WO2010135004A1 - Enzymatic degradation of a2e and other bisretinoid compounds of rpe lipofuscin - Google Patents

Abstract

The present invention provides methods for reducing the amount of lipofuscin compounds present in a cell which comprises contacting the cell with an amount of an enzyme sufficient to directly or indirectly degrade the amount of lipofuscin compounds, so as to reduce the amount of lipofuscin compounds, wherein the enzyme is a carotenoid cleaving dioxygenase or an oxo-iron heme-based enzyme. The invention also provides methods for. treating a subject suffering from a lipofuscin- associated disorder which comprises administering to the subject a composition comprising an amount of an enzyme sufficient to directly or indirectly degrade lipofuscin compounds associated with the disorder in cells of the subject, so as to thereby treat subject's the lipofuscin- associated disorder, wherein the enzyme is a carotenoid cleaving dioxygenase or an oxo-iron heme-based enzyme.

Description

Enzymatic Degradation of A2E and Other Bisretinoid Compounds of RPE Lipofuscin

This application claims priority of U.S. Provisional Application No. 61/216,914, filed May 22, 2009, the entire content of which is hereby incorporated by reference herein.

Throughout this application various publications and published patents are referenced. The disclosures of these 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 .

The invention disclosed herein was made with government support under NIH-National Eye Institute grant EY12951 (JRS) . Accordingly, the U.S. Government has certain rights in this invention.

Background of Invention

Beginning in childhood, Retinal pigment epithelial (RPE) cells in the eye accumulate fluorescent bisretinoid compounds that are housed within lysosomal organelles and that constitute the lipofuscin of the cell. Three groups of these bisretinoid pigments have been identified in RPE lipofuscin: 1) 2- (2, 6- dimethyl-8- (2,6, 6-trimethyl-l-cyclohexen-l-yl) - IE, 3E, 5E, 7E-octatetraenyl) -1- (2-hydroxyethyl) -4- (4-methyl-6- (2,6, 6-trimethyl-l-cyclohexen-l-yl) - IE, 3E, 5E-hexatrienyl) -pyridinium (A2E) and its isomers; 2) A2- dihydropryridine-phosphatidylethanolamine (A2-DHP-PE) and 3) compounds of the all- trans-retinal dimer series (Fishkin, 2005; Kim, 2007; Parish, 1998; Wu, 2009) . These compounds accumulate with age in all individuals and accumulate with particular abundance in individuals with some inherited forms of retinal degeneration including recessive Stargardt ' s disease and Best disease.

The bisretinoid pigments identified in RPE lipofuscin are derived in large part from reactions of all- trans-retinal, the retinoid that is generated when 11-cis-retinal, the chromophore of visual pigment isomerizes after absorbing a photon of light. Accordingly, when the 11-cis-retinal (11- cisRAL) and all- trans-retinal (atRAL) chromophores are absent, such as occurs with targeted deletion of Rpe65 in mouse or with RPE65 gene mutation in humans, the lipofuscin-specific autofluorescence emanating from RPE cells is dramatically reduced or absent (Katz, 2001; Lorenz, 2004) .

Since members of each of the three groups of bisretinoid pigments identified in RPE lipofuscin include isomers and photooxdized products, the content is a complicated mixture. For all of these groups of bisretinoids, biosynthesis begins in photoreceptor cells (Ben-Shabat, 2002; Liu, 2000; Fishkin, 2005). For instance, two all- trans-retinal molecules can react nonenzymatically to form all- trans-retinal dimer (λ_max 290, 432 nm) , the latter then reacting with phosphatidylethanolamine (PE) to generate the protonated Schiff base conjugate - all- trans-retinal dimer-PE (λ_max 290, 510 nm) (Fishkin, 2005; Kim, 2007) . Alternatively, two all- trans-retinal can condense with phosphatidylethanolamine to yield A2E via a multi-step pathway that includes the formation of NRPE (iV-retinylidene-phosphatidylethanolamine) , A2PE-H₂ and A2PE as intermediates (Ben-Shabat, 2002; Liu, 2000; Parish, 1998). When discarded photoreceptor outer segments are phagocytosed by RPE cells, A2PE is deposited into the lysosomal compartment of the cells. The inventor has previously shown that an enzyme activity present in RPE cell lysosomes can generate A2E (λ_raax 338, 439 nm) from A2PE by phosphate hydrolysis of the precursor A2PE (Ben-Shabat, 2002; Sparrow, 2008) . The facility with which phospholipase-D inhibitors suppress this hydrolytic activity in lysosomal fractions, together with the ability of purified phospholipase-D to produce A2E by cleaving A2PE, indicates that phospholipase-D is the enzyme in RPE cell lysosomes that converts A2PE to A2E (Ben-Shabat, 2002; Sparrow, 2008) . After A2E is released from A2PE by enzyme-mediated phosphate cleavage, it appears that little or no further enzyme degradation of the A2E molecule occurs and thus the pigment accumulates in the cell. The inventor has surmised that since the structure of A2E is unprecedented, it may not be recognized by the lysosomal enzymes of the RPE cell and thus is largely indigestible (Sparrow, 2005; Sparrow, 2008) . Phospholipase D can also release all- trans-retinal dimer- ethanolamine from al- trans-retinal dimer-PE by phosphate hydrolysis (Kim, 2007) .

In addition to the age-related accumulation, RPE lipofuscin forms in particular abundance in retinal disorders caused by mutations in ABCA4 (ABCR) (Allikmets, 1997), the gene that encodes a photoreceptor-specific ATP-binding cassette transporter (Ann, 2000; Sun, 1999) . These disorders include autosomal recessive (AR) Stargardt ' s disease, AR retinitis pigmentosa and AR cone-rod dystrophy (Shroyer, 1999) . Indeed, evidence on several fronts indicates that the amassing of this material in RPE cells is responsible for the RPE atrophy that characterizes these forms of retinal degeneration (Weng, 1999; Sparrow, 2007; Sparrow, 2007; Sparrow, 2003). The bisretinoid compounds of RPE lipofuscin may also contribute to complement activation (Zhou, 2006; Zhou, 2009), dysregulation of which may be the basis for the association between genetic variants in complement factors and susceptibility to age-related macular degeneration (Edwards, 2005; Gold, 2006; Hageman, 2005; Haines, 2005; Klein, 2005; Zareparsi, 2005; Yates, 2007) .

Several therapeutic approaches aimed at alleviating vision loss by retarding lipofuscin formation in ABCA4/ABCR- associated disorder have been tested in Abcr^'/' mice. These approaches have included gene therapies based on AAV- (Allocca, 2008) and lentiviral-vector (Kong, 2008) mediated delivery of the wild-type gene. Other preclinical studies have focused on systemic administration of compounds that limit the visual cycle including isotretinoin, an inhibitor of 11-cis-retinol dehydrogenase (Radu, 2003); the retinoid analog fenretinide that lowers serum vitamin A (Radu, 2005); and RPE65 inhibitors (Maiti, 2006; Maeda, 2008) . In all these preclinical studies HPLC quantitation of A2E served as the outcome measure and reductions in this lipofuscin pigment were observed. Nevertheless, neither gene nor drug therapy can reverse the accumulation of lipofuscin once it has already occurred. Thus, an alternative treatment method which can reverse the accumulation of lipofuscin post-formation is needed .

Lipofuscin-Associated Disorders

Lipofuscin accumulation has been implicated in macular degeneration, a degenerative disease of the eye (Lacey, 2006) . Macular degeneration is the primary cause of visual impairment in industrialized countries, with the dry form of age-related macular degeneration (dry AMD) accounting for the majority of cases and juvenile onset forms, such as Stargardt ' s disease and Best disease, being relatively rare. These forms of macular degeneration are associated with fatty accumulations of bisretinoid compounds of lipofuscin in RPE cells . Although lipofuscin accumulates in the RPE cells of all individuals as they age, they accumulate unusually rapidly and in high abundance in some individuals, causing vision impairment and blindness .

Abnormal accumulation of lipofuscin has also been associated with a group of neurodegenerative diseases called lipofuscinoses, e.g., neuronal ceroid lipofuscinosis, also known as Batten disease. Accumulation of lipofuscin has also been implicated in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, certain lysosomal diseases, acromegaly, denervation atrophy, lipid myopathy, chronic obstructive pulmonary disease, centronuclear myopathy (Allaire, 2002) .

As there is currently no treatment for these forms of macular degeneration, there is a significant unmet medical need for novel therapeutics, primarily among the growing aging population. Summary of the Invention

The subject invention provides a method for reducing the amount of lipofuscin compounds present in a cell which comprises contacting the cell with an amount of an enzyme sufficient to directly or indirectly degrade the amount of lipofuscin compounds, so as to reduce the amount of lipofuscin compounds, wherein the enzyme is a carotenoid cleaving dioxygenase or an oxo-iron heme-based enzyme.

The subject invention also provides a method for treating a subject suffering from a lipofuscin-associated disorder which comprises administering to the subject a composition comprising an amount of an enzyme sufficient to directly or indirectly degrade lipofuscin compounds associated with the disorder in cells of the subject, so as to thereby treat subject's the lipofuscin-associated disorder, wherein the enzyme is a carotenoid cleaving dioxygenase or an oxo-iron heme-based enzyme.

Description of the Figures

FIGURE 1: HPLC quantitation of A2E pigment after incubation in the presence and absence of horseradish peroxidase (HRP) . A:

Chromatographic overlay of HPLC profiles generated with samples of A2E incubated with H₂O₂, A2E incubated with HRP and

A2E incubated with H₂O₂ and HRP for 24 hours. Reverse phase

HPLC. B: Quantitation of A2E and A2E isomers. Chromatographic peaks areas were measured; A2E and isomers were summed and are presented as percent of pigment in absence of horseradish peroxidase. Mean ± SEM of 3 experiments. *, p<0.01.

FIGURE 2 : Intracellular horseradish peroxidase (HRP) can degrade A2E that is accumulated in ARPE-19 cells. A-C: Intracellular horseradish peroxide introduced to the cells via BioPORTER^®-delivery is detected immunocytochemically using primary antibody to HRP, an avidin-biotin-alkaline phosphatase-complex, and color development with Vector Red. The reaction product is visible with both fluorescence (A) and brightfield (B) optics. HRP is present ( +) or absent (-) . D-K-. Colocalization of HRP with a lysosomal marker in ARPE-19 cells. Imaging by laser scanning confocal microscopy (x-y scans). Lysotracker (D, H), HRP present ( +) or absent (-) (E, J), DAPI-stained nuclei (F, J^") and overlay (G, K). L-. HPLC quantitation of A2E in ARPE-19 cells to which HRP was delivered using BioPORTER^® reagent; +, presence of compound/reagent . Mean ± SEM of 4-7 experiments.

FIGURE 3: FAB-MS (fast atom bombardment mass spectrometry) analysis of samples of A2E following oxidation and cleavage by horseradish peroxidase (HRP) . A-C-. Representative FAB-MS profile obtained after 1-hour and 5-hour incubations of A2E, HRP and hydrogen peroxide (H₂O₂) . The FAB-MS pattern was similar across replicate samples and with/without H₂O₂. D: Structure of A2E. E-G: Proposed structures for HRP-associated cleavage products corresponding m/z 312, 340, and 380. FIGURE 4: ¹H NMR (Proton Nuclear Magnetic Resonance) spectrum. Spectra recorded in the region of 10-5 ppm (400 MHz,- MeOD) . A2E (A) and A2E incubated with HRP (and H₂O₂) (B) for 5 hours. The signal at ~ 9.4 ppm (arrow) is characteristic of the aldehyde proton.

FIGURE 5; Cell viability is retained with HRP-mediated degradation of intracellular A2E. A: Cell viability was probed by 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) assay. Bar height is indicative of MTT absorbance (at 570 nm) and reflects cell viability. Mean ± SEM of 3 experiments. B: A two-color fluorescence assay was employed to quantify percent nonviable cells. Mean ± SEM of 2 experiments. Cells accumulated A2E in culture and HRP was delivered to the cells using BioPORTER^® reagent. "+" indicates presence of compound/reagent . C: Cell viability 3 and 14 days after introduction of HRP was probed by MTT assay. Bar height is indicative of MTT absorbance (at 570 nm) and reflects cell viability. Mean ± SEM of 4 experiments. Cells accumulated A2E in culture and HRP was delivered to the cells using BioPORTER^® reagent. +, presence of compound/reagent.

FIGURE 6; Proposed mechanisms for the formation of adducts (m/z 620, 638 and 648) and cleavage products (m/z 312, 340 and 380) generated by HRP-catalyzed degradation of A2E. Mechanism described in detail in Example 1, discussion section. Dotted lines in B, C and D indicate cleavage sites; arrows in D indicate alternate positions for epoxidation.

FIGURE 7; A2E and A2E-bromine (A2E-Br) . Structures, mass-to- charge ratio (m/z) , UV-visible absorbance (nm) , and electronic transition assignments (<->•) .

FIGURE 8; HPLC quantitation of A2E pigment after incubation in the presence and absence of horseradish peroxidase (HRP)ZH₂O₂. A-D: HPLC chromatograms generated with samples of A2E, A2E incubated with H₂O₂, A2E incubated with HRP, and A2E incubated with H₂O₂ (0.2%) and HRP for 24 hours. Reverse phase HPLC. E: Quantitation of A2E and A2E isomers . Chromatographic peaks areas were measured, A2E and isomers were summed and are presented as percent of pigment in absence of HRP and H₂O₂. Mean ± SEM of 3 experiments. +, presence of compound/reagent.

FIGURE 9; HPLC quantitation of A2E pigment after incubation without HRP, with HRP and with Cathepsin D. Samples of A2E

(top) and A2E incubated with HRP and H₂O₂ (1%) (middle) and A2E incubated with cathepsin D (bottom) for 5 hours at room temperature were analyzed by reverse HPLC. Inset, UV-visible spectra of A2E and isoA2E; *, minor isomers of A2E. The decrease in peak height in the middle panel is consistent with

HRP-mediated degradation of A2E. AU, absorbance units.

FIGURE 10; UPLC-ESI-MS analysis of samples of A2E-Br following oxidation and cleavage by horseradish peroxidase (HRP) . A-C: Representative reverse phase UPLC profiles (ACQUITY BEH C18 column, monitoring at 430 nm) obtained after 3-hour and 18- hour incubations of A2E-Br, HRP and hydrogen peroxide (H₂O₂, 0.2%), as well as of A2E-Br alone. Insets in A and B (top): UV-visible absorbance spectra of A2E-Br, monooxo-A2E-Br and bisoxo-A2E-Br . Inset B (right) : chromatogram expanded between retention time 2-4 min for detection of HRP-associated cleavage products (Fl, F2 , F3 , and F4) ,^• chromatogram is magnified in Fig. 5A. D-H: MS spectra of peaks a-e present in A-C. The molecular ions at m/z 730/732, 746/748 and 762/764, correspond to A2E-Br, monooxo-A2E-Br and bisoxo-A2E-Br, respectively. Note bromine isotope (⁷⁹Br and ⁸¹Br) peaks differing by 2 m/z units in D-H.

FIGURE 11; UPLC-MS analysis of HRP-induced cleavage products of A2E-Br. A: UPLC chromatogram (monitoring at 430 nm) of A2E- Br incubated with HRP and H₂O₂ (0.2%) for 3 hrs; expanded between retention times 2-4 min (full chromatogram in Fig. 4B) . Top insets, UV-visible absorbance spectra of cleavage products F1-F4. B-G: Extracted ion monitoring chromatograms of A2E-Br incubated in water (B-D) and with HRP and H₂O₂ (E-G) for 3 hrs. Data were acquired in ESI mode with selection for mass- to-charge ratios (m/z) 610, 570 and 544, and recorded as a function of retention time in a reverse phase UPLC column. In E-G four prominent ion peaks corresponding to UPLC peaks F1-F4 (A) were observed. H-K-. Assignment of m/z to chromatographic peaks (F1-F4) by coupled electrospray ionization analyses. The bromine tag was indicative of cleavage products that included the pyridinium head group of A2E-Br. Insets, proposed structures of cleavage products and electronic transition assignments («→) of intact arms of A2E-Br.

FIGURE 12; UPLC-MS analysis of ¹⁸O-labeled cleavage products generated from A2E-Br incubated with HRP in ¹⁸0-labeled water (H₂ ¹⁸O) and hydrogen peroxide (H₂ ¹⁸O₂) . A and B: Selected ion chromatograms with detection set for mass-to-charge ratios (m/z) of 614 and 574. Two peaks (F2a and F3a) were observed that corresponded to ¹⁸0-labeled peaks F2 and F3 according to the identical retention time. C and D: ESI-MS spectra of the chromatographic peaks, F2a and F3a. Insets, structures of ¹⁸O- labeled cleavage products. In the use of H₂ ¹⁸O and H₂ ¹⁸O₂, the mass of F2 (m/z 610/612) and F3 (m/z 570/572) is shifted by 2 m/z units to 612/614 (F2a) and 572/574 (F3a) , respectively, due to ¹⁶O-¹⁸O exchange.

FIGURE 13; Proposed mechanisms for HRP-catalyzed oxidation and degradation of A2E. L, ligand. The HRP catalytic cycle involves two active species, Compound I (Cpdl) and Compound II (Cpd II) . Two-electron oxidation of HRP by H₂O₂ (step 1) , generates the first active species Cpd I; the latter mediates oxygen transfer reactions to yield C-H hydroxylation (step 2) and/or C=C epoxidation (step 3_) . Cpd I then returns to ground state (step 4_^) . At epoxidation sites, opening and hydration of the epoxide ring would give rise to a diol (step 5_^) , that would undergo a periodate-like enzymatic carbon-carbon fission reaction to form two carbon-centered alcoholic radicals (step 6_^) , one that includes the positively charged pyridinium ring (Φ) with the residual component being without charge (neutral) (step 6_) . Cpd I (step 1^) , initiates one-electron oxidation on one of these radicals to generate an aldehyde or methylketone- bearing products (step 1) and Cpd II (step 8) . The latter forms by transfer of a hydrogen atom from the hydroxyl group of the radical to the iron-oxo complex (Cpd I) . Subsequently one-electron oxidation of another radical by Cpd II yields an aldehyde or methylketone-bearing product (step 1) and the enzyme (Cpd II) returns to ground state (Ann, 1997) (step 9) . Note that enzyme cleavage at the 9-10 double bond would generate an aldehyde-bearing fragment and a ketone-bearing fragment; cleavage at 7-8, 7'-8' or 11 '-12' double bonds would lead to two aldehyde-bearing fragments.

Detailed Description of the Invention

Embodiments of the Invention

The subject invention provides a method for reducing the amount of lipofuscin compounds present in a cell which comprises contacting the cell with an amount of an enzyme sufficient to directly or indirectly degrade the amount of lipofuscin compounds, so as to reduce the amount of lipofuscin compounds, wherein the enzyme is a carotenoid cleaving dioxygenase or an oxo-iron heme-based enzyme.

In one embodiment, the cell is an eye cell, brain cell, nerve cell, kidney cell, liver cell, heart muscle cell, or adrenal cell. In another embodiment, the cell is an eye cell. In yet another embodiment, the eye cell is a retinal pigment epithelial (RPE) cell.

In one embodiment, the lipofuscin compounds comprise a bisretinoid. In another embodiment, the bisretinoid is 2- (2, 6- dimethyl-8- (2,6, 6-trimethyl-l-cyclohexen-l-yl) - IE, 3E, 5E, 7E-octatetraenyl) -1- (2-hydroxyethyl) -4- (4-methyl-6- (2,6, 6-trimethyl-l-cyclohexen-l-yl) - IE, 3E, 5E-hexatrienyl) -pyridinium, its isomer, or its precursor. In another embodiment, the bisretinoid is N- retinylidene-N-retinylethanolamine or a N-retinylidene-N- retinylethanolamine precursor. In yet another embodiment, the bisretinoid is an all- trans-retinal compound or dimer of an all- trans-retinal compound.

In one embodiment, the enzyme is a carotenoid cleaving dioxygenase. In another embodiment, the enzyme is an oxo-iron heme-based enzyme. In another embodiment, the oxo-iron heme- based enzyme is a cytochrome P450, chloroperoxidase, cytochrome c peroxidase, catalase, or horseradish peroxidase. In yet another embodiment, the oxo-iron heme-based enzyme is horseradish peroxidase.

The subject invention also provides a method for treating a subject suffering from a lipofuscin-associated disorder which comprises administering to the subject a composition comprising an amount of an enzyme sufficient to directly or indirectly degrade lipofuscin compounds associated with the disorder in cells of the subject, so as to thereby treat subject's the lipofuscin-associated disorder, wherein the enzyme is a carotenoid cleaving dioxygenase or an oxo-iron heme-based enzyme .

In one embodiment, the enzyme is a carotenoid cleaving dioxygenase. In another embodiment, the enzyme is an oxo-iron heme-based enzyme. In another embodiment, the oxo-iron heme- based enzyme is a cytochrome P450, chloroperoxidase, cytochrome c peroxidase, catalase, or horseradish peroxidase. In yet another embodiment, the oxo-iron heme-based enzyme is horseradish peroxidase.

In one embodiment, the lipofuscin-associated disorder is age- related macular degeneration, Stargardt ' s disease, Best disease, lipofuscinoses, Alzheimer's disease, or Parkinson's disease. In another embodiment, the lipofuscin-associated disorder is age-related macular degeneration. In another embodiment, the lipofuscin-associated disorder is Stargardt ' s disease. In yet another embodiment, the lipofuscin-associated disorder is Best disease.

In one embodiment, the cells are eye cells, brain cells, nerve cells, kidney cells, liver cells, heart muscle cells, or adrenal cells. In another embodiment, the cells are eye cells. Yet another embodiment, the eye cells are retinal pigment epithelial cells.

In one embodiment, the lipofuscin compounds comprise a bisretinoid. In another embodiment, the bisretinoid is 2- (2, 6- dimethyl-8- (2,6, 6-trimethyl-l-cyclohexen-l-yl) -

IE, 3E, 5E, 7E-octatetraenyl) -1- (2-hydroxyethyl) -4- (4-methyl-6-

(2,6, 6-trimethyl-l-cyclohexen-l-yl) -

IE, 3E, 5E-hexatrienyl) -pyridinium, its isomer, or its precursor. In another embodiment, the bisretinoid is N- retinylidene-N-retinylethanolamine or a N-retinylidene-N- retinylethanolamine precursor. In yet another embodiment, the bisretinoid is an all- trans-retinal compound or dimer of an all- trans-retinal compound.

Definitions

As used herein, "all- trans-retinal dimer-E" means all- trans- retinal dimer-ethanolamine; "all- trans-retinal dimer-PE" means all- trans-retinal dimer-phosphatidylethanolamine,- "NRPE" means i\7-retinylidene-phosphatidylethanolamine; and "PE" means phosphatidylethanolamine.

As used herein, "lipofuscin-associated disorder" shall mean disorders associated with an increased accumulation of lipofuscin in cells. These disorders may include, but are not limited to Age-related Macular Degeneration (AMD), Stargardt ' s disease, Best disease, lipofuscinoses, e.g., neuronal ceroid lipofuscinoses , also known as Batten disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, certain lysosomal diseases, acromegaly, denervation atrophy, lipid myopathy, chronic obstructive pulmonary disease, centronuclear myopathy or any disease which is correlated with an increased accumulation of lipofuscin.

As used herein, "oxo-iron heme-based enzyme" shall mean an enzyme either covalently or non-covalently bonded to a heme (iron-containing porphyrin) prosthetic group, and which mediates the transfer of molecular oxygen mediated by iron. These enzymes may include, but are not limited to, cytochrome P450, chloroperoxidase, cytochrome c peroxidase, catalase, β carotene 15, 15-monooxygenase and horseradish peroxidase (HRP) .

As used herein, "carotenoid cleaving dioxygenase" is an enzyme that carries out the oxidative cleavage of carotenoids. Carotenoids are organic pigments that are naturally occurring in chromoplasts of plants and some other photosynthetic organisms. In humans, carotenoids such as beta-carotene are a precursor to vitamin A.

As used herein, "degrades" or "degradation" refers to enzymatic degradation of an existing compound by an enzyme.

As used herein, "retinal pigment epithelial cells", or "RPE cells" shall mean the single layer of pigmented cells just outside the neurosensory retina that nourishes retinal visual cells. The RPE cells are densely packed with pigment granules and attached to the underlying choroid and overlying retinal visual cells. (Cassin, 2001; Boyer, 2000) The retinal pigment epithelium is involved in the phagocytosis of the outer segment of photoreceptor cells and it is also involved in the vitamin A cycle where it isomerizes all trans retinol to 11- cis retinal.

As used herein, "bisretinoid" shall mean a compound which has two retinoid molecules linked together and includes A2E, A2E isomers and compounds of the all- trans-retinal dimer series, i.e., dimers of trans isomers of the retinal compound.

As used herein, "A2E" shall mean 2- (2, 6-dimethyl-8- (2 , 6 , 6- trimethyl-1-cyclohexen-l-yl) -

IE, 3E, 5E, 7E-octatetraenyl) -1- (2-hydroxyethyl) -4- (4-methyl-6- (2,6, 6-trimethyl-l-cyclohexen-l-yl) -

IE, 3E, 5E-hexatrienyl) -pyridinium, the bisretinoid compound found in lipofuscin of RPE cells which can be formed when two all- trans-retinal molecules react nonenzymatically to form all- trans-retinal dimer followed by a reaction with phosphatidylethanolamine (PE) . Alternatively, two all- trans- retinal molecules can condense with phosphatidylethanolamine to yield A2E via a multi-step pathway that includes the formation of NRPE (iV-retinylidene-phosphatidylethanolamine) , A2PE-H₂ and A2PE as intermediates.

"Administering" a compound can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, nasally, via the cerebrospinal fluid, via implant, transmucosally, transdermalIy, intramuscularly, intraocularly, topically and subcutaneousIy. The following delivery systems, which employ a number of routinely used pharmaceutically acceptable carriers, are only representative of the many embodiments envisioned for administering compositions according to the instant methods .

Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering compounds (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA' s). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone.

Oral delivery systems include tablets and capsules . These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating compounds (e.g., starch polymers and cellulosic materials) and lubricating compounds (e.g., stearates and talc) .

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid) .

Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrrolidone) . In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer .

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending compounds (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine) , preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking compounds, coating compounds, and chelating compounds (e.g. , EDTA) .

In the practice of the method, administration may comprise daily, weekly, monthly or hourly administration, the precise frequency being subject to various variables such as age and condition of the subject, amount to be administered, half-life of the compound in the subject, area of the subject to which administration is desired and the like.

Experimental Details

EXPERIMENT 1

In the first experiment described herein, an alternative treatment method for lipofuscin-associated disorder was tested. Specifically, this experiment sought to determine whether enzymes can be delivered to RPE cells for the purpose of safely degrading the bisretinoid constituents of RPE lipofuscin. In this experiment, a well known enzyme, horseradish peroxidase (HRP) was used. HRP is a heme- containing redox enzyme that functions as an excellent electrophile in oxygen transfer reactions (van Rantwijk, 2000) . HRP is one of a group of oxo-iron hem based enzymes that also include cytochrome P450, chloroperoxidase, cytochrome c peroxidase and catalase. On the basis of non- cellular and cell-based assays and by employing quantitative HPLC (high performance liquid chromatography) , MS (mass spectroscopy) , NMR (nuclear magnetic resonance) spectroscopy and immunocytochemical techniques, enzymatic degradation of A2E was demonstrated.

METHODS

Cell-Free Enzyme Assay

A2E (20 μM from a 20 mM stock in DMSO) and horseradish peroxidase (HRP type IV; 200 Units; Sigma-Aldrich Corp, St. Louis MO) were incubated at room temperature in citrate buffer (68 mM citric acid, 136 mM sodium phosphate; pH 6.5) containing 0.03% EDTA and 0.4% Tween® 80 and with and without hydrogen peroxide (H₂O₂) (2%) . Control samples included A2E incubated in the absence of HRP. After 24 and 48 hours, the reaction mixtures were extracted 3 times with chloroform/methanol (1:1) and after combining and evaporation were re-dissolved in 100% methanol for HPLC quantitation.

To test extraction efficiency when A2E was combined with HRP, mixtures of A2E alone and A2E plus HRP in buffer as described above were prepared and immediately extracted with chloroform/methanol (1:1) 3 times, combined, evaporated and re-dissolved in 100% methanol. HPLC analysis showed that the amount of A2E recovered was not reduced by the presence of HRP when the extraction was performed immediately after mixing.

As an additional control, HRP and cathepsin D activity on A2E were compared. To this end, 20 μM A2E was incubated in phosphate buffered saline (with 0.2% DMSO) containing 200 units/mL cathepsin D (from human liver; Sigma-Aldrich Corp,

St. Louis MO) or 200 units/mL HRP for 5 hours at room temperature. Subsequently, the mixture was extracted with chloroform, dried under argon and analyzed by HPLC as described below.

Cell-based assay A human adult RPE cell line (ARPE-19; American Type Culture Collection, Manassas VA) that is devoid of endogenous A2E (Parish, 1998) was cultured in 35 mm cell culture dishes (Corning NY) to confluence as previously reported (Sparrow, 2000; Sparrow, 1999). The cells were allowed to accumulate synthesized A2E (Parish, 1998) for 3 weeks from a 10 μM concentration in culture media (Zhou, 2006) that included 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) . Incubation of ARPE-19 cells with A2E at higher concentrations perturbs cell membranes (Sparrow, 1999; Sparrow, 2006). Following accumulation, the cells were allowed to quiesce for at least 3-7 days. Subsequently, HRP was delivered to the cells using a BioPORTER^® protein delivery system (Sigma-Aldrich Corp, St. Louis MO) as vehicle. Briefly, 100 ng of HRP in 40 μL serum-free Dulbecco's Modified Eagle medium (DMEM) was combined with BioPORTER^® reagent in the reaction tube provided and after mixing was incubated at room temperature for 5 minutes . After bringing the final volume of the BioPORTER^®/HRP mixture to 1 iriL with serum-free medium, the mixture was added to the DMEM-washed cultures and incubation was implemented for 4 hours at 37°C. Subsequently, FBS was added to a final concentration of 5% and incubation was continued for 20 hours. BioPORTER^®/HRP loading was repeated two additional times and two days after the final BioPORTER^®/HRP treatment, cells were harvested for HPLC analysis. Controls included cells incubated with BioPORTER^® in the absence of HRP and cells not incubated.

Immunocytochemical detection of HRP

ARPE-19 cells to which HRP was delivered via the BioPORTER^® reagent were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 and washed (3 changes, 5 minutes each) . The cultures were incubated with mouse anti-HRP antibody (Abeam, Cambridge MA; 1:100 dilution) for 2 hours at room temperature, biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame CA) and Vectastain ABC-alkaline phosphatase (Vector Laboratories) with washing between each step. Vector Red (Vector Laboratories) was used as alkaline phosphatase substrate, generating a reaction product that can be viewed with both brightfield and fluorescence microscopy.

To examine for co-localization of HRP and a lysosomal marker, ARPE-19 cells that had accumulated HRP by means of the BioPORTER^® delivery system were incubated with 50 nM LysoTracker Red (Invitrogen, Carlsbad, CA) for 30 minutes at 37°C. The cells were then fixed with 2% paraformaldehyde (Tousimis, Rockvill MD) and permeabilized with 0.2% Triton-X- 100 (Sigma-Aldrich, St. Louis MO) . After blocking with 5% normal goat serum the slide was incubated with mouse anti-HRP monoclonal antibody (Abeam, Cambridge MA) followed by DyLight 488-conjugated affinity purified goat anti-mouse IgG (Jackson Iitimunoresearch Laboratories, West Grove PA). The cells were stained with DAPI (4', 6-diamidino-2-phenylindole; Invitrogen, Carlsbad, CA) before coverslipping. Images were captured with a Zeiss multiphoton confocal microscope (LSM 510 NLO: Carl Zeiss, Heidelberg, Germany) using 488 ran excitation and 500- 550 nm emission for HRP, 543 run excitation and 565-615 ran emission for Lysotracker Red and 2-photo excitation at 780 nm for DAPI with emission at 435-485 nm.

Cell Viability Assays

Cytotoxicity was assayed by MTT (3- (4, 5-dimethylthiazol-2-yl) - 2,5-diphenyl tetrazolium bromide) a colorimetric assay (Roche Diagnostics, Basel, Switzerland) , that measures the ability of viable cells to cleave the yellow tetrazolium salt MTT to purple formazan crystals. Briefly, 20 ml of MTT reagent was added to 0.2 ml of culture medium in each well and after incubating for 4 hours 200 ml of solubilization solution was added and incubation was continued overnight . After centrifugation, supernatants were measured spectrophotometrically, a decrease in the 570 nm absorbance indicating a loss in cellular viability.

Percent of nonviable cells was also determined by labeling the nuclei of nonviable cells with Dead Red (Molecular Probes, Eugen OR) and nuclei of all cells with DAPI (Sparrow, 2002) . Values in each experiment were based on the sampling of 5 fields per well.

HPLC analysis

Samples were analyzed by reverse-phase HPLC using an Alliance System (Waters Corp, Milford MA) equipped with 2695 Separation Module, 2996 Photodiode Array Detector and a 2475 Multi λ Fluorescence Detector. For chromatographic separation, an analytical scale Atlantis^® dC18 (3 μm, 4.6 x 150 mm, Waters) column was utilized with an acetonitrile and water gradient and 0.1% trifluoroacetic acid (85-15%, 0.8 mL/min, 0-15 min; 100% acetonitrile, 0.8-1.2 mL/min 15-20 min,- monitoring at 430 ran,- 30 μL injection volume) . Extraction and injection for HPLC was performed under dim red light. Integrated peak areas were determined using Empower^® software, and picomolar concentrations were calculated by reference to an external standard of synthesized compound and by normalizing to the ratio of the HPLC injection volume versus total extract volume .

Mass spectrometry analysis

A2E (1 μL of 10 mM stock in DMSO) was added to 500 μL of DPBS buffer containing 200 units/mL of HRP and 5 μL H₂O₂. This mixture was incubated for 1 hour and 5 hours at room temperature, respectively. The HRP/A2E reaction mixtures were extracted with chloroform, dried under argon, and subjected to fast atom bombardment mass spectrometry (FAB-MS) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) . Tween® 80 was not added to reaction mixtures intended for MS since in the presence of this surfactant, multiple peaks differing in m/z 44 were observed in the mass profile.

Statistical Analysis Data were analyzed by one-way ANOVA and Newman-Keul Multiple Comparison Test (Prism, GraphPad Software, San Diego, CA) .

RESULTS

HRP Degrades A2E in a Cell -Free Assay

The study first probed for evidence that HRP could act on A2E by incubating HRP and A2E in a cell-free system, with and without H₂O₂. Shown in Figure IA is a representative overlay of HPLC chromatograms generated from various mixtures of A2E, HRP and H₂O₂. The height of the A2E and isoA2E peaks were clearly reduced in the presence of HRP and further reduction occurred with the addition of H₂O₂. Quantitation by integrating peak areas (Fig. IB) also demonstrated that after a 24-hour incubation with HRP the levels of A2E in the mixture had decreased by 40% and were down to 20% of starting levels in the presence of HRP and hydrogen peroxide. To test extraction efficiency when A2E was combined with HRP, mixture of A2E alone and A2E plus HRP in buffer were prepared and immediately extracted with chloroform/methanol (1:1) 3 times, combined, evaporated and re-dissolved in 100% methanol. HPLC analysis showed that the amount of A2E recovered in the extract was not reduced by the presence of HRP. In initial experiments Tween® 80 was included in the reaction mixtures, but it was later found that addition of this reagent was not a requisite for this enzyme-mediated reaction.

Cathepsin D is a ubiquitous lysosomal enzyme that is known to play a role in the lysosomal digestion of phagocytosed photoreceptor outer segments by the retinal pigment epithelium (Rakoczy, 2002) Thus, in additional experiments, the results obtained from incubating A2E with HRP versus with the lysosomal enzyme cathepsin D were compared. In contrast to the HPLC profile generated when A2E was incubated in cells in the absence of enzymes (Fig. 1C, top) , incubation of A2E with HRP for 5-hours at room temperature resulted in decreases in the chromatographic peak areas of both A2E and isoA2E. Moreover, the peaks attributable to A2E and isoA2E were often bifurcated after HRP incubation, suggesting that the compounds had been modified such as by enzyme activity or oxidation. No changes were observed in A2E levels when incubated with cathepsin D (Fig. 1C, bottom) .

HRP and A2E In A Cell-Based Assay

HRP was delivered to ARPE-19 cells in culture, by employing the BioPORTER^® system, an approach that uses a lipid-based reagent to encapsulate molecules, thus forming complexes that attach to negatively charged cell surfaces and are internalized. To confirm uptake of HRP, the cells were permeabilized and immunostained with antibodies to HRP and detected labelling using a chromagen that can be visualized with both fluorescence and brightfield microscopy. As shown in Figure 2A and 2B, 3 days after exposing the cells to BioPORTER^®/HRP, HRP-specific immunoreactivity was observed in association with all of the cells in the cultures.

The inventor has previously shown that when A2E accumulates in cultured RPE, the pigment is deposited into the lysosomal compartment (Sparrow, 1999). As for the fate of protein delivered by the BioPORTER^® system, it has been reported that uptake into cells leads to a endosomal, cytosolic and nuclear distribution of the carried-protein (Zheng, 2003); however, lysosomal deposition is also indicated by studies showing that the degradation of proteins delivered into the cell by BioPORTER^® system is reduced by inhibitors of lysosomal proteolytic activity (Marques, 2004). Thus, to determine whether HRP, when delivered to the cells, is also located in lysosomes, evidence is sought for co-localization of HRP and a lysosomal probe, Lysotracker Red, a membrane-diffusible acidophilic fluorophore. To this end, ARPE-19 cells that had acquired intracellular HRP were incubated with Lysotracker Red, fixed and immunostained for HRP. On examination by confocal microscopy, co-localization of HRP and Lysotracker Red was evident from the merging of red (Lysotracker) and green (HRRP) signals to yield yellow (Fig. 2 D-K) . These results indicated that a portion of the HRP taken up into the cell resided with lysosomes .

To test for the ability of the internalized HRP to degrade A2E, ARPE-19 cells were first allowed to accumulate A2E in culture over a 3 week period. After the 3 week period, the A2E was withdrawn. After another 7 days, HRP was delivered via the BioPORTER^® system. Three days after introducing HRP to the cultures, the cells were harvested and quantified A2E by HPLC (Fig. 2L) . In cells that had been incubated with BioPORTER^® and HRP, A2E levels were reduced by 40% as compared to BioPORTER^® system alone. After 7 and 14 days, the decrease was not appreciably different (range, 35-45%) . BioPORTER^® system in the absence of HRP did not confer significant changes in A2E levels (Fig. 2L) , indicating the HRP exclusively contributes to the degradation and oxidation of A2E.

Mass Spectrometric Detection of A2E Degradation

To determine whether cleavage of A2E followed from the oxidation, samples of A2E that had been incubated with HRP for 1 and 5 hours were subjected to mass spectrometry including FAB-MS, a relatively soft ion source technology that produces primarily intact charged molecules, and APCI-MS. Accordingly, FAB-MS spectra (Figs. 3B and C) generated from mixtures of A2E and HRP revealed several molecular ion peaks that were not present in the sample of A2E incubated in the absence of HRP. (Fig. 3A) Specifically, in addition to a molecular ion peak at a mass-to-charge ratio (m/z 592) attributable to A2E (structure shown in Fig. 3D), four lower mass peaks (m/z 312, 342, 380 and 530) and three higher (m/z 619, 637 and 646) mass peaks were prominent. The peak at m/z 638 was readily detectable in samples incubated for 1 hour but 4 hours later was barely visible, which can be a indication that further degradation of this product had occurred. Some of these peaks were also observed in spectra derived by APCI-MS. It should be mentioned that in the untreated sample of A2E examined by FAB- MS, molecular ion peaks corresponding to m/z 288, 306, 338 and 391 constitute inherent background since all of these peaks were present when methanol only was injected into the HPLC. The peak at m/z 608 {m/z 592 +16) is a frequently observed photooxidized product of A2E.

NMR Detection of Aldehyde Moiety

On the basis of the inventor's previous finding that opening of epoxide rings and cleavage at sites of photooxidation or MCPBA oxidation of A2E can lead to the formation of aldehyde groups (29), the inventor also surmised that enzyme-catalyzed oxidation of A2E at double bonds along the polyene chains followed by cleavage would generate aldehyde moieties . To test for this mechanism, the reaction mixture of A2E and HRP by ¹H NMR spectroscopy was analyzed. Accordingly, after incubating A2E with HRP at 37⁰C for 5 hours, ¹H NMR revealed a new resonance at 9.0-9.5 ppm indicative of a specific aldehyde proton signal that was not present with untreated A2E (Fig. 4) .

HRP-Mediated Degradation of Intracellular A2E Does Not Impair Cell Viability

Finally, the health of ARPE-19 cells in the presence of HRP mediated A2E cleavage was assayed. In this experiment HRP was delivered via the BioPORTER^® system to ARPE-19 cells that had accumulated A2E in culture. Two days after introducing HRP to the cultures, A2E was quantified by HPLC and in companion cultures assayed cell viability by MTT assay. In cells that had been incubated with BioPORTER^® and HRP, A2E levels were reduced by 40% versus incubation with BioPORTER^® only. A concomitant loss of cell viability was not observed, since MTT absorbance readings in cultures that had accumulated A2E versus cultures that had accumulated both A2E and HRP were not significantly different (Fig. 5A) . Similarly, loss of cell viability was not observed with a fluorescence assay that detects dead cells (Fig. 5B) DISCUSSION

This first set of experiments showed that A2E, compound that contains multiple C=C functionalities, can serve as a substrate for HRP-mediated degradation. HRP derived from the root of horseradish (Armoracia ruεticana) , is one of a group of oxo-iron heme-based enzymes that also includes cytochrome P450, chloroperoxidase, cytochrome c peroxidase and catalase. Like other metalloenzymes, HRP utilizes iron ions in a high valence state to store electrons that are used to oxidize substrates (Derat, 2006). The active site of HRP is an iron- containing porphyrin (heme) - that in the resting state consists of Fe(III) embedded in the central nitrogen-bordered (N4) cavity of the heterocyclic molecule (porphyrin) .

HRP typically acts as an electron sink epoxidizing double bonds. Indeed, the affinity of HRP for C=C double bonds in small molecules (Kumar, 2005) would explain the ease with which this electrophile degraded A2E. Conversely, the topology of the protein pocket hinders the access of some substrates to the oxy-ferryl centre of HRP and it is likely for this reason that the ability of HRP to hydroxylate at C-H bonds is limited

(Veitch, 2004) . It has been reported that HRP can also bind molecular oxygen and convert it to hydrogen peroxide (de

Visser, 2007) . Such a mechanism could explain HRP-mediated A2E oxidation in the above assays even in the absence of exogenous H₂O₂.

Epoxidation of A2E by HRP could occur at any number of the C=C double bonds along the side-arms of the molecule. At these epoxidation sites, opening of the C-O bond and cleavage of the C-C bond could generate any number o products with shortened side-arms. Moreover, it has previously been noted that HRP- mediated epoxidation is frequently followed by the formation of aldehydes (van Rantwijk, 2000) . Thus it is not surprising that it was bound by ¹H NMR, that a series of aldehydes are derived from cleavage of double bonds in A2E following HRP- associated oxidation. In addition to catalyzing epoxidation at double bonds (Kumar, 2005) , HRP can remove a single electron from a substrate molecule (Fig. 6A) ; acting on A2E in this way, HRP would convert the ethyl alcohol moiety extending from the pyridinium ring into an aldehyde group (Figure 6) . The active oxidant is expected to be compound I, an iron (IV) oxo complex with porphyrin IX (Por) as radical cation (Por^*+—Fe^IV=O) . HRP- catalyzed oxidation of the ethyl alcohol (Fig. 6 A, II, red) moiety of A2E would involve the transfer of a hydrogen atom to the ferryl oxygen of the iron oxo complex (compound I) , accompanied by formation of a α-hydroxy carbon radical and the iron hydroxy complex (Por-Fe^IV-OH) (compound II) (Fig. 6 A, II, path a) . This could lead to the hydrated form (Fig. IA, II, blue) of the ethyl aldehyde moiety directly (Fig. 6 A, II, path b) or stepwise formation (Fig. IA, II path c and d) (Baciocchi, 1999; Baciocchi, 2001) via the intermediate α- hydroxyethylate cation with subsequent dehydration yielding an aldehyde moiety (Fig. 6 A, II, green) extending from the pyridinium ring.

The MS profile exhibited peaks with mass greater than 592 (m/z 620, 638, and 648) that were indicative of adduct formation. Reaction amongst HRP-generated fragments could produce a complex mixture of higher mass adducts but since FAB-MS detection is restricted to charged compounds, only adducts that included the pyridinium moiety were observed in this analysis. Possible mechanisms for the formation of HRP- associated cleavage and adduction products are summarized in Fig. 6. Following epoxidation at double bonds, opening at the C-O bond and cleavage of the C-C bond could result in the release of HC=O radicals (Fig. 6B, I) . Subsequent reaction of the HC=O radical with the α-hydroxy carbon radical intermediate could generate the fragment ion at m/z 620. Furthermore, one-electron elimination of the hydroxyl group on the m/z 620 species would give rise to an intermediate radical that could bind a second molecule of the HC=O radical to afford the fragment ion at m/z 648 (Fig. 65, II) . The peak at m/z 638 could be generated following epoxidation of the cleavage product at m/z 620. Subsequent ring opening at the C-O bond would provide two free electrons that could accept a hydrogen atom and hydroxyl radical (Fig. 6C) . Nevertheless, this arrangement would be unstable and the elimination of one molecule of water would permit reversion to the double bond; dehydration of the m/z 638 compound with reformation of m/z 620 might explain the above FAB-MS data indicating that the signal at m/z 638 was observed in 1-hour HRP/A2E incubation mixtures, whereas four hours later it had almost disappeared.

In addition to peaks with m/z greater than the mass of A2E

(m/z 592) , other peaks were observed with m/z species that were smaller (m/z 312, 340 and 380) and thus indicative of A2E cleavage products (Fig 3 and Fig. 6 D) . For the m/z 312 and 340 peaks, since epoxidation could occur at any of the double bonds along the side-arms of the molecule, as many as eight candidate structures are possible; two of these structures are shown in Fig. 3E-F and Fig. SD. With epoxidation occurring at the 7,8 and 9'10' double bonds as well as in a third position, opening of the epoxide ring at the C7/10'—O bond and cleavage of the C7/C9'—C8/C10' bond would give rise to an intermediate with an aldehyde moiety at the end of the short arm and a methine radical at the end of the long arm. The intermediate radical could bind two hydrogen atoms to generate the fragment at m/z 312, or could in step-wise fashion bind one hydrogen atom and then one molecule of the HC=O radical to afford the fragment ion at m/z 340. Alternatively, the m/z 312 product could form by the same mechanism but with epoxidation beginning at the 7' 8' and 9, 10 double bonds. The inventor has previously showed, with corroboration by NMR spectroscopy, that following the formation of a 5, 6 epoxide along the side- arms of A2E, rearrangement to a 5,8 furanoid occurs (Dillon, 2004; Jang, 2005). Thus an exclusive candidate for the aldehyde-bearing m/z 380 species is likely to be a fragment that includes a 5, 8 furanoid as presented in Fig. 3 and Fig. SD. At this time, a structure for the peak at m/z 530 cannot be determined, but it is expected that it could be an adduct, the formation of which would involve reactions amongst radical bearing cleavage products followed by complex rearrangement.

The mechanisms involved in HRP-mediated A2E cleavage appear to be different from those observed with A2E photooxidation and cleavage. In particular, photooxidation of A2E occurs primarily via singlet oxygen addition at C=C bonds to form endoperoxides , cleavage of which generates O-centered radicals that accept hydrogen atoms to become nonradical species . In the case of HRP-mediated epoxidation of A2E, ring-opening and cleavage of the C-C bond would result in the formation of aldehyde carbon-centered radicals (HC=O) that would immediately seek out other radicals with which to form stable compounds. As discussed above, reaction of these cleavage products likely explains the generation of higher mass adducts. Another difference between HRP-mediated versus photooxidation, is that cell death accompanies A2E photooxidation and cleavage yet cell death was not observed in the presence of HRP-mediated A2E degradation. Although details are not understood, it is expected that the mechanistic differences account for the absence of cell death under the latter conditions.

In summary, it was found that HRP can serve to degrade the bisretinoid A2E. As a form of medical bioremediation (de Grey, 2005) , this approach is similar to the use of microbial catabolic enzymes to degrade 7-ketocholesterol, an oxidized derivative of cholesterol considered to be involved in that pathogenesis of atherosclerosis (Mathieu, 2008) HRP has several auspicious properties, including good stability at 37⁰C, high activity at neutral pH and a lack of inherent toxicity, that could facilitate its harnessing for clinical application. HRP can also be conjugated to antibodies or other cell-recognition molecules (Veitch, 2004) . For example, it has been suggested that tumor-targeted HRP could be employed together with a pro-drug to generate cytotoxic products for use in cancer therapy (Folkes, 2002). There is also interest in using HRP as a biocatalyst in the commercial production of drugs. Towards these ends, the HRP gene has been synthesized and expressed in Escherichia coli and the chemistry of HRP has been successfully modified via site- directed mutagenesis (Smith, 1990) .

EXPERIMENT 2

In the second set of experiments described herein, an alternative treatment method for lipofuscin-associated disorder is tested. Specifically, these experiments seek to determine whether carotenoid cleaving dioxygenase can be delivered to RPE cells for the purpose of safely degrading the bisretinoid constituents of RPE lipofuscin. On the basis of non-cellular and cell-based assays and by employing quantitative HPLC (high performance liquid chromatography) , MS (mass spectroscopy) , NMR (nuclear magnetic resonance) spectroscopy and immunocytochemical techniques, enzymatic degradation of the bisretinoid constituents of RPE lipofuscin is demonstrated.

RESULTS

It was found that carotenoid cleaving dioxygenase can serve to degrade the bisretinoid constituents of RPE lipofuscin.

EXPERIMENT 3

Gene and drug-based therapies are being investigated as treatments for forms of macular degeneration characterized by excessive accumulation of bisretinoid lipofuscin compounds in retinal pigment epithelial (RPE) cells of the eye. However, neither of these approaches can reverse the accumulation of these bisretinoids once it has already occurred. The experiment described below examined the feasibility of degrading these compounds by enzyme activity. It was found that horseradish peroxidase (HRP) can cleave A2E, a well-known lipofuscin compound. In a cell-free assay it was found that incubation of A2E with HRP and hydrogen peroxide (H₂O₂) lead to diminished levels of A2E as measured by HPLC. When HRP was delivered to ARPE-19 cells in culture, the enzyme was observed by immunocytochemistry and confocal imaging to co-localize with a lysosomal probe. Subsequently, in cells that had accumulated A2E followed by HRP, HPLC quantitation revealed a ~ 40% reduction in A2E versus cells not receiving HRP. To facilitate the detection of HRP-associated cleavage products, bromine-tagged A2E (A2E-Br) was utilized with ultra performance liquid chromatography (UPLC) coupled to electrospray ionization-mass spectrometry (ESI-MS) to observe ion peaks indicative of A2E-Br oxidation (m/z 746/748 and 762/764) and cleavage (m/z 610/612, 570/572 and 544/546). Moreover, structures of the cleavage products were elucidated with the assistance of ¹⁸O-labelling, UV-visible absorbance and ¹H NMR spectroscopy to detect aldehyde signals. These findings indicate that the RPE lipofuscin pigment A2E can serve as a substrate for HRP-mediated epoxidation, hydroxylation and degradation.

Synthesis of Compounds

A2E (Parish, 1998) and bromine tagged-A2E (A2E-Br) (Wu, 2010) were synthesized as described. The structures are presented in Figure 7.

Cell-free enzyme assay

A2E (20 μM from a 10 mM stock in DMSO) and horseradish peroxidase (HRP type IV; 100 Units; Sigma-Aldrich Corp, St. Louis MO) were incubated at room temperature in 500 μL of citrate buffer (68 mM citric acid, 136 μM sodium phosphate; pH 6.5) containing 0.03% EDTA and 0.4% Tween® 20 and with and without hydrogen peroxide (H₂O₂, 0.2%). Control samples included A2E incubated in the absence of HRP and H₂O₂. After 24 and 48 hours, the reaction mixtures were subjected to HPLC quantitation. In other experiments, 20 μM A2E was incubated in phosphate buffered saline (with 0.2% DMSO) containing 200 units/mL cathepsin D (from human liver; Sigma-Aldrich Corp, St. Louis MO) or 200 units/mL HRP in the presence of 1% H₂O₂ for 5 hours at room temperature. Subsequently, the mixture was extracted with chloroform, dried under argon and analyzed by HPLC as described below.

Cell-based assay, Immunocytochemical detection of HRP, Cell viability assays and HPLC quantitation

See methods as described in Experiment 1.

UPLC-MS analysis

A2E-Br (3 μL of 10 mM stock in DMSO) , which imparted enhanced sensitivity, or A2E was added to 500 μL of water containing 200 units/mL of HRP and 1 μL H₂O₂. This reaction mixture was incubated for 3 h and 18 h at room temperature and then subjected to UPLC-MS using a Waters SQD single quadrupole mass spectrometer that was coupled on-line to a Waters Acquity UPLC system (Waters, New Jersey, USA) with PDA eλ detector, sample manager and binary solvent manager. The mass spectrometer was equipped with ESCi multi-mode ionization and ion trap analyzer operating in full scan mode from m/z 200 ~ 1200. For compound elution, an Acquity BEH C18 (1.7um, 2.1x50 mm) reverse phase column was used for the stationary phase and for the mobile phase a gradient of acetonitrile in water with 0.1% formic acid: 50-70% acetonitrile (0-3 min) ; 70-100% acetonitrile (3- 30 min) with a flow rate of 0.5 ml/min) . Detection at 430 ran was by photodiode array.

¹⁸O-labelled assay

A2E-Br (20 μM) was incubated in 500 μL of ¹⁸O-labelled water (H₂ ¹⁸O, 99 atom% ¹⁸O, Sigma-Aldrich) (with 0.2% DMSO) containing 200 units/mL HRP (type IV; Sigma-Aldrich Corp, St. Louis MO) in the presence of 5 μL of ¹⁸O-labelled hydrogen peroxide (H₂ ¹⁸O₂, 90 atom% ¹⁸O, Sigma-Aldrich) for 18 hours at room temperature. Subsequently, the 10 μL of mixtures were analyzed by UPLC-MS as described above.

Statistical analysis Data were analyzed by one-way ANOVA and Newman-Keul Multiple Comparison Test (Prism, GraphPad Software, San Diego, CA) .

RESULTS

HRP degrades A2E in a cell-free assay

The inventor first probed for evidence that HRP can act on A2E by incubating HRP and A2E in a cell-free system with and without H₂O₂. Shown in Fig. 8A-D are representative HPLC chromatograms generated from various mixtures of A2E, HRP and H₂O₂. Quantitation by integrating peak areas (Fig. 8E) demonstrated that after a 24-hour incubation with HRP and H₂O₂, levels of A2E in the mixture were reduced by 75% relative to starting levels. HRP or H₂O₂ alone did not diminish A2E (Fig. 8B, C, E) . Cathepsin D is a ubiquitous lysosomal enzyme that is known to play a role in the lysosomal digestion of phagocytosed photoreceptor outer segments by RPE cells (Rakoczy, 2002). In contrast to the effects observed with HRP and H₂O₂ wherein incubation for 5 hrs resulted in decreases in A2E levels (Fig. 9, middle), no changes were observed in A2E when incubated with cathepsin D (Fig. 9, bottom). Interestingly, the peaks attributable to A2E and isoA2E were often bifurcated after HRP incubation, suggesting that the compounds had been modified, such as by oxidation.

HRP and A2E In A Cell-Based Assay

See Experiment 1 Results .

UPLC-MS detection of A2E degradation

Since quantitative HPLC analysis indicated that A2E could serve as a substrate for HRP, the inventor also sought to determine whether the enzyme activity resulted in both oxidation and cleavage of A2E. To facilitate the detection of oxidation and cleavage products, A2E-Br was used. The bromine tag in this analog imparts a characteristic isotope pattern (two peaks of similar intensity separated by 2 m/z units due to isotopes ⁷⁹Br and ⁸¹Br) that makes the signal in the mass spectrum more readily distinguishable. Importantly, molecular ions featuring the bromine isotope peaks could be recognized as cleavage products that contained the pyridinium head group of A2E-Br. Accordingly, UPLC chromatograms of A2E-Br incubated with HRP and H₂O₂ for 3 hrs exhibited four prominent peaks (Fig. 1OB: peaks b, c, d and e) and several minor peaks (Fig. 1OB, inset e.g. Fl-4) that were not present in the sample of A2E-Br incubated in the absence of HRP (Fig. 10A) . These peaks eluted earlier than A2E-Br (Fig. lOA-C, peak a) and thus are consistent with oxidized and cleaved products that are more polar than the parent compound (Wu, 2010) . Subsequent analysis of the prominent UPLC peaks (Fig. 1OB: peaks b-e) by coupled mass spectrometry revealed ion signals with m/z +16 (b:746/748, c:746/748) and m/z +2x16 (d:762/764) and e:762/764) (Fig. IQE-H)₁ relative to A2E-Br {m/z 730/732) (Fig. 10D). These peaks were indicative of the addition of oxygen atoms with the formation of monooxo-A2E-Br (b and c) and bisoxo-A2E- Br (d and e) . Interestingly, following an 18-hour incubation period, the heights of the peaks corresponding to these oxidized forms of A2E-Br were significantly diminished, suggesting cleavage of these species (Fig. 10C).

The inventor anticipated that oxidation on the long arm of A2E-Br would alter the longer wavelength absorbance (absorbance maximum, λ_max 446 run) while oxidation on the short arm would change the shorter wavelength absorbance (λ_max 337 run) of A2E-Br. For instance, the loss of one carbon-carbon double bond through epoxidation is responsible for a UV-visible absorbance blue shift of 25-40 run. Accordingly, since the UV-visible absorbances of two of the oxidized products (Fig 1OB: peak b, λ_max 327 and 444 nm,- and Fig. 1OB: peak d, λ_max 334 and 441 nm) were similar to that of A2E-Br (λ_max 337 and 446 nm) (Fig. 10A) it is likely that these compounds are characterized by methyl hydroxylation (see Fig. 13) rather than C=C bond epoxide since hydroxylation of a methyl moiety is unlikely to have an appreciable effect on UV-visible absorbance. In contrast, two other oxidized products (λ_max 338 and 410 nm, Fig 1OB: peak c; and λ_max 334 and 420 nm, Fig. 1OB: peak e) presented with UV-visible absorbance spectra whereby the absorbance of the long arm exhibited a blue shift of 36 nm (from 446 nm in A2E-Br to 410 nm) ; this shift is indicative of the loss of one carbon-carbon double bond via epoxidation. Interestingly, the second of these oxidized products was identified as a bisoxo-A2E-Br (λ_max 334 and 420 nm) with the absorbance generated on the long-arm exhibiting a blue shift of 26 nm (from 446 nm in A2E-Br to 420 nm) , indicative of the loss of one C=C bond via epoxidation together with hydroxylation of one methyl moiety.

Several minor peaks (Fig. 10B) eluted earlier than the oxidized products in the UPLC chromatogram; four of these were examined (Fig. 1OB, inset: F1-F4; Fig. HA) . As shown in Fig. WE-G, extracted ion chromatograms with selection for mass-to- charge ratios (m/z) 610, 570 and 544 revealed four (4) prominent ion chromatographic peaks, that on the basis of retention time corresponded to UPLC peaks F1-F4 (Fig. HA) . These ion chromatographic peaks were not observed in samples of A2E-Br incubated in the absence of HRP (Fig. 1IB-D) . Analysis- of UPLC peaks F1-F4 by ESI-MS (Fig. 11H-K) corroborated the presence of ion signals at m/z 610/612 (Fl and F2), 570/572 (F3), and 544/546 (F4) ; all were lower in mass than m/z 730/732 attributable to A2E-Br (Fig. 10D) and thus were indicative of HRP-induced cleavage products. These cleavage products of A2E were not visible after incubation for 18 hours (Fig. 10C) probably because of further HRP-mediated degradation. It is also worth mentioning here that cleavage of untagged A2E (Fig. 7) by the HRP-H₂O₂ system takes place in the same fashion as A2E-Br since the inventor also detected the product ions at m/z 472, 432, and 406 that matched cleavage products generated with A2E-Br (m/z 610/612, 570/572 and 544/546) after the mass of bromine tag was accounted for (data not shown) .

¹⁶O-¹⁸O exchange To further elucidate reaction mechanisms involved in HRP- mediated A2E cleavage, the inventor utilized oxygen isotope labeling to determine the number of oxygen atoms added to the cleavage products. To this end, the inventor incubated A2E-Br with HRP in ¹⁸O-labelled water (H₂ ¹⁸O) in the presence of ¹⁸O- labelled hydrogen peroxide (H₂ ¹⁸O₂) for 18 hrs, and monitored relevant cleavage products by UPLC-MS. As depicted in Fig. 12, selected ion monitoring at m/z 614 and 574 to detect the heavy isotope (+2 m/z units) revealed prominent ion chromatographic peaks, F2a (Fig. 12A) and F3a (Fig. 12B), indicative of the inclusion of ¹⁸O in the cleavage product. Examination of these two ion chromatographic peaks (F2a and F3a) by ESI-MS divulged m/z signals at 612/614 for F2a (Fig. 12C) and 572/574 for F3a (Fig. 12D) . Again the +2 m/z units {m/z 610/612 for F1/F2 and m/z 570/572 for F3), verified the presence of one ¹⁸O atom in each A2E-Br cleavage product. Since the ¹⁶O-¹⁸O exchange does not alter retention time, the inventor could readily identify compounds F2a (Fig. 12C, inset) and F3a (Fig. 12D, inset) as being the same as F2 and F3, respectively. ¹⁸O-labelled species (m/z 612/614 and 546/548) corresponding to cleavage products Fl and F4 were not observed probably because heavy isotope H₂ ¹⁸O₂ oxidizes with less efficiency than H₂O₂ and the ratio of H₂ ¹⁸O₂ to H₂O₂ in the mixture was 90:10.

NMR detection of aldehyde moiety

The inventor surmised that enzyme-catalyzed oxidation of A2E- Br at double bonds along the polyene chains followed by cleavage would in some cases generate aldehyde-bearing products . Thus to corroborate the generation of aldehyde moieties, the inventor also analyzed the reaction mixture of A2E and HRP in the presence of H₂O₂ by ¹H NMR spectroscopy. Accordingly, after incubating A2E with HRP and H₂O₂ at 37⁰C for 5 hrs, ¹H NMR revealed a new resonance at 9.0-9.5 ppm indicative of a specific aldehyde proton signal that was not present with untreated A2E (Fig. 4) .

Assignment of structures Structures were proposed for the cleavage products F1-F4 (Fig. 1OB, inset; Fig. HA, H-K, insets) on the basis of m/z values together with data from the NMR and oxygen isotope studies indicating that some cleavage products would bear a single oxygen atom residing within an aldehyde moiety. In addition, the inventor could be certain that the bromine isotope peaks signalled the presence of the pyridinium ring in the A2E-Br fragment. Two of the ion peaks, specifically Fl and F2 (m/z 610/612) , presented with the same mass but different retention times. Examination of UV-visible absorbance spectra (Fig. 11) (Jang, 2005), revealed that the Fl-cleavage product exhibited a single absorbance maxima (λ_max) of 338 nm indicative of an intact short arm while the absence of the longer wavelength absorbance of A2E-Br (λ_max -446 nm) was a sign that cleavage had occurred on the long arm. Conversely, the F2 fragment retained the longer wavelength absorbance (λ_max 443 nm) indicating that the fragment consisted of the long-arm of A2E-Br. Since the UV-visible absorbance spectra of F3 and F4 were characterized by λ_max (440 and 443 nm, respectively) similar to the 446 nm absorbance attributable to the long arm of A2E-Br, it was clear that the short-arms had been cleaved while the long-arms were intact .

Effects of HRP-mediated degradation of intracellular A2E on cell viability

The inventor also assayed the health of ARPE-19 cells in the presence of HRP-mediated A2E cleavage. In these experiments the inventor took ARPE-19 cells that had accumulated A2E in culture and delivered HRP via the BioPORTER^® system. Three and fourteen days after introducing HRP to the cultures, the inventor quantified A2E by HPLC and in companion cultures assayed cell viability by MTT assay. In cells that had been incubated with BioPORTER^® and HRP, A2E levels were reduced by ~ 40% versus incubation with BioPORTER^® only. A concomitant loss of cell viability was not observed within 3 days, since MTT absorbance readings in cultures that had accumulated A2E versus cultures that had accumulated both A2E and HRP were not significantly different (Fig. 5C) . After 3 days, loss of cell viability was also not observed with a fluorescence assay

(Fig. 5A) . However, after 14 days, cell viability by MTT assay was decreased by 10.5% as compared to cultures receiving BioPORTER^® without HRP (Fig. 5C).

DISCUSSION

The inventor has shown here that A2E, a compound that contains multiple C=C functionalities, can serve as a substrate for HRP-mediated oxidation and degradation and the inventor has proposed a mechanism by which this would occur (Fig. 13). The inventor has characterized the structures of 4 of the largest degradation products; continued decline with time in the peak intensities attributable to these products indicated that enzyme-mediated cleavage was progressive. In the acellular assays, the addition of H₂O₂ was a co-requisite to HRP-mediated A2E cleavage. However, in the cell-based experiments since exogenous H₂O₂ was not added, it is likely that mitochondrial metabolism served as a source of H₂O₂. Of course HRP is an enzyme that RPE cells would not normally produce. It was observed that 3 days after the introduction of HRP to the cells, A2E levels were reduced by approximately 40%. At longer intervals (7 and 14 days) no further decrease was observed. This limited period of degradation may reflect the life-time of the enzyme under intracellular conditions and could be an advantage since persistence of the therapeutic enzyme longer than required may be undesirable. Measured as a decline in amounts of A2E, the therapeutic effect observed with small molecules that target the visual cycle in mouse models, have ranged from 30-60% (Radu, 2005; Radu, 2003; Maiti, 2006; Maeda, 2009).

The active site of HRP is an iron-containing porphyrin (heme) that in the resting state consists of Fe (III) embedded in the central nitrogen-bordered (N4) cavity of the heterocyclic molecule (porphyrin) (Derat, 2006). Like other metalloenzymes, the HRP catalytic cycle involves two active species (Compound I, Cpd I; and Compound II, Cpd II) that possess high valence iron ions storing two and one oxidation equivalents, respectively (de Visser, 2003; Derat, 2006) (Fig. 13). Given that HRP has a particular affinity for C=C double bonds in small molecules (Kumar, 2005) , it is not surprising that HRP can act on A2E. Two oxygen-transfer reactions of the enzyme HRP are C-H hydroxylation of alkanes and C=C epoxidation of alkenes (Kumar, 2005; de Visser, 2002). Since the side-arms of A2E consist of alternating carbon-carbon double bonds together with methyl groups, it follows that HRP/H₂O₂ mediated oxidation of A2E would involve both hydroxylation of methyl groups and epoxidation of C=C double bonds. It has previously been noted that HRP-mediated epoxidation is frequently followed by the formation of aldehydes (van Rantwijk, 2000) . Thus it is not surprising that the inventor found by ¹H-NMR, that a series of aldehydes are generated upon cleavage of carbon-carbon double bonds in A2E following HRP-associated oxidation and degradation. The formation of aldehyde moieties (Fig. 13) is also supported by the ¹⁸O-labeling experiments (Fig. 12).

It was found that both of the acyclic polyenic side-arms of A2E-Br are susceptible to HRP-mediated epoxidation and degradation. For the ions at m/z 610/612 attributable to the Fl and F2 cleavage products (Fig. 11) , epoxidation occurred at the C7, C8 (short-arm) or C7',C8' (long-arm) carbon-carbon double bonds. Following opening and hydration of the epoxide ring, a diol would have formed followed by enzymatic cleavage of the C-C single bond to yield two alcoholic radicals. Subsequently, two-electron oxidation involving Cpd I and Cpd II would convert these two radicals to a pair of aldehyde- bearing products (Fig. 13) (Ann, 1997), Fl and 2,6,6- trimethylcyclohex-1-enecarbaldehyde. The latter is a small and volatile aldehyde-bearing compound that would be difficult to detect by direct LC-MS without trapping (Wu, 2010) . The HRP- catalyzed pathways for formation of ion signals at m/z 570/572 and 544/546 attributable to cleavage products F3 and F4, respectively (Fig. 11), were similar to that of Fl and F2. The minor differences were that epoxidation sites of the parent compound were at the C9-C10 and C11-C12 double bonds, respectively.

The mechanisms involved in HRP-mediated A2E cleavage, as described above, appear to be different from those the inventor observed with A2E cleavage following photooxidation. In particular, photooxidation of A2E occurs primarily via singlet oxygen addition at C=C bonds to form endoperoxides , cleavage of which generates aldehyde or methylketone-bearing fragments (Wu, 2010) . In the present work the inventor focused on A2E, just one of the bisretinoids of RPE lipofuscin. Nevertheless, these findings are applicable to the other lipofuscin bisretinoids the inventor has characterized (discussed above) since all of these compounds present with polyene side-arms that the inventor has shown here, can be degraded by certain enzymes. Bisretinoids appears to be the major constituents of RPE lipofuscin since when they do not form, the autofluorescence originating in RPE lipofuscin is absent (Katz, 2001; Lorenz, 2004).

In summary, the inventor has found that HRP can serve to degrade the bisretinoid A2E. The HRP gene has been synthesized and expressed in Escherichia coli and the chemistry of HRP has been successfully modified via site-directed mutagenesis (Smith, 1990) .

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Claims

ClaimsWhat is claimed is:

1. A method for reducing the amount of lipofuscin compounds present in a cell which comprises contacting the cell with an amount of an enzyme sufficient to directly or indirectly degrade the amount of lipofuscin compounds, so as to reduce the amount of lipofuscin compounds, wherein the enzyme is a carotenoid cleaving dioxygenase or an oxo-iron heme-based enzyme.

2. The method of claim 1, wherein the cell is an eye cell, brain cell, nerve cell, kidney cell, liver cell, heart muscle cell, or adrenal cell.

3. The method of claim 2, wherein the cell is an eye cell.

4. The method of claim 3 , wherein the eye cell is a retinal pigment epithelial (RPE) cell.

5. The method of any one of claims 1-4, wherein the lipofuscin compounds comprise a bisretinoid.

6. The method of claim 5, wherein the bisretinoid is 2- (2, 6- dimethyl-8- (2,6, 6-trimethyl-l-cyclohexen-l-yl) -

IE, 3E, 5E, 7E-octatetraenyl) -1- (2-hydroxyethyl) -4- (4- methyl-6- (2,6, 6-trimethyl-l-cyclohexen-l-yl) -

IE, 3E, 5E-hexatrienyl) -pyridinium, its isomer, or its precursor.

7. The method of claim 5, wherein the bisretinoid is an all- trans-retinal compound or dimer of an all- trans-retinal compound.

8. The method of any one of claims 1-7, wherein the enzyme is a carotenoid cleaving dioxygenase.

9. The method of any one of claims 1-7, wherein the enzyme is an oxo-iron heme-based enzyme.

10. The method of claim 9, wherein the oxo-iron heme-based enzyme is a cytochrome P450, chloroperoxidase, cytochrome c peroxidase, catalase, or horseradish peroxidase.

11. The method of claim 10, wherein the oxo-iron heme-based enzyme is horseradish peroxidase.

12. A method for treating a subject suffering from a lipofuscin-associated disorder which comprises administering to the subject a composition comprising an amount of an enzyme sufficient to directly or indirectly degrade lipofuscin compounds associated with the disorder in cells of the subject, so as to thereby treat subject's the lipofuscin-associated disorder, wherein the enzyme is a carotenoid cleaving dioxygenase or an oxo-iron heme- based enzyme.

13. The method of claim 12 , wherein the enzyme is a carotenoid cleaving dioxygenase.

14. The method of claim 12, wherein the enzyme is an oxo-iron heme-based enzyme .

15. The method of claim 14, wherein the oxo-iron heme-based enzyme is a cytochrome P450, chloroperoxidase, cytochrome c peroxidase, catalase, or horseradish peroxidase.

16. The method of claim 15, wherein the oxo-iron heme-based enzyme is horseradish peroxidase.

17. The method of any one of claims 12-16, wherein the lipofuscin-associated disorder is age-related macular degeneration, Stargardt ' s disease, Best disease, lipofuscinoses, Alzheimer's disease, or Parkinson's disease.

18. The method of claim 17, wherein the lipofuscin-associated disorder is age-related macular degeneration.

19. The method of claim 17, wherein the lipofuscin-associated disorder is Stargardt ' s disease.

20. The method of claim 17, wherein the lipofuscin-associated disorder is Best disease.

21. The method of any one of claims 12-20, wherein the cells are eye cells, brain cells, nerve cells, kidney cells, liver cells, heart muscle cells, or adrenal cells.

22. The method of claim 21, wherein the cells are eye cells.

23. The method of claim 22, wherein the eye cells are retinal pigment epithelial cells.

24. The method of any one of claims 12-23, wherein the lipofuscin compounds comprise a bisretinoid.

25. The method of claim 24, wherein the bisretinoid is 2- (2, 6-dimethyl-8- (2,6, 6-trimethyl-l-cyclohexen-l-yl) -

IE, 3E, 5E, 7E-octatetraenyl) -1- (2-hydroxyethyl) -4- (4- methyl-6- (2,6, 6-trimethyl-l-cyclohexen-l-yl) -

IE, 3E, 5E-hexatrienyl) -pyridinium, its isomer, or its precursor.

26. The method of claim 24, wherein the bisretinoid is an all- trans-retinal compound or dimer of an all- trans- retinal compound.