Pharmacotherapy for metabolic and cellular stress in degenerative retinal diseases.

Retinal photoreceptors continually endure stresses associated with prolonged light exposure and the metabolic demands of dark adaptation. Although healthy photoreceptors are able to withstand these stresses for several decades, the disease-affected retina functions at a reduced capacity and is at an increased risk for dysfunction. To alleviate cellular and metabolic stressors in degenerative retinal diseases, a new class of drugs that modulate the metabolic activity of the retina have been developed. A clinical candidate in this class (emixustat) has been shown to reduce retinal pathology in various animal models of human retinal disease and is currently under clinical study. Here, we describe the pharmacological properties of emixustat, its mechanisms of action, and potential for use in the treatment of specific retinal diseases.

Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets ß-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1.

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide's various therapeutic uses will begin to resolve the pleotropic nature of this compound.

Investigation of oral fenretinide for treatment of geographic atrophy in age-related macular degeneration.

BACKGROUND: Excessive accumulation of retinol-based toxins has been implicated in the pathogenesis of geographic atrophy (GA). Fenretinide, an orally available drug that reduces retinol delivery to the eye through antagonism of serum retinol-binding protein (RBP), was used in a 2-year trial to determine whether retinol reduction would be effective in the management of geographic atrophy. METHODS: The efficacy of fenretinide (100 and 300 mg daily, orally) to slow lesion growth in geographic atrophy patients was examined in a 2-year, placebo-controlled double-masked trial that enrolled 246 patients at 30 clinical sites in the United States. RESULTS: Fenretinide treatment produced dose-dependent reversible reductions in serum RBP-retinol that were associated with trends in reduced lesion growth rates. Patients in the 300 mg group who achieved serum retinol levels of ≤ 1 µM (≤ 2 mg/dL RBP) showed a mean reduction of 0.33 mm in the yearly lesion growth rate compared with subjects in the placebo group (1.70 mm/year vs. 2.03 mm/year, respectively, P = 0.1848). Retinol-binding protein reductions <2 mg/dL correlated with further reductions in lesion growth rates (r = 0.478). Fenretinide treatment also reduced the incidence of choroidal neovascularization (approximately 45% reduction in incidence rate in the combined fenretinide groups vs. placebo, P = 0.0606). This therapeutic effect was not dose dependent and is consistent with anti-angiogenic properties of fenretinide, which have been observed in other disease states. CONCLUSION: The findings of this study and the established safety profile of fenretinide in chronic dosing regimens warrant further study of fenretinide in the treatment of geographic atrophy.

Assay of retinol-binding protein-transthyretin interaction and techniques to identify competing ligands.

The principles of fluorescence resonance energy transfer have been utilized to develop a high-throughput assay which detects compounds that interfere with interaction between retinol-binding protein (RBP) and transthyretin (TTR). In this assay, the intrinsic fluorescence from the RBP-retinol complex excites a probe molecule which is covalently coupled to TTR. Generation of an emission signal from the TTR probe indicates interaction between RBP-retinol and TTR. Importantly, the inclusion of retinol in the assay allows discrimination of test compounds which bind RBP versus those which bind to TTR. Thus, compounds which bind to RBP must compete with retinol in order to affect RBP-TTR interaction. This feature of the assay will be useful to identify test compounds which are more likely to have an effect in vivo.

Pharmacologic treatment of atrophic age-related macular degeneration.

PURPOSE OF REVIEW: To review and compare the various therapeutic platforms, investigational drugs, and clinical trials targeting geographic atrophy in age-related macular degeneration. RECENT FINDINGS: Investigational agents based on hypothesized causes are being developed to treat geographic atrophy. These platforms are designed to attack the disease on several different fronts. SUMMARY: As knowledge of geographic atrophy pathophysiology advances, targeted pharmacotherapies may well be able to mitigate the retinal damage and vision loss associated with geographic atrophy.

Retinal pigment epithelium-retinal G protein receptor-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells.

Visual perception begins with the absorption of a photon by an opsin pigment, inducing isomerization of its 11-cis-retinaldehyde chromophore. After a brief period of activation, the resulting all-trans-retinaldehyde dissociates from the opsin apoprotein rendering it insensitive to light. Restoring light sensitivity to apo-opsin requires thermal re-isomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle in retinal pigment epithelial (RPE) cells. Vertebrates can see over a 10(8)-fold range of background illumination. This implies that the visual cycle can regenerate a visual chromophore over a similarly broad range. However, nothing is known about how the visual cycle is regulated. Here we show that RPE cells, functionally or physically separated from photoreceptors, respond to light by mobilizing all-trans-retinyl esters. These retinyl esters are substrates for the retinoid isomerase and hence critical for regenerating visual chromophore. We show in knock-out mice and by RNA interference in human RPE cells that this mobilization is mediated by a protein called "RPE-retinal G protein receptor" (RGR) opsin. These data establish that RPE cells are intrinsically sensitive to light. Finally, we show that in the dark, RGR-opsin inhibits lecithin:retinol acyltransferase and all-trans-retinyl ester hydrolase in vitro and that this inhibition is released upon exposure to light. The results of this study suggest that RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters from a storage pool in lipid droplets to an "isomerase pool" in membranes of the endoplasmic reticulum. This translocation permits insoluble all-trans-retinyl esters to be utilized as substrate for the synthesis of a new visual chromophore.

Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.

PURPOSE: Autosomal dominant Stargardt disease-3 (STGD3) is caused by mutations in elongase of very long chain fatty acids-4 (ELOVL4). The goal of this study was to generate and characterize heterozygous and homozygous knockin-mice that carry a human STGD3 pathogenic mutation in the mouse Elovl4 gene. METHODS: Recombinant Stgd3-knockin mice were generated using a DNA construct which introduced a pathogenic five-base pair deletion and two point mutations in exon 6 of the Elovl4 gene. Stgd3-mouse genotypes were confirmed by Southern blot analysis and expression of wild-type (wt) and mutated Elovl4 mRNAs assayed by nuclease protection assay. The retinal phenotype of heterozygous Stgd3 mice was characterized by morphological studies, elecroretinographic (ERG) analysis and assay of lipofuscin accumulation. Homozygous Stgd3 mice were examined for both retinal and gross morphology. They were also analyzed for skin morphology and skin barrier function, and for epidermal lipid content using high performance liquid chromatography (HPLC) combined with mass spectrometry (MS). RESULTS: The Stgd3 allele codes for a truncated mouse Elovl4 protein, which also contains the same aberrant 8-amino acid C-terminus encoded by the human pathogenic STGD3 allele. Heterozygous Stgd3 mice expressed equal amounts of both wt and mutant Elovl4 mRNAs in the retina, showed no significant changes in retinal morphology, but did show accumulation of lipofuscin and reduced visual function. Homozygous Stgd3 mice were born with an expected Mendelian frequency, without any initial gross anatomical or behavioral abnormalities. By 6-12 h postpartum, they became dehydrated and died. A skin permeability assay detected a defect in epidermal barrier function. Homozygous mutant epidermis expressed a normal content of mutated Elovl4 mRNA and contained all four epidermal cellular layers. HPLC/MS analysis of epidermal lipids revealed the presence of all barrier lipids with the exception of the complete absence of acylceramides, the critical lipids for barrier function of the skin. CONCLUSIONS: The generated Stgd3-knockin mice are a genetic model of human STGD3 and reproduce features of the human disease: accumulation of lipofuscin and reduced visual functions. Homozygous Stgd3 mice showed a complete absence of acylceramides from the epidermis. Their absence suggests a role for Elovl4 in acylceramide synthesis, and in particular, a role in the synthesis of the unique very long chain C30-C40 fatty acids present in skin acylceramides.

Characterization of native retinal fluorophores involved in biosynthesis of A2E and lipofuscin-associated retinopathies.

Mutations in the photoreceptor-specific ABCA4 gene are associated with several inherited retinal and macular degenerations. A prominent phenotype of these diseases is the accumulation of cytotoxic lipofuscin fluorophores such as A2E within the retinal pigment epithelium. Another compound, dihydro-N-retinylidene-N-retinylphosphatidyl-ethanolamine (A2PE-H(2)), also accumulates in retinas of mice and humans harboring ABCA4 mutations and was proposed to be a precursor of A2E. The role of A2PE-H(2) in the biogenesis of A2E and its relationship to other retinal fluorophores has not been previously investigated. We report spectral properties and structural relationships of the principal retinal fluorophores that accumulate in retina and retinal pigment epithelium of abca4(-/-) mice. A long wavelength fluorescence emission intrinsic to abca4(-/-) retinal explants is shown to emanate from A2PE-H(2). All-trans retinal dimer conjugates, which were also identified in the retinal explants, possessed distinct fluorescence and structural properties and, unlike A2PE-H(2), did not accumulate in an age-dependent manner. Derivative absorbance and fluorescence spectroscopy revealed that A2PE-H(2), A2E, and N-retinylidene-N-retinyl-phosphatidylethanolamine (A2PE), a known precursor of A2E, share common electronic and resonant structures. Importantly, collision-induced dissociation of A2PE-H(2) produced daughter ions that were identical to authentic A2E and its daughter ions. Finally, intravitreal administration of A2PE-H(2) to wild-type mice resulted in the formation of A2PE and A2E. These data validate a previously hypothesized biosynthetic pathway for A2E and implicate A2PE-H(2) as a precursor in this pathway. Fluorescence properties of A2PE-H(2) and other related fluorophores characterized in this report have significance for evaluation of human retinal diseases characterized by aberrant fundus autofluorescence.

Choroidal neovascularization in transgenic mice expressing prokineticin 1: an animal model for age-related macular degeneration.

The prognosis of choroidal neovascularization (CNV) in age-related macular degeneration (AMD) is poor and existing treatments are limited in retarding the progression of disease. The development of an animal model for AMD will be beneficial for finding potential treatments, including gene therapy. Recently prokineticin 1 (hPK1) was identified as a mitogen of fenestrated endothelium. We hypothesized that hPK1 could induce CNV, a hallmark of the exudative or wet form of AMD, since the endothelium of the choriocapillaris, but not retinal endothelium, has fenestration. We generated transgenic mice expressing hPK1 in the retina using the rhodopsin promoter. In these transgenic mice, an enlarged vascular bed of choroid resembling CNV was observed without any morphological changes in the retinal vasculature. In addition, the major fluorophore of lipofuscin, N-retinylidene-N-retinylethanolamine, which has several potential cytotoxic effects on the RPE, was accumulated approximately twice as much in the transgenic mouse eyes compared to controls. hPK1 could be one of the causative factors of AMD and the transgenic mouse exhibiting CNV may be useful to establish treatments for the wet form of AMD.

Reductions in serum vitamin A arrest accumulation of toxic retinal fluorophores: a potential therapy for treatment of lipofuscin-based retinal diseases.

PURPOSE: Excessive accumulation of lipofuscin is observed in numerous degenerative retinal diseases. A toxic vitamin A-based fluorophore (A2E) present within lipofuscin has been implicated in the death of RPE and photoreceptor cells. Here, we used an animal model that manifests accelerated lipofuscin accumulation (ABCA4-/- mutant) to evaluate the efficacy of a therapeutic approach based on reduction of serum retinol. METHODS: N-(4-hydroxyphenyl)retinamide (HPR) potently and reversibly reduces serum retinol. The interaction of HPR with retinol binding protein (RBP) and transthyretin was studied by spectrofluorometry and size-exclusion chromatography. To assess the effects of HPR on visual cycle retinoids and A2E biosynthesis, HPR was chronically administered to ABCA4-/- mice. Mice were evaluated using biochemical, electrophysiological, and morphologic techniques. RESULTS: Administration of HPR to ABCA4-/- mice caused immediate, dose-dependent reductions in serum retinol and RBP. Chronic administration produced commensurate reductions in visual cycle retinoids and arrested accumulation of A2E and lipofuscin autofluorescence in the RPE. Physiologically, HPR treatment caused modest delays in dark adaptation. Chromophore regeneration kinetics, light sensitivity of photoreceptors, and phototransduction processes were normal. Histologic examinations showed no alteration of retinal cytostructure or morphology. CONCLUSIONS: These findings demonstrate the vitamin A-dependent nature of A2E biosynthesis and validate a novel therapeutic approach with potential to halt the accumulation of lipofuscin fluorophores in the eye.

Chicken retinas contain a retinoid isomerase activity that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol.

Vertebrate retinas contain two types of light-detecting cells. Rods subserve vision in dim light, while cones provide color vision in bright light. Both contain light-sensitive proteins called opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde by absorption of a photon. Restoration of light sensitivity requires chemical re-isomerization of retinaldehyde by an enzymatic pathway called the visual cycle in the retinal pigment epithelium. The isomerase in this pathway uses all-trans-retinyl esters synthesized by lecithin retinol acyl transferase (LRAT) as the substrate. Several lines of evidence suggest that cone opsins regenerate by a different mechanism. Here we demonstrate the existence of two catalytic activities in chicken retinas. The first is an isomerase activity that effects interconversion of all-trans-retinol and 11-cis-retinol. The second is an ester synthase that effects palmitoyl coenzyme A-dependent synthesis of all-trans- and 11-cis-retinyl esters. Kinetic analysis of these two activities suggests that they act in concert to drive the formation of 11-cis-retinoids in chicken retinas. These activities may be part of a new visual cycle for the regeneration of chromophores in cones.

Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration.

Recessive Stargardt's macular degeneration is a blinding disease of children caused by mutations in the ABCA4 (ABCR) gene. Mice with a knockout mutation in abcr accumulate toxic lipofuscin pigments in ocular tissues, similar to affected humans. The major fluorophore of lipofuscin is the bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E). In the current study, we sought to define the effect of increasing light on A2E accumulation. We crossed the abcr(-/-) mutation onto an albino background. The retinoid profiles in albino mice indicated higher retinal illuminance than in pigmented mice exposed to similar ambient light. Unexpectedly, A2E levels were not higher in the albino mice. Also, A2E levels in abcr(-/-) mice reared under cyclic light at 30, 120, or 1,700 lux were similar. Thus, increased retinal illuminance was not correlated with higher A2E. A2E has been shown to undergo light-dependent oxidation to yield a series of A2E epoxides or oxiranes. These oxiranes react with DNA in vitro, suggesting a potential mechanism for A2E cytotoxicity. We analyzed ocular tissues from abcr(-/-) mice for A2E oxiranes by mass spectrometry. Unlike A2E, the oxiranes were more abundant in albino vs. pigmented abcr(-/-) mice, and in abcr(-/-) mice exposed to increasing ambient light. These observations suggest that both the biosynthesis of A2E and its conversion to oxiranes are accelerated by light. Finally, we showed that the formation of A2E oxiranes is strongly suppressed by treating the abcr(-/-) mice with Accutane (isotretinoin), an inhibitor of rhodopsin regeneration.

Isotretinoin treatment inhibits lipofuscin accumulation in a mouse model of recessive Stargardt's macular degeneration.

Recessive Stargardt's macular degeneration is an inherited blinding disease of children caused by mutations in the ABCR gene. The primary pathologic defect in Stargardt's discase is accumulation of toxic lipofuscin pigments such as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium (RPE). This accumulation appears to be responsible for the photoreceptor death and severe visual loss in Stargardt's patients. Here, we tested a novel therapeutic strategy to inhibit lipofuscin accumulation in a mouse model of recessive Stargardt's disease. Isotretinoin (Accutane) has been shown to slow the synthesis of 11-cis-retinaldehyde (11cRAL) and regeneration of rhodopsin by inhibiting 11-cis-retinol dehydrogenase (11cRDH) in the visual cycle. Light activation of rhodopsin results in its release of all-trans-retinaldehyde (atRAL), which constitutes the first reactant in A2E biosynthesis. Accordingly, we tested the effects of isotretinoin on lipofuscin accumulation in abcr-/- knockout mice. Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pigments by electron microscopy. We observed no significant visual loss in treated abcr-/- mice by electroretinography. Isotretinoin also blocked the slower, age-dependent accumulation of lipofuscin in wild-type mice. These results corroborate the proposed mechanism of A2E biogenesis. Further, they suggest that treatment with isotretinoin may inhibit lipofuscin accumulation and thus delay the onset of visual loss in Stargardt's patients. Finally, the results suggest that isotretinoin may be an effective treatment for other forms of retinal or macular degeneration associated with lipofuscin accumulation.

Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells.

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.

Treatment with isotretinoin inhibits lipofuscin accumulation in a mouse model of recessive Stargardt's macular degeneration.

Recessive Stargardt's macular degeneration is an inherited blinding disease of children caused by mutations in the ABCR gene. The primary pathologic defect in Stargardt's disease is accumulation of toxic lipofuscin pigments such as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium. This accumulation appears to be responsible for the photoreceptor death and severe visual loss in Stargardt's patients. Here, we tested a therapeutic strategy to inhibit lipofuscin accumulation in a mouse model of recessive Stargardt's disease. Isotretinoin (Accutane) has been shown to slow the synthesis of 11-cis-retinaldehyde and regeneration of rhodopsin by inhibiting 11-cis-retinol dehydrogenase in the visual cycle. Light activation of rhodopsin results in its release of all-trans-retinaldehyde, which constitutes the first reactant in A2E biosynthesis. Accordingly, we tested the effects of isotretinoin on lipofuscin accumulation in abcr(-/-) knockout mice. Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pigments by electron microscopy. We observed no significant visual loss in treated abcr(-/-) mice by electroretinography. Isotretinoin also blocked the slower, age-dependent accumulation of lipofuscin in wild-type mice. These results corroborate the proposed mechanism of A2E biogenesis. Further, they suggest that treatment with isotretinoin may inhibit lipofuscin accumulation and thus delay the onset of visual loss in Stargardt's patients. Finally, the results suggest that isotretinoin may be an effective treatment for other forms of retinal or macular degeneration associated with lipofuscin accumulation.

Isomerization and oxidation of vitamin a in cone-dominant retinas: a novel pathway for visual-pigment regeneration in daylight.

The first step toward light perception is 11-cis to all-trans photoisomerization of the retinaldehyde chromophore in a rod or cone opsin-pigment molecule. Light sensitivity of the opsin pigment is restored through a multistep pathway called the visual cycle, which effects all-trans to 11-cis re-isomerization of the retinoid chromophore. The maximum throughput of the known visual cycle, however, is too slow to explain sustained photosensitivity in bright light. Here, we demonstrate three novel enzymatic activities in cone-dominant ground-squirrel and chicken retinas: an all-trans-retinol isomerase, an 11-cis-retinyl-ester synthase, and an 11-cis-retinol dehydrogenase. Together these activities comprise a novel pathway that regenerates opsin photopigments at a rate 20-fold faster than the known visual cycle. We suggest that this pathway is responsible for sustained daylight vision in vertebrates.

Vitamin A Homeostasis in the Diabetic Rat.

The concentrations of vitamin A (retinol) and retinyl ester in the plasma and liver of normal and diabetic rats were measured by HPLC (high-performance liquid chromatography). Diabetic rats had severe hyperglycemia, induced by a single streptozotocin injection 5 weeks prior to sampling. In the normal rats, plasma retinyl palmitate was very low, and the level was increased 10-fold by diabetes. Detailed time-course studies showed that rats became hyperglycemic within 48 h of streptozotocin injection, yet the plasma retinyl palmitate level was not elevated until some three weeks later. Severe diabetes did not significantly influence plasma retinol: however. free retinol in the liver was elevated within 10 days of initiation of the disease and continued to increase for the duration of the study. These results show that streptozotocin-induced diabetes significantly alters the concentrations of hepatic retinol and plasma retinyl ester. The biochemical mechanism(s) of this altered vitamin A homeostasis in diabetes and its possible relationship to tissue pathogenesis are not known at present.