WO2013166040A1

WO2013166040A1 - S-fta and s-fta analogues capable of inhibiting retinol-dependent rbp4-ttr interaction for treatment of age-related macular degeneration, stargardt disease, and other retinal disease characterized by excessive lipofuscin accumulation

Info

Publication number: WO2013166040A1
Application number: PCT/US2013/038908
Authority: WO
Inventors: Konstantin Petrukhin; Nicoleta DOBRI; Donald W. Landry
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2012-05-01
Filing date: 2013-04-30
Publication date: 2013-11-07

Abstract

A method for treating a disease characterized, by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith comprising administering to the mammal an effective amount of a compound having the structure (I), wherein X is O or S, or an ester or a pharmaceutically acceptable salt thereof.

Description

S-FTA AND S-FTA ANALOGUES CAPABLE OF INHIBITING RETIHOL- DEPENDEN RBP -TTR INTERACTION FOR TREATMENT OF AGE-RELATED

MACULAR DEGENERATION, STARGARDT DISEASE, AND OTHER RETINAL

DISEASE CHARACTERIZED BY EXCESSIVE LIPOFUSCIN ACCUMULATION

This invention was made with government support under grant numbers NS067594 and NS074476 awarded by the National Institutes of Health. The government has certain rights in the invention .

Throughout this application, certain publications are referenced, including referenced in parenthesis. Full citations for these publications may be found immediately preceding the claims. The disclosures of all referenced publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains .

Background of the Invention

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. It is estimated that 62.9 million individuals worldwide have the most prevalent atrophic (dry) form of AMD; 8 million of them are Americans. Due to increasing life expectancy and current demographics this number is expected to triple by 2020. There is currently no FDA-approved treatment for dry AMD. Given the lack of treatment and high prevalence, development of drugs for dry AMD is of upmost importance. Clinically, atrophic AMD represents a slowly progressing neurodegenerative disorder in which specialized neurons (rod and cone photoreceptors) die in the central part of the retina called macula (1) . Histopathological and clinical imaging studies indicate that photoreceptor degeneration in dry AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath photoreceptors and provides critical metabolic support to these light-sensing neuronal cells. Experimental and clinical data indicate that excessive accumulation of cytotoxic autofluorescent lipid-protein-retinoid aggregates (lipofuscin) in the RPE is a major trigger of dry AMD (2-9) . In addition to AMD, dramatic accumulation of lipofuscin is the hallmark of Stargardt Disease (STGD) , an inherited form of juvenile-onset macular degeneration. The major cytotoxic component of RPE lipofuscin is pyridinium bisretinoid A2E (Fig. 1) . Additional cytotoxic bisretinoids are isoA2E, atRAL di-PE, and A2-DHP-PE (40, 41) .

A2E is a product of condensation of all-trans retinaldehyde with phosphatidyl-ethanolamine which occurs in the retina in a non-enzymatic manner and, as illustrated in Fig. 4, can be considered a by-product of a properly functioning visual cycle (10) . Light-induced isomerization of 11-cis retinaldehyde to its all-trans form is the first step in a signaling cascade that mediates light perception. The visual cycle is a chain of biochemical reactions that regenerate visual pigment (11-cis retinaldehyde conjugated to opsin) following exposure to light.

As cytotoxic bisretinoids are formed during the course of a normally functioning visual cycle, partial pharmacological inhibition of the visual cycle may represent a treatment strategy for dry AMD and other disorders characterized by excessive accumulation of lipofuscin (25-27, 40, 41). Summary of the Invention

The present invention relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith comprising administering to the mammal an effective amount of a compound having the structure:

wherein X is 0 or S, or an ester or a pharmaceutically acceptable salt thereof.

The present invention also provides a compound having the structure :

or an ester or a pharmaceutically acceptable salt thereof.

Brief Description of the Figures

Figure 1. Structure of bisretinoid A2E, a cytotoxic component of retinal lipofuscin.

Figure 2. Structure of bisretinoid atRAL di-PE (all-trans- retinal dirtier-phosphatidyl ethanolamine) , a cytotoxic component of retinal lipofuscin . Rl and R2 refer to various fatty acid constituents.

Figure 3, Structure of bisretinoid A2-DHP-PE, a cytotoxic component of retinal lipofuscin.

Figure 4. Visual cycle and biosynthesis of A2E . A2E biosynthesis begins when a portion of all-trans- retinal escapes the visual cycle (yellow box) and non-enzymatically reacts with phosphatidyl - ethanolamine forming the A2E precursor, A2-PE. Uptake of serum retinol to the RPE (gray box) fuels the cycle.

Figure 5. Three-dimensional structure of the RBP4-TTR-retinol complex. Tetrameic TTR is shown in blue, light blue, green and yellow. RBP is shown in red and retinol is shown in gray (28) .

Figure 6. Structure of fenretinide, [N- (4-hydroxy- phenyl) retinamide, 4HRP] , a retinoid RBP4 antagonist .

Figure 7. Schematic depiction of the HTRF-based assay format for characterization of RBP4 antagonists disrupting retinol-induced RBP4-TTR interaction. Figure 8. Analysis of 76 compounds from the nuclear receptor ligand library in the HTRF- ased RBP4-TTR interaction assay (antagonist format) . S-F A identified as a compound disrupting retinol-induced RBP4-TTR interation after screening the collection of compound library from Biomol/Enzo .

Figure 9. Dose-response curve for S-FTA in the retinol-induced

RBP4-TTR interaction Assay (HTRF) . Titration of S- FTA in the HTRF RBP4-TTR interaction assay in the presence of 1 μΜ retinol .

Figure 10. Titration curves of S-FTA in the SPA-based RBP4 binding assay. Dose-response curve for S-FTA in the SPA-based RBP4 binding assay: S-FTA is a bona fide RBP4 ligand.

Figure 11. RBP4 levels in mouse serum after intraperitoneal injection of 0.6 mg S-FTA (data from two animals). S-FTA exhibits in vivo activity: after intraperitoneal injection compound reduces the level of serum RBP4.

Figure 12. Titration of three compounds in the HTRF-based

RBP4-TTR interaction assay in the presence of 1 μΜ retinol. Three commercially available farnesoids do not disrupt retinol-induced RBP4-TTR interaction.

Figure 13. Titration of three compounds in the HTRF-based

RBP4-TTR interaction assay in the presence of 1 μΜ retinol. Three additional commercially available farnesoids do not disrupt retinol-induced RBP4-TTR interaction. Figure 14. Titration of three compounds in the HTRF-based R3P4-TTR interaction assay in the presence of 1 M retinol . Three additional commercially available farnesoids do not disrupt retinol-induced RBP4-TTR interaction.

Figure 15. Effect of S-FTA analogs on retinol-induced RBP4-TTR interaction: Compound titration in the HTRF RBP4-TTR interaction assay in the presence of 1 μΜ retinol . Compound 3 , an analog of S-FTA, disrupts retinol- induced RBP4-TTR interaction. The S-FTA analogs were synthesized as described herein.

Figure 16. Effect of S-FTA analogs on retinol-induced RBP4-TTR interaction: Compound titration in the HTRF RBP4-TTR interaction assay in the presence of 1 μΜ retinol . Repeated titration of Compound 3 along with additional farnesoid compounds confirms Compound 3 activity. No new active compounds identified.

Figure 17. Comparison of S-FTA and Compound 3 in the HTRF

RBP4-TTR interaction assay. Head-to-head comparison of S-FTA and Compound 3. Compound 3 is not more active than S-FTA in this assay.

Figure 18. Comparison of S-FTA and Compound 3 in SPA-based

RBP4 binding assay. Head-to-head comparison of S-FTA and Compound 3 in binding assay; Compound 3 is proven to be an RBP4 ligand that is slightly less active than S-FTA in this assay.

Figure 19. Titration of S-FTA in the HTRF-based RBP4-TTR assay in the presence of 4.5 μΜ retinol. Figure 20. Titration of S-FTA and FTA compounds in the HTRF RBP4-TTR assay in the presence of 4.5 uM retinol.

Detailed Descrlption of the Invention

The present invention relates to a method for treating bisretinoid-mediated macular degeneration in a mammal afflicted therewith comprising administering to the mammal an effective amount of a compound having the structure:

wherein X is O or S, or an ester or a pharmaceutically acceptable salt thereof.

termed "S-FTA" herein, an ester or a pharmaceutically acceptable salt thereof .

termed "Compound

3" herein, an ester or a pharmaceutically acceptable thereof . The present invention relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith comprising administering to the mammal an effective amount of a compound having the structure:

wherein X is 0 or S, or an ester or a pharmaceutically acceptable salt thereof .

The present invention relates to a method for treating a disease characterized by excessive lipofuscin accumulation in the retina a mammal afflicted therewith comprising administering to the mammal an effective amount of a compound having the structure:

termed "S-FTA" herein, an ester or a pharmaceutically acceptable salt thereof.

termed "Compound

3" herein, an ester or a pharmaceutically acceptable salt thereof.

In some embodiments , the disease is further characterized by bisretinoid-mediated macular degeneration.

The amount of a compound of the invention may be effective to lower the serum concentration of RBP4 in the mammal .

In some embodiments of the invention, the amount of a compound of the invention may be effective to lower the retinal concentration of a bisretinoid in lipofuscin in the mammal.

In some embodiments, the bisretinoid is A2E. In some embodiments the bisretinoid is isoA2E. In some embodiments the bisretinoid is A2-DHP-PE. In some embodiments the bisretinoid is atRAL di-PE.

In some embodiments, bisretinoid-mediated macular degeneration may be Age-Related Macular Degeneration or Stargardt Disease.

In some embodiments, the bisretinoid-mediated macular degeneration is Age-Related Macular Degeneration.

In some embodiments, the bisretinoid-mediated macular degeneration is dry (atrophic) Age-Related Macular Degeneration. In some embodiments, the the bisretinoid-mediated macular degeneration is Stargardt Disease.

In some embodiments , the bisretinoid-mediated macular degeneration is Best disease.

In some embodiments , the bisretinoid-mediated macular degeneration is adult vitelliform maculopathy .

In some embodiments, the bisretinoid-mediated macular degeneration is Stargardt-like macular dystrophy.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina may be Age-Related Macular Degeneration or Stargardt Disease.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is Age-Related Macular Degeneration .

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is dry (atrophic) Age- Related Macular Degeneration.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is Stargardt Disease.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is Best disease.

In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is adult vitelliform maculopathy . In some embodiments, the disease characterized by excessive lipofuscin accumulation in the retina is Stargardt-like macular dystrophy.

The bisretinoid-mediated macular degeneration may comprise the accumulation of lipofuscin deposits in the retinal pigment epi helium.

The present invention also provides a compound having the structure:

or an ester or a pharmaceutically acceptable salt thereof.

In emdodiments of the invention which encompass an ester of a compound having the structure:

the ester may have the structure:

wherein

X is 0 or S; and

i is alkyl.

or a pharmaceutically acceptable salt thereof.

Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

It is understood that where a parameter range is provided, all integers wi hin that range, and tenths thereof, are also provided by the invention. For example, "0.2-5 mg/kg/day" is a disclosure of 0.2 mg/kg/day, 0.3 mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/day etc . up to 5.0 mg/kg/day.

Terms

As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.

As used herein, "about" in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.

As used herein, "bisretinoid lipofuscin" is lipofuscin containing a cytotoxic bisretinoid. Cytotoxic bisretinoids include but are not necessarily limited to A2E, isoA2E, atRAL di-PE, and A2-DHP-PE (Fig. 1-3).

As used herein, "treating" means reducing, slowing, stopping, or preventing the progression of a disease. An embodiment of "treating bisretinoid-mediated macular degeneration" is delaying or preventing the onset, progression, or mitigating or reducing vision loss or the severity of vision loss .

The compounds and compositions of the present invention are useful for the treatment of lipofuscin-mediated macular degeneration. Compounds of the present invention include those in Table 1. Table 1. Structure of Farnesoids .

S-Farnesyl Thioacetic Acid, or "S-FTA", has the structure:

The CAS Registry No. of S-FTA is 135784-48-4. The formal name of S-FTA is (3,7,ll-trimethyl-2E,6E,10- dodecatrienyl) thioacetic acid. A synonym of "S-FTA" is "FTA" . The molecular formula of S-FTA is C₁7H₂8O2S . The molecular weight of S-FTA is 296.5.

S-FTA may be purchased from Cayman Chemical Company as a solution in ethanol (Ann Arbor, Michigan, USA; Catalog No. 63260) . Cayman Chemical Company suggests that S-FTA be stored as supplied at -20°C, and that it should be stable for at least one year. To change the solvent, the ethanol may be evaporated under a gentle stream of nitrogen and the solvent of choice immediately added. Solvents such as ethanol, DMSO, and dimethyl formamide purged with an inert gas can be used. The solubility of S-FTA in these solvents is about 20 mg/ml. Further dilutions of the stock solution into aqueous buffers or isotonic saline may be made prior to performing biological experiments. Since organic solvents may have physiological effects at low concentrations, it should be ensured that the residual amount of organic solvent is insignificant. If an organic solvent-free solution of S-FTA is needed, it can be prepared by evaporating the ethanol and directly dissolving the neat oil in aqueous buffers. For increased aqueous solubility, S-FTA may be directly dissolved in 0.1 M Na₂C0₃ (63 mg/ml) and then diluted with PBS (pH 7.2) to achieve the desired concentration or pH. Cayman Chemical Company does not recommend storing the aqueous solution for more than one day.

S-FTA is an analog of S-farnesyl cysteine which can behave as a competitive inhibitor of isoprenylated protein methyltransferase (also known as S-adenosylmethionine- dependent methyltransferase) , and it can inhibit methylation of both farnesylated and geranylgeranylated substrates (41- 43) . Farnesyl thiosalicylic acid (Catalog No, 10010501) , N-acetyl- S-farnesyl-L-cysteine (Catalog No. 63270) and N-acetyl-S- geranykgeranyl-L-cysteine (Catalog No. 63340) may be purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA; Trans, trans-Farnesyl acetate (Catalog No. 340480), Farnesol (Catalog No. F203), Geranykgeraniol (Catalog No. G3278) and Tetradecylthioacetic acid (Catalog No, T1698) may be purchased from Sigma-Aldrich Company (St . Louis, MO, USA) ; Tetradecylthioacetic acid (Catalog No. BML-GR236-0010) , N- acetyl-S-geranyl-L-cysteine (Catalog NO.BML-G221-0005) and farnesyl thiotriazole (Catalog No. BML-PE190-0010) may be purchased f om Enzo Life Sciences (Farmingdale, NY, USA) .

As used herein, the description "pharmaceutically active" is used to characterize a substance, compound, or composition suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians' Desk Reference ( DR Network, LLC; 64th edition,- November 15, 2009) and "Approved Drug Products with Therapeutic Equivalence Evaluations" (U.S. Department of Health and Human Services, 30^th edition, 2010) , which are hereby incorporated by reference .

Ester derivatives of compounds may be generated from a carboxylic acid group in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Ester derivatives may serve as pro- drugs that can be converted into compounds of the invention by serum esterases.

As used herein, "alkyl " includes both branched and straight- chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and may be unsubstituted or substituted. Thus, C_x-C_a as in *Ci-C_n alkyl * is defined to include groups having 1, 2, n-1 or n carbons in a linear or branched arrangement. For example, Ci-Ce, as in "Ci-Cs alkyl" is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl , hexyl , and octyl .

Except where otherwise specified, when the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemical^ controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as ¹²C, ^UC, or ¹C. Furthermore, any compounds containing ¹³C or ¹⁴C may specifically have the structure of any of the compounds disclosed herein.

The compounds used in the method of the present invention may be prepared by techniques well know in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

The compounds used in the method of the present invention may be prepared by techniques described in Vogel's Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, P.W.G. Smith, (Prentice Hall) 5^th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5^th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as ^lH, ²H, or ³H. Furthermore, any compounds containing ³H or Ή may specifically have the structure of any of the compounds disclosed herein.

Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically- labeled reagents in place of the non-labeled reagents employed.

A compound, may be in a salt form. As used herein, a "salt* is a salt of the instant compound which has been modified by making acid or base salts of the compounds . In the case of the use of compounds of the invention for treatment of bisretinoid-media ed macular degeneration, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the invention . These salts can be prepared in situ during the final isolation and purification of a compound, or by separately reacting a purified compound in its free acid form with a suitable organic or inorganic base, and isolating the salt thus formed.

A compound may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the mammal in need of the drug is treated or given another drug for the disease in conjunction with a compound of the invention. This combination therapy can be sequential therapy where the mammal is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the mammal. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier.

The dosage of a compound of the invention administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of the compound and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds of the invention may comprise a compound alone, or mixtures of a compound with additional compounds used to treat lipofuscin-mediated macular degeneration. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous {bolus or infusion) , intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection or other methods, into the eye, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

A compound of the invention can be administered in a mixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices . The unit will be in a form suitable for oral , rectal , topical , intravenous or direct injection or parenteral administration. The compounds can be administered alone but are generally mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. In one embodiment the carrier can be a monoclonal antibody. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants , diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents . Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen. Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U. S. Pat. No. 3,903,297, issued Sept. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al . , 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976) ; Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992),- Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.) Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, ol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, manni ol , sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

A compound of the invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.

A compound of the invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol,

polyhydroxyethylasparta-midephenol , or polyethyleneoxide- polylysine substituted with palmitoyl residues. Furthermore, a compound may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters , polyacetals, polydihydropyrans , polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels . A compound can be administered orally in solid dosage forms, such as capsules , tablets, and powders , or in liquid dosage forms, such as elixirs, syrups, and suspensions. It can also be administered parentally, in sterile liquid dosage forms.

Gelatin capsules may contain a compound of the invention and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours . Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

For oral administration in liquid dosage form, a compound may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general , water, a suitable oil, saline, aqueous dextrose (glucose) , and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions . Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient , suitable stabilizing agents , and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol . Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

A compound may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

The compounds and compositions thereof of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject. Compounds of the present invention may be stored, for example, at the following concentrations in dimethyl sulfoxide (DMSO) .

Synthesis of Compounds of the Invention

Scheme 1: Synthesis of Compound 1, Compound 2 and Compound 3

Compound 3 Compound 2 -9

ompoun

This invention will be better understood by reference to the Examples which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter .

Examples

Example 1. TR-FRET assay for antagonists of retinol-induced RBP4-TTR interaction

TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) is an assay format that can be used in characterization of compounds affecting protein-protein interactions (31-33) . The HTRF (Homogeneous Time-Resolved Fluorescence) variant of TR- FRET is the most advanced as it has improved light capturing due to the use of Eu³* cryptates . In the presence of retinol , RBP4-TTR interaction induces FRET that can be registered as increased ratio of 668/620 fluorescence signals. Binding of a desired RBP4 antagonist displaces retinol and induces hindrance for RBP4-TTR interaction resulting in the decreased FRET signal (Fig. 7) .

An assay was developed using E. coli-expressed MBP-tagged RBP4 and commercially available TTR labeled directly with Eu3+ cryptate. In addition to MBP-RBP4 and Eu³* (K) -TTR, a detector reagent anti-MBP-d2 was present in the mix. The assay was first optimized in the agonist mode; sensitivity and dynamic range of the assay was first mode in respect to RBP4, TTR and detection reagent concentrations .

Given that retinol is present in serum at micromolar concentrations and taking into account the results of retinol titrations, the assay was converted to the antagonist mode by testing fixed concentration of retinol within the 1-10 μΜ range. The optimum retinol concentration in the antagonist mode in regard of assay sensitivity and dynamic range was found to be in the 4.5-6.5 μΜ range. TR-FRET assay for retinol-induced RBP4-TTR interaction

Bacterial ly expressed MBP-RBP4 and untagged TTR were used in this assay. For the use in the TR-FRET assay the maltose binding protein (MBP ) -tagged human RBP4 fragment (amino acids 19-201) was expressed in the Gold(DE3 IpLysS E. coli strain (Stratagene) using the pMAL-c4x vector. Following cell lysis, recombinant RBP4 was purified from the soluble fraction using the ACTA FPLC system (GE Healthcare) equipped with the 5 -ml the MBP Trap HP column. Human untagged TTR was purchased from Calbiochem. Untagged TTR was labeled directly with Eu³⁺ Cryptate-NHS using the HTRF Cryptate Labeling kit from CisBio following the manufacturer's recommendations. HTRF assay was performed in white low volume 384 well plates (Greiner-Bio) in a final assay volume of 16 μΐ per well. The reaction buffer contained 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% NP-40, 0.05% Prionex, 6% glycerol, and 400 mM KF . Each reaction contained 60 nM MBP-RBP4 and 2 nM TTR-Eu along with 26.7nM of anti-MBP antibody conjugated with d2 (Cisbio) . Titration of test compounds in this assay was conducted in the presence of 1 μΜ retinol. All reactions were assembled in the dark under dim red light and incubated overnight at +4°C wrapped in aluminum foil . TR-FRET signal was measured in the SpectraMax M5e Multimode Plate Reader (Molecular Device) . Fluorescence was excited at 337 nm and two readings per well were taken: Reading 1 for time-gated energy transfer from Eu(K) to d2 (337 nm excitation, 668 nm emission, counting delay 75 microseconds, counting window 100 microseconds) and Reading 2 for Eu(K) time-gated fluorescence (337 nm excitation, 620 nm emission, counting delay 400 microseconds, counting window 400 microseconds) . The TR-FRET signal was expressed as the ratio of fluorescence intensity: Flue6s/Flu62o x 10,000. Example 2. S-ΓΤΑ administration and Serum RBP4 measurements

S-FTA was purchase from Cayman Chemical Company; 6 mg/ml solution of S-FTA in sesame oil or ethanol was prepared. One hundred microliters of S-FTA solution was administered intraperitoneally to a mouse (weight of the mouse: 20 g) to accomplish 30 mg/kg dosing. Blood samples were collected from a tail vein before dosing and at 5 min, 30 min, 1 hr, 2 hr, 4 hr, and 6 hr timepoints. Whole blood was drawn into a centrifuge tube and was let clot at room temperature for 30 min followed by centrifugation at 2,000 x g for 15 minutes at +4°C to collect serum. Serum RBP4 was measured using the RBP4 (mouse/rat) dual ELISA kit (Enzo Life Sciences) following the manufacturer's instructions.

Example 3. Scintillation proximity RBP4 binding assay

Untagged human RBP4 purified from urine of tubular proteinuria patients was purchased from Fitzgerald Industries International . It was biotinylated using the EZ-Link Sulfo- HS-LC-Biotinylation kit from Pierce following the manufacturer's recommendations. Binding experiments were performed in 96-well plates (OptiPlate, PerkinElmer) in a final assay volume of 100 μΐ per well in SPA buffer (IX PBS, pH 7.4, ImM EDTA, 0.1%BSA, 0.5%CHAPS). The reaction mix contained 10 nM ³H-Retinol (48.7Ci/mmol; PerkinElmer), 0.3 mg/well Streptavidin-PVT beads, 50 nM biotinylated RBP4 and a test compound. Nonspecific binding was determined in the presence of 20 μΜ of unlabeled retinol. The reaction mix was assembled in the dark under dim red light. The plates were sealed with clear tape (TopSeal-A: 96-well microplate, PerkinElmer), wrapped in the aluminum foil, and allowed to equilibrate 6 hours at room temperature followed by overnight incubation at +4°C. Radiocounts were measured using a TopCount NXT counter (Packard Instrument Company) . Discussion

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. Its prevalence is higher than that of Alzheimer's disease. There is no treatment for the most common dry form of AMD. Dry AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells and provides critical metabolic support to these light-sensing cells. RPE dysfunction induces secondary degeneration of photoreceptors in the central part of the retina called the macula. Experimental data indicate that high levels of lipofuscin induce degeneration of RPE and the adjacent photoreceptors in atrophic AMD retinas. In addition to AMD, dramatic accumulation of lipofuscin is the hallmark of Stargardt disease (STGD), an inherited form of juvenile onset macular degeneration. The major cytotoxic component of RPE lipofuscin is a pyridinium bisretinoid A2E. A2E formation occurs in the retina in a non-enzymatic manner and can be considered a byproduct of a properly functioning visual cycle. Given the established cytotoxic affects of A2E on RPE and photoreceptors, inhibition of A2E formation could lead to delay in visual loss in patients with dry AMD and STGD. It was suggested that small molecule visual cycle inhibitors may reduce the formation of A2E in the retina and prolong RPE and photoreceptor survival in patients with dry AMD and STGD. Rates of the visual cycle and A2E production in the retina depend on the influx of all-trans retinol from serum to the RPE. Pharmacological downregulation of serum retinol is a valid treatment strategy for dry AMD and STGD. Serum retinol is maintained in circulation as a tertiary complex with retinol-binding protein (RBP4) and transthyretin (TTR) . Without interacting with TTR, the RBP4-retinol complex is rapidly cleared due to glomerular filtration. Retinol binding to RBP4 is required for formation of the RBP4-TTR complex; apo-RBP4 does not interact with TTR. Importantly, the retinol - binding site on RBP4 is sterically proximal to the interface mediating the RBP4-TTR interaction. Without wishing to be bound by any scientific theory, the data herein show that small molecule RBP4 antagonists displacing retinol from RBP4 and disrupting the RBP4-TTR interaction will reduce serum retinol concentration, inhibit retinol uptake into the retina and act as indirect visual cycle inhibitors reducing formation of cytotoxic A2E .

Serum RBP4 as a drug target for pharmacological inhibition of the visual cycle

As rates of the visual cycle and A2E production in the retina depend on the influx of all-trans retinol from serum to the RPE (Fig. 4), it has been suggested that partial pharmacological down-regulation of serum retinol may represent a target area in dry AMD treatment (11) . Serum retinol is bound to retinol-binding protein (RBP4) and maintained in circulation as a tertiary complex with RBP4 and transthyretin (TTR) - Fig. 5. Without interacting with TTR, the RBP4-retinol complex is rapidly cleared from circulation due to glomerular filtration. Additionally, formation of the RBP4-TTR-retinol complex is required for receptor-mediated all-trans retinol uptake from serum to the retina.

Without wishing to be bound by any scientific theory, visual cycle inhibitors may reduce the formation of toxic bisretinoids and prolong RPE and photoreceptor survival in dry AMD. Rates of the visual cycle and A2E production depend on the influx of all-trans retinol from serum to the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4 ) - transthyretin (TTR) -retinol complex in serum is required for retinol uptake from circulation to the RPE . Retinol-binding site on RBP4 is sterically proximal to the interface mediating the RBP4--TTR interaction. RBP4 antagonists that compete with serum retinol for binding to RBP while blocking the R3P4-TTR interaction would reduce serum retinol, slow down the visual cycle, and inhibit formation of cytotoxic bisretinoids . The present invention relates to new classes of RBP4 antagonists .

RBP4 represents an attractive drug target for indirect pharmacological inhibition of the visual cycle and A2E formation. The retinol-binding site on RBP4 is sterically proximal to the interface mediating the RBP4-TTR interaction. Retinol antagonists competing with serum retinol for binding to RBP4 while blocking the RBP4-TTR interaction would reduce serum RBP4 and retinol levels which would lead to reduced uptake of retinol to the retina. The outcome would be visual cycle inhibition with subsequent reduction in the A2E synthesis .

A synthetic retinoid called fenretinide [N- (4-hydroxy- phenyl) retinamide, 4HRP] previously considered as a cancer treatment (29) was found to bind to RBP4, displace all-trans retinol from RBP4 (13) , and disrupt the RBP4-TTR interaction (13,14) .

Fenretinide was shown to reduce serum RBP4 and retinol (15) , inhibit ocular all-trans retinol uptake and slow down the visual cycle (11) . Importantly, fenretinide administration reduced A2E production in an animal model of excessive bisretinoid accumulation, Abca4 -/- mice (11) . Pre-clinical experiments with fenretinide validated RBP4 as a drug target for dry AMD. However, fenretinide is non-selective and toxic. Independent of its activity as an antagonist of retinol binding to BP4, fenretinide is an extremely active inducer of apoptosis in many cell types (16-19), including the retinal pigment epithelium cells (20) . It has been suggested that fenretinide' s adverse effects are mediated by its action as a ligand of a nuclear receptor RAR (21-24) . Additionally, similar to other retinoids, fenretinide is teratogenic.

The TR-FRET assay was developed and optimized for compounds antagonizing retinol-dependent RBP4-TTR interaction using purified MBP-tagged RBP4 and apo-TTR directly labeled with Eu3+-cryptate. The assay was used to screen several commercial libraries of compounds with drug-like properties. Positive compounds were further evaluated using the developed competition binding assays which unutilized the scintillation proximity (SPA) format for probing the displacement of tritiated retinol from MBP-RPB4. Medicinal chemistry optimization of one positive compound was attempted in order to establish the SAR, structure-activity relationship, in this structural series.

As disclosed herein, the TR-FRET assay for the library screening, identified a farnesoid derivative, a non-retinoid compound, as a low micromolar inhibitor of the retinol- dependent RBP4-TTR interaction. Using the competition binding assay, the compound's ability to directly compete with all- trans retinol for binding to RBP4 was established. The two assays were used to perform characterization of 9 newly synthesized compounds along with 11 commercially available structures in order to establish SAR in this structural series . The 11 commercially available compounds (Fig. 12-14) were S- farnesyl thioacetic acid, farnesyl thiosalicylic acid, S- farnesyl-L-cysteine methyl ester, farnesyl acetate, farnesyl thiotriazole, farnesol, geranylgeraniol , acetyl -geranyl- cysteine, N-acetyl -S-farnesyl-L-cysteine, N-acetyl-S- geranylgeranyl-L-cysteine, tetradecylthioacetic acid.

The present invention relates to a new class of compounds , S- FTA, S-farnesyl thioacetic acid, and analogues thereof, for treatment of macular degeneration and Stargardt Disease. As disclosed herein, S-FTA was discovered to strongly and specifically bind to RBP4 using a scintillation proximity assay. S-FTA binding competes wi h retinol binding indicating that S-FTA interacts with the retinol-binding pocket in RBP4. The TR-FRET RBP4-TTR interaction assay disclosed herein established that S-FTA prevents retinol-induced interaction of RBP4 with TTR in a dose-dependent way. This implies that S-FTA will act as a visual cycle inhibitor in vivo which would reduce levels of serum RBP4 and retinol and inhibit the rate of the visual cycle. The effect of S-FTA on RBP4 is specific; among 6 farnesoids with related structures, S-FTA is the only one that binds to RBP4 and disrupts RBP4-TTR interaction. As disclosed herein, this in vitro data established S-FTA and related compounds as a potential small molecule treatment for dry AMD and Stargardt disease.

Currently, there is no FDA-approved treatment for dry AMD or Stargardt disease, which affect millions of patients. An over the counter, non FDA-approved cocktail of antioxidant vitamins and zinc (AREDS formula) is claimed to be beneficial in a subset of dry AMD patients. There are no treatments for Stargardt disease. The present invention provides a new class of non-retinoid RBP4 antagonists that are useful for the treatment of dry AMD and other conditions characterized by excessive accumulation of lipofuscin. Without wishing to be bound by any scientific theory, as accumulation of lipofuscin seems to be a direct cause of RPE and photoreceoptor demise in AMD and STGD retina, the compounds described herein are disease-modifying agents since they directly address the root cause of these diseases . Experiments which were conducted and that are described herein defined the path for further SAR optimization in this structural series . The present invention provides compounds that will preserve vision in AMD patients , Stargardt disease patients , and patients suffering from conditions characterized by excessive accumulation of lipofuscin.

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Claims

la claimed is:

A method for treating a disease characterized by excessive lipofuscin accumulation in the retina in a mammal afflicted therewith comprising administering to the mammal an effective amount of a compound having the structure:

wherein X is 0 or S, or an ester or a pharmaceutically acceptable salt thereof.

2. The method of claim 1 or 2 , wherein X is 0.

3. The method of claim 1 or , wherein X is S .

4. The method of any of claims 1-3 wherein the disease is further characterized by bisretinoid-mediated macular degeneration.

5. The method of any one of claims 1 to 4, wherein the amount of the compound is effective to lower the serum concentration of RBP4 in the mammal .

6. The method of any one of claims 1 to 4, wherein the amount of the compound is effective to lower the retinal concentration of a bisretinoid in lipofuscin in the mammal .

7. The method of claim 4, wherein the bisretinoid is A2E.

8. The method of claim 4, wherein the bisretinoid is isoA2E.

9. The method of claim 4, wherein the bisretinoid is A2-DHP- PE.

The method of claim 4, wherein the bisretinoid is atRAL di-PE.

The method of any one of claims 1-10, wherein the disease characterized by excessive lipofuscin accumulation in the retina is Age-Related Macular Degeneration.

The method of any one of claims 1-10, wherein the disease characterized by excessive lipofuscin accumulation in the retina is dry (atrophic) Age-Related Macular Degeneration .

The method of any one of claims 1-10, wherein the disease characterized by excessive lipofuscin accumulation in the retina is Stargardt Disease.

The method of any one of claims 1-10, wherein the disease characterized by excessive lipofuscin accumulation in the retina is Best disease.

15. The method of any one of claims 1-10, wherein the disease characterized by excessive lipofuscin accumulation in the retina is adult vitelliform maculopathy.

16. The method of any one of claims 1-10, wherein the disease characterized by excessive lipofuscin accumulation in the retina is Stargardt-like macular dystrophy.

17. A compound having the structure:

or an ester or a pharmaceutically acceptable salt thereof .