR wherein at least one R group is —OR; wherein each R is independently: alkyl: aryl; -alkyl-N(R): O -aryl-N(R);-alkyl-COH:-aryl-COH:—O C(O)— O R; -P(O)(OR); -S(O)(OR); an amino acid; a | -OMe, peptide, a carbohydrate; C(O) (CH), COR: a o1 \ nucleoside reside, or a co-antioxidant; where R is OMe hydrogen, alkyl, or aryl; wherein R is hydrogen, alkyl, O aryl, benzyl, or a co-antioxidant; and where R is hydro gen; alkyl: aryl; -P(O)(OR): NH2: —S(O)(OR); an amino acid; a peptide, a carbohydrate; a O nucleoside, or a co-antioxidant; and where n is 1 to 9. US 2009/O124574 A1 May 14, 2009 57

-continued -continued

O O H NHHCl; O N O NYS NHHCI O O NH2: NH2 NH2, H O NH" -- "S N ~" O O and wherein each R is independently H, alkyl, aryl, benzyl, Group IA metal, or co-antioxidant. -- N~~ NH2: 19. The composition of claim 18, where each —OR is NH2 independently O H O N OH

O NH2: O H O N OH: O NH2 O or a co-antioxidant; wherein R is hydrogen, alkyl, aryl, ben Zyl. Group IA metal, or a co-antioxidant; wherein R' is CH: and wherein n is 1 to 9. 20. The composition of claim 18, where each —OR is independently

O

O -- NH2:

O

21. The composition of claim 12, wherein the carotenoid analog or derivative has the structure US 2009/O124574 A1 May 14, 2009 58

wherein each R is independently hydrogen or methyl, and 22. The composition of claim 12, wherein non-carotenoid wherein each R" and Rare independently: medicament or composition Suitable for the treatment of a disorder associated with platelet aggregation comprises one or more anticoagulants. 23. The composition of claim 22, wherein said anticoagul lants comprise aspirin. 24. The composition of claim 12, wherein non-carotenoid medicament or composition Suitable for the treatment of a disorder associated with platelet aggregation comprises one or more non-fractionated or fractionated heparins. 25. The composition of claim 12, wherein non-carotenoid medicament or composition Suitable for the treatment of a disorder associated with platelet aggregation comprises one or more statins. 26. The composition of claim 25, wherein said statins comprise simvastatin. 27. The composition of claim 12, wherein non-carotenoid medicament or composition Suitable for the treatment of a disorder associated with platelet aggregation comprises one or more ADP receptor inhibitors. 28. The composition of claim 27, wherein said ADP recep tor inhibitors comprise clopidogrel. 29. The composition of claim 12, wherein non-carotenoid medicament or composition Suitable for the treatment of a disorder associated with platelet aggregation comprises one or more thrombin inhibitors. 30. The composition of claim 12, wherein non-carotenoid medicament or composition Suitable for the treatment of a disorder associated with platelet aggregation comprises one or more factor Xa inhibitors. 31. The composition of claim 12, wherein non-carotenoid medicament or composition Suitable for the treatment of a where each R is independently hydrogen, -OH, disorder associated with platelet aggregation comprises one or more agonists of purinergic receptors. —CH-OH, or —OR, wherein at least one R group is 32. The composition of claim 12, wherein non-carotenoid —OR; wherein each R is independently: alkyl: aryl; medicament or composition Suitable for the treatment of a -P(O)(OR); an amino acid; a peptide, a carbohy disorder associated with platelet aggregation comprises one drate;—C(O)–(CH), CO.R. a nucleoside residue, or more antagonists of CD40 or CD40 ligand (CD40L) or or a co-antioxidant; wherein R is hydrogen, alkyl, aryl, compounds that disrupt the interaction of CD40 and CD40L. benzyl or a co-antioxidant; wherein R is hydrogen; 33. The composition of claim 12, wherein non-carotenoid alkyl: aryl; -P(O)(OR); an amino acid; a peptide, a medicament or composition Suitable for the treatment of a carbohydrate; a nucleoside, or a co-antioxidant; and disorder associated with platelet aggregation comprises one wherein n is 1 to 9. or more PGE1 agonists, PG synthase inhibitors, TX synthase inhibitors, and TXA2 antagonists); glycoprotein IIb/IIIa antagonists, and any combination thereof. 34. The composition of claim 12, further comprising one or more pharmaceutically acceptable carriers. 35. A method of treating a disorder associated with platelet aggregation in a Subject comprising administering to a Sub ject who would benefit from such treatment a pharmaceutical composition comprising: a therapeutically effective amount of a one or more caro tenoid analogs or derivatives; and atherapeutically effective amount of at least one additional non-carotenoid medicament or composition Suitable for the treatment of a disorder associated with platelet or a co-antioxidant; wherein R is hydrogen, alkyl, aryl, ben aggregation. Zyl. Group IA metal, or a co-antioxidant; wherein R' is CH: 36-57. (canceled) and wherein n is 1 to 9.