TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of methods for detecting oxidative stress, and more particularly, to novel methods for detecting oxidative stress in body fluids.

BACKGROUND OF THE INVENTION

Retinitis Pigmentosa (RP) is the term used for a genetically heterogenous group of inherited retinal degenerations. Findings may be limited to the eyes or the eye findings may be part of a syndrome the most common of which is Usher's Syndrome in which deafness accompanies the retinal disease. In each disorder the inciting event is a mutation that leads to the death of rod photoreceptors, initially causing night blindness. Rods are the major consumers of oxygen in the retina and the loss of rods causes an increase in the tissue oxygen level in the outer retina. This activates NADPH oxidase causing accumulation of superoxide radicals in the cytosol and also increases their generation in mitochondria of cones. The excess superoxide radicals overwhelm superoxide dismutase 1 (SOD1) and SOD2 and cause a chain reaction by which other free radicals are generated including some that are even more damaging than superoxide radicals, such as hydroxyl radicals and peroxynitrite. The free radicals attack proteins, lipids, and DNA causing specific modifications that indicate that oxidative damage has occurred. Oxidative damage to lipids results in lipid hydroperoxides that break down to form 4-hydroxynonenal, malondialdehyde (MDA), and acrolein. The most common modification to proteins from oxidative damage is the formation of carbonyl adducts. These modifications can impair the function of macromolecules and while there are endogenous repair processes, they are overwhelmed by severe oxidative stress resulting in reduced cellular function and eventually apoptosis. After rods are eliminated from the photoreceptor layer, oxidative stress in the outer retina is severe and leads to gradual cone cell death usually starting in the midperiphery where cone density is low and then spreading peripherally and posteriorly. The posterior spread of cone death results in constriction of the visual field and eventually a central island of vision and its elimination causes blindness.

Clinical signs of RP include pigmentary changes in the retina, often around blood vessels and characterized as “bone spicule-like pigmentation”, constriction of retinal vessels, and optic disc pallor. Spectral domain optical coherence tomography can show thinning of the retina in areas of photoreceptor cell loss and with segmentation the loss is seen in the outer nuclear layer.

Visual field testing shows constriction of the visual fields and electroretinograms show reduced a- and b-wave amplitudes.

Currently, there is no approved therapy that stops the evolution of the disease or restores vision. The therapeutic approach is restricted to slowing down the degenerative process by sunlight protection and vitamin A supplementation, treating complications (cataract and macular edema), and helping patients to cope with the social and psychological impact of blindness. Although the Argis II Retinal Prosthesis System was approved by FDA in 2013 as an implanted device to treat adults with severe RP, it only produces the sensation of light, thereby helping patients identify the location or movement of objects and people; the device is not disease modifying.

In order to test new treatments, it is necessary to have robust measures of disease progression. The most widely accepted functional measure of disease progression is loss of visual field; however, there are several ways to assess visual fields and their value may differ at different stages of the disease. Goldman visual fields provide a good assessment of peripheral visual fields and are useful during the early stages of cone cell loss. Assessment of retinal function by full field ERGs is also most useful in early stage disease because signals are low and often unrecordable in later stage disease. Automated measures of retinal sensitivity provide measurements on posterior retina, which is normal early in the disease, but in later stage disease is more sensitive and quantitative than Goldman visual fields.

Determining the annual rate of change in the width of the central area of intact inner segment ellipsoid zone by spectral domain OCT provides a fairly sensitive anatomic measure of disease progression in relatively advanced disease. A problem shared by all of these outcome measures is that their rate of change over time is low necessitating long term clinical trials to have a chance of detecting treatment effects.

As such, the identification and development of biomarkers that could serve as interim readouts of drug activity, bioavailability, and compliance would be a major benefit for planning and carrying out clinical trials.

SUMMARY OF THE INVENTION

The present inventors have determined that the ongoing oxidative stress in patients with RP causes oxidative damage to secreted macromolecules as well as those that are retained within cells. Therefore novel markers of oxidative stress should be detectable in aqueous humor and serum of patients with, e.g., retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, myopia, high myopia, Fuchs' dystrophy, diabetic macular edema (DME), geographic atrophy, Stargardt's disease, or retinal vein occlusion (RVO), and should be greater than the baseline levels in aqueous humor and serum from normal control patients. Furthermore, as cone death progresses, oxidative stress should increase and therefore the level of any particular marker of oxidative damage is likely to increase in an RP patient over time, and in a population of RP patients there is a positive correlation between level of an oxidative damage marker and stage of disease.

Thus, in one aspect, the present invention provides methods for assessing the severity or stage of RP in a patient diagnosed with the disease by measuring markers, which indicate increasing or decreasing oxidative stress in the aqueous humor and serum of patients. The methods disclosed herein can also be used to monitor the progression of the disease in a patient over time and determine whether a course of treatment is effective in slowing or stopping the progression of the disease in the eye.

In accordance with an embodiment, the present invention provides a method for quantifying or staging the severity of disease of the eye in a subject diagnosed as having an eye disease caused by oxidative stress or damage, including but not limited to, retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, myopia, high myopia, Fuchs' dystrophy, diabetic macular edema (DME), geographic atrophy, Stargardt's disease, or retinal vein occlusion (RVO), comprising: a) obtaining a biological sample from the subject; b) providing a control biological sample; c) measuring one or more of the following: cupric reducing antioxidant capacity, GSH/GSSG ratio, and carbonyl adduct level in the sample of a) and b); d) comparing the one or more measurements of c) in the sample from the subject to the control sample; and e) quantifying or staging the severity of disease in the subject as progressing in stage, or increasing in severity, if the levels of cupric reducing antioxidant capacity and/or GSH/GSSG ratio is reduced relative to the control sample, and/or if the levels of carbonyl adduct level is elevated relative to the control sample.

Therefore, in accordance with another embodiment, the present invention provides a method for treating a subject having eye damage caused by oxidative stress or damage, e.g., retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, myopia, high myopia, or Fuchs' dystrophy, comprising: a) obtaining a biological sample from the subject; b) providing a control biological sample; c) measuring one or more of the following: cupric reducing antioxidant capacity, GSH/GSSG ratio, carbonyl adduct level in the sample of a) and b); d) comparing the one or more measurements of c) in the sample from the subject to the control sample; e) quantifying or staging the severity of disease in the subject as progressing in stage, or increasing in severity, if the levels of cupric reducing antioxidant capacity and/or GSH/GSSG ratio is reduced relative to the control sample, and/or if the levels of carbonyl adduct level is elevated relative to the control sample; and f) selecting a course of treatment for the disease in the subject which is based on the stage or severity of disease indicated in e).

Therefore, in accordance with another embodiment, the present invention provides a method for quantifying oxidative stress comprising: a) obtaining a biological sample from the subject; b) providing a control biological sample; c) measuring one or more of the following: cupric reducing antioxidant capacity, GSH/GSSG ratio, and carbonyl adduct level in the sample of a) and b); and d) comparing the one or more measurements of c) in the sample from the subject to the control sample, wherein an increase in carbonyl adduct level, a ratio of GSH to GSSG is reduced, and/or a reduction in cupric antioxidant capacity is indicative of oxidative stress. In one aspect, the sample is from the aqueous humor of the eye of the subject. In another aspect, the sample is from the blood or plasma of the subject. In another aspect, the sample is obtained from a subject suspected of having retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, myopia, high myopia, Fuchs' dystrophy, diabetic macular edema (DME), geographic atrophy, Stargardt's disease, or retinal vein occlusion (RVO). In another aspect, the levels of protein carbonyl levels are determined by EIA (enzyme immunoassay), ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), indirect competitive immunoassay, direct competitive immunoassay, non-competitive immunoassay, sandwich immunoassay, agglutination assay, Western blot assay, slot blot assay, fluorescence polarization, latex agglutination, lateral flow assay, immunochromatographic assay, immunochips, dip stick immunotesting, or bead-based technology. In another aspect, the method further comprises the step of measuring cone cell death upon a finding of an increase in carbonyl adduct level, a ratio of GSH to GSSG is reduced, and/or a reduction in cupric antioxidant capacity is indicative of oxidative stress in the subject.

Therefore, in accordance with another embodiment, the present invention provides a kit for determining the oxidative stress comprising: a) a first container for a biological sample from the subject; b) a second container for a control biological sample; c) one or more vials that comprise reagents to determine: cupric reducing antioxidant capacity, GSH/GSSG ratio, and carbonyl adduct level in the sample of a) and b); and d) instructions for comparing the one or more measurements of c) in the sample from the subject to the control sample, wherein an increase in carbonyl adduct level, a ratio of GSH to GSSG is reduced, and/or a reduction in cupric antioxidant capacity is indicative of oxidative stress. In one aspect, the sample is from the aqueous humor of the eye of the subject. In another aspect, the biological is from a subject suspected of having retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, myopia, high myopia, Fuchs' dystrophy, diabetic macular edema (DME), geographic atrophy, Stargardt's disease, or retinal vein occlusion (RVO). In another aspect, the levels of protein carbonyl levels are determined by EIA (enzyme immunoassay), ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), indirect competitive immunoassay, direct competitive immunoassay, non-competitive immunoassay, sandwich immunoassay, agglutination assay, Western blot assay, slot blot assay, fluorescence polarization, latex agglutination, lateral flow assay, immunochromatographic assay, immunochips, dip stick immunotesting, or bead-based technology. In another aspect, the instructions are used to determine if the levels of cupric reducing antioxidant capacity and/or GSH/GSSG ratio is reduced relative to the control sample, and/or if the levels of carbonyl adduct level is elevated relative to the control sample then the disease is identified as progressing in stage, or increasing in severity, or if the levels of cupric reducing antioxidant capacity and/or GSH/GSSG ratio is elevated relative to the control sample, and/or if the levels of carbonyl adduct level is reduced relative to the control sample then the disease is identified, the decease is seen as reducing in stage, or the disease is decreasing in severity. In another aspect, the increase in carbonyl adduct level, the ratio of GSH to GSSG is reduced, and/or a reduction in cupric antioxidant capacity is indicative of cone cell death in Retinitis Pigmentosa.

