LV13564B - Methods and compositions for treating ophtalmic conditions with retinyl derivatives - Google Patents

Description

METHODS AND COMPOSITIONS FOR TREATING OPHTHALMIC CONDITIONS VVITH RETINYL DERIVATIVES

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application serial number 60/582,293, filed on June 23, 2004, U.S. Provisional Application serial number 60/629,695, filed on November 19, 2004, U.S. Provisional Application serial number 60/660,904, filed on March 11, 2005, U.S. Provisional Application serial number 60/672,405, filed on April 18, 2005, the disclosures of ali of vvhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The methods and compositions described herein are directed to the treatment of ophthalmic conditions.

BACKGROUND OF THE INVENTION

The visuai cycle or retinoid cycle is a series of light-driven and enzyme catalyzed reactions in which the active visuai chromophore rhodopsin is converted to an all-zranr-isomer that is then subsequently regenerated. Part of the cycle occurs vvithin the outer segment of the rods and part of the cycle occurs in the retinal pigment epithelium (RPE). Components of this cycle include various dehydrogenases and isomerases, as vvell as proteins for transporting intermediates betvveen the photoreceptors and the RPE.

Other proteins associated vvith the visuai cycle are responsible for transporting, removing and/or disposine of compounds and toxic products that accumulate from excess produetion of visuai cycle retinoids, such as all-iran.tretinal (atRAL). For example, /V-retinylidene-A-retinylethanolamine (A2E) arises from the condensation of all-f/anj -retinal vvith phosphatidylethanolamine. Although certain Ievels of this orange-emitting fluorophore are tolerated by the photoreceptors and the RPE, excessive quantities can lead to adverse effects, including the produetion of lipofuscin, and potentially drusen under the macula. See, e.g., Finnemann, S.C., Proc. Nati. Acad. Sei., 99:3842-47 (2002). In addition, A2E can be cytotoxic to the RPE, vvhich can lead to retinal damage and destruction. Drusen are extracellular deposits that accumulate below the RPE and are risk factors for developing age-related macular degeneration. See, e.g., Crabb, J. W., et al., Proc. Nati. Acad. Sei., 99:14682-87 (2002). Thus, removal and disposal of toxic products that arise from side reactions in the visuai cycle are important because several lines of evidence indicate that the over-accumulation of toxic products is partially responsible for the symptoms associated vvith the macular degenerations and retinal dystrophies.

There are tvvo general categories of age-related macular degeneration: the wet and dry forms. Dry macular degeneration, vvhich accounts for about 90 percent of ali cases, is also knovvn as atrophic, nonexudative, or drusenoid macular degeneration. With dry macular degeneration, drusen typically accumulate beneath the RPE tissue in the retina. Vision loss can then occur vvhen drusen interfere vvith the funetion of photoreceptors in the macula. This form of macular degeneration results in the gradual loss of vision over many years.

Wet macular degeneration, vvhich accounts for about 10 percent of cases, is also knovvn as choroidal neovascularization, subretinal neovascularization, exudative, or disciform degeneration. In vvet macular degeneration, abnormal blood vessel grovvth can form beneath the macula; these vessels can leak blood and fluid into the macula and damage photoreceptor celis. Studies have shovvn that the dry form of macular degeneration can lead to the vvet form of macular degeneration. The vvet form of macular degeneration can progress rapidly and cause severe damage to Central vision.

Stargardt Disease, also knovvn as Stargardt Macular Dystrophy or Fundus Flavimaculatus, is the most frequently encountered juvenile onsst fona of macular dystrophy. Research indicates that this condition is transmitted as an autosomal recessive trait in īheABCA4 gene (also known as the ABCR gene). This gene is a member of the ABC Super Family of genes that encode for transmembrane proteīns involved in the energy dependent transport of a wide spectrum of substances aeross membranes.

Symptoms of Stargardt Disease include a decrease in Central Vision and difficulty vvith dark adaptation, problems that generally vvorsen vvith age so that many persons afHicted vvith Stargardt Disease experience visual loss of 20/100 to 20/400. Persons vvith Stargardt Disease are gsnerally encouraged to avoid bright light because of the potential over-produetion of all-ircns-ietinal.

Methods for diagnosing Stargardt Disease include the observation of an atrophic oi beaten-bronze appearance of deterioration in the macula, and the presence of numerous vellovvish-vvhite spots that occur vvithin the retina suirounding the atrophic-appearing central macular lesion. Other diagnostic tests include the use of an electroretinogram, eiectrooculogram, and dark adaptation testing. In addition, a flnorescein angiogram can. be used to confirm the diagnosis. In this latter tēst, observation of a “dark” or “silent” ehoroid appears associated vvith the accumulation of hpofuscin in the retinal pigment epithelium of the patient, one of the eariy symptoms of macular degeneration.

Cuirently, treatment optīons for the macular degenerations and macular dystrophies are limited. Some patients vvith dry fonu AMD have responded to high doses of vitamīns and minerāls. In addition, a fevv studies have indicated that laser photocoagulation of drusen prevents or delays the development of drusen that can lead to the more severe symptoms of dry form AMD. Finally, certain studies have shovvn that extracorporeal rheopheresis benefīts patients vvith dry form AMD.

Hovvever, snccesses have been limited and there continues to be a strong desire for nevv methods and treatments to manage and limit vision loss associated vvith the macular degenerations and dysirophies.

SUMMAlRY OF THE INVENTION

Presented herein are methods, compostions and formulations for (a) treating ophthalmic conditions, and (b) controlling symptoms that presage (e.g., risk factors) or are associated vvith such ophthalmic conditions. In one aspect, such methods and formulations comprise the use of retinyl derivatives. In other aspects the ophthalmic conditions are macular degenerations, macular dystrophies and retinal dystrophies. In other aspects, the methods and formulations are used to protect eyes of a mammai from light, in other aspects the methods and formulations are used to limit the formation of all-zranj-retinal, M-retinyhdene-Vreiinylethanolaniine, Vretinylidene-phosphatidylethanolamine, dūydro-A’-retinylidene-A'-retmyl-phosphatidylethanolainme, NTetinylidene-jV-retinyl-phosphatidylethanoIannne, dihydro-A^r-retinylidene-/V’-retinyI-ethanolamine, Nretinylidene-phosphatidylethanolamine, lipofuscin, geographic atrophy (of vvhich scotoma is one non-limiting example), photoreceptor degeneration and/or drusen in the eye of a mammai. In other aspects, such methods and formulations comprise the use of aģents that can irnpair night vision. In other aspects, such methods and formulations comprise the nse of aģents to treat ophthalmic conditions by (a) lovvering the Ievels of serum retinol in the body of a patient, (b) modnlating the activity of enzymes or proteīns in the eye of a patient vvherein such enzvmes or proteīns are involved in the visual cycle, such as, by way of example, lecithin-retinol acvltransferase and/or cellular retinaldehyde binding protein, or (c) combining the effects of (a) and (b). In yet otbe · aspects, the methods and formulations are used in combiņatioņ vvith other treatment modalities.

In one aspect are methods for reducing the formation of all-ft-azzj-retina] in an eye of a mammai comprising administering to the mamma! at least once an effective amount of a first compound having the structure of Formula (I):

wherem X, is selected from the group consisting of NR², 0, S, CHR²; R¹ is fCHR^j-L'-R³, wherem x is 0, 1, 2, or 3; L¹ is a single bond or -C(0)-; R² is a moiety selected from the group consisting of H, (CjC4)alkyl, F, (Ci-C4)fluoroalkyl, (CrC4)alkoxy, -C(O)OH, -C(O)-NH2, -(Ci-C4)alkylamine, -C(O)-(C1-C4)aIkyl, C(O)-(CrC4)fluoroalkyl, -C(O)-(CI-C4)ālkyIaiiiine, and -C(O)-(Ci-C4)aIkoxy; and R³ is H or a moiety, optionally substituted with 1-3 independently selected substituents, selected from the group consisting of (C-iC7)alkenyl, (C₂-C₇)alkynyl, aryl, (C₃-C₇)cycloalkyl, (Cs-C₇)cycloalkenyl, and a heterocycle, provided that R³ is not H when both x is 0 and L¹ is a single bond; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

In another aspect are methods for reducing the formation of JV-retinylidene-jV-retinylethanolainine, Nretinylidene-phosphatidylethanolamine, dihydro-iV-retinyhdene-jV-retinyl-phosphatidylethanolāmine, Nretinylidene-jV-retinyl-phosphatidylethanolainme, dmy±o-A'-retmyIidene-//-retmyI-ethanolaniLrie, and/or Nretinylidene-phosphatidylethanolamine, in an eye of a mammai comprising administering to the mammai at least once an effective amount of a first compound having the structure of Formula (I).

In another aspect are methods for reducing the formation of lipofuscin in an eye of a mammai comprising administering to the mammai an effective amount of a first compound having the. structure of Formula (I).

In another aspect are methods for reducing the formation of drusen in an eye of a mammai comprising administering to the mammai an effective amount of a first compound having the structure of Formula (I).

In another aspect are methods for modulatmg lecithin-retinol acyltransferase in an eye of a mammai comprising administering to the mammai an effective amount of a first compound having the structure of Formula (I).

In another aspect are methods for treating macular deģenerātiem in an eye of a mammai comprising administering to the mammai an effective amount of a first compound having the structure of Formula (I). In a further embodiment of this aspect, the macular degeneration is juvenile macular degeneration, including Stargardt Dīsease. In a further embodiment of this aspect, (a) the macular degeneration is dry form age-related macular degeneration, or (b) the macular degeneration is cone-rod dysttophy. In a further embodiment of this aspect, the macular degeneration is the wet form of age-related macular degeneration. In a further embodiment of this aspect, the macular degeneration is choroīdal neovascularization, subretinal neovascularization, exudative, or disciform degeneration.

In another aspect are methods for reducing the formation or limiting the spread of geographic atrophy (of which scotoma is one non-Iimiting example) and/or photoreceptor degeneration in an eye of a mammai comprising administering to the mammai an effective amount of a first compound having the structure of Formula (I).

In another aspect are methods for reducing the formation of abnormal blood vessel growth beneath the macula in an eye of s mammai comprising administering to the mammai an effective amount of a first compound having the strueture of Formula (I).

In another aspect are methods for protecting the photoreceptors in any eye of a mammai comprising administering to the mammai an effective amount of a first compound having the strueture of Formula (i).

In another aspect are methods for protecting an eye of a mammai from lighi comprising administering to the mammai an effective amount of a compound having the strueture of Formula (I).

In another aspect are methods for disrupting the visual cycle in an eye of a mammai comprising administering to the mammai an effective amount of a compound having the strueture of Formula (I).

In another aspect is the use of a compound of Formula (I; in the manufacture of a medicament for treating an ophthalmic disease or condition in an animal in vvhich the activity of at least one visual cycle protein contributes to the pathology and/or sytnptoms of the disease or condition. In one embodiment of this aspect, the visual cycle protein is selected from the group consisting of lecithin-retinol acyltransferase and cellular retinaldehyde binding protein. In another or further embodiment of this aspect, the ophthalmic disease or condition is a retinopathy. In a further or altemative embodiment, the retinopathy is a macular degeneration. In a further or altemative embodiment, the symptom of the disease or condition is formation of all-n-mzs-retinal, yVretmvlidene-.V-retinvlethanolamme, N-retinylidene-phosphatidylethanolamme, dihydro-/V-retmylidens-Nretinyl-ph.osphatidylethanolamine, /v^z-retinyIidene-A-retinyl-phosphatidyleihanolainine, dihydro-.V-retuiylidenejV-retinyl-ethanolamine, 7v^r-retinylidene-phosphatidyleihanolamine, lipofuscin, photoreceptor degeneration, geographic atrophy (of vvhich scotoma is one non-limiting example), choroidal neovascularization, and/or drusen in the eye of a mammai.

In any of the aforementioned aspects are ftniher embodiments in vvhich (a) X¹ is NR², vvherein R² is H or (Ci-C4)alkyl; (b) vvherein x is 0; (c) x is 1 and L¹ is -C(O)-; (d) R³ is an optionally substituted aryl; (e) R° is an optionally substituted heteroaryl; (f) X¹ is NH and R³ is an opdonally substituted aiyl, including yet further embodiments in vvhich (i) the aryl group has one substituent, (ii) the aryl group has one substituent selected from the group consisting ofhalogen, OH, O(C[-C4)alkyl, NH(Ci-C4)alkyl, O(C,-C₄)fluoroaIkYL andN[(C[C«)alkyl]₂, (iii) the aryl group has one substituent, vvhich is OH, (v) the aryl is a phenyl, or (vi) the aiyl is

naphthyl; (g) the compound is OH , or an active metabolite, or a phannaceuticall}' acceptable prodrug or solvate thereof; (h) the compound is 4-hydroxyphenylretinamide, or a metabolite, or a pharmaceutically acceptable prodrug or solvate thereof; (i) the compound is 4methoxyphenvlretmamide, or (jj 4-oxo fenretinide, or a metabolite, or a phaimaceutically acceptable prodrug or solvate thereof.

In any of the aforementioned aspects are furthei embodiments in vvhich (a) the effective amount of the compound is systemically administered to the mammai; (b) the effective amount ofthe compound is administered orallv to the mammai; (c) the effective amount of the compound is intravenously administered to the mammai; (d) the effective amount of the compound is ophthahmcal]y administered to the mammai; (e) the effective amount of the compound is ādmini.stgred by iontophoresis; or (f) the effective amount of the compound is administered by injection to the mammai.

In any of the aforementioned aspects are further embodiments in vvhich the mammai is a human, including embodiments vvherein (a) the human is a carrier of the mutant AS CA4 gene for Stargardt Disease or the human has a mutant ELOV4 gene for Stargardt Disease, or has a genetic variation in complement facror H associated with age-related macular degeneration, or (b) the human has an ophthalmic condition or trait selected from the group consisting of Stargardt Disease, recessive retinitis pigmentosa, geographic atrophy (of vvhich scotoma is one non-limiting example), photoreceptor degeneration, diy-form AMD, recessive cone-rod dystrophy, exudative age-related macular degeneration, cone-rod dystrophy, and retinitis pigmentosa. In any of the aforementioned aspects are further embodiments in vvhich the mammai is an animal modei for retina] degeneration, examples of vvhich are provided herein.

In any of the aforementioned aspects are further embodiments comprising multiple administrations of the effective amount of the compound, including further embodiments in vvhich (i) the time betvveen multiple administrations is at least one week; (ii) the time betvveen multiple administrations is at least one day; and (iii) the compound is administered to the mammai on a daily basis; or (iv) the compound is administered to the mammai every 12 hours. In further or altemative embodiments, the method comprises a drug holiday, vvherein the administration of the compound is temporarily suspended or the dose of the compound being administered is temporarily reduced; at the end of the drug holiday, dosing of the compound is resumed. The length of the drug ho!iday can vary from 2 days to 1 year.

In any of the aforementioned aspects are further embodiments comprising administering at least one additional aģent selected from the group consisting of an inducer of nitric oxide produetion, an antiinflammatory aģent, a physiologically acceptable antioxidant, a physiologically acceptable mineral, a negatively charged phospholipid, a carotenoid, a statiņ, an anti-angiogenic drug, a matrix metalloproteinase inhibitor 13czs-retinoic acid (including derivatives of 13-czr-retinoic acid), 11-cn-rennoic acid (including derivatives of 11czr-retinoīc acid), 9-cis-retinoic acid (including derivatives of 9-cis-retinoic acid), and retinylamine derivatives.

In further embodiments:

(a) the additional aģent is an inducer of nitric oxide produetion, including embodiments in vvhich the inducer of nitric oxide produetion is selected from the group consisting of ciirulline, omīthine, nitrosated Z-arginine, nitrosylated Z-arginine, mtrosated A^z-hydroxy-Z-arginine, nitrosylated Nhydroxy-Z-arginine, nitrosated Z-homoarginine and nitrosylated Z-homoarginine;

(b) the additional aģent is an anti-inflanimatory aģent, including embodiments in vvhich the antiinflammatorv aģent is selected from the group consisting of a non-steroidal anti-inflanunatory drug, a lipoxygenase inhibitor, piednisonc, dexamethasone, and a cyclooxygenase inhibitor;

(c) the additional aģent is at least one physiologically acceptable anooKĪdanĻ including embodiments in vvhich the physiologicaliy acceptable antioxidant is selected from the group consisting of Vitamin C, Vitamin E, beta-carotene, Coenzyme Q, and 4-hydroxy-2-2,6,6-teiramethylpiperadine7Z-oxyl, or embodiments in vvhich (i) the at least one physiologically acceptable antioxidant is administered vvith the compound having the structure of Formula (I), or (ii) at least tvvo physiologically acceptable antioridants are administered vvith the compound having the structure of Formula (I);

(d) the additional aģent is at least one physiologically acceptable mineral, including embodiments in which the physiologically acceptable mineral is selected from the group consisting of a zinc (Π) compound, a Cu(II) compound, and a selenium (H) compound, or embodiments further comprising administering to the mammai at least one physiologically acceptable antioridant;

(e) the additional aģent is a negatively charged phospholipid, including embodiments in which the negatively charged phospholipid is phosphatidylglycerol;

(i) the additional aģent is a carotenoid, including embodiments in vvhich the carotenoid is selected from the group consisting of lutein and zeaxanthin;

(g) the additional aģent is a statiņ, including embodiments in vvhich the statiņ is selected from the group consisting of rosuvastatin, pitivastatin, simvastatin, pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin, compactin, lovastatin, dalvastatin, fiuindostatin, atorvastatin, atorvastatin calcium, and dihydrocompactin;

(h) the additional aģent is an anti-angiogenic drug, including embodiments in vvhich the the antiangiogenic drug is Rhufab V2, Tiyptophanyl-tRNA synthetase, an Anti-VEGF pegylated antaruer, Squalamine, anecortave acetate, Combretastatin A4 Prodrug, Macugen™, mifepristone, subtenon triamcinolone acetonide, intravitreal crystalline triamcinolone acetonide, AG3340, fluocinolone acetonide, and VEGF-Trap;

(i) the additional aģent is a matrix metalloproteinase inhibitor, including embodiments in which the matrix metalloproteinase inhibitor is a tissue inhibitors of metalloprotemases, os-toiacroglobulin, a tetracycline, a hydroxamate, a chelator, a synthetic MMP fragment, a succinyl mercaptopurine, a phosphonamidate, and a hydroxarrnmc acid;

Q) the additional aģent is 13-cfr-retinoic acid (including derivatives of 13-cfr-rsiinoic acid), 11-cirretinoic acid (including derivatives of 11-ciī-retinoic acid), or 9-cis-retinoic acid (including derivatives of 9-cfr-rerinoic acid);

(k) the additional aģent is a retmylamine derivative, including an all-iVaus-rermvlairune derivative, a 13-cri-retinylamine derivative, a 11 -cri-retinylamine derivative, or a 9-cri-retinylamine derivative;

(l) the additional aģent is administered (i) prior to the administration ofthe compound having the structure of Formula (I), (ii) subsequent to the administration of the compound having the structure of Formula-fī), (iii) simultaneously vvith the administration of the compound having the structure of Formula (I), or (iv) both prior and subsequent to the administration of the compound having the structure of Formula (I); or (m) the additional aģent and the compound having the structure of Formula (I),are administered in the same pharmaceutical composition.

In any of the aforementioned aspects are further embodiments comprising administering eztracorporeal rheopheresis to the mammai.

In any of the aforementioned aspects are further embodiments comprising administering to the mammai a therapy selected from the group consisting of limited retinal translocation, photodynamic therapv, orusen iasering, macular hole surgery, macular translocation surgery, Phi-Motion, Proton Beam Therapv,

Retinal Detachment and Vitreous Surgery, Scleral Buckle, Submaculai Surgery, Transpupillary Theimotbei£py, Photosystem I therapy, MicroCurrent Stimulation, anti-ini]ammaiory aģents. RNA interference, administration of eye medications such as phospholine iodide or echothiophate or carbonic anhydrase inhibitors, īrncrochip implantation, stem celi therapy, gene replacement therapy, ribozyme gene therapy, photoreceptor/retinal celis transplantation, and acupuncture.

In any of the aforementioned aspects are further embodiments comprising the use of laser photocoagulation to remove drusen from the eye of the mammai.

In any of the aforementioned aspects are further embodiments comprising administering to the mammai at least once an effective amount of a second compound having the structure of Formula (I), vvherein the first compound is different from the second compound.

In any of the aforementioned aspects are further embodiments comprising (a) monitoring formation of drusen in the eye of the mammai; (b) measuring Ievels of lipofuscin in the eye of the mammai by autofluorescence; (c) measuring visual acuīty in the eye of the mammai; (d) conducting a visual field examination on the eye of the mammai, inciuding embodiments in vvhich the visual field examination is a Humphrey visual field exam; (e) measuring the autofluorescence or absoīption spectra of A-retinylidenephosphatidylethanolamine, dihydro-?/-retinylidene-jV-retinyl-phosphatidyle£hanolamine, .'V-reiinvlidene-.Vretinyl-phosphatidylethanolamine, dihydro-N-retmyhdene-V-retinyl-ethanolamine, and/or /V-retmyIidenephosphatidylethanolamine in the eve of the mammai; (i) conducting a reading speed and/or reading acuitv examination; (g) measuring scotoma size; or (h) measuring the size and number of the geographic atrophy lesions.

In any of the aforementioned aspects are further embodiments comprising determining vvhether the mammai is a carrier of the mutant ĀBCA4 allele for Stargardt Disease or has a mutant ELO V4 allele for Stargardt Disease or has a genetic variation in complement factor H associated vvith age-related macular degeneration.

In any of the aforementioned aspects are further embodiments comprising an additional treatment for retinal degeneiation.

In another aspect are pharmaceutical compositions comprising an effective amount of compound having the structure:

vvherein X] is selected from the group consisting of NR², 0, S, CHR²; R^] is (CHR²)I-L^,-R²ļ vvherein x is 0, 1, 2, or 3; L¹ is a single bond or -C(O)-; R² is a moiety selected from the group consisting of H, (Cp

C₄)alkyi, F, (CpC4)fluoroaIkyl, (Ci-C₄)alkoxy, -C(O)OH, -C(0)-NH2, -(CpCJaU^lamine, -C(O)-(C_rC4)alkyl, C(O)-(C|-C4)fiuoroalkyl. -C(O)-(C_rC4)alkylanune, and -C(O)-(CpC.4)alkoxy; and R^J is H or a moiety, optionally substituted vvith 1-3 independently selected substituents, selected from the group consisting of (C₂C₇)alkenyl, (C2-C₇)aihynyl, aryl, (C₃-C7)cycloalkyl, (Cj-C₇)cycloalkenyĻ and a beterocycle; provided that R is not H vvhen both x is 0 and L¹ is a single bond; or an active metabolite, or a phaimaceutically acceptable prodrug or solvate thereof; and a phannaceutically acceptable carrier.

In fuither embodiment of the pharmaceutical composition aspect, (a) the pharmaceutical)y acceptabie carrier is suitable for ophthalmic administration; (b) the pharmaceutically acceptabie carrier comprises lysophospbatidylcholine, monoglyceride and a fatty acid; (c) the phamnaceutically acceptabie carrier further comprises flour, a sweetener, and a humectant; (d) the pharmaceutically acceptabie carrier comprises com oil and a non-ionic surfactant; (e) the pharmaceuticallv acceptabie carrier comprises dimyristoyl phosphatidylcholine, soybean oil, t-butyl alcohol and water; (f) the pharmaceutically acceptabie carrier comprises ethanol, alkoxylated caster oil, and a non-ionic surfactant; (g) the pharmaceutically acceptabie carrier comprises an erfended release formulation; or (h) the phannaceutically acceptabie carrier comprises a rapid release formulation.

In further embodiment of the pharmaceutical composition aspect, the pharmaceutical composition further comprising an effective amount of at least one additional aģent selected from the group consisting of an inducer of nitric oxide production, an anti-inflaniinatory aģent, a physiologically acceptabie antioxidant, a physiologically acceptabie mineral, a negatively charged phospholipid, a carotenoid, a statiņ, an anti-angiogenic drug, a matrix metalloproteinase inhibitor, 13 -cis-retinoic acid (including derivatives of 13-cis-retinoic acid),

11-cri-rennoic acid (including derivatives of 1 l-cri-retinoic acid), 9-czj-retinoic acid (including derivatives of 9cis-retinoic acid), and retinylantine derivatives. In further embodiments, (a) the additional aģent is a physiologically acceptabie antiorfdant; (b) the additional aģent is an inducer of nitric oxide production; (c) the additional aģent is an anti-mflammatory aģent; (d) the additional aģent is a physiologically acceptabie mineral; (e) the additional aģent is a negatively charged phospholipid; (f) the additional aģent is a carotenoid; (g) the additional aģent is a statiņ; (h) the additional aģent is an anti-angiogenic aģent; (i) he additional aģent is a matris metalloproteinase inhibitor; or Q) the additional aģent is a 13-ciy-retmoic acid.

In another aspect are methods for treating a retinopathy comprising modulating the serum Ievel of retinol in the body of a mammai, including embodiments vvherein (a) the retinopathy is juvenile macular degeneration, including Stargardt Disease; (b) the retinopathy is dry form age-related macular degeneration; (c) the retinopathy is cone-rod dystrophy; (d) the ietinopathy is retinitis pigmentosa; (e) the retinopathy is wet-fonn age-related macular degeneration; (i) the retinopathy is or presents geographic atrophy and/or photoreceptor degeneration; or (g) the retinopathy is a lipofuscin-based retina! degeneration.

In a embodiment of the aforementioned aspect, the method further comprises administering to the mammai at least once an effective amount of a first compound having the structure:

vvherein X, is selected from the group consisting of NR², O, S, CHR²; R¹ is (CHR²)x-L'-R³, wherein x is 0, 1, 2, or 3; L¹ is a single bond or -C(O)-; R² is a moiety selected from the group consisting ofH, (Cr C₄)alkyl, F, (C_rC₄)fluoroalkyl, (C_rC₄)alkoxy, -C(O)OH, -C(O)-NHi, -(Ci-C₄)alkylamine, -C(OXC_rC₄)alkyl, C(O)-(C|-C₄)fluoroalkyl, -C(O)-(C|-C₄)alkylamine, and -C(O)-(Ci-C₄)alkoxy; and R³ is H or a moiety, optionally substituted vvith 1-3 independently selected substituents, selected from the group consisting of(C₂C₇)alkenyl, (C₂-C₇)alkynyl, aryl, (C₃-C₇)cycloalkyl, (C5~C₇)cycloalkenyl, and a heterocycle; provided that R is not H when both x is 0 and L¹ is a single bond; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

In yet a further embodiment, the method further comprises administering at least one additional aģent selected from the group consisting of an inducer of nitric oxide produetion, an anti-inflammatory aģent, a physiologically acceptable antioxidant, a physiologically acceptable mineral, a negatively charged phospholipid, a carotenoid, a statiņ, an anti-angiogenic drug, a matrix metalloproteinase inhibitor, 13-c/s-retmoic acid (including derivatives of 13-czj-retmoic acid), 1 l-czj-retinoic acid (including derivatives of 11-cfr-retinoic acid), 9-cir-retinoīc acid (including derivatives of 9-czj-retinoic acid), and retmylamine derivatives. Further embodiments include methods wherein: (a) the additional aģent is an inducer of nitric oxide produetion; (b) the additional aģent is an anti-inflammatory aģent; (c) the additional aģent is at least one physiologically acceptable antioxidant; (d) the additional aģent is at least one physiologically acceptable mineral; (e) the additional aģent is a negatively charged phospholipid; (f) the additional aģent is a carotenoid; (g) the additional aģent is a statiņ; (h) the additional aģent is an anti-angiogenic drug; (i) the additional aģent is a matrix metalloproteinase inhibitor; or (j) the additional aģent is 13-czj-ieiinoic acid.

In a further embodiment of the aforementioned aspect, the method for treating a retinopathy further comprises modulating lecithin.-retinol acyltransferase in an eye of a mammai, including embodiments wherein (a) the retmopathy is juvenile macular degeneration, including Stargardt Disease; (b) the retmopathy is dry fonn age-related macular degeneration; (c) the retinopathy is cone-rod dystrophy; (d) the retinopathy is retinitis pigmentosa; (e) the retinopaihy is wet-form age-related macular degeneration; (i) the retinopathy is or presents geographic atrophy and/or photoreceptor degeneration; or (g) the retinopathv is a līpofuscin-based retinal degeneration. In yet a further embodiment the method further comprises administering to the mammai at least once an effective amount of a first compound having the structure:

wherein Xj is selected from the group consisting of NR², O, S, CHR²; R¹ is (CHR²)x-L‘-R^j, wherein x is 0, 1, 2, or 3; L¹ is a single bond or -C(O)-; R² is a moiety selected from the group consisting of H, (Cr C4)alkyl, F, (C_rC₄)fluoroalkyl, (Ci-C₄)alkoxy, -C(O)OH, -C(O)-NH₂, -(C_rC₄)alkylamine, -C(O)-(Ci-C₄)alkyl, C(O)-(C,-C₄)fluoroalkyl, -C(O)-(C₁-C₄)alkylamine, and -C(O)-(C_rC₄)aIkoxy; and R³ is H or a moiety, optionally substituted with 1-3 independently selected substituents, selected from the group consisting of (C₂C_;)alkenyl, (C₂-C₇)alfcynyl, aryl, (C₃-C₇)cyc]oalkyl, (C₅-C₇)cycloalkenyl, and a heterocycle; provided that R is not H when both x is 0 and L¹ is a single bond; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

In stili a further embodiment, the method further comprises administering at least one additional aģent selected from the group consisting of an inducer of nitric oxide produetion, an anti-mflaiiiīnatory aģent, a physiologically acceptable antioTcdant, a physiologically acceptable mineral, a negatively charged phospholipid, a carotenoid, a statiņ, an anti-angiogenic drug, a matots metalloproteinase inhibitor, 13-cri-retinoic acid (including derivatives of 13-cij-retinoic acid), 1 l-czj-retinoic acid (including derivatives of 1 l-czs-retinoic acid). 9-czj-rerinoic acid (including derivatives of 9-czr-retinoic acid), and retinylamine derivatives. Further embodiments include methods wherein: (a) the additional aģent is an inducer of nitric oxide production; (b) the additional aģent is an anti-inflammatorv aģent; (c) the additional aģent is at least one pbysiologically acceptable antioxidant; (d) the additional aģent is at least one physiologically acceptable mineral; (e) the additional aģent is a negatively charged phospholipid; (i) the additional aģent is a carotenoid; (g) the additional aģent is a statiņ; (h) the additional aģent is an anti-angiogenic drug; (i) the additional aģent is a matrix metalloproteinase inhibitor; or 0) the additional aģent is 13-czs-retinoic acid.

In another aspect are methods for treating retinopathy comprising administering to a mammai an aģent that impairs the night vision of the mammai, including embodiments v/herem (a) the retinopathy is juvenils macular degeneration, including Stargardt Disease; (b) the retinopathy is dry form age-related macular degeneration; (c) the retinopathy is cone-rod dystrophy; (d) the retinopathy is retmitis pigmentosa; (e) the retinopathy is v/et-foim age-related macular degeneration; (f) the retinopathy is or presents geographic atiophy and/or photoreceptor degeneration; or (g) the retinopathy is a lipofuscin-based retina! degeneration. In yer a further embodiment, the method further comprises administering to the mammai at least once an effective amount of a first compound having the structure:

wherein Xi is selected from the group consisting of NR², 0, S, CHPri; R¹ is (CīīRŅ-L’-R³, wherein x is 0, 1, 2, or 3; L¹ is a single bond or -C(O)-; R² is a moiety selected from the group consisting of H. (Cr C4)alkyl, F, (CrC4)fluoroalkyl, (CrC4)alkoxy, -C(O)OH, -C(O)-NH2, -(Cj-Cņaūļdainine, -C(O)-(C,-C4)alkyl, C(0)-(Ci-C4)fluoroaIkyl, -C(O)-(Ci-C4)alkylan3ine, and -C(O)-(Ci-C4)alkoxy; and R^J is K or a moiety, optionally substituted with 1-3 independently selected substituents, selected from the group consisting of (C2C₇)alkenyl, (C₂-C₇)alkynyl, aryl, (C₃-C₇)cycloa]kyl, (C5-C₇)cycloalkenyl, and a heterocycle; provided that Ris not H when both x is 0 and L¹ is a single bond; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

In stili a further embodiment, the method further comprises administering at least one additional aģent selected from the group consisting of an inducer of nitric oxide production, an anti-inflammatory aģent, a physiologically acceptable antioxidant, a physiologically acceptable mineral, a negatively charged phospholipid, a carotenoid, a statiņ, an anti-angiogenic drug, a matrix metalloproteinase inhibitor, 13-cis-retīnoic acid (including derivatives of 13-ci'j-retinoic acid), 1 l-czs-retinoīc acid (including derivatives of 11-cis-retinoic acid),

9-cis-retinoic acid (including derivatives of 9-cfr-retinoic acid), and retinylamine derivatives. Further embodiments include methods wherein: (a) the additional aģent is an inducer of nitric oxide production; (b) the additional aģent is an anti-inflammatorv aģent; (c) the additional aģent is at least one pbysiologically acceptable antioxidant; (d) the additional aģent is at least one physiologically acceptable mineral; (e) the additional aģent is a negatively charged phospholipid; (f) the additional aģent is a carotenoid; (g) the additional aģent is a statiņ; (h) the additional aģent is an anti-angiogenic drug; (i) the additional aģent is a maīrix metalloproteinase inhibitor; or (j) the additional aģent is 13-cis-retinoic acid.

Ln another aspect are phannaceutical compositions for (a) reducing the formation of 7V-retinylidene-iVretinvlerhanolamine, jV-retmvIidene-phosphatidvle&anolarnine, dih.ydro-//-retinyhdene-jV-retinYlLV 13564 phosphatidylethanolamine, A-rstinylidene-/Lretinyl-phosphatidylethanolaniine, dihydro-.V-retinylidene-./'/retinyl-ethanolamine, and/or N-retmvīidene-phosphatidvlethanolaniine. in an eys of a mammai, (b) reducing the formation of lipofuscin in an eye of a mammai, (c’j reducing the formation of drusen in an eye of a mammai, (d) preventing macular degeneration in an eye of a mammai, (e) reducing the formation of all-trans-retinal in an eye of a mammai, (i) disrupting the visuai cycle in an eye of a mammai, and/or (g) protecting an eye of a mammai from light, comprising an effective amount of at least one compound having the structure of Formula (1) and a pharmaceutically acceptable carrier.

Compounds, including, but not limited to those having the structure of Formula (I), that find use in (a) reducing the formation of A-retinyhdene-ALntinylethanolamine, /V-retinylidene-phosphatidylethano3amine, dihydro-/V-retinylidene-A''-retinyl-phosphatidylethanolamine, jV-retinyIidene-N’-retinylphosphatīdylethanolamine, dihydro-jV-retinyiidene-jV-retinyl-ethanoIamine, and/or jV-retinylidenephosphatidylethanolamine, in an eye of a mammai, (b) reducing the formation of lipofuscin in an eye of a mammai, (c) reducing the formation of drusen in an eye of a mammai, (d) preventing macular degeneration in an eye of a mammai, (e) reducing the formation of all-trans-retinal in an eye of a mammai, and/or (f) protecting an eye of a mammai from light, have at least one of the follovring properties: the ability to disrupt the visuai cycle in the eye of a mammai, the ahility to cause reversible night blindness in a mammai, acceptable bioavailability to the eye of a mammai, and the abiliiy to cause only limited and acceptable irritation to the eye of a mammai,

In another or further aspect are methods for reducing the formation of or limiting the spread of geographic atrophy and/or photoreceptor degeneration in an eye of a mammai comprising administering to the mammai at least once an effective amount of a first compound having the structure of Formula (I). In further or alternative embodiments are methods further comprising admūnistering at least one additional aģent selected from the group consisting of an inducer of nitric oxide produetion, an anti-inflai±imatory aģent a physiologically acceptable antioxidant a physiologically acceptable mineral, a negatively charged phospholipid, a carotenoid, a statiņ, an anti-angiogenic drug, a matrix metalloproteinase inhibitor, 13-cis-retinoic acid (including derivatives of 13-cis-retinoic acid), 11-ori-retinoic acid (including derivatives of 11-ris-retinoic acid), S-rir-retinoic acid (including derivatives of 9-cri-retinoīc acid), and retinylamine derivatives.

In further or alternative embodiments of any of the aforementioned methods involving the administration of a compound having the structure of Formula (I) are methods further comprising measuring the reading speed and/or reading acuity of the mammai.

In further or alternative embodiments of any of the aforementioned methods involving the administration of a compound having the structure of Formula (I) are methods further comprising measuring the number and/or size of the scotoma in the eye of the mammai.

In further or alternative embodiments of any of the aforementioned methods involving the administration of a compound having the structure of Formula (I) are methods further comprising measuring the size and/or number of the geographic atrophy lesions in the eye of the mammai.

In further or alternative embodiments of any of the aforementioned methods involving the administration of a compound having the structure of Formula (I) are methods further comprising reducing the esterification of vitamin A in the eye of the mammai.

In further or alternative embodiments of any of the afcrementioned methods involving the administration of a compound having the structure of Formula (I) are methods further comprising īsing lovvering the autofluorescence of lipofuscin in the retinal pigment epithelium in the eye of the mammai.

In further or alternative embodiments of any of the afcrementioned methods involving the administration of a compound having the structure of Formula (I) are methods further comprising reducing the concentration of a substrate for a visual cycle protein dovvnstream from LRAT in the eye of the mammai. In further or alternative embodiments, the dovvnstream visual cycle protein is selected from the group consisting of a chaperone protein, an isomerase, and a dehydrogenase.

In a further aspect are methods for reducing the concentration of a substrate for a visual cycle protein downstream from LRAT in an eye of a mammai comprising administering to the mammai at least once an effective amount of a first compound having the structure of Formula (I). In further or alternative embodiments, fhe dovvnstream visual cycle protein is selected from the group consisting of a chaperone protein, an isomerase, and a dehydrogenase.

In a further aspect are methods for reducing the esterification of vitamin A in an eye of a mammai comprising administering to the mammai at least once an effective amount of a first compound having the structure of Formula (Ί).

In another aspect are methods for modulatīng the activity of Cellular Reunaldehvde Binding Protein (CRALBP) comprising contacting CRALBP vvith compounds having the structure of Formula (I). In a further embodiment, the compound directly contacts CellularRetinaldehyde Binding Protein. Ina further embodiment, such modulation occurs in vivo. In an alternative embodimenp such modulation occurs in viiro. In a further embodiment, such modulation occurs in the eys of a mammai. In a further embodiment, such modulation provides therapeutic benefit to a mammai having an ophthalmic disease or condition. In a further embodimenf such modulation improves or othervvise alleviates at least one symptom associated vvith an ophthalmic disease or condition in a mammai. In further or alternatīvs embodiments, the disease or condition is selected from the group consisting of a macular degeneration, a macular dystrophy, a ±etinopafhy. In further or alternative embodiments, the compound is 4-hydroxyphenylretmamide; or a metabolite, or a pharmaceutically acceptable prodrug or solvate thereof. In further or alternative embodiments, the compound is 4-methoxyphenylretinamide; or a metabolite, or a phannaceutically acceptable prodrug or solvate thereof.

In another aspect are methods for indirectiy modulatīng the activity of visual cycle proteīns that are not directly modulated by the compounds of Formula (T). In one embodiment of such an aspect, the compounds of Formula (I) directly modulate one of the visual cycle proteīns (by binding to such a protein or by binding to the ligand of such a protein, vvherein the binding may be Chemical binding, physical binding, or a combination thereof, including hydrogen bonding) so as to reduce the concentration of the expected reaction product of the that visual cvcle protein. In a further embodiment, the visual cycle protein that is directly modulated by the compounds of Formula (I) is LRAT. In a further embodiment, the direct modulation of LRATby a compound of Formula (I) reduces the concentration of all-Zrany-retinyl esters. In a further embodiment, reduction in the concentration of all-ira7ij-rstinyl esters indirectly modulates the activity of dovvnstream visual cycle protems by lovvering the concentration of substrates for such downsfream visual cycle proteīns. In further embodiments, such dovvnstream visual cycle proteīns include isomerases, chaperone proteīns, and dehydrogenases.

Other objecrs, features and advantages of the methods and compositions described herein vvill become apparent from the follovving detailed description. It should be understood, hovvever, that the detailed description and the specific examples, vvhile indicating specific embodiments, are given by way of illustration only, since various changes and modifications vvithin the spirit and scope of the invention vvill become apparent to those skilled in the art from this detailed description.

AH references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURĒS

FIGS. la-1 c illustrate various reverse phase LC analyses of acetonitrile extracts of seram. The seram vvas obtained from mice administered vvith either DMSO (FIG. la), 10 mg/kg 7/-4-(hydroxyphenyl)retinaimde (HPR) (FIG lb), or 20 mg/kg HPR (FIG. 1c) for 14 davs.

FIG. 2a illustrates ocular concentrations of all-tranj· retinol (atROL) and HPR as a function of time in mice follovving injection of 10 mg/kg HPR

FIG. 2b illustrates serum concentrations of all-iram retinol and HPR in mice following 14-day treatment vvith DMSO, 10 mg/kg HPR, or 20 mg/kg HPR; see FIG. 11 for an updated and corrected version of this figurē.

FIG. 3a illustrates a control binding assav for the interaction betvveen retinol and retinol-bīnding protein as measured by fluorescence quenching.

FIG. 3b illustrates a binding assay for the interaction betvveen retinol and retinol-binding protein in the presence of HPR (2 μΜ) as measured by fluorescence quenching.

FIG. 4a illustrates the effect of HPR on A2PE-H₂ biosynthesis in abca4 null mutant mice.

FIG. 4b illustrates the effect of HPR on ARE biosvnthesis in abca4 null mutant mice.

FIG. 5 illustrates the effect of HPR dosage on LRAT activity in the RPE using an in vitro biochemical assay.

FIG. 6a illustrates the effect of HPR on all-zranr retinyl ester biosynthesis using an in viiro biochemical assay.

FIG. 6b illustrates the effect ofHPR on Tl-cri retinol biosynthesis using min vitro biochemical assay. FIG. 6c illustrates the effect ofHPR on all-trans retinol utilization using an in viīro biochemical assay. FIG. 7 illustrates the interaction of Cellular Retinaldehyde Binding Protein (CRALBP) vvith various ligands as measured by fluorescence quenching.

FIG. S illustrates the interaction of CRALBP vvith various ligands as measured by size exclusion chromatography and UV/Visible spectrophotometry.

FIG. 9 iUustrates the binding of 7/-4-(methoxyphenyl)retīnamide (MPR) to retinol binding protein (RBP) as measured by fluorescence quenching.

FIG. 10 illustrates the modulation of TTR binding to RBP-MPR as measured by size exclusion chromatography and UV/Visible spectrophotometry.

FIG. 11 iUustrates the analysis of serum retinol as a function of feuretinide concentration.

FIG. 12 illustrates a correlaiion plot relating fenretinide concentration to reductions in retinol, A2PE-H and A2E in A3CA4 null mutant mice.

FIG. 13 illustrates (A) the quenching of CRALBP protein fluorescence with 11-cis-retinal (3 3 cRAL), and (B) the auenching of CRALBP protein fluorescence with fenretinide.

FIG. 14 illustrates a spectroscopic analysis of fenretinide binding to CRALBP.

FIG. 15 illustrates the fluorescence quenching of apo-CRALBP as a function of the concentration of either 11 cRAL or fenretinide.

FIG. 16 illustrates the effect of fenretinide on the esterification of vitamin A in the retinal pigment epithelium.

FIG. 17 illustrates retinoid composition in light adapted DMSO- and HPR-treated mice (panei A); the affect of HPR on the regeneration of visual chromophore (panei B); the effect of HPR on bleached chromophore recycling (panei C); and electrophysiological measurements ofrod function (panei D), rod and cone function (panei E), and recovery from photobleaching (panei F).

FIG. 18 illustrates the analysis of A2?E-H₂ and A2E Ievels as a function of fenretinide dose and treatment period (panels A-F) and lipofhscin autofluorescence in the RPE of ASG44 null mutant mice as a function of fenretinide treatment (panels G-I).

FIG. 19 illustrates light microscopy images ofthe retinās from DMSO- and HPR-treated animals.

FIG. 20 illustrates absorbance and fluorescence chromatograms from eyecup extracts of control mice (panei A), and of mice previousiy maintained on HPR therapy (panei B) following a 12-day drug holiday; absorbance and fluorescence chromatograms from eyecup extracts of control mice (panei C), and of mice previously maintained on HPR therapy (panei D) follosving a 28-day drug hoiiday; the histogram presents the reiative A2E Ievels for the mice described in panels A-D.

DETAILED DESCRIPTION OF THE INVENTION

Compounds having the structure of Formula (I) have been used for the treatment of cancer. In particular, the compound V-(4-hydroxyphenyl)retinamide, also known as fenretinide, HPR orAHPR, has been extensively tested for the treatment of breast cancer. Moon, et al., Cancer Res., 39.Ί339Α6 (1979). Fenretinide is described in U.S. Pat. Nos. 4,190,594 and 4,323,581. In addition, other methods for preparing fenretinide are known, and further, numerous analogs of fenretinide have been prepared and tested for their effectiveness in treating cancer. See, e.g., U.S. Patent Application Publication 2004/0102650; U.S. Patent No. 6,696,606; Villeneuve & Chan, Tetrahedron Letters, 38:6489-92 (1997); Um, S. J-, et al., Chem. Phann. Buli., 52:501-506 (2004). Of concem, however, has been the general tendency of such compounds to producē certain side-effects in human patients, including impairment of night vision. See, e.g., Decensi, A., et al., J. Nati. Cancer Inst., 86:1-5-110 (1994); Mariani, L., Tumori., 82:444-49 (1996). A recent study has also provided some evidence that /V-(4-hydroxyphenyI)retinamide can inducē neuronal-like differentiation in certain cultured human RPE celis. See Chen, S., et al., J. Neurochem., 84:972-81 (2003).

Surprisingly, the compounds of Formula (I) can be used to provide benefit to patients šuffering from or susceptible to various macular degenerations and dystrophies, including but not limited to dry-form age-related macular degeneration and Stargardt Disease. Specifically, compounds of Formula (I) provide at least some of the following benefits to such human patients: reduction in the amount of all-tzans-retmal (atRAL), reduction in the formation of APĒ, reduction in the formation of lipofuscin, reduction in the formation of drusen, and reduction in light sensitivity. There is a reduced tendency to fonn A2E in ophthalmic and ocular tissues caused, in part. by a reduction in the over-accumulation of all-Zrmrj-retinal in these tissues. Because A2E itself is cytotoxic to the RPE (which can lead to retina celi death), administration of compounds having the structure of Formula (I) (alone, or in combination with other aģents, as described herein) reduces the rāte of accumulation of A2E, a cytotoxic aģent thus providing patient benefīt. In addition, because A2E is the major fluorophore of lipofuscin, reduced quantities of A2E in ophthalmic and ocular tissues also results in a reduced tendency to accumulate lipofuscin in such tissues. Thus, in some respects the methods and compositions described herein can be considered to be lipofoscin-based treatments because administration of compounds having the structure of Formula (I) (alone, or in combination with other aģents, as described herein) reduces, lovters or ofhenvise impacts the accumulation of lipofuscin in ophthalmic and/or ocular tissues. A reduction in the rāte of accumulation of lipofuscin in opbthalnhc and/or ocular tissues benefīts patients that have diseases or conditions such as macular degenerations and/or dystrophies.

In addition, because dry-form age-related macular degeneration is often a precursor to wet-fonn agerelated macular degeneration, the use of compounds of Formula (I) can also be used as a preventative therapy for this latter ophthalmic condition.

Interestingly, the compounds of Formula (ī) and/or its derivatives also have an effect on enzymes or proteīns in the visual cycle. For example esterification in the retinal pigment epithelium involves lecithinreiinol acyltransferase (LRAT) which catalyzes the transfer of an acyl group from lecithin to retinot Administration of Formula (I) and/or its derivatives modifies the activity of LRAT which could benefit patients suffering from or susceptible to various macular degenerations and dystrophies.

Vitaττπτι A in serum is delivered to exira-hepatic target tissues and immediately esterified by the membrane-bound enzvme LRAT. LRAT catalyz.es the transfer of a fatty acid from membrane phospholīpids to retinol fhereby generating all-trans retinyl esters, the principal storage form of vitamin A in ali tissues. In the RPE, all-rianj rennyl esters are the sols substrate for a unique isomerase enzyme wbich generaies a lightsensitive visual chromophore precursor, 11-cis retinol. Subsequent oxidation of this retinoid and conjugation to the opsin apoprotein in the retina yields rhodopsin.

/V-4-(hydroxyphenyl)retinamīde has been shown to cause marked inhibition of LRAT activity in membranes prepared from liver and small intestine. Additionally, we have demonstrated (e.g., Example 13) for the first time that LRAT activity in the RPE of the eye is inhibited by HPR As discussed in the Examples, administration of HPR is also associated with decieased serum retinol and retinol binding protein (RBP). Thus, in addition to the systemic effects of HPR (e.g., decreased serum retinol Ievels), there is also an intracellular, enzyme-specific effect (e.g., LRAT activity in RPE celis). The fact that vitamin A homeostasis in the eye relies not only upon delivery of retinol from serum but also upon intracellular stores of retinyl esters to provide visual chromophore, suggests that effects of HPR may be most pronounced in this organ.

In addition, compounds having the structure ofFormula (I) also bind to Cellular Retinaldehvde Binding Protein (CRALBP), which is another visual cycle protein. To illustrate this effect, and by way of example only, the data presented in FIGS. 7 & 8 demonstrate that HPR binds to CRALBP. Thus, in ophthalmic tissues, where CRALBP can be found, compounds having the structure of Formula (I) are expected to bind to CRALBP, and consequently, (a) modulate the binding of other compounds, such as retinaldehyde, to CRALBP, (b) modulate the activity of CRALBP, (c) serve as a ligand to CRALBP, (d) undergo activity catalynedby CRALBP, indludmg transport activity, aud/οι (e) serve as a therapeutic aģent in the methods and compositions described herein.

The Visual Cvcle. The vertebrate retina contains two types of photoreceptor celis - rods and cones. Rods are specializēti for vision under low light conditions. Cones are less sensitive, provide vision at high temporai and spatial resolutions, and afford color perception. Under daylight conditions, the rod response is saturated and vision is mediated entirely by cones. Both celi types contain a structure called the outer segment comprising a stack of membranous discs. The reactions of visual transduction take place on īhe surfaces of these discs. Tne first step in vision is absoīption of a photon by an opsin-pigmeni molecule (rhodopsin), which involves 11-cis to all-n-cms isomerization of the chromophore. Before light sensitivity can be regained, the resulting all-huns-retmal must he converted back 11-cir-retinal in a multi-enzyme process vvhich takes place in the retinal pigment epithelium, a monolayer of celis adjacent to the retina.

Macular or Retinal Degenerations and Dvstrophies. Macular degeneration (also referred to as retinal degeneration) is a disease of the eye that involves deterioration of the macula, the Central portion of the retina. Approximately 85% to 90% of the cases of macular degeneration are the “dry” (atrophic or non-neovascular) type. In dry macular degeneration, the deterioration of the retina is associated vvith the formation of small yellow deposits, knovvn as drusen, under the macula; in addition. the accumulation of lipofuscin in the RPE leads to photoreceptor degeneration and geographic atrophy. This phenomena leads to a thinning and drying out of the macula. The location and amount of thinning in the retina caused by the drusen directly correlates to the amount of centra! vision loss. Degeneration of the pigmented layer of the retina and photoreceptors overlying drusen become atrophic and can cause a slow loss of centra! vision. Ultimately, loss of retinal pigment epithelium and underlying photoreceptor celis results in geographic atrophy. Administration of at least one compound having the structure of Formula (I) to a mammai can reduce the formation of, or limit the spread of, photoreceptor degeneration and/or geographic atrophy in the eye of the mammai. By way of example only, administration of

HPR and/or MPR to a mammai, can be used to treat photoreceptor degeneration and/or geographic atrophy in the eye of the mammai.

In “wet macular degeneration new blood vessels form (i.e., neovascularization) to improve the blood supply to retinal tissue, specificallv beneath the macula, a portion of the retina that is responsible for our sharp centra! vision. The nevv vessels are easily damaged and sometimes ruptare, causing bleeding and injury to the surrounding tissue. Although vvet macular degeneration only occurs in about 10 percent of ali macular degeneration cases, it accounts for approximately 90% of macular degeneration-related blindness. Neovascularization can lead to rapid loss of vision and eventual scarring of the retinal tissues and bleeding in the eye. This scar tissue and blood producēs a dark, distorted area in the vision, often rendering the eye legally blind. Wet macular degeneration usually starts vvith distortion in the centra! field of vision. Straight lines become wavy. Many people vvith macular degeneration also report having blurrea vision and blank spots (scotoma) in their visual field. Grovvth promoting proteins called vascular endothelial grovvth factor, or VEGF, have been targeted for triggering this abnonnal vessel grovvth in the eye. This discovery has lead to aggressive research of experimental drugs that inhibit or block VEGF. Studies have shovvn that anti-VEGF aģents can be used to block and prevent abnonnal blood vessel grovvth. Such anti-VEGF aģents stop or inhibit VEGF stimulation, so there is less grovvth of blood vessels. Such anti-VEGF aģents may also be successful in antiansiogenesis or blocking VEGF’s abiiity to inducē blood vessel grovvth beneath the retina, as vvell as blood vessel leakiness. Administration of at least one compound having the structure of Formula (I) to a mammai can reduce the formation of, or limit the spread of, vvet-fonn age-related macular degeneration in the eye of the mammai. By wav of example only, administration of HPR and/or MPR to a mammai, can be used to treat vvetfonn age-related macular degeneration in the eye of the mammai. Similarly, the compounds ofFormula (I) (including by way of example only, HPR and/or MPR) can be used to treat choroidal neovascularization and the formation of abnormal blood vessels beneath the macula of the eye of a mammai.

Stargardt Disease is a macular dystrophy that manifests as a recessive form of macular degeneration vvith an onset during childhood. See e.g., Allikmets et al., Science, 277:1805-07 (1997); Levvis etal., Am. J. Hum. Genet., 64:422-34 (1999); Stone et al., Nature Genetics, 20:328-29 (1998); Allikmets, Am. J. Hum. Gen., 61:793-799 (2000); Klevering, et al, Ophthalmology, 111:546-553 (2004). Stargardt Disease is characterized clinically by Progressive loss of central vision and progressive atrophy of the RPE overlying the macula. Mutatīons in the human ABCA4 gene for Rim Protein (RmP) are responsible for Stargardt Disease. Early in the disease course, patients shovv delayed dark adaptation but othervvise normai rod function. Histologically, Stargardt Disease is associated vvith deposition of lipofuscin pigment granules in RPE celis.

Mutations in A.BCA.4 have also been implicated in recessive retinitis pigmentosa, see, e.g., Cremers et al., Hum. Mol. Genet., 7:355-62 (1998), recessive cone-rod dystrophy, see īd., and non-exudative age-related macular degeneration, see e.g., Allikmets et al., Science, 277:1805-07 (1997); Lewis et al., Am. J. Hum. Genet., 64:422-34 (1999), although the prevalence OĪABCA4 mutations in AMD is stili uncertain. See Stone et ai, Nature Genetics, 20:328-29 (1998); Allikmets, Am. J. Hum. Gen., 67:793-799 (2000); Klevering, et al, Ophthalmology, 111:546-553 (2004). Similar to Stargardt Disease, these diseases are associated vvith delayed rod dark-adaptation. See Steinmetz et al., Brit. J. Ophthalm., 77:549-54 (1993). Lipofuscin deposition in RPE celis īs-alsG seen prominentlyin AMD, see Kīifien et al., Microsc. Res. Tech., 36:106-22 (1997) and some cases of retinitis pigmentosa. See Bergsma et al., Nature, 265:62-67 (1977). In addition, an autosomal dominant form of Stargardt Disease is caused by mutations in the ELO'/4 gene. See Karan, etal., Proc. Nati. Acad. Sci.

(2005).

In addition, there are several types of macular degenerations that affect children, teenagers or adults that are commonly knovvn as early onset or juvenile macular degeneration. Many of these types are hereditary and are looked upon as macular dystrophies instead of degeneration. Some examples of macular dystrophies include: Cone-Rod Dystrophy, Comeal Dystrophy, Fuch’s Dystrophy, Sorsby’s Macular Dystrophy, Best Disease, and Juvenile Retinoschisis, as vvell as Stargardt Disease.

CHEMICAL TERMINOLOGY

An “alkoxy” group refers to a (aīkyl)O- group, vvhere alkyl is as defined herein.

An “alkvl” group refers to au aliphatic hydrocarbon group. The alkyl moiety may be a “saturated alkyl” group, vvhich means that it does not contain any aikene or alkyne moieties. The alkyl moiety may also be an “unsaturated alkyl” moiety, vvhich means that it contains at least one aikene or alkyne moiety. An “aikene” moiety refers to a group consisting of at least tvvo carbon atoms and at least one carbon-carbon double bond, and an “alkyne” moiety refers to a group consisting of at least tvvo carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, vvhether saturated or unsaturated, may be branched, straight chain, or cyclic.

The “alkyl” moiety may have 1 to 10 carbon atoms (vvhenever it appears herein, a numerical range such as “1 to 10” refers to each integer in the given range; e.g., “i to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present defīnition also covers the occuirence of the term “alkyl” vvhere no numerical range is designated). The alkyi group could also be a “lovver alkyl” having 1 to 5 carbon atoms. The allcvl group of the compounds deseribed herein may be designated as “C,-C₄ alkyl” or similar designations. By vvay of example only, “Ci-C₄ alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typīcal alkyl groups include, but are in no way limited to, methvl, ethyl, propyl, isopropyl, butvl, īsobutyl, tertiary butyl, pentyl, hexyl, etbenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

The tenn “alkylamine” refers to the -N(alkyl)_zH_y group, vvhere x and y are selected from the group x=l, y=l and x=2, y=0. When x=2, the alkyl groups, taken together, can optionally form a cyclic ring system.

The term “alkenyl” refers to a type of aūcyl group in vvhich the first two atoms of the alkyl group form a double bond that is not part of an aromatic group. That is, an alkenyl group begins vvith the atoms -C(R)=CR, vvherein R refers to the remaining portions of the alkenyl group, vvhich may be the same or different. Nonlimiting examples of an alkenyl group include -CH=CH, -C(CH₃)=CH, -CH=CCH₃ and —C(CH₃)=CCH₃. The alkenyl moiety may be branched, straight chain, or cyclic (in vvhich case, it vvould also be knovvn as a “cycloalkenyl” group).

The term “alkynyl” refers to a type of alkyl group in vvhich the first tvvo atoms of the alkyl group form a triple bond. That is, an alkynyl group begins vvith the atoms —C =C-R, vvherein R refers to the remaining portions of the alkynyl group, vvhich mav be the same or different. Non-limiting examples of an alkynyl group include -C =CH, -C ^CCH₃ and -C <JCH₂CH₃. The “R” portion of the a!kynyl moiety may he branched, straight chain, or cyclic.

An “amide” is a Chemical moiety vvith formula -C(O)NHR or -NHC(O)R, vvhere R īs selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyciic (bonded through a ring carbon). An amide may be an amino acid or a peptide molecule attached to a compound of Formula (I), thereby forming a prodrug. Any amine, hydroxy, or carboxyl side chain on the compounds deseribed herein can be amīdined. The procedures and specific groups to make such amides are knovvn to those of skill in the art and can readīly be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, Nevv York, ΝΎ, 1999, vvhich īs incorporated herein by reference in its entirety.

The tenn “aromatic” or “aryl” refers to an aromatic group vvhich has at least one ring having a conjugated pi electron system and includes both carbocvclic aryl (e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine). The term includes monocyclīc or fused-ring polycyclic (i.e., rings vvhich share adjacent pairs of carbon atoms) groups. The term “carbocyclic” refers to a compound vvhich contains one or more covalently closed ring structures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in vvhich the ring backbone contains at least one atom vvhich is different from carbon.

A “cyano” group refers to a -CN group.

The tenn “cycloalkyl” refers to a monocvclic orpolycyclic radical that contains only carbon and hydrogen, and may be saturated, partial]y unsaturated, or fully unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include the follovving moieties:

The term “ester” refers to a Chemical moiety vvith formula -COOR, vvhere R is selected from the grotrp consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). Any amine, hydroxy, or carboxyl side chain on the compounds described herein can be esterified. The procedures and specific groups to make such esters are knovvn to those of skill in the art and can readily be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, Nevv York NY, 1999, vvhich is incoīporaied herein by reference in its entirety.

The term “halo” or, altemativelv, “halogen” means fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro, chloro and bromo.

The terms “haloalkyL,” “hāloalkenyl,” “haloalkynyl” and “haloalkoxy” include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted vvith one or more halo groups or vvith combinations thereof. The terms “fluoroalkyl” and “fluoroalkoxy include haloalkyl and haloalkoxy groups, respectively, in vvhich the halo is fluorine.

The terms “heteroalkyl” “heteroalkenyl” and “heteroalkynyl” include optionally substituted atkyl, alkenvl and alkynyl radicals and vvhich have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof.

The terms “heteroaryl” or, altematively, “heteroaromatic” refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur. An iV-containing “heteroaromatic” or ¹ 'heteroaryl” moiety refers to an aromatic group in vvhich at least one of the skeletal atoms of the ring is a nitrogen atom. The polycyclic heteroaryl group may be fused ornon-fused. Illusirative examples ofheteroaryl groups include the follovving moieties:

The term heterocycle refers to heteroaromatic and heteroalicyclic groups contain ing one to four heteroatoms each selected from 0, S and N, wherein each heterocyclic group has žorn 4 to 10 atoms in its ring system, and with the proviso that the ring of said group does not contain two adjacent 0 or S atoms. Nonaromatic heterocyclic groups include groups having only 4 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. The heterocyclic groups include benzo-fused ring systems. An example of a 4-membered heterocyclic group is azetidinyl (derived from azetidine). An ezample of a 5-membered hetsrocyclic group is thiazolyL An erample of a 6-membered heterocyciic group is pyriayl, and an example of a 10-membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are pyrroiidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyf ietrahydropyianyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino. morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyi, £hiepanyl, oxazepīnyl, diazepinyl, ihiazepinyl, l,2,3,6-teirahydropyridinyl, 2-pyxrolinyļ 3-pyirolinyļ indolinyl, 2H-pyranyl, 4H-pyranyI, dioxanyl, 1,3dioxolanyl, pyrazolinyļ, dithianyl, dithiolanyl, dihydropyranyļ, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolīdinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-mdolyl and quinolizinyl. Examp]es of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyiazmyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyirolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzoferanyl, cinnolīnyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyļ oxaaiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridmyl, and furopyridinyl. The foregoing groups, as derived from the groups listed above, may be C-attached or //-attached where such is possible. For instance, a group derived frompyrrole may be pyrrol-l-yl (//-attached) or pyrrol-3-yl (Cattached). Further, a group derived from imidazole may be imidazol-l-yl or imidazol-3-yl (both //-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (ali C-attached). The heterocyclic groups include benzo-fused nng systems and ring svstems substituted with one or two oxo (=0) moieties such as pyrrolidin-2-one.

A “heteroa!icyclic” group refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen and sulfur. The radicals may be fused with an aryl or heteroaiyl. Illustrative examples of heterocycloalkyl groups include:

Ο

Theteim heteroalicyclic also ineludes ali ring fonus of the carbohydrates, including but not limited to the monosaccharides, the disaccharides and the oligosaccharides.

The term “membered ring” can embrace any cyclic structure. The term “membered” is meant to denote the number of skeleial atoms that constitute the ring. Thus, for example, cyclohexyļ, pyridine, pyran and thiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

An “isocyanato” group refers to a -NCO group.

An “isothiocyanato” group refers to a -NCS group.

A “mercaptyT group refers to a (alkyl)S- group.

The terms “nucleophile” and “electropbile” as used herein have their usual meanings familiar to synthetic and/or physical organic chemistry. Carbon elecirophiles typically comprise one or more alkyl, alkenyl, alkynyl or aromatic (sp^J, sp², or sp hybridized) carbon atoms substituted with any atom or group having a Pauling electronegativity greater than that of carbon itself. Examples of carbon electrophiles include but are not limited to carbonyls (aldehydes, ketones, esters, amides), oximes, hydrazones, epoxides, aziridines, alkyl-, alkenvl-, and aryl halides, acyls, sulfonates (aryl, alkyl and the Iike). Other examples of carbon electrophiles include unsaturated carbon atoms electronically conjugated with electron withdrawing groups, examples being the 6-carbon in alpha-unsaturated ketones or carbon atoms in fluorine substituted aryl groups. Methods of generating carbon electrophiles, especially in ways which yield precisely controlled products, are known to those skilled in the art of organic synthesis.

The relative disposition of aromatic substituents (ortho, meta, and para) imparts distinetive chemistry for such stereoisomers and is χνείΐ recognized within the field of aromatic chemistry. Para- and metasubstitutional pattems project the two substituents into different orientations. Ortho-disposed substituents are oriented at 60° vvith respect to one another; meta-disposed substituents are oriented at 120° vvith respect to one another; para-disposed substituents are oriented at 180° vvith respect to one another.

ortho

60° meta

120° para

180°

Relative dispositions of substituents, viz, ortho, meta, para, also affect the electronic properties of the substituents. Witnout being bound to any particular type or Ievel of theory, it is knovvn that ortho- and paradisposed substituents electronically affect one another to a greater degree than do corresponding meta-disposed substituents. Meta-disubstituted aromaiics are often synthesized using different routes than are the corresponding ortho and para-disubstituted aromatics.

The term “moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized Chemical entities embedded in or appended to a molecule.

The term “bond” or “single bond” refers to a Chemical bond betvveen tvvo atoms, or tvvo moieties when the atoms joined by the bond are considered to be part of larger substructure.

A “sulfīnyl group refers to a -S(=O)-R, vvhere R is selected from the group consisting of a!kyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon)

A “sulfonyl group refers to a -S(=O)2-R vvhere R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon)

A “thiocyanato” group refers to a -CNS group.

The term “optionally substituted” means that the referenced group may be substituted vvith one or more additional group(s) individually and independently selected from alkyl, cycloalkyl, aryļ heteroaryl, heteroalicyc!ic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloaIkyĻ, perfiuoroalkyi, silyl, and amino, including monoand di-substituted amino groups, and the protected derivatives thereof. The protecting groups that may form the protective derivatives of the above substituents are knovvn to those of skill in the art and may be found in references such as Greene and Wuts, above.

The compounds presented herein may possess one or more chiral centers and each center may exist in the R or S confīguration. The compounds presented herein include ali diastereomeric, enantiomeric, and epimeric forms as vveil as the appropriate mixtures thereof. Stereoisomers may be obtained, if desired, by methods knovvn in the art as, for example, the separation of stereoisomers by chiral chromatographic columns.

The methods and formulations described herein include the use of A-oxides, crystallme fonus (also knovvn as polymoxphs), or pharmaceutically acceptable salts of compounds having the structure of Formula (I), as vveil as active metabolites of these compounds having the same type of activity. By way of example only, a knovvn metabolite of fenretinide is N-(4~methoxyphenyI)retinamide, also knovvn as 4-MPR or MPR. .Another knovvn metabolite of fenretinide is 4-oxo fenretinide. In some situations, compounds may exist as tautomers.

Ali tautomers are included vvithin the scope of the compounds presented herein. In addition, the compounds described herein can exist in unsoivated as vvell as solvated forms vvith pharmaceutically acceptable solvents such as vvater, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

PHARMACEUTICAL COMPOSITIONS

Another aspect are pharmaceutical compositions comprising a compound of Formula (I) and a pharmaceutically acceptable diluent, excipient, or carrier.

The term “pharmaceutical composition” refers to a mbtture of a compound of Fonnula (I) with other Chemical components, such as carriers, stabilīzers, diluents, dispersing aģents, suspending aģents, thickening aģents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

The term “carrier” refers to relatively nontoxic Chemical compounds or aģents that facilitate the incorporation of a compound into celis or tissues.

The term “diluent” refers to Chemical compounds that are used to dilute the compound of interesi prior to delivery. Diluents can also be used to stabilizē compounds because they can provide a more stable environment. Salts dissolved in buffered Solutions (vvhich also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution.

The term “physiologically acceptable” refers to a material, such as a carrier or diluent, that does not abrogate the biological activity oi properties of the compound, and is nontoxic.

The term “pharmaceutically acceptable salt” refers to a formulation of a compound that does not cause significant irritation to an organism to vvhich it is administered and does not abrogate the biological activitv and properties of the compound. Pharmaceutically acceptable salts may be obtained by reacting a compound of Formula (I) vvith acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Phaimaceutically acceptable salts may also be obtained by reacting a compound of Fonnula (I) vvith a base to form a salt such as an ammonium salt, an alkali mētai salt, such as a sodium or a potassium salt, an alkaline earth mētai salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine₃ V-methyl-D-gIucamine. tris(hydroxymethyl)methylamine, and salts vvith amino acids such as arginine, lysine, and the like, or by other methods knovvn in the art

A “metabolite” of a compound disclosed herein is a derivative of that compound that is formed vvhen the compound is metabolized. The term “active metabolite” refers to a biologically active derivative of a compound that is formed vvhen the compound is metabolized. The term “metabolized” refers to the sum of the processes (including, but not limited to, hydrolysis reactions and reactions catalyzed by enzymes) by vvhich a particular substance is changed by an organism. Thus, enzymes may producē specific structural alterations to a compound. For ezample, cytochrome P450 cataiyzes a variety of oxidative and reductive reactions vvhile uridine diphospbate glucuronvltransferases catalyze the transfer of an activated glucuronic-acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines and free sulphydryl groups. Further infonnation on metabolism may be obtained from The Pharmacological Basis ofTherapeuti.es, Pth Edition, McGravv-Hill (1996).

Metabolites of the compounds disclosed herein can be identified either by administration of compounds to a host and analysis of tissue samples from the Liesi, or by incnbation of compounds vvith hepatic celis in vitro and analysis of the resulting compounds. Both methods are vvell knovvn in the art.

By way of example only, MPR is a knovvn metabolite of HPR, both of vvhich are contained vvithin the structure of Formula (I). MPR accumulates systemically in patients that have been chronically treated vvith HPP_ One of the reasons that MPR accumulates systemically is that MPR is only (if at ali) slowly roetabolized, vvhereas HPR is metabolized to MPR. In addition, MPR may undergo relatively slow clearance. Thus, (a) the pharmacokinetics and pharmacodynamics of MPR must be taken into consideration vvhen administering and detennining the bioavailabilitv of HPR, (b) MPR is more stable to metabolism than HPR, and (c) MPR can be more immediately bioavailable than HPR follovving absorption. Another knovvn metabolite of fenretinide is 4oxo fenretinide.

MPR may also be considered an active metabolite. As shovvn in FIGS. 9 and 10, MPR (like HPR) can bind to Retinol Binding Protein (RBP) and prevent the binding ofRBP to Transeryūiiia (TTR). As a result, vvhen either HPR or MPR is administered to a patient, one of the resulting expected features is that MPR vvill accumulate and bind to RBP and inhibit binding of retinol to RBP, as vvell as the binding ofRBP to TTR. Accordingly, MPR can (a) serve as an inhibitor of retinol binding to RBP, (b) serve as an inhibitor ofRBP to TTR, (c) limit the transport of retinol to certain tissues, including ophthalmic tissues, and (d) be transported by RBP to certain tissues, including ophthalmic tissues. MPR appears to bind more weakly to RBP than HPR, and is thus a-less strong inhibitor of retinol binding to RBP. Nevertheless, both MPR and HPR are expected to inhibit approximately equivalently, the binding of RBP to TTR. Furthermore, it is expected that MPR (like HPR) vvūl bind to visual cycle proteīns, including LRAT and CRALBP. MPR has, in these respects, the same mode of action as HPR and can serve as a therapeutic aģent in the methods and compositions described herein.

A “prodrug” refers to an aģent that is converted into the parent drug zn vivo. Prodrugs are often useful because, in some situ2tions, they may.be easier to administer than the parent drug. Ihey may, for instance, be bioavailable by oral administration vvhereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. An example, vvithout limitation, of a prodrug would be a compound of Formula (I) vvhich is administered as an ester (the “prodrug”) to facilitate transmittal aeross a celi membrane vvhere vvater solubility is detrimental to mobility but vvhich then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the celi vvhere water-solubility is beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group vvhere the peptide is metabolized to reveal the active moiety.

The compounds described herein can be administered to a human patient per se, or in pharmaceutical compositions vvhere they are mixed vvith other active ingredients, as in combiņatioņ therapy, or suitable carrier(s) or excipicnt(s). Techniques for formulation and administration of the compounds of the instant application may be found in “Remington: The Science and Practice of Phatmacy,” 20th ed. (2000).

Routes Of Administration

Suitable routes of administration may, for example, include oral, rectal, transmucosal, transdermal, pulmonarv, ophthalmic or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intrameduilary injections, as vvell as intrathecal, direct intraventricular, intraperitoneal, intranasal, or mtraocular injections.

Altemately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directlv into an organ, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated vvith organ-specific aniiboay. The liposomes vvill be targeted to and taken up selectivelv by the organ. In addition, the drug may be provided in the form of a rapid release formulation, in the form of an extended release fonnulation, or in the form of an intermediate release formulation.

Composition/F ormulaiion

Pharmaceutical compositions comprising a compound of Formula (I) may be manufactured in a manner that is itself knovvn, e.g., by means of conventionai mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

Pharmaceutical compositions may be formulated in conventionai manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries vvhich facilitate processing of the active compounds into preparations vvhich can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the vvell-knovvn techmques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington’s Pharmaceutical Sciences, above.

The compounds of Formula (I) can be administered in a variety of vvays, including ali forms of local delivery to the eye. Additionally, the compounds of Formula (I) can be administered systemically, such as oially or intravenously. The compounds of Formula (I) can be administered topically to the eye and can be formulated into a variety of topically administrable ophthalmic compositions, such as Solutions, suspensions, gels or ointments. Thus, “ophthalmic administration” encompasses, but is not limited to, intraocular injection, subretinal injection, intravitreal injection, periocuiar administration, subconjuctival injections. retrobulbar injections, intracameral injections (including into the anterior or vitreous chamber), sub-Tenon’s injections or implants, ophthalmic Solutions, ophthalmic suspensions, ophthalmic ointments, ocular implants and ocular inserts, intraocular Solutions, use of iontophoresis, incorporation in surgical iirigating Solutions, and packs (by vvay of example only, a saturated cotton pledget inserted in the fomix).

Administration of a composition to ihe eye generally results in direct contact of the aģents vvith the comea, through vvhich at least a portion of the administered aģents pass. Often, the composition has an effective residence time in the eye of about 2 to about 24 hours, more typically about 4 to about 24 hours and most typically about 6 to about 24 hours.

A composition comprising a compound of Formula (I) can illustratively take the form of a Iiquid vvhere the aģents are present in solution, in suspension or both. Typically vvhen the composition is administered as a solution or suspension a first portion of the aģent is present in solution and a second portion of the aģent is present in particulate form, in suspension in a liquid matrix. In some embodiments, a liquid composition may include a gel formulation. In other embodiments, the liquid composition is aqueous. Altematively, the composition can take the form of an ointment

Useful compositions can be an aqueous solution, suspension or solution/suspension, vvhich can be presented in the form of eye drops. A desired dosage can be administered via a knovvn number of drops into the eve. For example, for a drop volume of 25 μΐ, administration of 1-6 drops vvill deliver 25-150 μ] of the composition. Aqueous compositions typically contain from about 0.01% to about 50%, more typically about

0.1% to about 20%, stili more typically about 0.2% to about 10%, and most typically about 0.5% to about 5%, weighu volume of 2 compound ofFonnula (I).

Typically, aqueous compositions have ophthalmically acceptable pH and osmolality. “Ophihalmicallv acceptable” vvith respect to a formulation, composition or ingredient typicaliy means having no persistent detrimental effect on the treated eye or the īuncboniag thereof, or on the general health of the subject being treated. Transient effects such as minor irritation or a stinging sensation are common vvith topical ophthalmic administration of aģents and consistent vvith the fonnulation, composition or ingredient in questīon being “ophthalmically acceptable.”

Useful aqueous suspension can also contain one or more polymers as suspending aģents. Useful polymers include vvater-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose, and vvater-insoluble polymers such as cross-Iinked carboxyl-containing polymers. Useful compositions can also comprise an ophthalmically acceptable mucoadbesive polymer, selected for example from carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophīl, acrylic acid/butyl acrylate copolymer, sodium alginate and dextran.

Useful compositions may also include ophthalmically acceptable solubilizing aģents to aid in the solubility of a compound ofFonnula (I). The term “solubilizing aģent” generally includes aģents that result in formation of a miceilar solution or a true solution of the aģent Certain ophthalmicafly acceptable nonionic surfactanis, for example polysorbate 80, can be useful as solubilizing aģents, as can ophthalmically acceptable glycols, polyglycols, e.g., polyethylene glycol 400, and glycoi ethers.

Useful compositions may also inciude one or more ophthalmically acceptable pH adjusting aģents or buffering aģents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids: bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomeĪhane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an oph.thalmically acceptable range.

Useful compositions may also include one or more ophthalmically acceptable salts in an amount reauired to bring osmolality of the composition into an ophihahnically acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

Other useful compositions may also include one or more ophthalmically acceptable preservatives to inhibit microbial activīty. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; siabilized chlorine dioxide; and quatemary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridhnum chloride.

Stili other useful compositions may include one or more ophthahnically acceptable surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkvlethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.

Stili other usefiil compositions may include one or more antioxidauts to enhance Chemical stability vvhere required. Suitable antioxidants include, by vvay of example only, ascorbic acid and sodium metabisulfite.

Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Altematively, multiple-dose reclosable containers can be used, in vvhich case it is typical to include a preservative in the composition.

The ophthalmic composition may also take the form of a solid article that can be inserted betvveen the eye and eyelid or in the conjunctival sac, vvhere it reļeases the aģent. Release is to the lacrimaJ fluid that bathes the surface of the comea, or directly to the comea itself, vvith vvhich the solid article is generally in intimate contact Solid articles suitable for implantation in the eye in such fashion are generally composed primarily of polymers and can be biodegradable or non-biodegradable.

For intravenous injections, compounds of Formula (I) may be formulated in aqueous Solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally knovvn in the art. For other parenteral injections, appropriate formulations may include aqueous or nonaqueous Solutions, preferably vvith physiologically compatible buffers or excipients. Such excipients are generally knovvn in the art.

One useful formulation for solubilizing higher quantities of the compounds of Formula (I) are, by vvay of example only, positively, negatively or neutrally charged phospholipids, or bile salt/phosphatidylcholine mixed.lipid aggregate systems, such as those described in Li, C.Y., et al., Pharm. Res. 13.-907-913 (1996). An additional formulation that can be used for the same purpose vvith compounds having the stmcture of Formula (I) involves use of a solvent comprising an alcohoĻ such as ethanol, in combination vrith an alkoxylated caster oil. See, e.g., U.S. Patent Publication Number 2002/01S3394. Or, altematively, a formulation comprising a compound of Formula (I) is an emulsion composed of a lipoid dispersed in an aaueous phase, a stabilizing amount of a non-ionic surfactant, optionaily a solvent, and optionally an isotonic aģent See id. Yet another formulation comprising a compound of Formula (I) includes com oil and a non-ionic surfactant See U.S. Patent No. 4,665,098. Stili another formulation comprising a compound ofFormuIa (I) includes lysophosphatidylcholine, monoglyceride and a fatty acid. See U.S. Patent No. 4,874,795. Stili another formulation comprising a compound of Formula (I) includes flour, a svveetener, and a humectant. See International Publication No. WO 2004/069203. And stili another formulation comprising a compound of Formula (I) includes dimyristoyl phosphatidylcholine, soybean oil, t-butyl alcohol and vvater. See U.S. Patent Application Publication No. US 2002/0143062.

For oral administration, compounds of Formula (Γ) canbe formulated readilyby combining the active compounds vvith pharmaceutically acceptabie carriers or excīpients vvell knovvn in the art. Such carriers enable the compounds described herein to be formulated as tablets, povvders, pilis, dragees, capsules, liquids, gels, syrups, elirfrs, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by miring one or more solid excipient vvith one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable atmliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as: for example, maize starch, vvheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polvvinvlpvrrolidone (PVP orpovidone) or calcium phosphate. If desired, disintegrating aģents may be added, such as the cross-liuked croscarmellose sodium, polyvinylpyiTolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided vvith suitable coatings. For this purpose, concentrated sugar Solutions may be used, vvhich may optionally contain gum arabic, talc, polyviaylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer Solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for Identification or to characterizs different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fīt capsules made of gelatin, as vvell as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fīt capsules can contain the active ingredients in admixture vvith filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liguids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Ali formulations for oral administration should be in dosages suitable for such administration.

For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in conventional manner.

Another useful formulation for administration of compounds having the structure of Formula (I) employs transdermal delivery devices (patches). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical aģents is vvell knovvn in the art See, e.g., U.S. Pat. No. 5,023,252. Such patches may be constructed for continuous, pulsatile, or on demand deliverv of pharmaceutical aģents. Stili further, transdermal deiivery of the compounds of Formula (I) can be accomplished by means of iontophoretic patches and the like. Transdermal patches can provide controlled delivery of the compounds. The rāte of absorption can be slovved by using rate-controllmg membranes or by trapping the compound vvithin a polymer matrix or gel. Conversely, absorption enhancers can be used to increase absorption. Formulations suitable for transdermal administration can be presented as discrete patches and can be Iipophiiic emulsions or buffered, aqueous Solutions, dissolved and/or dispersed in a polymer or an adhesive. Transdermal patches may be placed over different portions of the patient’s body, including over the eye.

Additional iontophoretic devices that can be used for ocular administration of compounds having the structure of Formula (I) are the Eyegate applicator, created and patented by Optis France S.A., and the Ocuphor™ Ocular iontophoresis system developed Iomed, Inc.

For administration by inhalation, the compounds of Formula (Tj are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, vvith the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafhioroethane, carbon diozids or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufffator may be formulated containing a povvder mix of the compound and a suitable povvder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, vvith an added preservative. The compositions may take such forms as suspensions.

Solutions or emulsions in oily or aqueous vehicles, and may contain foim.ulatory aģents such as suspending, stabilizing and/or dispersing aģents.

Pharmaceutical formulations for parenteral administration include aqueous Solutions of the active compounds in vvater-soluble form Additīonally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters. such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances vvhich increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or aģents vvhich increase the solubility of the compounds to aliovv for the preparation of highly concentrated Solutions.

Altematively, the active ingredient may be in povvder form for constitution vvith a suitable vehicle, e.g., steriie pyrogen-free vvater, before use.

The compounds may also be formulated in rectal compositions such as rectal gels, rectal foam, rectal aerosols, suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerīdes.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or iniramuscularly) or by intramuscular injection. Thus, for ezample, the compounds may be formulated with suitable polymeric or hydrophobic materiāls (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Injectable depot forms may be made by forming microencapsulated matrices (also knovvn as microencapsule matrices) of the compound of Formula (I) in biodegradable polymers. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, die rāte of drug release can be controlled. Depot injectable formulations may be also prepared by enirapping the drug in liposomes or microemulsions. By way of example only, posterior juxtascleral depots may be used as a mode of administration for compounds having the structure of Formula (I). The sclera is a thin avascular layer, comprised of highly ordered collagen netvvoik surrounding most of vertebrate eye. Since the sclera is avascular it can be utilized as a natūrai storage depot from vvhich injected material cannot rapidly removed or cleared from the eye. The formulation used for administration of the compound into the scleral layer of the eye canbe any form suitable for application into the sclera by injection through a cannula vvith small diameter suitable for injection into the scleral layer. Examples for injectable application forms are Solutions, suspensions or colloidal suspensions.

A pharmaceutical carrier for the hydrophobic compounds of Formula (I) īs a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a vvater-miscible organic polymer, and an aqueous phase. The cosolvent svstem may be a 10% ethanol, 10% polyethylene glvcol 300, 10% polyethylene glycol 40 castor oil (PEG-40 castor oil) vvith 70% aqueous solution. This cosolvent system dissolves hydrophobic compounds vvell, and itself producēs lovv toxicity upon systemic administration. Naturally, the proportions of a cosolvent system may be varied considerably vvithout destroying its solubility and toxicity characteristics. Furthermore, the identity of the cosolvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of PEG-40 castor oil, the fraction size of polyethylene glycol 300 may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyirolidone; and other sugars or polysaccharides maybe included in the aqueous solution.

Altematively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are vvell knovvn examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as jV-methylpyrrolidone also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, snch as semipermeable matrices of solid hydrophobic polymers containing the therapeutic aģent Various sustained-release materiāls have been established and are vvell knovvn by those skilled in the art. Sustained-release capsules may, depending on their Chemical nature, release the compounds for a fevv vveeks up to over 3 00 days. Depending on the Chemical nature and the biological stability of the therapeutic reaģent additional strategies for protein stabilization may be employed.

One formulation for the administration of compounds having the structure of Formula (I) has been used vvith fenretinide in the treatment of neuroblastoma, prostate and ovarian cancers, and is marketed by Avanti Polar Lipids, Inc. (Alabaster, Alabama) under the name Lym-X-Sorb™. This formulation, vvhich comprises an organizēti lipid matrix that includes lysophosphatidylcholine, monoglyceriae and fatty acid, is designed to improve the oral availability of fenretinide. Such a formulation, z.e., an oral formulation that includes lysophosphatidylcholine, monoglyceride and fatty acid, is proposed to also provide improved bioavailability of compounds having the structure of Formula (I) for the treatment of ophthalmic and ocular diseases and conditions, inciuding but not limited to the macular degenerations and dystrophies.

Ali of the formulations described herein may benefit from antioxidants, mētai chelating aģents, thiol containing compounds and other general stabilizing aģents. Examples of such stabilizing aģents, inciude, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (h) about 0.1% to about 1% vv/v methionine, (c) about 0.1% to about 2% vv/v monothioglycerol, (d) about I mM to about 10 mM EDTA, (e) about 0.01% to about 2% vv/v ascorbic acid, (f) 0.003% to about 0.02% vv/v polysorbate S0, (g) 0.002% to about 0.05% vv/v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

Many of the compounds of Formula (I) may be provided as salts vvith phannaceutically compatible counterions. Pharmaceutically compatible salts may be formed vvith many acids, inciuding but not limited to hycfrochioric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free acid or base forms.

TREATMENTMETHODS. DOSAGES AND COMBINATION TBERAPIES

The term “mammai” means ali mammals inciuding humāns. Mammals inciude, by way of example only, humāns, non-hum3n primates, cows, dogs, cats, goats, sheep, pigs, rats, mice and rabbits.

The term “effective amount” as used herein refers to that amount of the compound being administered vvhich vvill relieve to some extent one or more of the symptoms of the disease, condition or disorder being treated.

The compositions containing the compound(s) described herein can be administered for prophylactic and/or iherapeutic treatments. The term “treating” is used to refer to either prophylactic and/or therapeutic treatments. In therapeutic applications, the compositions are administered to a patient already suffering from a disease. condition or disorder, in an amount sufficient to cure or at least partially arrest the symptoms of the disease, disorder or condition. Amounts effective for this use vvill depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. It is considered vvell vvithin the skill of the art for one to determine such therapeutically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).

In prophylactic applications, compositions containing the compounds described herein are administered to a patient susceptible to or othervvise at risk of a particular disease, disorder or condition. Such an amount is defined to be a prophylactical]y effective amount or dose. In this use, the precise amounts also depend oh the patient's State of health, vveight, and the like. It is considered vvell vvithin the skill of the art for one to determine such prophylactically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).

The terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect Thus, in regard to enhancing the effect of therapeutic aģents, the term “enhancing” refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic aģents on a system.

An “enhancing-effective amount” as used herein, refers to an amount adequate to enhance the effect of another therapeutic aģent in a desired system. When used in a patient amounts effective for this use vvill depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

In the case vvherein the patient’s condition does not improve, upon the doctor’s discretion the administration of the compounds may be administered chronically, that is, for an extended period of time, including throughout the duration of the patient’s life in order to ameliorate or othervvise control or limit the symptoms of the patient’s disease or condition.

In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the compounds may be given continuously; altematively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). The length of the drug holiday can vary between 2 days and 1 year, including by vvay of example only, 2 days, 3 days, 4 days, days, 6 days, 7 days, 10 davs, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dose reduction during a drug holiday may be frotn 10%-100%, including by vvay of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once improvement of the patient’s conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a funetion of the symptoms, to a Ievel at vvhich the improved disease, disorder or condition is retained. Patients can, hovvever, require intermittent treatment on a long-tenn basis upon any recutrence of symptoms.

The amount of a given aģent that vvill correspond to such an amount vvill vary depending upon factors such as the particular compound, disease condition and its severity, the īdentity (e.g., vveight) ofthe subject or host in need of treatment, but can nevertheless be routinely determined in a manner knovvn in the art according to the particular circumstances surrounding the case, including, e.g., the specific aģent being administered, the route of administration, the condition being treated, and the subject or host being treated. In general, hovvever, doses employed for adult human treatment vvill typically be in the range of 0.02-5000 mg per day, preferably 11500 mg per day. The desired dose may convenienūy be presented in a single dose or as divided doses administered sīmultaneously (or over a short period of time) or at appropriate intervāls, for exanrple as tvvo, three, four or more sub-doses per day.

In certain instances, it may be appropriate to administer at least one of the compounds described herein (or a pharmaceutically acceptable salt, ester, amide, prodrug, or solvate) in combination with another therapeutic aģent. By way of example only, if one of the side effects experienced by a patient upon receiving one of the compounds herein is inflammation, then it may be appropriate to administer an anti-inflammatory aģent in combination vvith the initial therapeutic aģent. Or, by way of example only, the therapeutic effectiveness of one of the compounds described herein may be enhanced by administration of an adjuvant (i.e., by itself the adjuvant may onl.y have minimal therapeutic benefit, but in combination with another therapeutic aģent the overall therapeutic benefit to the patient is enhanced). Or, by vvay of example only, the benefit of experienced by a patient may be increased by administering one of the compounds described herein with another therapeutic aģent (vvhich also inciudes a therapeutic regimen) that also has therapeutic benefit. By way of example only, in a treatment for macular degeneration involving admimstration of one of the compounds described herein, increased therapeutic benefit may result by also providing the patient vvith other therapeutic aģents or therapies for macular degeneration. In any case, regardless of the disease, disorder or condition being treated, the overall benefit experienced by the patient may simrp]y be additive of the tvvo therapeutic agenis or the patient may experience a synergistic benefit.

Specific, non-limiting examples of possfole combination therapies include use of at least one compound of formula (I) vvith nitric oidde (NO) inducers, statins, negatively charged phospholipids, antioxidants, minerāls, anti-inflammatory aģents, anti-angiogenic aģents, matrix metalloproteinase inhibitors, and carotenoids. In several instances, suitable combination aģents may fall vvithin multiple categories (hy way of example only, lutein is an anti-oxidant and a carotenoid). Further, the compounds of Formula (I) may also be administered vvith additional aģents that may provide benefit to the pacient including by way of example only cyclosporin A.

In addition, the compounds of Formula (I) may also be used in combination vvith procedures that may provide additional or synergistic benefit to the patient, including, by way of example only, the use of extracoiporeal rheopheresis (also knovvn as membrane differential filtration), the use of implantable miniature telescopes, laser photocoagulation of drusen, and microstimulation therapy.

The use of anti-oxidants has been shovvn to benefit patients vvith macular degenerations and dystrophies. See, e.g., Arch. Ophthalmol., 119:1417-36 (2001); Sparrovv, ef al., J. Biol. Chem., 278:18207-13 (2003). Examples of suitable anū-oxidants that could be used in combīnation vvith at least one compound having the structure of Formula (I) include vitamin C, vitamin E, beta-carotene and other carotenoids, coenzyme Q, 4-hydroxy-2,2,6,6-tetramethylpiperidine-Z/-oxyl (also knovvn as Tempol), lutein, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract.

The use of certain minerāls has also been shovvn to benefit patients vvith macular degenerations and dysirophies. See, e.g., Arch. Ophthalmol., 119: 1417-36 (2001). Examples of suitable minerāls that could be used in combination vvith at least one compound having the structure of Formula (I) include copper-containing minerāls, such as cupnc oxide (by way ofexample only); zinc-containing minerāls, such as zinc oxide (by way ofiAample only); and selenium-containing compounds.

The use of certain negatively-charged phospholipids has also been shovvn to benefit patients witb macular degenerations and dystrophies. See, e.g., Shaban & Richter, Biol. Chem., 383:537-45 (2002); Shaban, et al., Exp. Eye Res., 75:99-108 (2002). Examples of suitable negatively charged phospholipids that could be used in combination vvith at least one compound having the structure of Formula (I) inelude cardiolipin and phosphatidylglycerol. Positively-charged and/or neutral phospholipids may also provide benent for pahents vvith macular degenerations and dystrophies vvhen used in combination vvith compounds having the structure of Formula (I).

The use of certain carotenoids has been correlated vvith the maintenance of photoprotection uecessary in photoreceptor celis. Carotenoids are naturally-occuning yellow to red pigments of the terpenoid group that can be found in plants, algae, bacteria, and certain animals, such as birds and shellfish. Carotenoids are a large class of molecules in vvhich more than 600 naturally occurring carotenoids have been identified. Carotenoids inelude hydrocarbons (carotenes) and their oxygenated, alcoholic derivatives (xanthophylls). They inelude actinioerythrol, astaxanthin, canthaxanthin, capsanthin, capsorubin, u-8'-apo-caroteual (apo-carotenal), $-12'apo-carotenal, α-carotene, β -carotene, carotene (a mixture of a- and $-carotenes), γ-carotenes, β cyrptoxanthin, Iutein, lycopene, violerythrin, zeaxanthin, and esters of hydroxyl- or carboxyl-containing members thereof. Many of the carotenoids occur in nature as cis- and rrunr-isomeric forms, vvhile synihetic compounds are frequently racemic mistures.

In humāns, the retina selectively accumulates mainly tvvo carotenoids: zeaxanthin and Iutein. These tvvo carotenoids are thought to aid in protecting the retina because they are povverful antīoxidants and absorb blue light Studies vvith quails establish that groups raised on carotenoid-deficient diets had retinās vvith lovv concentrations of zeaxantinn and suffered severe light damage, as evidenced by a very high nurnber of apoptotic photoreceptor celis, vvhile the group with high zeaxanthin concentrations had minimal damage. Examples of suitable carotenoids for in combination vvith at least one compound having the structure of Formula (I) inelude Iutein and zeaxanthin, as vvell as any of the aforementioned carotenoids.

Suitable nitric oxide inducers inelude compounds that stimulate endogenous NO or elevate Ievels of endogenous endothelium-derived relaxing factor (EDRF) in vivo or are substraies for nitric oxide synthase.

Such compounds inelude, for example, L-arginine, L-homoarginine, and N-hydroxy-L-arginine, including their nitrosated and nitrosylated analogs (e.g., nitrosated L-arginine, nitrosylated L-arginine, nitrosated //-hydroxy-Larginine, nitrosylated A^r-hydroxy-L-arginine, nitrosated L-homoargimne and nitrosylated L-homoarginine), precursors of L-arginine and/or physiologically acceptable salts thereof) including, for example, citrulline, omithine, glutamine, lysine, polypeptides comprising at least one of these amino acids, inhibitors of the enzyme arginase (e.g., jV-hydroxy-L-arginine and 2(S)-amino-6-boronohexanoic acid) and the substrates for nitric oxide synthase, cytokīnes, adenosine, bradyfcimn, calreticuliu, bisacodyļ and phenolphthalein. EDRF is a vascular relaxing factor secreted by the endothehum, and has been identified as nitric oxide or a closely related derivative thereof (Palmer et al, Nature, 327:524-526 (1987); Ignarro etal, Proc. Nati. Acad. Sci. USA, 84:9265-9269 (1987)).

Statiņš serve as lipid-lowering aģents and/or suitable nitric oxide inducers. In addition, a relationship has been demonstrated betvveen statiņ use and delayed onset or development of macular degeneration. G.

McGvvin, er al, British Journal of Ophthahnology, 87:1121-25 (2003). Statiņš can thus provide benefit to a patient suffering from an ophtbalmic condition (such as the macular degenerations and dystrophies, and the retina] dystrophies) vvhen administered in combination with compounds ofFormula (I). Suitable statins include, by way of esamņle only, rosuvastatin, pitivastatin, simvastatin, pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin, compactin, lovastatin, dalvastatin, fluindostatin, atorvastatin, atorvastatin calcium (vvhich is the hemicalcium salt of atorvastatin), and dihydrocompactm.

Suitable anti-inflammatory aģents vvith which the Compounds ofFormula (I) may be used include, by way of example only, aspirin and other salicylates, cromolyn, nedocromil, tbeophylline, zileuton, zafirlukast, montelukast, pranlukast, indomethacin, and lipoxygenase inhibitors; non-steroidal antiinflammatory drugs (NSAIDs) (such as ibuprofen and naproxin); prednisone, dexamethasone, cyclooxygenase inhibitors (i.e., COX1 and/or C0X-2 inhibitors such as Naproxen™, or Celebrex™); statins (by way of example only, rosuvastatin, pitivastatin, simvastatin, pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin, compactin, lovastatin, dalvastatin, fluindostatin, atorvastatin, atorvastatin calcium (vvhich is the hemicalcium salt of atorvastatin), and dihydrocompactin); and disassociated steroids.

Suitable matrix metalloproteinases (MMPs) inhibitors may also be administered in combination vvith compounds ofFormula (I) in order to treat ophthalmic conditions or symptoms associated vvith macular or retinal degenerations. MMPs are knovvn to hydrolyze most components of the extracellular matrix. These proteinases play a Central role in many biological processes such as normai tissue remodeling, embryogenesis, vvound healing and angiogenesis. Hovvever, excessive expression of MMP has been observed inmany disease States, including macular degeneration. Many MMPs have been identified, most of vvhich are multidomain zinc endopeptidases. A number of metalloproteinase inhibitors are knovvn (see for example the revievv of MMP inhibitors by Whittaker M. etal, ChemicalReviews 99(9):2735-2776 (1999)). Representative examples ofMMP Inhibitors include Tissue Inhibitors of Metalloproteinases (TIMPs) (e.g., ΊΤΜΡ-1, TIMP-2, TIMP-3, or ΊΊΜΡ4), os-macroglobulin, tetracyclines (e.g., tetracycline, minocycline, and doxycycline), hydroxamates (e.g., BATĪMASTAT, MARIMISTAT and TROCADE), chelators (e.g., EDTA, cysteine, acetylcysteine, Dpenicillamine, and gold salts), synthetic MMP fragments, succinyl mercaptopurines, phosphonamidates, and hydroxaminic acids. Examples ofMMP inhibitors that may be used in combination vvith compounds of Formula (I) include, by vvay of example only, any of the aforementioned inhibitors.

The use of antiangiogenic or anti-VEGF drugs has also been shovvn to provide benefit for patients vvith macular degenerations and dystrophies. Examples of suitable antiangiogenic or anti-VEGF drugs that could be used in combination vvith at Ieast one compound having the structure ofFormula (I) include Rhufab V2 (Lucentis™), Tryptophanyl-tRNA synthetase (TrpRS), Eye001 (Anti-VEGF Pegylated Aptamer), squalamine, Retaane™ 15mg (anecortave acetate for depot suspension; Alcon, Inc.), Combretastatin A4 Prodrug (CA4P), Macugen™, Mifeprex™ (mifepristone - ru486), subtenon triamcinolone acetonide, intravitreal crystalline triamcinolone acetonide, Prinomastat (AG3340 - synthetic matrix metalloproteinase inhibitor, Pfizer), fluocmolone acetonide (including fluocinolone intraocular implant, Bansch & Lomb/Control Delivery Svstems), VEGFR inhibitors (Sugen), and VEGF-Trap (Regeneron/Aventis).

Other pharmaceutical therapies that have been used to relieve visual impairment can be used in combination vvith at Ieast one compound ofFormula (I). Such treatments include but are not limited to aģents such as Visudyne™ vvith use of a non-thermal laser, PKC 412, Endovion (NeuroSearch A/S), neurotrophic factors, including by way of example Glial Derived Neurotrophic Factor and Ciliary Neurotrophic Factor, diatazem. dorzolanude, Phototrop, 9-cfr-retinal, eve medication (including Echo Therapy) including phospholine iodide or echothiophate or carbonic anhydrase inhibitors, AE-941 (AEtema Laboratories, Inc.), Sima-027 (Sirna Therapeutics, Inc.), pegaptanib (NeXstar Pharmaceuticals/Gilead Sciences), neurotropbins (including, by vvay of example only, NT-4/5, Genentech), Cand5 (Acuity Pharmaceuticals), ranibizumab (Genentech), INS-37217 (Inspirē Pharmaceuticals), integrin antagonists (including those from Jēriņi AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd,), BDM-E (BioDiem Ltd.), thalidomide (as used, for example, by EntreMed, Inc.), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan/Oculex), DL-S234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University ofMichigan), LYN-002 (Lynkeus Biotech), microalgal compound (Aquasearch/Albany, Mēra Pharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme/Celtrix), tyrosine kinase inhibitors (Allergan, SUGEN, Pfizer), ΝΧ-278-L (NeXstar Pharmaceuticals/Gilead Sciences), Opt-24 (OPTIS France SA), retinal celi ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), and cyclosporin A. See U.S. Patent Application Publication No. 20040092435.

In any case, the multiple therapeutic aģents (one of vvhich is one of the compounds described herein) may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic aģents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pili or as tvvo separate pilis). One of the therapeutic aģents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing betvveen the multiple doses may vary from more than zero vveeks to less than four vveeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only tvvo aģents; we envision the use of multiple therapeutic combinations. By way of example only, a compound having the structure of Formula (I) may be provided with at least one antioxidant and at least one negatively charged pbospholipid; or a compound having the structure of Formula (I) may be provided vvith at least one antioxidant and at least one inducer of nitric oxide production; or a compound having the structure of Formula (I) may be provided vvith at least one inducer of nitric oxide produchons and at least one negatively charged phospholipid; and so forth.

īņ addition, the compounds of Formula (I) may also be used in combination vvith procedures that may provide additional or synergistic benefit to the patient. Procedures knovvn, proposed or considered to relieve visual impairment include but are not limited to ‘limited retinal translocation’, photodynamic therapy (including, by way of example only, receptor-targeted PDT, Bristo 1-Myers Squibb, Co.; porfimer sodium for injection vvith PDT; verteporfin, QLT Inc.; rostaporfin vvith PDT, Miravent Medical Technologies; talaporfīn sodium vvith PDT, Nippon Petroleum;^_motexafin lutetium, Pharmacychcs„ Inc.), antisense oligonucleotides (including, by vvay of example, products tested by Novagali Phaima SA and ISIS-13650, Isis Pharmaceuticals), laser photocoagulation, drusen lasering, macular hole surgery, macular translocation surgery, implantable miniature telescopes, Phi-Motion Angiography (also knovvn as Micro-Laser Therapy and Feeder Vessei Treatment),

Proton Beam Therapy, roicrostimulation therapy, Retinal Detachment and Vitreous Surgery, ScleraI Buckle, Submacular Surgery, Transpupillary Thermotherapy, Photosystem I therapy, use of RNA interference (RNAi), extracorporeal rheopheresis (also knovvn as membrane differential filtration and Rheotherapy), microchip implantation, stem celi therapy, gene replacement therapy, ribozyme gene therapy (including gene therapy for hypoxia response element, Oxford Biomedica; Lentipak, Genetix; PDEF gene therapy, GenVec), photoreceptor/retinal celis transplantātiem (including transplantable retinal epithelial celis, Diacrin, lnc.; retinal celi transplant, Celi Genesys, lnc.), and acupuncture.

Further combinations that may be used to benefit an individual include using genetic testing to determine vvhether that individual is a carrier of a mutant gene that is knovvn to be correlated vvith certain ophthalmic conditions. By way of example only, defeets in the human ABCA4 gene are thought to be associated vvith five distinet retinal phenotypes including Stargardt disease, cone-rod dystrophy, age-related macular degeneration and retinitis pigmentosa. See e.g., Allikmets et al., Science, 277:1805-07 (1997); Levvis et al., Am. J. Hum. Genet., 64:422-34 (1999); Stone et al., Nature Genetics, 20:328-29 (1998); Allikmets, Am. J. Hum. Gen., 67:793-799 (2000); Klevering, et al, Ophthalmology, 111:546-553 (2004). In addition, an autosomal dominant form of Stargardt Disease is caused by mutations in the ELOV4 gene. See Karan, et al., Proc. Nati. Acad. Sci. (2005). Patients possessing any of these mutations are expected to find therapeutic and/or prophylactic benefit in the methods described herein.

SYNTHESIS OF THE COMPOUNDS OF FORMULA (TV

Compounds ofFormula (ī) may be synthesized using Standard synthetic techniques knovvn to those of skill in the art or using methods knovvn in the art in combination vvith methods described herein. See, e.g., U.S. Patent Application Publication 2004/0102650; Um, S. I, et al., Chem. Pharm. Buli., 52:501-506 (2004). In addition, several of the compounds ofFormula (I), such as fenretinide, may be purchased from various commercial suppliers. As a further guide the follovving syuthetic methods may also be utilized.

Formation of Covalent Linkages by Reaction of an Electrophile vvith a Nucleophile

Selected examples of covalent linkages and precursor functional groups vvhich yield them are given in the Table entitled “Examples of Covalent Linkages and Precursors Thereof.” Precursor functional groups are shovvn as electrophilic groups and nucleophilic groups. The functional group on the organic substance may be attached directly, or attached via any useful spacer or linker as defined belovv. Table 1: Examnles of Covalent Linkages and Precursors Thereof

Covalent Linkage Product	Electrophile	Nucleophile
Carboxamides	Activated esters	amines/anilines
Carboxamides	acyl azides	amines/anilines
Carboxamides	acyl halides	amines/ anilines
Esters	acyl halides	alcohols/phenols
Esters	acyl nitriles	alcohols/phenols
Carboxamides	acyl nitriles	amines/anilines
Imines	Aldehydes	amines/anilines
Hydrazones	aldehydes or ketones	Hydrazines
Oximes	aldehydes or ketones	Hydroxylamines
Alkyl amine s	alkyl halides	amines/anilines
Esters	alkyl halides	carboxylic acids
Thioethers	alkyl halides	Thiols
Ethers	alkyl halides	alcohols/phenols
Thioethers	alkyl sulfonates	Thiols
Esters	alkyl sulfonates	carboxylic acids
Ethers	alkvl sulfonates	alcohols/phenols
Esters	Anhydrides	alcohols/phenols
Carboxamides	Anhydrides	amines/anilines
Thiophenols	aryl halides	Ihiols
Aryl amines	aryl halides	Amines
Thioethers	Azindines	Thiols
Boronate esters ]	. Boronates	Glvcols

Carboxamides	carboxylic acids	arnines/anilines
Esters	carboxylic acids	Alcohols
hydrazines	Hydrazides	carboxylic acids
jV-acylureas or Anhydrides	carbodiimides	carboxylie acids
Esters	diazoalkanes	carboxylic acids
Thioethers	Epoxides	Thiols
Thioethers	haloacetamides	Thiols
Ammotriazines	halotriazines	amines/anilines
Triazinyl ethers	halotriazines	aicohols/phenols
Amidines	imido esters	amines/anilines
Ureas	Isocyanates	amines/ānilines
Urethanes	Isocyanates	aicohols/phenols
Thioureas	isothiocyanates	amines/anilines
Thioethers	Maleimides	Thiols
Phosphite esters	phosphoramidites	Alcohols
Silyl ethers	silyl halides	Alcohols
Alkyl amines	sulfonate esters	amines/anilines
Thioethers	sulfonate esters	Thiols
Esters	sulfonate esters	carboxylic acids
Ethers	sulfonate esters	Alcohols
Sulfonamides	sulfonyl halides	amines/anilines
Sulfonate esters	sulfonyl halides |	. phenols/alcohols

In general, carbon electrophiles are susceptible to attack by complementary nucleophīles, including carbon nucleophiles, wherein an attaclting nucleophile brings an electron pair to tbe carbon electrophile in order to form a new bond betvveen the nucleophile and the carbon electrophile.

Suitable carbon nucleophiles include, but are not limited to alkyl, alkenyl, aryl and alkynyl Grignard, organolithium, organozinc, alkyl-, alkenyl, aryl- and alkynyl-tin reaģents (organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reaģents (organoboianes and organoboronates); these carbon nucleophiles have the advantage of being kinetically stable in vvater or polar organic solvents. Other carbon nucleophiles include phosphorus ylids, enol and enolate reaģents; these carbon nucleophiles have the advantage of being relatively easy to generate from precursors well knovvn to those skilled in the art of synthetic organic chemistry. Carbon nucleophiles, when used in conjunction vvith carbon electrophiles, engender nevv carbon-carbon bonds betvveen the carbon nucleophile and carbon electrophile.

Non-carbon nucleophiles suitable for coupling to carbon electrophiles include but are not limited to primary and secondary amines, thiols, thiolates, and thioethers, alcohols, alkoxides, azides, semicarbazides, and the like. These non-carbon nucleophiles, vvhen used in conjunction vvith carbon electrophiles, typically generate heteroatom linkages (C-X-C), vvherein X is a hetereoatom, e. g, oxygen or nitrogen.

Use of Protecting Groups

The term “protecting group” refers to Chemical moieties that block some or ali reactive moieties and prevent such groups from participating in Chemical reactions until the protective group is removed. It is preferred that each protective group be removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal. Protective groups can be removed by acid, base, and hydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal and tbutyldimethylsilyl are acid labtie and mav be used to protect carboxy and bydroxy reactive moieties in the presence of amino groups protected vvith Cbz groups, vvhich are removable by hydrogenolysis, and Fmoc groups, vvhich are base labtie. Carboxylic acid and hydroxy reactive moieties may be blocked vvith base labtie groups such as, without limitation, methyl, ethyl, and acetyl in the presence of amines blocked vtith acid labile groups such as t-butyl carbamate or vvith carbamates that are both acid and base stable but hydrolytically removable.

Carboxylic acid and hydroxy reactive moieties may also be blocked vvith hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding vvith acids maybe blocked vvith base labile groups such as Fmoc. Carboxylic acid reactive moieties may he protected by conversion to simple ester derivatives as exempiifīed herein, or they may be blocked vvith oxidativelyremovable protective groups such as 2,4-dimethoxybenzyl, while co~existing amino groups may be blocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in then presence of acid- and base- protecting groups since the former are stable and can be subsequently removed by mētai or pi-acid catalysts. For example, an allyl-blocked carboxylic acid can be deprotected vvith a Pd/j-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to vvhich a compound or intermediate may be attached. As long as the residue is attached to the resin, that functional group is blocked and cannot react. Once released from the resin, the functional group is available to react.

Typically blocking/protecting groups may be selected from:

allyl Bn Cbz alloc Me

H₂

HaC'

Et (H₃C)₃C' t-butyl

H₃c_s ch₃ (H₃C)₃C-^Si\

TBDMS (CH₃)₃C

Teoc (CH₃)₃C^/OT^ 0

Boc

(C_sH₅)₃Ctrityl

H,C acetyl

Fmoc

Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New YoTk, NY, 1999, which is incorporated herein by reference in its entirety.

ILLUSTRATIVE EXAMPLES

The follovving examples provide illustrative methods for testing the effectiveness and safety of the compounds of Formula (I). These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

HUMAN STUDIES

Detection of Macular or Retinal Degeneration. Identification of abnormal blood vessels in the eve can be done vvith an angiogram. This Identification can help determine vvhich patients are candīdates for the use of a candidate substance or other treatment method to hinder or prevent further vision loss. Angiograms can also be useful for follow-up of treatment as vvell as for future evaluation of any nevv vessel grovvth.

A fluorescein angiogram (fluorescein angiography, fluorescein angioscopy) is a techxuque for the visualization of choroidal and retinal circulation at the back of the eye. Fluorescein dye is injected intravenously follovved by multiframe photography (angiography), ophthalmoscopic evaluation (angioscopy), or by a Heidelberg retina angiograph (a confocal seanning laser system). Additionally, the retina can be examined by OCT, a non-invasive way to obtain high-resolution cross-sectional images of the retina. Fluorescein angiograms are used in the evaluation of a vvide range of retinal and choroidal diseases through the analysis of leakage or possible damage to the blood vessels that feed the retina. It has also been used to evaluate abnormalities of the optic nerve and iris by Berkovv et al., Am. J. Ophthalmol. 97:143-7 (1984).

Similarly, angiograms using indocyanine green can be used for the visualization circulation at the back of the eye. Wherein fluorescein is more efficient for studying retinal circulation, indocyanine is better for ohserving the deeper choroidal blood vessel layer. The use of indocyanine angiography is helprul vvhen neovascularization may not be observed vvith fluorescein dye alone.

Appropriate human doses for compounds having the structure of Formula (I) vvill be determined using a Standard dose esealation study. Hovvever, some guidance is available from studies on the use of such compounds in the treatment of cancer. For example, a 4800 mg/m² dose of fenretinide, vvhich is a compound having the structure of Formula (I), has been administered to patients vvith a variety of cancers. Such doses vvere administered three times daily and observed toxicities vvere minimal Hovvever, the recommended dose for such patients vvas 900 mg/m² based on an observed ceiling on achievable plasma Ievels. In addition, the bioavaīlability of fenretinide is increased vvith meals, vvith the plasma concentration being three times greater after high fat meals than after carbohydrate meals.

The observation of occasional night blindness in humāns suggests to us significant impairment of rhodopsin regeneration at normai therapeutic doses. Based on these data, we propose that inhibitory concentrations of fenretinide in RPE tissue is achieved at doses similar to, or possibly belovv, human therapeutic doses for the treatment of cancer.

Example 1: Testing for the Effīcacy of Compounds of Formula (I) to Treat Macular Degeneration

For pre-testing, all human patients undergo a routine ophthalmologic examination including fluorescein angiography, measuremeut of visual acuity, electrophysiologic parameters and biochenhcal and rheologic parameters. Inclusion criteria are as follovvs: visual acuity betvveen 20/160 and 20/32 in at least one eye and signs of AMD such as drusen, areolar atrophy, pigment clumping, pīgment epithelium detachment, or subretinal neovascularization. Patients that are pregnant or actively breast-feeding children are excluded from the study.

Tvvo hundred human patients diagnosed vvith macular degeneration, or vvho have progressive formatjons of A2E, lipofuscin, or drusen in their eyes are divided into a control group of about 100 patients and an experimental group of 100 patients. Fenretinide is administered to the experimental group on a dailv basis. A placebo is administered to the control group in the same regime as fenretinide is administered to the experimental group.

Administration of fenretinide or placebo to a patient can be either orally or parenterally administered at amounts effective to inhibit the development or reoccurrence of macular degeneration. Effective dosage amounts are in the range of from about 1-4000 mg/irf up to three times a day.

One method for measuring progression of macular degeneration in both control and experimental groups is the best corrected visual acuity as measured by Early Treatment Diabetic Retinopathy Study (ETDRS) charts (Lighthouse, Long Island, NY) using line assessment and the forced choice method (Ferris et al. Am J Ophthalmol, 94:97-98 (1982)). Visual acuity is recorded in logMAR. The change of one line on the ETDRS chart is equivalent to 0.1 logMAR. Further typical methods for measuring progression of macular degeneration in both control and experimental groups include use of visual fīeld examinations, including but not linhted to a Humphrey visual field ezamination, and measuring/monitoring the autofluorescence or absorption spectra of Nretmylidene-phosphatidylethanolamine, dihydro-A-retinylidene-N-retinyl-phosphatidylethanolamine, Nretinylidene-V-retin.yl-phosphatidylethanolamine, dihydro-V-retinylidene-A-retinyl-ethanolam!ne, and/or Nretinylidene-phosphatidylethanolamine in the eye of the patient. Autofluorescence is measured using a variety of equipment, including but not limited to a confocal scanning laser ophthalmoscope. See Bindevvald, et al.,

Am. J. Ophthalmol., 137:556-8 (2004).

Additional methods for measuring progression of macular degeneration in both control and experimental groups include taking fundus photographs, observing changes in autofluorescence over time using a Heidelberg retina angiograph (or altematively, techniques described in M. Hammer, et al. Ophthalmologe 2004 Apr. 7 [Epub ahead of patent)), and taking fluorescein angiograms at baseline, three, six, nine and twelve months at follovv-up visits. Documentation of morphologic changes include changes in (a) drusen size, character, and distribution; (b) development and progression of choroidal neovascularization; (c) other interval fundus changes or abnormalities; (d) reading speed and/or reading acuity; (e) scotoma size; or (f) the size and number of the geographic atrophy lesions. In addition, Amsler Grid Tēst and color testing are optionally administered.

To assess statistically visual improvement during drug administration, examiners use the ETDRS (LogMAR) chart and a standardīzed refraction and visual acuity protocol. Evaluation of the mean ETDRS (LogMAR) best corrected visual acuity (BCVA) from baseline through the available post-treatment interval visits can aid in determining statistical visual improvement.

To assess the ANOVA (analysis of variance between groups) betvveen the control and experimental group, the mean changes in ETDRS (LogMAR) visual acuity from baseline through the available post-treatment interval visits are compared using two-group ANOVA vvith repeated measures analysis vvith unstructured covariance using SAS/STAT Softvvare (SAS Institutes Inc, Cary, North Carolina).

Toxicity evaluation after the commencement of the study include check ups every three months during the subsequent year, every four months the year after and subsequently every six months. Plasma Ievels of fenretinide and its metabolite N-(4-methoxyphenyl)-retinamide can also be assessed during these visits. The toxicity evaluation includes patients using fenretinide as vvell as the patients in the control group.

Example 2: Testing for the Efficacy of Compounds of Formula (I) to Reduce A2E Production The same protocol design, including pre-testing, administration, dosing and toxicity evaluation protocols, that are described in Example 1 are also used to tēst for the efficacy of compounds of Formula (I) m reducing or othervvise limiting the production of A2E in the eye of a patient.

Methods for measuring or monitoring formation of A2E include the use of autofluorescence measurements of ?/-retinylidene-phosphatidylethanolamine, dihydro-//-retinylidene-yV-retinylphosphatidylethanolamine, 7V-retinylidene-//-retiny]-phosphatidylethanolamine, dihydro-A^f-retinylidene-Nretinyl-ethanolamine, and/or yV'-reiinyiidene-phosphatidylethanolamine in the eye of the patient Autofluorescence is measured using a variety of equipment, including but not limited to a confocal scanning laser ophthalmoscope, see Bindewald, et al., Am. J. Ophthalmol., 137:556-8 (2004), or the autofluorescence or absorption spectra measurement techniques noted in Example 1. Other tests that can be used as surrogate markers for the effīcacy of a particular treatment include the use of visual acuity and visual field examinations, reading speed and/or reading acuity examinations, measurements on the size and number of scotoma and/or geographic atrophic lesions, as described in Example 1. The statistical analyses described in Example 1 is employed.

Example 3: Testing for the Effīcacy of Compounds of Formula (I) to Reduce Lipofuscin Production

The same protocol design, including pre-testing, administration, dosing and toxicity evaluation protocols, that are described in Example 1 are also used to tēst for the efficacy of compounds of Formula (ī) in reducing or othervvise limiting the production of lipofuscin in the eye of a patient. The statistical analyses described in Example 1 may also be employed.

Tests that can be used as surrogate markers for the efficacy of a particular treatment include the use of visual acuity and visual field examinations, reading speed and/or reading acuity examinations, measurements on the size and number of scotoma and/or geographic atrophic lesions, and the measuring/monitoring of autofluorescence of certain compounds in the eye ofthe patient, as described in Example 1.

Example 4: Testing for the Efficacy of Compounds of Formula (I) to Reduce Drusen Production

The same protocol design, including pre-testing, administration, dosing and toxicīty evaluation protocols, that are described in Example 1 are also used to tēst for the efficacy of compounds of Formula (I) in reducing or othervvise limiting the production or formation of drusen in the eye of a patient. The statistical analyses described in Example 1 may also be employed.

Methods for measuring progressive formations of drusen in both control and experimental groups include taking fondus photographs and fluorescein angiograms at baseline, three, six, nine and tvvelve months at follovv-up visits. Documentation of morphologic changes may include changes in (a) drusen size, character, and distribution (b) development and progression of choroidal neovascularization and (c) other interval fundus changes or abnorroalities. Other tests that can be used as surrogate markers for the efficacy of a particular treatment include the use of visual acuity and visual field examinations, reading speed and/or reading acuity examīnations, measurements on the size and number of scotoma and/or geographic atrophic lesions, and the measuring/monitoring of autofluorescence of certain compounds in the eye of the patien t, as described in Ezample 1.

Eiample 5: Genetic Testing for Macular Dystrophies

Defects in the human AJ3CA4 gene are thought to be associated with five distinct retina! phenotypes including Stargardt Disease, cone-rod dystrophy, age-related macular degeneration (both dry form and wet form) and retinitis pigmentosa. See e.g., Allikmets el al., Science, 277:1805-07 (1997); Levvis et al., Am. J. Hum. Genet., 64:422-34 (1999); Stone et al., Nature Genetics, 20:328-29 (1998); Allikmets, Am. J. Hum. Gen., 67:793-799 (2000); Klevering, et al, Ophthalmology, 111:546-553 (2004). In addition, an autosomal dominant form of Stargardt Disease is caused by mutations in the ELOV4 gene. See Karan, ei ak, Proc. Nati. Acad. Sci. (2005). Patients can be diagnosed as having Stargardt Disease by any of the follovving assays: (a) A directsequencing mutation detection strategy vvhich can involve sequencing ali exons and flanking īntron reģions of ABCA4 or ELOV4 for sequence mutation(s); (b) Genomic Southern analysis; (c) Microanay assays that include ali knovvn ABCA4 or ELOV4 variants; and (d) Analysis by Iiquid chiomatography tandem mass spectrometry coupled vvith immunocytochemical analysis using antibodies and Westem analysis. Funaus photographs, fluorescein anigograms, and scanning Iaser ophthalmoscope imaging along vvith the histoiy of the patient and his or her family can anticipate and/or confirm diagnosis.

MICE AND RAT STUDIES

The optimal dose of compounds of Formula (ī) to block formation of A2E in abca4T mice can be detennined using a Standard dose escalatīon study. One illustrative approach, utilizing fenretinide, vvhich is a compound having the structure of Formula (I) is presented belovv. Hovvever, similar approaches may be utilized for other compounds having the structure of Formula (I).

The effects of fenretinide on all-rrmtj-retinal in retinās from light-adapted mice vvould preferahly be determined at doses that bracket the human therapeutic dose. The preferred method iņcludes treating mice vvith a single moming intraperitoneal dose. An inereased frequency of injections may be requiied to maintain reduced Ievels of all-ft-mis-retinal in the retina throughout the day.

ABCA4 Knockout Mice. ABCA4 encodes rim protein (RmP), an ATP-binding cassette (ABC) transporter in the outer-segment dises of rod and cone photoreceptors. The transported substrate for RmP is unknovvn. Mice generated vvith a knockout mutation in the abca4 gene, see Weng ei al., Celi, 98:13-23 (1999), are useful for the study of RmP function as vvell as for an in vivo screening of the effectiveness for candidate substances. These animals manifest the complex ocular phenotype: (i) slovv photoreceptor degeneration, (ii) delayed recoveiy of rod sensitivity follovving light exposure, (iii) elevated atRAL and reduced atROL in photoreceptor outer-segments follovving a photobleach, (iv) constitutively elevated phosphatidylethanolamine (PE) in outer-segments, and (v) accumulation oflipofuscin inRPE celis. See Weng et al., Celi, 98:13-23 (1999).

Rātes of photoreceptor degeneration can be monitored in treated and untreated wild-type and abcaPi mice by tv/o techniques. One is the study of mice at different times by ERG analysis and is adopted from a clinical diagnostic procedure. See Weng et al., Celi, 98:13-23 (1999). An electrode is placed on the comeal surface of an anesthetized mouse and the electrical response to a light flash is recorded from the retina.

Amplitude of the α-vvave, v/hich results from light-induced hyperpolanzation of photoreceptors, is a sensitive indicator of photoreceptor degeneration. See Kedzierski et al., Invest. Ophthalmol. Vis. Sci., 38:498-509 (1997). ERGs are done on live animals. The same mouse can therefore be analyzed repeatedly during a time-course study. The defmitive technique for quantitating photoreceptor degeneration is histological analysis of retina!

sections. The number of photoreceptors remaining in the retina at each time point vvill be determined by counting the rovvs of photoreceptor nuclei in the outer nuclear layer,

Tissue Extraction. Eye samples vvere thavved on ice in 1 ml of PBS, pH 7.2 and homogenized by hand using a Duāli glass-glass homogenizer. The sample vvas further homogenized follovving the addition of 1 ml chloro form/methanol (2:1, v/v). The sample vvas transferred to a boroscilieate tabe and lipids vvere extracied into 4 mis of chloroform. The organic extract vvas vvashed vvith 3 mis PBS, pH 7.2 and the samples vvere then centrifiiged at 3,000 x g, 10 min. The choloroform phase vvas decanted and the aqneous phase vvas re-extracted vvith another 4 mis of chloroform. Follovving centrifugation, the chloroform phases vvere combined and the samples vvere taken to dryness under nitrogen gas. Samples residues vvere resuspended in 100 μΐ hexane and analyzed by HPLC as described belovv.

HPLC Analvsis. Chromatographic separations vvere achieved on an Agilent Zorbax Rx-Sil Column (5 /un, 4.6 X 250 mm) using an Agilent 1100 series liquid chromatograph equipped vvith fluorescence and diode array detectors. The mobile phase (hexane/2-propanol/ethanol/25 mM KH₂PO₄, pH 7.0/acetic acid;

485/376/100/50/0.275, v/v) vvas delivered at 1 ml/min. Sample peak Identification was made by comparison to retention time and absorbance spectra of authentic standards. Data are reported as peak fluorescence (L.U.) obtained from the fluorescence detector.

Example 6: Effect of Fenretinide on A2E Accumulation

Administration of fenretinide to an experimental group of mice and administration of DMSO alone to a control group of mice is performed and assayed for accumulation of A2E. The experimental group is given 2.5 to 20 mg/ kg of fenretinide per day in 10 to 25 μΐ of DMSO. Higher dosages are tested if no effect is seen vvith the highest dose of 50 mg/kg. The control group is given 10 to 25 /tl injections of DMSO alone. Mice are administered either experimental or control substances by intraperitoneal (i.p.) injection for various experimental time periods not to exceed one month.

To assay for the accumulation of A2E in abcaft mice RPE, 2.5 to 20 mg/kg of fenretinide is provided by i.p, injection per day to 2-month old abca4T mice. After 1 month, both experimental and control mice are killed and the Ievels of A2E in the RPE are determined by HPLC. In addition, the autofhiorescence or absorption spectra of M-retinylidene-phosphatidylethanolamine, dihydro-A-retinylidene-Mretinylphosphatidylethanolamine, N-retinylidene-//-retinyl-phosphatidylethanolamine, dihydro-/V-retinylidene-//retinyl-ethanolamine, and/or 77-retinylidene-phosphatidylethanolamine may be monitored using a UV/Vis spectrophotometer.

Example 7: Effect of Fenretinide on Lipofuscin Accumulation

Administration of fenretinide to an experimental group of mice and administration of DMSO alone to a control group of mice is performed and assayed for the accumulation of lipofuscin. The experimental group is given 2.5 to 20 mg/ kg of fenretinide per day in 10 to 25 μΐ of DMSO. Higher dosages are tested if no effect is seen vvith the highest dose of 50 mg/kg. The control group are given 10 to 25 μΐ injections of DMSO alone.

Mice are administered either experimental or control substances by i.p. injection for various experimental time periods not to exceed one month. Altematively, mice can be implanted vvith a pump vvhich delivers either experimental or control substances at a rāte of 0.25 μΐ/hr for various experimental time periods not to exceed one month.

To assay for the effects of fenretinide on the fonnation of hpofuscin in fenretinide treated and untreated abcaJT mice, eyes can be examined by electron or fluorescence microscopy.

EsampJe 8: Effect of Fenretinide on Rod Celi Death or Rod Functional Impairment

Administration of fenretinide to an experrmental group of mice and administration of DMSO alone to a control group of mice is perfonned and assayed for the effects of fenretinide on rod celi death or rod functional impairment. The experimental group is given 2.5 to 20 mg/kg of fenretinide per day in 10 to 25 pl of DMSO. Higher dosages are tested if no effect is seen vvith the highest dose of 50 mg/kg. The control group is given 10 to 25 pl injections of DMSO alone. Mice are administered either experimental or control substances by i.p. injection for various experimental time periods not to exceed one month. Altematively, mice can be implanted with a pump vvhich delivers either experimental or control substances at a rāte of 0.25 pl/hr for various experimental time periods not to exceed one month.

Mice that are treated to 2.5 to 20 mg/kg of fenretinide per day for approximately 8 vveeks can be assayed for the effects of fenretinide on rod celi death or rod functional impairment by monitoring ERG recordings and performing retinal histology.

Example 9: Testing for Protection from Light Damage

The following study is adapted from Sieving, P.A., et al, Proc. Nati. Acad. Sci., 98:1835AO (2001).

For chronic Iight-exposure studies, Sprague-Dawley male 7-wk-old albino rats are housed in 12:12 h light/dark cycle of 5 lux fluorescent vvhite light. Injections of20-50 mg/kg fenretinide by i.p. injection in 0.1S ml DMSO are given three times daily to chronic rats for 8 wk. Controls receive 0.18 ml DMSO by i.p. injection. Rats are killed 2 d after final injections. Higher dosages are tested if no effect is seen with the highest dose of 50 mg/kg.

For acute light-exposure studies, rats are dark-adapted ovemight and given a single i.p. injection of fenretinide 20-50 mg/kg in 0.18 ml DMSO under dim red light and ķept in darkness for 1 h before being exposed to the bleaching light before ERG measurements. Rats exposed to 2,000 lux vvhite fluorescent light for 48 h. ERGs are recorded 7 d later, and histology is performed immediately.

Rats are euthanized and eyes are removed. Column celi counts of outer nuclear layer thickness and rod outer segment (ROS) length are measured every 200 pm across both hemispheres, and the numbers are averaged to obtain a measure of cellular changes across the entire retina. ERGs are recorded from chronic rats at 4 and 8 wks of treatment In acute rodents, rod recovery from bleaching light is tracked by dark-adapted ERGs by using stimuli that elicit no cone contribution. Cone recovery is tracked vvith photopic ERGs. Prior to ERGs, animals are prepared in dim red light and anaesthetized. Pupils are dilated and ERGs are recorded from both eyes simultaneously by using gold-wire comeal Ioops.

Example 10: Combination Therapy Involving Fenretinide and Accutane

Mice and/or rats are tested in the manner described in Examples 6-9, but vvith an additional tvvo arms.

In one of the additional arms. groups of mice and/or rats are treated vvith increasing doses of Accutane, from 5 mg/kg per day to 50 mg/kg per day. In the second additional arm, groups of mice and/or rats are treated vvith a combination of 20 mg/kg per day of fenretinide and increasing doses of Accutane, from 5 mg/kg per day to 50 mg/kg per day. The benefits of the combination therapy are assayed as described in Examples 6-9.

Erample 11: Efficacy of Fenretinide on the Accumulation of Lipofuscin (and/or A2E) in abca4 null Mutant Mice: Phase I - Dose Response and Effect on Serum Retinot

The effect ofHPR on reducing serum retinol in animals and human subjects led us to explore the possibility that reductions in lipofuscin and the toxic bis-retinoid conjugate, A2E, may also be realized. The rationale for this approach is based upon tvvo independent lines of scientific evidence: 1) reduction in ocular vitamin A concentration via inhibition of a knovvn visual cycle enzyme (1l-cis retinol dehydrogenase) results in profound reductions in lipofuscin and ARE; 2) animals maintained on a vitamin A deficient diet demonstrate dramatic reductions in lipofuscin accumulation. Thus, the objective for this example vvas to examine the effect ofHPR in an animal modei vvhich demonstrates massive accumulation of lipofuscin and A2E in ocular tissue, the abca4 null mutant mouse.

Initial studies began by examining the effect ofHPR on serum retinol. Animals vvere divided into three groups and given either DMSO, 10 mg/kg HPR, or 20 mg/kg HPR for 14 days. At the end of the study period, blood vvas collected from the animals, sera were prepared and an acetonitrile extract of the serum vvas analyzed by reverse phase LC/MS. UV-visible spectra! and mass/charge analyses vvere performed to coufrrm the identity of the eluted peaks. Sample chromatograms obtained from these analyses are shovvn: Fig. la. - extract from an abca4 null mutant mouse receiving HPR vehicle, DMSO; Fig. Ib. -10 mg/kg HPR; Fig. Ic. - 20 mg/kg HPR. The data clearly shovv a dose-dependent reduction in serum retinol. Ģuantītative data indicate that at 10 mg/kg HPR, all-O-a/u retinol is decreased 40%, see Fig. 11. For 20 mg/kg HPR, serum retinol is decreased 72%, see Fig. 11. The steady state concentrations of retinol and HPR in seram (at 20mg/kg HPR) vvere determined to be 2.11 pM and 1.75 pM, respectively.

Based upon these fīndings, we sought to further explore the mechanism(s) of retinol reduction during HPR treatment. A tenable hypothesis is that HPR may displace retinol by competing at the retinol binding site on RBP. Like retinol, HPR vvill absorb (quench) light energy in the region of protein fluorescence; hovvever, unlike retinol, HPR does not emit fluorescence. Therefore, one can measure displacement of retinol from the RBP holoprotein by observing decreases in both protein (340 nm) and retinol (470 nm) fluorescence. We performed a competition binding assay using RBP-retinol/HPR concentrations vvhich vvere similar to those determined from the 14 day triai at 20 mg/kg HPR described above. Data obtained from these analyses reveal that HPR effīciently displaces retinol from the RBP-retinol holoprotein at physiological temperature, see Fig.

3b. The competitive binding ofHPR to RBP vvas dose-dependent and saturable. In the control assays, decreases in retinol fluorescence vvere associated vvith concomitant increases in protein fluorescence, see Fig.

3a. This effect vvas determined to be due to temperature effects as the dīssociation constant of RBP-retinol increases (decreased affinity) vvith increased time at 37C. In summary, these data suggest that increases ofHPR beyond equimolar equivalents, relative to RBP holoprotein (e.g., 1.0 pM HPR 0.5 pM RBP), vvill cause a significant fraction of retinol to be dispiaced from RBP in vivo.

Esaniple 12: Efficacy of Fenretinide on the Accumulation of Lipofuscin (and/or A2E) in abca4 null Mutant Mice: Phase Π - Chronic Treatment of abca4 Null Mutant Mice.

We initiated a one-month study to evaluate the effects ofHPR on reduction of A2E and ARE precursors in abca4 null mutant mice. HPR was administered in DMSO (20 mg/kg, ip) to abca4 null mutant mice (BL6/129, aged 2 months) daily for apcriod of28 days. Control age/strain matched mice received only the

DMSO vehicle. Mice vvere sampled at 0, 14, and 28 days (n = 3 per group), the eyes vvere enucleated and chloroform-soluble constituents (lipids, retinoids and lipid-retinoid conjugates) vvere extracted. Mice vvere sacrificed by cervical dislocation, the eyes vvere enucleated and individually snap frozen in ciyo vials. The sample extracts were then analyzed by HPLC vvith on-line fluorescence detection. Results from this study shovv remarkable, early reductions in the A2E precursor, A2PE-H2, see Fig. 4a, and subsequent reductions in A2E, see Fig. 4b. Ģuantitative analysis revealed a 70% reduction of A2PE-H2 and 55% reduction of A2E follovving 28 days of HPR treatment. A similar study may be undertaken to ascertain effects of HPR treatment on the electroretinographic and morphological phenotypes.

Example 13: Effects of Fenretinide on Vitamin A Homeostasis in the Retinal Pigment Epithelium We examined the effects on HPR on enzymes or proteins of the visual cycle using ūz vitvo biochemical assays. Specifically, the utilization of exogenous all-izmzs retinol by membranes prepared frombovine RPE vvas studied. Representative data from our studies are shovvn in Fig. 5. Rinetic analyses of the inhibition data indicate that half-maximal inhibition of LRAT occurs at approximately 20 pM HPR. Steady-state Ievels of HPR in the RPE (determined from mice vvhich have been given 20 mg/kg HPR i.p., daily for 28 days) range from 5 10 μΜ. With tliis in mind, we examined the effects of 10 pM HPR on produetion ofall-fra/zj retiny] esters and 11-czs retinol in assays similar to those described above. In addition to decreases in all-trans retinol utilization (Fig. 6c) and all-nmzj retinyl ester synthesis (Fig. 6a), the data reveal a statistically significant inhibition of 11cis retinol biosynthesis (p< 0.05, indicated by asterisk), see Fig. 6b. ln the presence of endogenous retinoids, utilization of exogenous all-frazis retinol is extremely lovv and 11-cz's retinol is produced solely from the endogenous all-iznzzr retinyl esters. In fact, vvhen vve perform our experiments in the presence of endogenous retinyl esters vve do not ohserve an effect of HPR on 1 l-cis retinol produetion; hov/ever, inhibition of LRAT activity persists. Thus, the retinoic acids appear to affect at least tvvo targets in the visual cycle. We have determined that HPR-induced reduction of 11 -cis retinol biosynthesis occurs via LRAT inhibition and reduction in all-mmzj retinyl ester Ievels. In this situation, the isomerase enzyme is starved for substrate and 11 -cis retinol produetion declines.

In. the aggregate, it is clear from several studies that multiple targets exist for modulation of visual chromophore biosynthesis. Lovvered visual chromophore then leads to a consequent decrease in all-trans retinal, the retinoid from vvhich A2E is generated. Thus, treatment with HPR not only has systemic effects in lovvering the amount of retinol delivered to the eye, but also intracellular effects on lovvering steady State Ievels of all-trans retinal. The final outeome vvill be lovvered A2E in the RPE, as evidenced above.

Thus, one of the outeomes of this study is that the treatment of the macular degenerations and dystrophies, including but not limited to controlling the formation of all-trans retinal, /7-retinylidene-Aretinvlethanolamine, A^r-retinylidene-pbosphatidyletbanolamine, dihydro-?/-retinylidene-7/-retinylphosphatidylethanolamine, A^z-retinylidene-7/-retinyl-phosphatidy]ethanolainine, dihydro-/V-retinylidene-/Vretinvl-ethanolamme, Mretinylidene-phosphatidylethanolamine, geographic atrophy, scotoma, lipofuscin and drusen in the eye of a mammai, may be effected by administration of an aģent or aģents that can both lovver the Ievels of serum retinol and modulate at least one enzyme or protein in the visual cycle, including by way of example, LRAT activity. This dual action approach to the treatment of the macular or retinal dystrophies and degenerations, or the alleviation of symptoms associated vvith such diseases or conditions, is considered to be a generallv applicable approach, and has been observed, as described herein, vvith fenretinide. In addition, (a) admirustration of an aģent or aģents that lovver the Ievels of serum retinol in a patient vvithout modulating at least one enzyme in the visual cycle or (b) administration of an aģent or aģents that modulate at least one enzyme in the visual cycle vdthout lovvering Ievels of serum retinol in a patient, by themselves, may also provide a treatment for such dystrophies and degenerations or the symptoms associated thereof. Assays, such as those described herein, may be used to select further aģents possessing this dual action, including aģents selected from compounds having the structure ofFoimula (I) as vvell as other aģents. Putative lead compounds include other aģents knovvn or demonstrated to effect the serum Ievel of retinol.

In order to determine the effects of HPR on visual cycle enzymes or proteins in vivo, the regeneration of rhodopsin from endogenous retinoid stores may be examined in HPR-freated mice and age/strain matched Controls.

Esample 14: Combination Therapy Involving Fenretinide and a Statiņ

Mice and/or rats are tested in the manner described in Examples 6-9, but vvith an additional tvvo arms.

In one of the additional aims, groups of mice and/or rats are treated vvith a suitable statiņ such as: Lipitor® (Atorvastatin), Mevacor® (Lovastatin), Pravachol® (Pravastatin sodium), Zocor™ (Simvastatin), Leschol (fluvastatin sodium) and the like vvith optimal dosage based on vveight. In the second additional arm, groups of mice and/or rats are treated vvith a combination of 20 mg/kg per day of fenretinide and increasing doses of the statiņ used in the previous step. Suggested human dosages of such statins are for example: Lipitor® (Atorvastatin) 10-80 mg/day, Mevacor® (Lovastatin) 10-80 mg/day, Pravachol® (Pravastatin sodium) 10-40 mg/day, Zocor™ (Simvastatin) 5-S0 mg/day, Leschol (fluvastatin sodium) 20-80 mg/day. Dosage of statins for mice and/or rat subjects should be calculated based on vveight. The benefits of the combination therapy are assayed as described in Examples 6-9.

Example 15: Combination Therapy Involving Fenretinide, Vitamīns and Minerāls

Mice and/or rats are tested in the manner described in Example 14, but vvith selected vitamīns and minerāls. Administration of fenretinide in combination vvith vitamīns and minerāls can be either orally or parenterally administered at amounts effective to inhibit the development or reoccuirence of macular degeneration. Tēst dosages are initially in the range of about 20 mg/kg per day of fenretinide vvith 100-1000 mg vitamin C, 100 - 600 mg vitamin E, 10,000 — 40,000 IU vitamin A, 50-200 mg zinc and 1-5 mg copper for 15 to 20 vveeks. The benefits of the combination therapy are assayed as described in Examples 6-9.

Example 16: Fluorescence Quenching Stndy of Binding to Cellular Retinaldehyde Binding Protein (CRALBP)

Apo-CRALBP at 0.5 μΜ vvas incubated vvith 1 μΜ of 11-cis Retinal (1 lcRAL), all-trans retinal (atRAL) or N-4-hydroxyphenyl retinamide (HPR) in PBS at room temperature for 1 hour. As a control, same volume of DMSO vvas added to the Apo-CRABLP solution. The emission spectra vvere measured betvveen 290 nm to 500 nm vvith excitatīon vvavelength at 280 nm and 2 nmbandpass (See FIG. 7).

Compared to DMSO control, ali three retinoids signifīcantly quenched the fluorescence emission of CRALBP, vvith 1 lcRAL having the highest degree of quenching and HPR having the lovvest, suggesting ali three compounds bind to CRALBP. The fluorescence quenching likely results from the fluorescence resonance energy transfer betvveen protein aromatic residues and bound retinoids

Example 17: Size Exclusion Chromatography Study of Binding to CRALBP

Apo-CRALBP at 4 μΜ vvas incubated vvith 8 uM of 1 lcRAL, atRAL or HPR in PBS at room temperature for 1 hour. In control experiment, equivalent volume of DMSO vvas added to the CRALBP solution. 50 μΐ of each sample mixture vvas anaiyzed by BioRad Bio-Sil SEC125 Gel Filtration Column (300x7.8 min).

In DMSO control (see FIG. Sa), apo-CRALBP eluted as multimers (elution peak at 8.1 ml); vvhile ligand-bound holo-protein shifted to monomer form (elution peak at 9.4 ml). In the presence 1 lcRAL, a rnajcntv of the CRALBP is bound vvith ligand and displays strong 430 nm absorbance at the monomer elution position (see FIG. 8b). Less than half of the atRAL is bound to CRALBP (see FIG. 8c), and only small amount of HPR is bound to CRALBP, indicated by 350 nm absorbance peak (see FIG. 8d).

Eiampie 18: Fluorescence Ģuenching Study of MPR Binding to Retīnol Binding Protein (RBP)

Apo-RBP at 0.5 μΜ vvas incubated vvith 0, 0.25, 0.5, 1 and 2 μΜ of MPR in PBS at room temperature for 1 hour, respectively. As Controls, the same concentration of Apo-RBP vvas also incubated vvith 1 μΜ of HPR or 1 μΜ of atROL. Ali mixtures contained 0.2% Ethanol (v/v), The emission spectra vvere measured betvveen 290 nm to 550 nm vvith excitation vvavelength at 280 nm and 3 nm bandpass.

As shovvn inFIG. 9, MPR exhibited concentration-dependent quenching of RBP fluorescence, and the quenching saturated at 1 μΜ of MPR for 0.5 μΜ of RBP. Because the observed fluorescence quenching is likely due to fluorescence resonance energy transfer betvveen protein aromatic residues and bound MPR molecule, MPR is proposed to bind to RBP. The degree of quenching by MPR is smaller than those by atROL and HPR, tvvo other ligands that bind to RBP.

Eiampie 19: Size Eiclusion Study of Transthyretin (TTR) Binding to RBP

Apo-RBP at 10 μΜ vvas incubated vvith 50 μΜ of MPR in PBS at room temperature for 1 hour. 10 μΜ of TTR vvas then added to the solution, and fhe misture vvas incubated for another hour at room temperature. 50 μΐ of the sample mixiures vvith and vvithout TTR addition vvere analyzed by BioRad Bio-Sil SEC125 Gel Filtration Column (300x7.3 mm). In control expeiiments, atROL-RBP and atROL-RBP-TTR mixture vvere analyzed in the same manner.

As shovvn in FIG. 10a, the MPR-RB? sample exhibited an RBP elution peak (at 11 ml) vvith strong absorbance at 360 nm, indicating RBP binds to MPR; after incubation vvith TTR, this 360 run absorbance stayed vvith the RBP elution peak, vvhile TTR elution peak (at 8.6 ml) did not contain any apparent 360 nm absorbance (see FIG. 10b), indicating MPR-RBP did not bind to TTR. In atROL-RBP control experiment, RBP elution peak shovved strong 330 nm absorbance (see FIG. 10c); after incubation vvith TTR, more than half of this 330 nm absorbance shifted to TTR elution peak (see FIG. lOd), indicating atROL-RBP binds to TTR. Thus, MPR inhibits the binding of i'l'R to RBP.

Example 20: Analysis of serum retīnol as a function of HPR concentration

ABCA4 null mutant mice vvere given the indicated dose of HPR in DMSO (i.p.) daily for 28 days (n = mice per dosage group). At the end of the study period, blood samples vvere taken and serum vvas prepared. Follovving acetonitrile precipitation of serum proteins, the concentrations of retinol and HPR vvere determined from the soluble phase by LC/MS (see FIG. 11). Identity of the eluted compounds vvas confirmed by UV-vis absorption spectroscopy and co-elution of sample peaks vvith authentic standards.

Example 21: Correlation of HPR concentration to reductions in retinol, A2PE-H₂ and A2E in A-BCAJ null mutant mice

Group averages from the data shovvn in panels A - G of FIG. 18 in Exaruple 25 (28 day time points) are plottcd to illustrate the strong correlation betvveen increases in serum HPR and decreases in serum retinol (see FIG. 12). Reductions in serum retinol are highly correlated vvith reductions in A2E and precursor compounds (A/PE-FF). A pronounced reduction in A2PE-H₂ in the 2.5 mg/kg dosage group (-47%) is observed when the serum retinol reduction is only 20%. The reason for this disproportionate reduction is related to the inherently lovver ocnlar retinoid content in this group of 2-month old animals compared to the other groups. It is likely that if these animals had been maintained on the 2.5 mg/kg dose for a more prolonged period, a greater reduction in A2E would also be realized.

Esample 22: Fluorescence analysis ofHPR binding to cellular retinaldehyde binding protein (CRALBP)

Ouenching of CRALBP protein fluorescence vvith 11 -cis retinal (11 cRAL). The fluorescence emission of recombinant apo-CRALBP (0.5 μΜ) vvas measured using 280nm excitation (“no 1 IcRAL”). Addition of the native ligand (llcRAL) quenched CRALBP protein fluorescence in a concentration dependent manner (see FIG. 13A). These data validate the technical approach used to confirm protein-ligand interaction.

Ouenching of CRALBP -protein fluorescence vvith HPR. The data shovvn vvere obtained using an experimental design identical to that described above. The fluorescence emission of recombinant apo-CRALBP vvas measured using 280 nm excitation (“no HPR”). Addition of HPR quenched CRALBP protein fluorescence in a concentration dependent manner similar to that observed vvith the native ligand (see FIG. 13b). These data strongly suggest that CRALBP binds HPR at physiological concentrations.

Example 23: Spectroscopic analysis of HPR binding to celluiar retinaldehyde binding protein (CRALBP)

In order to confirm data obtained during fluorescence analysis of HPR binding to CRALBP, a second analysis vvas performed using affinity chromatography and spectroscopic analysis. The recombinant apoCRALBP was constructed vvith a histidine tag vvhich is utilized to purify the protein on a Ni+ affinity column follovving expression cloning. Here, vve utilized this feature of apo-CRALBP to specifīcally “trap” the protein and any protein-ligand species for spectroscopic analysis. Tvvo binding mixtures vvere prepared containing apoCRALBP (10 μΜ) and either 1 IcRAL (20 μΜ) or HPR (20 μΜ). In control experiments for the analysis of non-specific ligand binding onto the afnnity matrix, vve prepared tvvo additional mixtures containing only

I IcRAL (20 μΜ) or HPR (20μΜ) in binding buffer. The binding mixtures vvere passed through separate Ni+ affinity columns and the columns vvere vvashed extensively to elute unbound protein and ligand. Follovving the addition of elution buffer, the eluted fractions vvere analyzed by spectroscopy. Spectroscopic analysis of the

II cRAL + apo-CRALBP binding mixture (positive control) confirms that this technique is effective as the spectra are consistent vvith 1 IcRAL bound to CRALBP. frnportantly, the data also shovv that HPR binds apoCRALBP. If HPR did not bind apo-CRALBP only the protein absorbance (280 nm) vvould be observed in the eluted HPR + apo-CRALBP sample. Instead, tvvo absorption maxima are seen: one at 2S0 nm and a second at 360 nm, vvhich is attrihutable to the absorption ofHPR (see FIG. 14).

We performed an analysis of the dissociation constant (K_D) for 1 IcRAL and HPR binding to apoCRALBP (see FIG. 15). Transformation of the fluorescence quenching data revealed similar values (~ 30 nM) for each ligand. This calculation is based upon the ligand concentration necessary to fully quench the protein fluorescence. The data reveal that both 1 IcRAL and HPR quench apo-CRALBP fluorescence maximally at 1.5 μΜ. Thus, although apo-CRALBP is described as an 11 m-specifīc retinoid binding protein, it appears to bind HPR as vvell. The fact that concentrātions ofHPR in the RPE far exceed 30 nM during the animal trials (even at the lovvest therapeutic dose of 2.5 mg/kg), suggests that some degree of HPR-mediated inhibition vvill be expected during biosynthesis of visual chromophore in the visual cycle.

Example 24: Effects ofHPR on esterifīcation of vitamin A in the retinal pigment epithelium (RPE)

A second target for HPR in the visual cycle was identified using in vitro biochemical assays. Lecithin retinol acyl transferase (LRAT) catalyzes the conversion ofretinol into retinyl esters. LRAT is critical not only for retinol-retinvl ester homeostasis but also for generation of substrate for visual chromophore biosynthesis.

The data shovvn in panei A of FIG. 16 illustrate the inhibitory effect ofHPR on the rāte of retinyl ester synthesis. In this assay, bovine RPE microsomes are used as an enzyme source and all-trans retinol (atROL) is the substrate. HPR decreases net retinyl ester synthesis in a concentration-dependent manner. A secondary transformation (Eadie-Hofstee) of the kinetic data in panei A reveal that the mode of inhibition is competitive (see FIG. 16, panei B). Therefore, HPR competes vvith atROL for binding sites on LRAT. The apparent inhibition constant (Κ;) vvas determined to be ~ 6 μΜ. This means that at 6 μΜ HPR, the rāte of retinyl ester synthesis vvould be decreased by 50%. In a separate study, vve have determined that HPR concentrations in the RPE approach 10 μΜ vvith a 10 mg/kg dose ofHPR.

In summary, it is clear from the data described in experiments 20-24 that the pronounced effect ofHPR on reducing accumulation of A2E and its precursors during the animal trials vvas due to both systemic effects on lovvering serum retinol and intracellular effects vvithin the visual cycle.

Example 25: Effects of HPR on Steady State Concentrations of Retinoids, A2E Fluorophores, and Retinal Physiology

Analysis of retinoid composition in light adapted DMSO- and HPR-treated mice (FIG. 17, panei A) shovvs approximately 50% reduetion of visual cycle retinoids as a result ofHPR treatment (10 mg/kg daily for 28 days). Panels B and C of FIG. 17 show that HPR does not affect regeneration of visual chromophore in these mice (panei B is visual chromophore biosynthesis, panei C is bleached chromophore recycling). Panels D - F of FIG. 17 are electrophysiological measurements of rod function (panei D), rod and cone function (panei E) and recovery from photobleaching (panei F). The only notable difference is delayed dark adaptation in the HPRtreated mice (panei F).

ABCA4 null mutant mice vvere given the indicated dose ofHPR in DMSO or DMSO alone daily for 28 days (n = 16 mice per treatment group). At study onset, mice in the 2.5 mg/kg group vvere 2 months of age, mice in the other treatment groups vvere 3 months of age. At the indicated times, representative mice vvere taken from each group (n = 4) for analysis of A2E precursor compounds (see FIG. 18, A2PE-Hi, panels A,

C and E) and A2E (see FIG. 18, panels B, D and F). Eyes vvere enueleated, hemisected and lipid soluble components vvere extracied from the posterior pole by chloroform/methanol-vvater phase partitioning. Sample extracts vvere analyxed by LC. īdentity of the eluted compounds vvas confirmed by UV-vis absorption spectroscopy and co-elution of sample peaks vvith authentic standards. Note: limitations in appropriately age and strain-matehed mice in the 10 mg/kg group prevented analysis at the 14-day interval. The data show dosedependent reduetions of A2PE-H₃ and A2E during the study period.

Panels G - Γ in FIG. 18 shovv morphological/histological evidence that HPR significantly reduces lipofuscin autofluorescence in the RPE of aber null mutant mice (Stargardt’s animal modei). Treatment conditions are as described above. The Ievel of autofluorescence in the HPR-treated animal is comparable to that of an age-matched wild-type animal. FIG. 19 shovvs light microscopy images of the retinās from DMSOand HPR-treated animals. No aberrant morphclogy or compromise of the iniegrity in retinal cytostructure vvas observed.

Accumulation of Iipofuscin in the retinal pigment epithelium (RPE) is a common pathological feature observed in various degenerative diseases of the retina. A toxic vitamin A-based flubrophore (A2E) present vvithin Iipofuscin granules has been implicated in death of RPE and photoreceptor celis. In these experiments, we employed an animal modei vvhich manifests accelerated Iipofuscin accumulation to evaluate the efficacy of a therapeutic approach based upon reduction of serum vitamin A (retinol). Fenretinide potently and reversibly reduces serum retinol. Administration of HPR to mice harboring a null ruutation in the Stargardt’s disease gene (ABCA4) produced profound reductions in serum retinol/retinol binding protein and arrested accumulation of A2E and Iipofuscin autofluorescence in the RPE. Physiologically, HPR-induced reductions of visual chromophore vvere manifest as modest delays in dark adaptation; chromophore regeneration kinetics vvere normai. Importantly, specific intracellular effects of HPR on vitamin A esterification and chromophore mobilization vvere also identified. These findings demonstrate the vitamin A-dependent nature of ARE biosynthesis and validate a therapeutic approach vvhich is readily transferable to human patients suffering from lipofuscin-based retinal diseases.

Eīaniple 26: Benefits of HPR Therapy Persist During Drug Holiday

HPR (10 mg/kg in DMSO) vvas administered to ABCA4-/- mice daily for a period of 28 days. Control ABCA4-/- mice received only DMSO for the same period. Biochemical (HPLC) analysis ofthe A2E precursor (A2PE-Hi) and A2E follovving a 28-day treatment period revealed a reduction of these fLuorophores in tbe eyes of HPR-treated mice (FIG. 18). Further analysis by fluorescence roicroscopy corroborated the biochemical data and revealed that Iipofuscin autofluorescence Ievels of HPR-treated ABCA4-/- mice vvere comparable to Ievels observed in untreated wild type mice (FIG. 18). Histological examinations by light microscopy shovved no alteration of retina cytostructure ormorphology (FIG. 19). Importantly, the observed reductions in Iipofuscin autofluorescence persist long after cessation of HPR therapy. HPR (10 mg/kg), or DMSO, administration vvas discontinued follovving 28 days of treatment and re-evaluated ARE and precursor Ievels after 2 vveeks and after 4 vveeks.

We examined eyecup extracts by HPLC and employed detection by absorbance and fluorimetry.

Identity of the indicated peaks vvas confirmed by on-line spectral analysis and by co-elution vvith authentic standards. The data shovv that in animals that had been previously maintained on HPR therapy (FIG. 20, panei Α), A2E and precursor (ARPE-H₂ and A2PE) Ievels remain significantly reduced relative to control mice (FIG.

20, panei B) even after 12 days vvithout receiving a dose of EDPR (i.e., a 12-day drug holiday). Similar results vvere observed in mice follovving a 28-day drug holiday: A2E and precursor (A2PE-H₂ and A2PE) Ievels remain significantly reduced relative to control mice (compare FIG. 20, pane] C, treated mice, vvith FIG. 20, panei D, control mice). Further, the A2E and precursor (A2PE-H₂ and A2PE) Ievels after a 12- ot 28-day drug holiday remained at or near tbe Ievels immediately follovving 28 davs of treatment (i.e., ca. 50% reduction relative to control), although after the 2S-day drug holiday, the amount of ARE and precursor (ARPE-H₂ and A2PE)had increased by a fevv percentage poīnts relative to the 12-day drug holiday Ievels. Despite the persisfent reduction in the Ievels of .ARE and precursor (ARPE-H₂ and A2PE) in the eyes of animals on an HPR drug holidav, vve vvere unable to detect either HPR or HPR metabolites (e.g., MPR) in the eyes of the animals on a 28~day drug holiday. The trace in FIG. 20, panels C and D, shovvs the intensity of autofluorescence associated vvith the indicated peaks. It is cleaT that peak fluorescence tracks vvith the abundance of A2E, A2PE and A2PEH₂.

These data bear on toxicity during clinical trials by maintaining patients on a reduced HPR dose follovving proof of clinical efficacy at a higher dose. This analysis may obviate the need for additional corroboration by nucroscopy. To our knovvledge this effect has not been observed vvith other methods for treating an ophthalmic condition or trait selected from the group consisting of Stargardt Disease, dry-form agerelated macular degeneration, a bpofuscin-based retinal degeneration, photoreceptor degeneration, and geographic atrophy. Nor has this effect been observed vvith methods for reducing the formation of Nretmyhdene-N-retinylethanolamine in an eye of a mammai, or methods for reducing the formation of lipofuscin in an eye of a mammai.

This effect cannot be attributed to long-term reductions in serum retīnol as serum retinol had retumed to baseline 48 hours follovving the last HPR dose. The fact that HPR accumulates vvithin the RPE, and our Identification ofHPR-mediated inhibition of specific enzym.es and proteins of the visual cycle, suggest that the latent, beneficial effects of HPR during the. drug holiday are attributable to effects vvithin the visual cycle. Furthermore, HPR reduces sernm retinol Ievels, vvhich leads to a reduction in the Ievel of retinol in the eyes of treated animals. Once the Ievel of retinol has been reduced in the eye, there is a time lag in the snbsequent increase in retinol Ievels in the eye. Alone or in combination, the production of A2E, A2PE and A2PE-H₂ rn the eye remains lovv despite the absence of HPR in the serum or the eye.

Ali of the methods disclosed and claimed herein can be made and executed vvithout undue experimentation in light of the present disclosure. It vvill be apparent to those of skill in the ari that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein vvithout departing from the concept, spirit and scope of the invention. More specifically, it vvill be apparent that certain aģents that are both chemically and physiologically related may be substituted for the aģents described herein vvhile the same or similar results vvould be aehieved. Ali such similar substitutes and modifications apparent to those skilled in the art are deemed to be vvithin the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. Use to the mammai at least once of an effective amount of a first compound having the structure:

wherein Χ-ι is selected from the group consisting of NR², 0, S, CHR²; R¹ is (CHR²)X-L¹-R³, vvherein x is 0,1,2, or 3; L? is a single bond or -C(O)-; R² is a moiety selected from the group consisting of H, (CrC₄)alkyl, F, (Cr C4)fluoroalkyl, (Ci-C₄)alkoxy, -C(O)OH, -C(O)-NH₂, -(CrC4)alkylamine, -C(O)(Ci-C4)fluoroalkyl, -C(O)-(C-i-C4)alky!amine, and -C(O)-(C₁-C₄)alkoxy; and R³ is H or a moiety, optionally substituted vvith 1-3 independently selected substituents, selected from the group consisting of (C₂-C7)alkenyl, (C₂C₇)alkynyl, aryl, (C3-C₇)cycloalkyl, (C₅-C7)cycloa)kenyl, and a heterocycle; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof for reducing the formation of /V-retinylidene-/V-retinylethanolamine in an eye of a mammai comprising administering to the mammai;

provided that R is not H when both x is 0 and L¹ is single bond.

2. Use to the mammai of an effective amount of a first compound having the structure: „ „ ι ι o vvherein Xi is selected from the group consisting of NR², O, S, CHR²; R¹ is (CHR²)X-L¹-R³, vvherein x is 0,1,2 or 3; L¹ is a single bond or -C(O)-; R² is a moiety selected from the group consisting of H, (Ci-C4)alkyl, F, (Cr C₄)fluoroalkyl, (C_rC₄)alkoxy, -C(O)OH, -C(O)-NH₂, -(C₁-C4)alkylamine, -C(O)(CrC₄)alkyl, -C(O)-(C₁-C₄)fluoroalkyl, -C(O)-(C₁-C₄)alkylamine, and -C(O)(Ci-C₄)alkoxy; and R³ is H or a moiety, optionally substituted vvith 1-3 independently selected substituents, selected from the group consisting of (C₂-C7)alkenyl, (C₂-C₇)alkynyl, aryl, (C3-C₇)cycloalkyl, (C₅-C₇)cycloalkenyl, and a heterocycle; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof for reducing the formation of lipofuscin in an eye of a mammai;

provided that R is not H vvhen both x is 0 and L¹ is a single bond.

3. Use to the mammai of an effective amount of a first compound having the structure: , , o vvherein Χ-ι is seiected from the group consisting of NR², O, S, CHR²; R¹ is (CHR²)x-L¹-R³, vvherein x is 0,1,2 or 3; L¹ is a single bond or -C(O)-; R² is a moiety selected from the group consisting of H, (Ci-C4)alkyl, F, (C_r C₄)fluoroalkyl, (CrC₄)alkoxy, -C(O)OH, -C(O)-NH₂, -(CrC₄)alkylamine, -C(O)(C_rC₄)alkyl, -C(O)-(CrC₄)fluoroalkyl, -C(O)-(C_rC₄)alkylamine, and -C(O)(Ci-C₄)alkoxy; and R³ is H or a moiety, optionally substituted vvith 1-3 independently selected substituents, selected from the group consisting of (C₂-C₇)alkenyl, (C2-C₇)alkynyl, aryl, (C₃-C₇)cycloalkyl, (C₅-C₇)cycloalkenyl, and a heterocycle; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof for treating dry form age-related macular degeneration in an eye of a mammai;

provided that R is not H vvhen both x is 0 and L¹ is a single bond.

4. Use according to any of Claims 1-3, vvherein x is 0.

5. Use according to any of Claims 1-3, vvherein X¹ is NH and R³ is phenyl group, vvherein the phenyl group has one substituent.

6. Use according to Claim 5, vvherein the substituent is moiety selected from the group consisting of halogen, OH, O(Ci-C₄)alkyl, NH(C_rC₄)alkyl, O(C_r C₄)fluoroalkyl, and N[(C₁-C₄)aikyi]₂.

7. Use according to Claim 6, vvherein the substituent is OH or OCH3.

8. Use according to any of Claims 1-3, vvherein the compound is

OH ' or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

9. Multiple use according to any of Claims 1-3 of the effective amount of the compound, vvherein the time betvveen multiple administrations is at least one day.

10. Use according to Claim 9, further comprising a drug holiday, vvherein the administration of the compound is temporarily suspended or the dose of the compound administered is temporariiy reduced.

11. Use according to Claim 10, vvherein the drug holiday lāsts at least seven days.

12. Use according to Claim 10, vvherein the effective amount ofthe compound is administered orally to the mammai.

13. Use according to Claim 10, vvherein the mammai is a human having an ophthalmic condition or trait selected from the group consisting of Stargardt Disease, dry-form age-related macular degeneration, a lipofuscin-based retinal degeneration, photoreceptor degeneration, and geographic atrophy.

14. Use according to Claim 10, further comprising measuring the autofluorescence of A/-retinylidene-phosphatidylethanolamine, dihydro-A/retinylidene-/V-retinyl-phosphatidylethanolamine, A/-retinylidene-/V-retinylphosphatidylethanolamine, dihydro-A/-retinylidene-/V-retinyl-ethanolamine, and/or /V-retinylidene-phosphatidylethanolamine in the eye ofthe mammai.

15. Use to the human at least once an effective amount of a first compound having the structure;

vvherein Xi is selected from the group consisting of NR², 0, S, CHR²; R¹ is (CHR²)x-L¹-R³, vvherein x is 0,1,2 or 3; L¹ is a single bond or -C(O)-; R² is a moiety selected from the group consisting of H, (Ci-C4)alkyl, F, (C_r C₄)fluoroalkyl, (CrC₄)alkoxy, -C(O)OH, -C(O)-NH₂, -(Ci-C₄)alkylamine, -C(O)(CrC₄)alkyl, -C(O)-(Ci-C₄)fluoroalkyl, -C(O)-(Ci-C₄)alkylamine, and -C(O)(Cļ-C₄)alkoxy; and R³ is H or a moiety, optionally substituted vvith 1-3 independently selected substituents, selected from the group consisting of (C2-C₇)alkenyl, (C2-C₇)alkynyl, aryl, (C3-C₇)cycloalkyl, (C₅-C₇)cycloalkenyl, and a heterocycle; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof for reducing geographic atrophy in an eye of a human;

Claims (15)

1. Pretenzijas

   1. Efektīva daudzuma pirmā savienojuma ar strukturu

   R

   X kur

   X¹ ir izvēlēts no rindas: NR², O, S, CHR²;

   R¹ ir (CHR² )_X-L¹-R³, kur x ir 0, 1,2 vai 3;

   L¹ ir vienkāršā saite vai -C(O)-;

   R² ir fragments, kas ir izvēlēts no rindas: H, C₁.₄alkilgrupa, F, C-i_₄fluoralkilgrupa, Ci_₄ alkoksigrupa, -COOH, -C(O)NH₂, -Ci.₄alkilaminogrupa, -C(O)-Ci_₄alkiigrupa, C(O)-C₁.₄fluoralkilgrupa, -C(O)-Ci.₄alkilaminogrupa un -C(O)-C-|.₄alkoksigrupa; un

   R³ ir H vai fragments, neobligāti aizvietots ar 1-3 neatkarīgi izvēlētiem aizvietotājiem, kas ir izvēlēti no rindas: C2-7alkenilgrupa, C₂-7alkinilgrupa, arilgrupa, C3-7 cikloalkilgrupa, C₅.₇cikloalkenilgrupa un heterocikliska grupa;

   vai tā aktīva metabolīta, vai farmaceitiski pieņemamas priekštečvielas vai solvāta vismaz vienreizēja izmantošana zīdītājiem N-retinilidēn-N-retiniletanolamīna veidošanās samazināšanai zīdītāju acīs, pie nosacījuma, ka R nevar būt H, ja abi x ir 0 un L¹ ir vienkāršā saite.
2. 2. Efektīva daudzuma pirmā savienojuma ar struktūru kur:

   X¹ ir izvēlēts no rindas: NR², O, S, CHR²;

   R¹ ir (CHR² )_X-L¹-R³, kur x ir 0, 1, 2 vai 3;

   L¹ ir vienkāršā saite vai -C(O)-;

   R² ir fragments, kas ir izvēlēts no rindas: H, C^aikiigrupa, F, C^fluoralkilgrupa, Ci_₄ alkoksigrupa, -COOH, -C(O)NH₂, -Ci-₄alkilaminogrupa, -C(O)-C₁.₄alkilgrupa, C(O)-Ci_₄fluoralkilgrupa, -C(O)-CMalkilaminogrupa un -C(O)-Ci_₄aikoksigrupa; un

   R³ ir H vai fragments, neobligāti aizvietots ar 1-3 neatkarīgi izvēlētiem aizvietotājiem, kas ir izvēlēti no rindas: C₂-7alkenilgrupa, C₂-7alkinilgrupa, arilgrupa, C3-7 cikloalkilgrupa, Cs^cikloalkenilgrupa un heterocikliska grupa;

   vai tā aktīva metabolīta vai farmaceitiski pieņemamas priekštečvielas, vai solvāta izmantošana zīdītājiem lipofuscīna veidošanās samazināšanai zīdītāju acīs, pie nosacījuma, ka R nevar būt H, ja abi x ir 0 un L¹ ir vienkāršā saite.
3. 3. Efektīva daudzuma pirmā savienojuma ar strukturu o

   kur

   X¹ ir izvēlēts no rindas: NR², O, S, CHR²;

   R¹ ir (CHR² )_X-L¹-R³, kur x ir 0, 1,2 vai 3;

   L¹ ir vienkāršā saite vai -C(O)-;

   R² ir fragments, kas ir izvēlēts no rindas: H, Ci-₄alkilgrupa, F, Cļ^fluoralkilgrupa, C_b4 alkoaksigrupa, -COOH, -C(O)NH₂, -C_Malkilaminogrupa, -C(O)-CMalkilgrupa, C(O)-Ci.₄fluoralkilgrupa, -C(O)-C-|.₄alkilaminogrupa un -C(O)-Ci.₄alkoksigrupa; un

   R³ ir H vai fragments, neobligāti aizvietots ar 1-3 neatkarīgi izvēlētiem aizvietotājiem, kas ir izvēlēti no rindas: C₂-7alkenilgrupa, C₂-7aJkinilgrupa, arilgrupa, C3.7 cikloalkilgrupa, C₅.₇cikloalkenilgrupa un heterocikliska grupa;

   vai tā aktīva metabolīta, vai farmaceitiski pieņemamas priekštečvielas vai sofvāta izmantošana zīdītājiem sausas formas vecuma izraisītas makulas deģenerācijas ārstēšanai zīdītāju acīs, pie nosacījuma, ka R nevar būt H, ja abi x ir 0 un L¹ ir vienkāršā saite.
4. 4. izmantošana pēc jebkura no 1. līdz 3. punktam, kur x ir 0.
5. 5. Izmantošana pēc jebkura no 1. līdz 3. punktam, kur X¹ ir NH un R³ ir fenilgrupa, kurfenilgrupa satur vienu aizvietotāju.
6. 6. Izmantošana pēc 5. punkta, kur aizvietotājs ir izvēlēts no rindas: halogēna atoms, OH, O-Ci-₄alkilgrupa, NH-Ci.₄alkilgrupa, O-C^fluoralkilgrupa un N(Ci.₄alkilgrupa)2·
7. 7. Izmantošana pēc 6. punkta, kur aizvietotājs ir OH vai OCH3.
8. 8. Izmantošana pēc jebkura no 1. līdz 3. punktam, kur savienojums ir

   OH vai ta aktīvs matabolits, vai farmaceitiski pieņemama priekštečvieia vai solvāts.
9. 9. Efektīva daudzuma savienojuma daudzkārtēja izmantošana pēc jebkura no 1. līdz 3. punktam, kur laiks starp daudzkārtējām izmantošanām ir vismaz viena diena.
10. 10. Izmantošana pēc 9. punkta, kas papildus ietver zāļu brīvdienu, kurā savienojuma lietošana ir uz laiku atlikta vai lietotā savienojuma deva ir uz laiku samazināta.
11. 11. Izmantošana pēc 10. punkta, kur zāļu brīvdiena ilgst vismaz septiņas dienas.
12. 12. Izmantošana pēc 10. punkta, kur efektīvais savienojuma daudzums tiek lietots zīdītājam iekšķīgi
13. 13. Izmantošana pēc 10. punkta, kur zīdītājs ir cilvēks ar oftalmisku slimības stāvokli vai pazīmi, kas ir izvēlēta no rindas: Stargarda slimība, sausas formas vecuma izraisīta makulas deģenerācija, uz lipofuscīna bāzēta tīklenes deģenerācija, fotoreceptora deģenerācija un ģeogrāfiska atrofija.
14. 14. Izmantošana pēc 10. punkta, kas papildus ietver N-retinilidēn-Nfosfatidiletanolamīna, dihidro-N-retinilidēn-Nretinilfosfatidiletanolamīna, Nretinilidēn-N-retinilfosfatidiletanolamīna autofluorescences mērīšanu zīdītāja acīs.
15. 15. Efektīva daudzuma pirmā savienojuma ar struktūru:

    kur

    X¹ ir izvēlēts no rindas: NR², O, S, CHR²;

    R¹ ir (CHR² )_X-L¹-R³, kur x ir 0, 1, 2 vai 3;

    L¹ ir vienkāršā saite vai -C(O)-;

    R² ir fragments, kas ir izvēlēts no rindas: H, C1-4alkilgrupa, F, Ci-₄fluoralkilgrupa, Ci. ₄alkoksigrupa, -COOH, -C(O)NH₂, -Ci_₄alkilaminogrupa, -C(O)-C-i.4alkilgrupa, C(O)-Ci_₄fluoralkilgrupa, -CiOj-C^alkilaminogrupa un -C(O)-C₁.₄alkoksigrupa; un

    R³ ir H vai fragments, neobligāti aizvietots ar 1-3 neatkarīgi izvēlētiem aizvietotājiem, kas ir izvēlēti no rindas: C₂-7alkenilgrupa, C₂_₇alkinilgrupa, arilgrupa, C₃-₇ cikloalkilgrupa, C₅.₇cikloalkenilgrupa un heterocikliska grupa;

    vai tā aktīva metabolīta vai farmaceitiski pieņemamas priekštečvielas, vai solvāta vismaz vienreizēja izmantošana zīdītājiem ģeogrāfiskas atrofijas samazināšanai cilvēka acīs, pie nosacījuma, ka R nevar būt H, ja abi x ir 0 un L¹ ir vienkāršā saite.