WO2006105215A2

WO2006105215A2 - The use of a spin trap for enhancing retinal cell survival and treating retinal degenerative diseases

Info

Publication number: WO2006105215A2
Application number: PCT/US2006/011497
Authority: WO
Inventors: Ryo Kubota
Original assignee: Acucela, Inc.
Priority date: 2005-03-28
Filing date: 2006-03-28
Publication date: 2006-10-05
Also published as: WO2006105215A3

Abstract

A method for treating neurodegenerative diseases and conditions, particularly retinal neurodegenerative diseases by administering a spin-trap antioxidant compound is provided. In one embodiment, the compound is a spin-trap antioxidant such as alpha-phenyl-N-tert-butyl nitrone (PBN), a derivative or analogue thereof, that enhances survival of photoreceptor cells and may be used for treatment of retinal neurodegenerative diseases, including the dry form of macular degeneration. Also provided are methods for identifying and measuring the activity of spin-trap antioxidant compounds that enhance survival of retinal neuronal cells, such as photoreceptor cells.

Description

METHODS FOR ENHANCING NEURONAL CELL SURVIVAL AND TREATING NEURODEGENERATIVE DISEASES

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to enhancing retinal neuronal cell survival using a spin-trap antioxidant compound. The invention is particularly related to using a spin-trap antioxidant such as alpha-phenyl-N-tert-butyl nitrone (PBN), or a derivative compound thereof, or a structurally related compound, for enhancing survival of retinal neuronal cells, including photoreceptor cells. Enhancing survival of photoreceptor cells using a spin-trap antioxidant such as PBN is useful for treatment of retinal diseases such as macular degeneration and glaucoma.

Description of the Related Art

Neurodegenerative diseases, such as glaucoma, macular degeneration, and Alzheimer's disease, affect millions of patients throughout the world. Considering the loss of quality of life associated with these diseases, drug research and development in this area is of great importance. Macular degeneration is a disease that affects central vision. Macular degeneration affects between five and ten million patients in the United States, and it is the leading cause of blindness worldwide. Macular degeneration is a disease that causes the loss of photoreceptor cells in the central part of retina called the macula. Macular degeneration can be classified into two types: dry type and wet type. The dry form is more common than the wet, with about 90% of age-related macular degeneration (ARMD) patients diagnosed with the dry form. The wet form of the disease usually leads to more serious vision loss. The exact causes of age-related macular degeneration are still unknown. The dry form of ARMD may result from the aging and thinning of macular tissues and from deposition of pigment in the macula. In wet ARMD, new blood vessels grow beneath the retina and leak blood and fluid. This leakage causes the retinal cells to die, creating blind spots in central vision.

One Food and Drug Administration (FDA)-approved protocol for treating ARMD is a photodynamic therapy that uses a special drug combined with laser photocoagulation. This treatment, however, can only be applied to half of patients newly diagnosed with wet form of ARMD. Very recently, the FDA approved Macugen® (pegaptanib sodium injection) for the treatment of neovascular (wet) ARMD). For the vast majority of patients who have the dry form of macular degeneration, no treatment is available. Because the dry form precedes development of the wet form of macular degeneration, intervention in disease progression of the dry form could benefit patients that presently have dry form and may delay or prevent development of the wet form.

Declining vision noticed by the patient or by an ophthalmologist during a routine eye exam may be the first indicator of macular degeneration. The formation of exudates, or "drusen," from blood vessels in and under the macula is often the first physical sign that macular degeneration may develop. Symptoms include perceived distortion of straight lines, and, in some cases, the center of vision appears more distorted than the rest of a scene. A dark, blurry area or "white-out" appears in the center of vision, and/or color perception changes or diminishes. Different forms of macular degeneration may also occur in younger patients. Non-age related etiology may be linked to heredity, diabetes, nutritional deficits, head injury, infection, or other factors.

Glaucoma is a broad term used to describe a group of diseases that causes visual field loss, often without any other prevailing symptoms. The lack of symptoms often leads to a delayed diagnosis of glaucoma until the terminal stages of the disease. Prevalence of glaucoma is estimated to be three million in the United States, with about 120,000 cases of blindness attributable to the condition. The disease is also prevalent in Japan, which has four million reported cases. In other parts of the world, treatment is less accessible than in the United States and Japan, thus glaucoma ranks as a leading cause of blindness worldwide. Even if subjects afflicted with glaucoma do not become blind, their vision is often severely impaired.

The loss of peripheral vision is caused by the death of ganglion cells in the retina. Ganglion cells are a specific type of projection neuron that connects the eye to the brain. Glaucoma is often accompanied by an increase in intraocular pressure. Current treatment includes use of drugs that lower the intraocular pressure; however, lowering the intraocular pressure is often insufficient to completely stop disease progression. Ganglion cells are believed to be susceptible to pressure and may suffer permanent degeneration prior to the lowering of intraocular pressure. An increasing number of cases of normal tension glaucoma has been observed in which ganglion cells degenerate without an observed increase in the intraocular pressure. Because current glaucoma drugs only treat intraocular pressure, a need exists to identify new therapeutic agents that will prevent or reverse the degeneration of ganglion cells. Recent reports suggest that glaucoma is a neurodegenerative disease, similar to Alzheimer's disease and Parkinson's disease, except that it specifically affects retinal neurons. The retinal neurons of the eye originate from diencephalon neurons of the brain. Though retinal neurons are often mistakenly thought not to be part of the brain, retinal cells are key components of vision, interpreting the signals from the light sensing cells.

Alzheimer's disease (AD) is the most common form of dementia among the elderly. Dementia is a brain disorder that seriously affects a person's ability to carry out daily activities. Alzheimer's is a disease that affects four million people in the United States alone. It is characterized by a loss of nerve cells in areas of the brain that are vital to memory and other mental functions. Some drugs can prevent AD symptoms for a finite period of time, but are no drugs are available that treat the disease or completely stop the progressive decline in mental function. Recent research suggests that glial cells that support the neurons or nerve cells may have defects in AD sufferers, but the cause of AD remains unknown. Individuals with AD seem to have a higher incidence of glaucoma and macular degeneration, indicating that similar pathogenesis may underlie these neurodegenerative diseases of the eye and brain. (See, e.g., Giasson et al, Free Radio. Biol. Med. 32:1264-75 (2002); Johnson et al, Proc. Natl. Acad. ScL USA 99:11830-35 (2002); Dentchev et al., MoI. Vis. 9:184-90 (2003)).

Retinal neuronal cell death underlies the pathology of these diseases.

However, very few compositions and methods that enhance retinal neuronal cell survival, particularly photoreceptor cell survival, have been discovered. A need therefore exists to identify and develop compositions that that can be used for treatment and prophylaxis of retinal diseases and disorders.

SUMMARY OF THE INVENTION The present invention provides methods for inhibiting degeneration of a retinal cell and enhancing retinal cell survival, including retinal neuronal cell survival, using a spin-trap antioxidant compound or a derivative thereof. In certain embodiments, the compound is a spin-trap antioxidant such as alpha-phenyl-N-tert- butyl nitrone (PBN), or derivative compounds thereof, or structurally related compounds. In certain embodiments, a spin-trap antioxidant such as PBN enhances survival of retinal neuronal cells, and in a certain embodiment a spin-trap antioxidant such as PBN enhances survival of photoreceptor cells. The present invention also provides an in vitro cell culture system of neuronal cells, preferably retinal neuronal cells, for identifying a spin-trap antioxidant compound that alters neurodegeneration of retinal neuronal cells. In one embodiment, the spin-trap antioxidant compound enhances (i.e., promotes) survival of retinal neuronal cells, or inhibits (prevents, or slows the progression of) neurodegeneration of retinal neuronal cells, such as photoreceptor cells.

In a specific embodiment, a spin-trap antioxidant such as PBN may be used to enhance photoreceptor survival, which can result in slowing or even halting the progression of macular degeneration, or other neurodegenerative diseases of the eye that are related to other neurodegenerative diseases, for example, Alzheimer's disease. Even more specifically, in one embodiment, a spin-trap antioxidant such as PBN can be used to slow or inhibit photoreceptor cell death in dry form macular degeneration, which is directly related to photoreceptor death. Two preferred methods of administration are external application of a spin-trap antioxidant such as PBN in a form including but not limited to gel, eye drops, or a cream, and a systemic application by methods including but not limited to oral dosing, parenteral administration, eye drops, inhalation, suppository, transdermal delivery, or mucosal delivery. Also provided are methods for identifying and characterizing spin-trap antioxidant compounds, compounds, derivatives, and analogues that enhance survival of retinal neuronal cells comprising contacting retinal neuronal cells with the candidate compound and determining survival of retinal cells in the presence and in the absence of the candidate compound. In certain embodiments, the retinal neuronal cells comprise amacrine cells, horizontal cells, bipolar cells, ganglion cells, and photoreceptor cells. In specific embodiments survival of photoreceptor cell is determined. In another embodiment, the method further comprises a stressor, wherein the stressor is white light or blue light or the stressor is a compound such as A2E or cigarette smoke concentrate. In one embodiment, a method is provided for enhancing retinal cell survival wherein the method comprises contacting a retinal neuronal cell with a spin- trap antioxidant. In another embodiment, a method is provided for inhibiting degeneration of a retinal cell wherein the method comprises contacting a retinal cell with a spin-trap antioxidant, hi yet another embodiment, a method is provided for inhibiting degeneration of a retinal cell in a subject having a retinal disease or disorder comprising administering to the subject a composition comprising a spin- trap antioxidant and a pharmaceutically acceptable carrier. In still another embodiment, a method is provided for enhancing retinal cell survival in a subject having a retinal disease or disorder comprising administering to the subject a composition comprising a spin-trap antioxidant and a pharmaceutically acceptable carrier. In a particular embodiment, the retinal cell is a retinal neuronal cell, and in certain embodiments, the retinal neuronal cell is at least one of photoreceptor cell, a ganglion cell, an amacrine cell, a horizontal cell, and a bipolar cell. In certain specific embodiments, the retinal neuronal cell is a photoreceptor cell, hi one embodiment, the spin-trap antioxidant is alpha-phenyl-N-tert-butyl nitrone (PBN); 5,5-dimethylpyrroline-N-oxide (DMPO); alpha-(4-pyridyl l-oxide)-N-tert-butyl nitrone (POBN); or 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO). In a particular embodiment, the spin-trap antioxidant is alpha-phenyl-N-tert-butyl nitrone (PBN), or a derivative thereof. In another specific embodiment, the retinal disease or disorder is macular degeneration, glaucoma, diabetic retinopathy, diabetic maculopathy, retinal detachment, retinal blood vessel occlusion, hemorrhagic retinopathy, retinitis pigmentosa, retinopathy of prematurity, optic neuropathy, inflammatory retinal disease, proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusion related retinal injury, hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, a retinal disorder associated with Alzheimer's disease, a retinal disorder associated with multiple sclerosis, a retinal disorder associated with Parkinson's disease, a retinal disorder associated with viral infection, a retinal disorder related to light overexposure, or a retinal disorder associated with AIDS. In a specific embodiment, the retinal disease or disorder is dry form macular degeneration or wet form macular degeneration. The composition in certain embodiments is administered topically to an eye of the subject, orally, intravenously, intraocularly, or periocularly.

In another embodiment, a method is provided for treating a retinal disease in a subject, comprising administering to the subject in need thereof a composition that comprises a spin-trap antioxidant and pharmaceutically acceptable carrier. The composition in certain embodiments is administered topically to an eye of the subject, orally, intravenously, intraocularly, or periocularly. In certain embodiments, the retinal disease or disorder is macular degeneration, glaucoma, diabetic retinopathy, diabetic maculopathy, retinal detachment, retinal blood vessel occlusion, hemorrhagic retinopathy, retinitis pigmentosa, retinopathy of prematurity, optic neuropathy, inflammatory retinal disease, proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusion related retinal injury, hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, a retinal disorder associated with Alzheimer's disease, a retinal disorder associated with multiple sclerosis, a retinal disorder associated with Parkinson's disease, a retinal disorder associated with viral infection, a retinal disorder related to light overexposure, or a retinal disorder associated with AIDS. In certain embodiments, the retinal disease or disorder is dry form of macular degeneration or is wet form macular degeneration. In particular embodiments, the retinal neuronal cell is at least one of a photoreceptor cell, a ganglion cell, an amacrine cell, a horizontal cell, and a bipolar cell. In certain specific embodiments, the retinal neuronal cell is a photoreceptor cell. In one embodiment, the spin-trap antioxidant is alpha-phenyl-N-tert-butyl nitrone (PBN); 5,5-dimethylpyrroline-N-oxide (DMPO); alpha-(4-pyridyl l-oxide)-N-tert-butyl nitrone (POBN); or 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO). In a particular embodiment, the spin-trap antioxidant is alpha-phenyl-N-tert-butyl nitrone (PBN), or a derivative thereof.

In another embodiment, a method is provided for treating a retinal disease or disorder in a subject, comprising administering to the subject a composition that comprises alpha-phenyl-N-tert-butyl nitrone (PBN), or a derivative thereof, and pharmaceutically acceptable carrier. In certain embodiments, the retinal disease or disorder is any one of the retinal diseases or disorders described above. In a particular embodiment, the composition is administered topically to an eye of the subject, orally, intravenously, intraocularly, or periocularly.

Also provided herein is a use for a spin-trap antioxidant for the manufacture of a medicament for treating a retinal disease or disorder. In certain embodiments, the retinal disease or disorder is macular degeneration, glaucoma, diabetic retinopathy, diabetic maculopathy, retinal detachment, retinal blood vessel occlusion, hemorrhagic retinopathy, retinitis pigmentosa, retinopathy of prematurity, optic neuropathy, inflammatory retinal disease, proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusion related retinal injury, hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, a retinal disorder associated with Alzheimer's disease, a retinal disorder associated with multiple sclerosis, a retinal disorder associated with Parkinson's disease, a retinal disorder associated with viral infection, a retinal disorder related to light overexposure, or a retinal disorder associated with AIDS. In certain embodiments, the retinal disease or disorder is dry form of macular degeneration or is wet form macular degeneration. In one particular embodiment, the spin-trap antioxidant is alpha-phenyl-N-tert-butyl nitrone (PBN); 5,5-dimethylpyrroline-N-oxide (DMPO); alpha-(4-pyridyl l-oxide)-N-tert-butyl nitrone (POBN); or 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO). In a particular embodiment, the spin-trap antioxidant is alpha-phenyl-N-tert-butyl nitrone (PBN), or a derivative thereof.

As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "the cell" includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including" and the like) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein may "consist of or "consist essentially of the described features.

These and other embodiments of the invention will become evident through the following detailed description and attached drawings. In addition, documents set forth herein that describe in more detail certain embodiments of this invention are incorporated by reference in their entireties. AU U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an immunohistochemical analysis of representative rhodopsin-expressing photoreceptors before stress. Figure 2 illustrates an immunohistochemical analysis of representative rhodopsin-expressing photoreceptors after stress (25 μM A2E for 24 hours). The small dots are debris.

Figure 3 shows an immunohistochemical analysis rhodopsin-expressing photoreceptors under stress in the presence of PBN (100 nM) for 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the surprising discovery that spin-trap antioxidant compounds enhance {i.e., prolong, promote, improve, or increase) the survival of retinal cells, including photoreceptor cells. A spin trap antioxidant compound that enhances or prolongs survival thus promotes, increases, stabilizes or maintains cell viability, and thus delays injury and/or death of a retinal cell. Thus, neurodegeneration of stressed retinal neuronal cells, particularly photoreceptor cells, is decreased or inhibited in cells that are concurrently or subsequently exposed to a spin- trap antioxidant compound. In one embodiment, the spin-trap antioxidant compound is a spin-trap antioxidant such as alpha-phenyl-N-tert-butyl nitrone (PBN). As described in more detail herein, exposure of stressed retinal neuronal cells in culture to a spin-trap antioxidant such as PBN results in an increased number of surviving photoreceptor cells. A spin-trap antioxidant such as PBN, or derivative compounds or analogues thereof, or structurally related compounds, may be used in methods for treating neurological diseases or disorders in general, and for treating degenerative diseases of the eye and brain in particular.

As described herein a spin-trap antioxidant compound such as PBN is capable of inhibiting (i.e., impairing, preventing, abrogating, reducing, slowing the progression of, in a statistically or biologically significant manner) degeneration of a retinal cell, which includes a retinal neuronal cell (such as a photoreceptor cell, amacrine cell, horizontal cell, ganglion cell, and bipolar cell). A compound useful for treating a retinal disease or disorder preferably inhibits degeneration of a retinal cell, and may be capable of regenerating a retinal cell. At least two classes of spin trapping antioxidant agents have been used as analytical reagents, nitroso compounds and nitrones. Nitrones, such as alpha-phenyl- N-tert-butylnitrone (PBN) and 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), are known to be useful as analytical reagents for detecting free radicals. Free radicals are short- lived reactive chemical species having one or more electrons with unpaired spins, and which are generally highly reactive and participate in hydrogen abstraction, radical addition, bond scission, and annihilation reactions.

Free radicals are capable of independent existence and are produced in living cells. Most radicals that occur in vivo either are, or originate from, reactive oxygen species (ROS) or reactive nitrogen species. ■ ROS include oxygen-based free radicals, such as superoxide, hydroxyl (OH-), alkoxyl (RO"), peroxyl (ROO "), and hydroperoxyl (ROOH"). Reactive nitrogen species include the free radicals nitric oxide (NO") and nitrogen dioxide (NO ^a) and the potent oxidant peroxynitrite (ONOO^"). Free radicals can potentially react with a variety of chemical species and consequently function in cell signaling pathways. However, ROS are also inadvertently produced in the body by a variety of mechanisms. The majority of free radicals produced in vivo are oxidants, which are capable of oxidizing a range of biological molecules, including carbohydrates, amino acids, fatty acids, and nucleotides. Because all free radical production cannot be prevented in vivo, a number of antioxidant defenses have evolved in the body. Both enzymatic and non-enzymatic antioxidants are present in cells. Antioxidant enzymes, for example, include superoxide dismutase, glutathione peroxidase, and catalase. Exemplary non-enzymatic antioxidants include glutathione (GSH), vitamin C, and vitamin E. The antioxidant defenses of the body are usually adequate to prevent substantial tissue damage. However, an overproduction of free radicals or a drop in the level of the antioxidant defenses will lead to an imbalance and cause deleterious effects, a situation referred to as oxidative stress.

A spin-trap antioxidant acts as a spin trapping agent when a diamagnetic (i.e., slightly repelled by a magnet), for example, a nitrone compound (the "spin trap") reacts with a transient, unstable free radical species (having the "spin") to provide a relatively more stable radical species, which is referred to as a spin adduct. The spin adduct may be detectable by electron paramagnetic resonance spectroscopy, depending upon the lifetime of the adduct. The technique of spin trapping has been a method for gaining information about free radicals that are difficult or impossible to detect by direct spectroscopic observation. Nitrones have been used to study unstable free radicals in biological systems, including lipid peroxidation.

As described herein spin trap antioxidant compounds are useful for enhancing (prolonging) retinal cell survival; increasing retinal cell viability; and/or inhibiting (decreasing, preventing, slowing, or eliminating) retinal cell degeneration. A retinal cell includes a retinal neuronal cell, such as a photoreceptor cell, amacrine cell, bipolar cell, ganglion cell, and horizontal cell, and other mature retinal cells including retinal pigmented epithelial (RPE) cells and Mύller glial cells. Spin-trap antioxidant compounds include, but need not be limited to, nitrones, phenolic compounds, indole derivatives, indolines, imidazoles, pheothiazines, phenoxazines, phenazines, diphenylamines, or carbazoles. Other spin trap antioxidant compounds include vitamin C (ascorbic acid), vitamin E compounds (e.g., α-tocopherol and γ-tocopherol); one or more carotene derivatives, (e.g., α-, β-, γ-, δ-carotene); ethoxyquin; N-acetyl-cysteine; 2,2,5, 5,-tretramethyl-3-pyrroline-l-oxyl-3-carboxylic acid; an ubiquinone (e.g., QlO coenzyme); and a captodative compound (i.e., an unsaturated compound in which a captor (electron withdrawing) substituent and a dative (electron releasing) substituent are both attached to the same radical centered carbon). In certain embodiments, an antioxidant trap may include an enzyme that is capable of neutralizing one or more reactive oxygen species such as superoxide dismutase, catalase, or glutathione peroxidase.

Nitrone compounds useful for methods for enhancing retinal cell survival and/or inhibiting degeneration of a retinal cell include PBN, 5,5- dimethylpyrroline-N-oxide (DMPO), alpha-(4-pyridyl l-oxide)-N-tert-butyl nitrone (POBN), and 2,2,6,6-tetramethylpiρeridine-l-oxyl (TEMPO). Nitrones have been studied and used as therapeutic and diagnostic agents for a variety of diseases and disorders (see, e.g., U.S. Patent Nos. 6,083,988; 5,942,507; 6,051,571; 6,339,102; 5,972,977; 6,140,356; 6,455,589; 6,376,540; Markland et al., J Cereb. Blood Flow Metab. 21:1259-67 (2001); Schmid-Elsaesser et al., Exp. Brain Res. 130:60-6 (2000); Gray et al., Brain Res. 982:179-85 (2003); Barth et al., Exp. Neurol. 141:330-36 (1996); Pharmacol. Ther. 100:195-214 (2003)). For example, nitrone antioxidants may have neuroprotective activity in diseases such as stroke, in which oxidative stress plays a key role, and may also be useful to protect damage resulting from ischemia. Nitrones have also been studied as therapeutic agents for treating hair loss (see, e.g., U.S. Patent No. 5,723,502), neurodegenerative diseases (see, e.g., Socci et al., Brain Res. 693:88-94 (1995); Schulz et al., Neuroscience 71:1043-48 (1996); Floyd et al., Meek Ageing Dev. 123:1021-31 (2002)), such as Alzheimer's disease and Parkinson's disease (see, e.g., Floyd, Proc. Soc. Exp. Biol. Med. 222:236-45 (1999); Floyd et al., J Neural Transm. Suppl (6O):387-414 (2000)), and inflammatory diseases including ocular inflammation (see, e.g., U.S. Patent No. 6,140,356). By contrast, a spin-trap antioxidant is useful for treating retinal inflammatory diseases and disorders. As contemplated and described herein, the therapeutic uses of a spin-trap antioxidant, such as PNB or a derivative thereof, is not intended to include uses in treatment of ocular inflammation such as uveitis, which is not the same as nor is a type of retinal inflammation. Uveal blood circulation is distinguishable from retinal circulation in that retinal circulation occurs inside the blood-retinal barrier.

In certain embodiments, a spin-trap antioxidant molecule that enhances survival of retinal cells and/or inhibits degeneration of retinal cells is a derivative of a spin-trap antioxidant such as a derivative of PBN, DMPO, TEMPO, or POBN. A nitrone derivative may be a naturally occurring compound or a synthetically prepared compound, hi one embodiment, a PBN derivative, NXY-059 (Cerovive®, Renovis, South San Francisco, CA), which is being evaluated in clinical trials (AstraZeneca) for treatment of acute ischemic stroke, may be used to enhance survival and/or inhibits degeneration of retinal cells including photoreceptor cells. Derivatives of PBN (such as N-tert-butyl-α-(2-sulfophenyl)nitrone (S-PBN)) and other nitrones may be prepared according to methods with which a skilled artisan will be familiar (see, e.g., U.S. Patent Nos. 5,972,977; 6,376,540; 6,083,988, 6,051,571, 5,942,507; 6,339,102) and include but are not limited to derivatives described in Durand et al., Bioorg. Med. Chem. Lett. 13:859-62 (2003); Dhainaut et al, J Med. Chem. 43:2165-75 (2000); U.S. Patent Nos. 6,569,902; 6,403,627; 6,140,356; 6,002,001; 5,681,845; 5,622,994; and 5,036,097. In another embodiment, a compound for enhancing retinal cell survival (and/or inhibiting retinal cell degeneration) is structurally related to PBN such as furan nitrone compounds (see U.S. Patent Nos. 5,942,507 and 6,051,571).

As described herein, PBN alone, promotes survival of retinal cells, for example, when the cells are exposed to a retinal cell stressor, such as retinoid N- retinylidene-N-retinyl-ethanolamine (A2E). By contrast, a nitrone derivative of PBN, S-PBN, has been combined with other therapeutic agents, particularly brain-derived neurotrophic factor (BDNF) and investigated for treating retinal ganglion cell damage (see, e.g., U.S. Patent No. 6,339,102; Klocker et al., J. NeuroscL 18:1038-46 (1998); Isenmann et al., Eur. J. Neurosci. 10:2751-56 (1998)).

The use of a mature retinal cell culture system that can include one or more retinal cell stressors that are environmental or physical (e.g., light or pressure), chemical (e.g., A2E, cigarette smoke concentrate), or biological (e.g., toxins, beta- amyloid) stressors, which are believed to contribute to or cause neurodegenerative retinal diseases in humans, led to the discovery that PBN or a derivative thereof is capable of enhancing retinal cell survival.

A spin-trap antioxidant compound, or a derivative thereof, as described herein also includes a pharmaceutically acceptable salt of the compound. A pharmaceutically acceptable salt includes both acid and base addition salts as appropriate for the particular spin-trap antioxidant compound, pharmaceutical use, and pharmaceutical formulation. A pharmaceutically acceptable salt of a spin-trap antioxidant (e.g., PBN or a derivative thereof) is intended to encompass any and all pharmaceutically suitable salt forms.

A pharmaceutically acceptable acid addition salt refers to those salts that retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

A pharmaceutically acceptable base addition salt refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Retinal Cells

The exemplary long-term in vitro cell culture system described herein permits and promotes the survival in culture of mature retinal cells, including retinal neurons, for at least 2-4 weeks, over 2 months, or for as long as 6 months. Retinal cells are isolated from non-embryonic, non-tumorigenic tissue and have not been immortalized by any method such as, for example, transformation or infection with an oncogenic virus. The cell culture system may comprise all the major retinal neuronal cell types (photoreceptors, bipolar cells, horizontal cells, amacrine cells, and ganglion cells), and also may include other mature retinal cells such as retinal pigmented epithelial (RPE) cells and Muller glial cells.

The retina of the eye is a thin, delicate layer of nervous tissue. The major landmarks of the retina are the area centralis in the posterior portion of the eye and the peripheral retina in the anterior portion of the eye. The retina is thickest near the posterior sections and becomes thinner near the periphery. The area centralis is located in the posterior retina and contains the fovea and foveola and, in primates, contains the macula: The foveola contains the area of maximal cone density and, thus, imparts the highest visual acuity in the retina. The foveola is contained within the fovea, which is contained within the macula.

The peripheral or anterior portion of the retina increases the field of vision. The peripheral retina extends anterior to the equator of the eye and is divided into four regions: the near periphery (most posterior), the mid-periphery, the far periphery, and the ora serrata (most anterior). The ora serrata denotes the termination of the retina.

The term neuron (or nerve cell) as understood in the art and used herein denotes a cell that arises from neuroepithelial cell precursors. Mature neurons (i.e., fully differentiated cells from an adult) display several specific antigenic markers. Neurons may be classified functionally into three groups: (1) afferent neurons (or sensory neurons) that transmit information into the brain for conscious perception and motor coordination; (2) motor neurons that transmit commands to muscles and glands; (3) interneurons that are responsible for local circuitry; and (4) projection interneurons that relay information from one region of the brain to anther region and therefore have long axons. Interneurons process information within specific subregions of the brain and have relatively shorter axons. A neuron typically has four defined regions: the cell body (or soma); an axon; dendrites; and presynaptic terminals. The dendrites serve as the primary input of information from other cells. The axon carries the electrical signals that are initiated in the cell body to other neurons or to effector organs. At the presynaptic terminals, the neuron transmits information to another cell (the postsynaptic cell), which may be another neuron, a muscle cell, or a secretory cell. The retina is composed of several types of neuronal cells. As described herein, the types of retinal neuronal cells that may be cultured in vitro by this method include photoreceptor cells, ganglion cells, and interneurons such as bipolar cells, horizontal cells, and amacrine cells. Photoreceptors are specialized light-reactive neural cells and comprise two major classes, rods and cones. Rods are involved in scotopic or dim light vision, whereas photopic or bright light vision originates in the cones by the presence of trichromatic pigments. Many neurodegenerative diseases that result in blindness, such as macular degeneration, retinal detachment, retinitis pigmentosa, diabetic retinopathy, etc., affect photoreceptors. Photoreceptors are the primary cell type affected in macular degeneration, a leading cause of blindness. Ganglion cells, projection neurons in the retina, are affected in glaucoma patients, also a leading cause of blindness.

Extending from their cell bodies, the photoreceptors have two morphologically distinct regions, the inner and outer segments (see Figure 1). The outer segment lies furthermost from the photoreceptor cell body and contains disks that convert incoming light energy into electrical impulses (phototransduction). As shown in Figure 1, the outer segment is attached to the inner segment with a very small and fragile cilium. The size and shape of the outer segments vary between rods and cones and are dependent upon position within the retina. See Eye and Orbit, 8^th Ed., Bron et al., (Chapman and Hall 1997).

Ganglion cells are output neurons that convey information from the retinal interneurons (including horizontal cells, bipolar cells, amacrine cells) to the brain. Bipolar cells are named according to their morphology, and receive input from the photoreceptors, connect with amacrine cells, and send output radially to the ganglion cells. Amacrine cells have processes parallel to the plane of the retina and have typically inhibitory output to ganglion cells. Amacrine cells are often subclassified by neurotransmitter or neuromodulator or peptide (such as calretinin or calbindin) and interact with each other, with bipolar cells, and with photoreceptors. Bipolar cells are retinal interneurons that are named according to their morphology; bipolar cells receive input from the photoreceptors and sent the input to the ganglion cells. Horizontal cells modulate and transform visual information from large numbers of photoreceptors and have horizontal integration (whereas bipolar cells relay information radially through the retina).

Other retinal cells that may be present in the retinal cell cultures described herein include glial cells, such as Mϋller glial cells, and retinal pigmented epithelial cells (RPE). Glial cells surround nerve cell bodies and axons. The glial cells do not carry electrical impulses but contribute to maintenance of normal brain function. Muller glia, the predominant type of glial cell within the retina, provide structural support of the retina and are involved in the metabolism of the retina {e.g., contribute to regulation of ionic concentrations, degradation of neurotransmitters, and remove certain metabolites {see, e.g., Kljavin et al., J Neurosci. 11 :2985 (1991)). Muller's fibers (also known as sustentacular fibers of retina) are sustentacular neuroglial cells of the retina that run through the thickness of the retina from the internal limiting membrane to the bases of the rods and cones where they form a row of junctional complexes. RPE cells form the outermost layer of the retina, nearest the blood vessel-enriched choroids. RPE cells are a type of phagocytic epithelial cell, functioning like macrophages, that lies below the photoreceptors of the eye. The dorsal surface of the RPE cell is closely apposed to the ends of the rods, and as discs are shed from the rod outer segment they are internalized and digested by RPE cells. RPE cells also produce, store, and transport a variety of factors that contribute to the normal function and survival of photoreceptors. Another function of RPE cells is to recycle vitamin A as it moves between photoreceptors and the RPE during light and dark adaptation.

In Vivo and In Vitro Systems for Determining Effect of Spin-Trap Antioxidant Compounds In one embodiment, methods are provided for enhancing (prolonging) retinal cell survival and/or inhibiting degeneration of a retinal cell using spin-trap antioxidant compounds, such as PBN or a derivative thereof. These compounds are useful for enhancing retinal cell survival and inhibiting degeneration of a retinal cell, including a photoreceptor cell, which can result in slowing or halting the progression of macular degeneration, or retinal blood vessel occlusion, or other neurodegenerative ophthalmic diseases described herein such as those related to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The compounds may be useful for treating and/or preventing other retinal diseases including retinal glaucoma, diabetic retinopathy, diabetic maculopathy, retinal detachment, hemorrhagic retinopathy, retinitis pigmentosa, retinopathy of prematurity, optic neuropathy, inflammatory retinal disease, proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusion related retinal injury, hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, a retinal disorder associated with multiple sclerosis, a retinal disorder associated with viral infection, a retinal disorder related to light overexposure, and a retinal disorder associated with AIDS.

The effect of spin-trap antioxidant compounds on retinal cell survival may be determined by using cell culture models, animal models, and other methods that are described herein and practiced by persons skilled in the art. By way of example, and not limitation, such methods and assays include those described in Oglivie et al., Exp. Neurol. 161:675-856 (2000); U.S. Patent No. 6,406,840; U.S. Patent Application No. 2002/0009713; U.S. Patent No. 6,117,675; U.S. Patent No. 5,736,516; U.S. Patent No. 6,183,735; U.S. Patent No. 6,090,624; International Patent Publication Nos. WO 01/09327, WO 01/81551, WO 98/12303, WO 00/40699, WO 99/29279, WO 01/83714, WO 01/42784; U.S. Patent No. 5,641,750; and U.S. Patent Application Publication No. 2005/0059148.

The lack of a good animal model has proved to be a major obstacle for developing new drugs to treat retinal diseases and disorders. For example, macula exist in primates (including humans) but not in rodents. A recently developed animal model may be useful for evaluating treatments for macular degeneration (Ambati et al. Nat. Med. 9:1390-97 (2003); Epub 2003 Oct 19). This animal model is one of only a very few exemplary animal models presently available for evaluating a compound or any molecule for use in treating (including preventing) progression or development of a neurodegenerative disease, especially an ophthalmic neurodegenerative disease. Accordingly, cell culture methods, such as the methods described herein, are particularly useful for determining the effect of on retinal neuronal cell survival.

Cell Culture System In one embodiment, a cell culture system is provided for determining the effects of a spin-trap antioxidant compound on survival of retinal cells, including retinal neuronal cells. The term "neuron" as understood in the art and used herein refers to a cell that arises from neuroepithelial cell precursors. In one embodiment, the neuronal cells are mature retinal neuronal cells. The exemplary cell culture model described herein is useful for determining the capability of the spin-trap antioxidant compounds to enhance or prolong survival of neuronal cells and/or inhibit degeneration of these cells, particularly retinal neuronal cells, and which compounds are useful for treating macular degeneration, such as dry form macular degeneration (see also U.S. Patent Application Publication No. 2005/0059148). The cell culture model comprises a long-term or extended culture of mature retinal cells that is a mixture of mature retinal neuronal cells and non-neuronal retinal cells. The cell culture system may comprise all the major retinal neuronal cell types (photoreceptors, bipolar cells, horizontal cells, amacrine cells, and ganglion cells), and also includes other mature retinal cells such as RPE and Mϋller glial cells. The retinal cell culture system may also comprise a cell stressor. The application or the presence of the stressor affects the mature retinal cells, including the retinal neuronal cells, in vitro in a manner that is useful for studying disease pathology that is observed in a retinal disease or disorder. The cell culture model described herein provides an in vitro neuronal cell culture system that will be useful in biological testing and analysis and identification of spin-trap antioxidant compounds (such as PBN and derivatives thereof) that are suitable for treatment of neurological diseases or disorders in general, and for treatment of degenerative diseases of the eye and brain in particular. The ability to obtain primary cells from mature, fully-differentiated retinal cells, including retinal neurons for culture in vitro over an extended period of time in the presence of a stressor enables examination of cell-to-cell interactions, selection and analysis of neuroactive compounds and materials, use of a controlled cell culture system for in vivo CNS and ophthalmic tests, and analysis of the effects on single cells from a consistent retinal cell population.

Accordingly, the cell culture system described herein and the retinal cell stress model may comprise cultured mature retinal cells, retinal neurons, and a retinal cell stressor, which are particularly useful for screening bioactive agents capable of inducing or stimulating regeneration of CNS tissue that has been damaged by disease. The cultured mature retinal neurons comprise all the major retinal neuronal cell types including photoreceptors, amacrine cells, ganglion cells, horizontal cells, and bipolar cells.

The in vitro cell culture system permits and promotes (or extends) the survival in culture of mature retinal cells, including retinal neurons, for over 2 months and for as long as 6 months. In other cell culture systems, the ability to screen drug candidates using mature retinal cells has been limited to the life span of the retinal cells (between one and two weeks), particularly the retinal neurons, in primary culture. See also, e.g., Luo et al., Invest. Ophthalmol. Vis. Sci. 42:1096-1106 (2001); Gaudin et al, Invest. Ophthalmol. Vis. Sci. 37:2258-68 (1996). Delays in enucleation and delays in tissue dissociation have a severe deleterious effect on recovery and survival of neurons (see, for example, Gaudin et al., supra). Neurons begin to deteriorate immediately after being dissociated from the animal body, and the resulting deterioration precludes adequate and reliable compound screening to identify agents that may be used for treating retinal diseases. Also, without the ability to maintain a long-term retinal cell culture, performing various analyses related to either projection neurons or photoreceptor cells is difficult. This cell culture system comprises the culture of retinal cells including retinal neurons in vitro for extended periods of time, thus providing viable, fully mature retinal cells and neurons for a period greater than 2 months. Also provided herein is a method for producing the cell culture system comprising isolating mature retinal cells from a biological source and culturing the mature retinal cells under conditions that maintain viability of the mature retinal cells. Viability of the retinal cells in the cell culture system means that all or a portion of the cells that are isolated and plated for tissue culture as described herein metabolize and exhibit structure and functions of a healthy, thriving cell that is characteristic for the particular cell type. Viability of one or more of the mature retinal cell types is maintained for an extended period of time, for example, at least 4 weeks, 2 months (8 weeks), or at least 4-6 months, for at least 10%, 25%, 40%, 50%, 60%, 70%, 80%, or 90% of the mature retinal cells that are isolated (harvested) from retinal tissue and plated for tissue culture. Viability of the retinal cells may be determined according to methods described herein and known in the art. Retinal neuronal cells, similar to neuronal cells in general, are not actively dividing cells in vivo and thus cell division of retinal neuronal cells would not necessarily be indicative of viability. An advantage of the cell culture system is the ability to culture amacrine cells, photoreceptors, and associated ganglion projection neurons for extended periods of time, thereby providing an opportunity to determine the effectiveness of spin-trap antioxidant compounds described herein for treatment of retinal disease. The cell culture system described herein thus serves as a model for chronic retinal diseases or disorders. Such a chronic disease model is of particularly importance because most neurodegenerative diseases are chronic. In addition, through use of this in vitro cell culture system, the earliest events in long-term disease development processes may be identified because an extended period of time is available for cellular analysis. The long-term mature retinal culture system described herein also is useful for experiments that are relatively short term in duration (e.g., 3-14 days) because the baseline for survival and viability is more stable than in short-term culture models heretofore developed in which the cells are progressively dying.

The mature retinal cells and retinal neurons may be cultured in vitro for extended periods of time, longer than 2 days or 5 days, longer than 2 weeks, 3 weeks, or 4 weeks, and longer than 2 months (8 weeks), 3 months (12 weeks), and 4 months (16 weeks), and longer than 6 months, thus providing a long-term culture. At least 20-40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of one or more of the mature retinal cell types remain viable in this long-term cell culture system. The biological source of the retinal cells or retinal tissue may be mammalian (e.g., human, non-human primate, ungulate, rodent, canine, porcine, bovine, or other mammalian source), avian, or from other genera. Retinal cells including retinal neurons from postnatal non-human primates, post-natal pigs, or post-natal chickens may be used, but any adult or post-natal retinal tissue may be suitable for use in this retinal cell culture system. The types of retinal neuronal cells that may be cultured in vitro by this method include ganglion cells, photoreceptors, bipolar cells, horizontal cells, and amacrine cells. Non-neuronal retinal cells that are cultured with the retinal neurons are cells that are derived from the original retinal tissue, and include, for example, RPE cells and Muller glial cells. By incorporating these different types of cells into the in vitro culture system, the system essentially resembles an "artificial organ" that is more akin to the natural in vivo state of the retina.

The cell culture system described herein provides for robust long-term survival of retinal cells without inclusion of cells derived from or isolated or purified from non-retinal tissue. The cell culture system comprises cells isolated solely from the retina of the eye, and thus the cell culture is substantially free of types of cells from other parts or regions of the eye that are separate from the retina, such as ciliary bodies and vitreous. A retinal cell culture that is substantially free of non-retinal cells contains retinal cells that comprise at least 80-85% of the cell types in culture, at least 90%-95%, or at least 96%- 100% of the cell types. Retinal cells in the cell culture system are viable and survive in the cell culture system without added purified (or isolated) glial cells or stem cells from a non-retinal source, or other non-retinal cells. As described herein the retinal cell culture system is prepared from isolated retinal tissue only, thereby rendering the cell culture system substantially free of non-retinal cells.

Persons skilled in the cell culture art will appreciate that successfully obtaining a long-term or extended culture of cells derived directly from a tissue source (i.e., a primary cell culture) and maintaining viability of the cells (e.g., retinal cells) in culture depends on several factors. Similar to establishing a long-term culture of any tissue-derived cell population (even including tumor tissue for propagation of immortalized cancer cells), the length of time that passes between harvesting of a retinal tissue and plating of the cells can particularly affect successful establishment of a long term culture. Neurons begin to deteriorate immediately after being dissociated from neural tissue. Delays in enucleation and delays in tissue dissociation have a severe deleterious effect on recovery and survival of neurons {see, for example, Gaudin et al., supra). Accordingly, methods for producing an extended retinal cell culture may benefit from minimizing the time periods between harvesting the tissue (which also includes minimizing the time between the death of the source animal and when the tissue is harvested) and dissecting the tissue, and the time between initiation and completion of the dissection and dissociation procedures and plating of the cells. For example, to prepare the retinal cell culture, the eyes that are dissected are preferably obtained and dissected within 12 hours of harvesting the organ. In addition, the dissection methods are performed more quickly than previously described methods for culturing retinal cells. The efficiency of this method is improved over methods for production of other retinal cell culture systems that combine retinal cells with other cell types from the eye or other regions of the CNS, by eliminating those additional cell preparation steps. Other factors that can affect successful culturing of tissue-derived cells include the temperature at which the tissues are maintained during and after transport, the health and age of the tissue donor, the skill of the animal handler, surgeon, and/or cell culturist, and similar factors appreciated by those skilled in the art. Dissection of the eye may be performed according to standard procedures known in the art and described herein. By way of example, eyes obtained from a donor animal are enucleated, and muscle and other tissue are cleaned away from the eye orbit. In one cell culture system, the peripheral retina is dissected from other portions or regions of the eye. The eyes are cut in half along their equator, and the neural retina is dissected from the anterior part of the eye. The retina, ciliary body, and vitreous are dissected away from the anterior half portion of the eye in a single piece, followed by gentle detachment of the opaque retina from the clear vitreous. In another cell culture system, the posterior portion of the retina containing the area centralis is isolated from other regions of the eye by dissection. The posterior portion of the retina contains the fovea (and the macula in primates), with a higher concentration of cone photoreceptors, whereas the anterior portion of the retina has a higher concentration of rod photoreceptors. Pigmented epithelial cells may or may not be totally separated from the dissected retina.

Retinal cells may be isolated from retinal tissue by mechanical means, such as dissection and teasing (trituration). Tissues of the eye may also be treated with one or more enzymes including but not limited to papain, hyaluronidase, collagenase, trypsin, and/or a deoxyribonuclease, to dissociate the cells and remove undesired cellular components. The cell culture system may be prepared by a combination of mechanical methods and enzymatic digestion. The cell culture systems and methods described herein may employ use of any plastic or glass surface (including, for instance, coverslips), preferably surfaces that are manufactured for cell culture use for providing a surface to which the retinal cells can adhere. The surface may also be coated with an attachment-enhancing substance or a combination of such substances, such as poly-lysine, Matrigel, laminin, poly ornithine, gelatin, and/or fibronectin, or the like. Retinal cells prepared from an eye as described herein may be plated onto one surface, such as a glass coverslip, which is then placed in a tissue culture container and immersed in tissue culture media. The tissue culture container may be, for example, a multi-well plate such as a 24-well tissue culture plate. Alternatively, one or more surfaces onto which the retinal cells are plated (and to which the cells will adhere) may be placed in one or more tissue culture flasks, which are familiar to persons in the art. Alternatively, the retinal cells may be applied to and maintained in standard tissue culture multi-well dishes and/or tissue culture flasks. Feeder cell layers, such as glial feeder layers, epithelial cell layers, or embryonic fibroblast feeder layers, may also find use within the methods and systems provided herein.

For maintaining viability of the retinal cells in the cell culture system, the system also comprises components and conditions known in the art for proper maintenance of cells in culture, including media (with or without antibiotics) that contains buffers and nutrients (e.g., glucose, amino acids (e.g., glutamine), salts, minerals (e.g., selenium)) and also may contain other additives or supplements (e.g., fetal bovine serum or an alternative formulation that does not require a serum supplement; transferrin; insulin; putrescine; progesterone) that are required or are beneficial for in vitro culture of cells and that are well known to a person skilled in the art (see, for example, Gibco media, Invitrogen Life Technologies, Carlsbad, CA). Similar to standard cell culture methods and practices, the retinal cell cultures described herein are maintained in tissue culture incubators designed for such use so that the levels of carbon dioxide, humidity, and temperature can be controlled. The cell culture system may also comprise addition of exogenous (i.e., not produced by the cultured cells themselves) cell growth factors or neurotrophic factors, such as including but not limited to ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), and glial cell line-derived neurotrophic factor (GDNF), which may be provided, for example, in the media or in the substrate or surface coating.

The disclosed retinal neuronal cell culture system may also be useful for the identification of both direct and indirect pharmacologic agent effects. For example, some drug candidates such as a spin-trap antioxidant such as alpha-phenyl-N-tert-butyl nitrone (PBN) or other spin-trap antioxidant compounds may stimulate one cell type in a manner that enhances or decreases the survival of other cell types. Cell/cell interactions and cell/extracellular component interactions may be important in understanding mechanisms of disease and drug function. For example, one neuronal cell type may secrete trophic factors that affect growth or survival of another neuronal cell type (see, e.g., PCT Publication No. WO 99/29279).

Retinal Neuronal Cell Culture Stress Model

The in vitro retinal cell culture systems described herein may serve as a physiological retinal model that can be used to characterize the physiology of the retina. This physiological retinal model may also be used as a broader general neurobiology model. A cell stressor, which as described herein is a retinal cell stressor, adversely affects the viability or reduces the viability of one or more of the different retinal cell types, including types of retinal neuronal cells, in the cell culture system. A person skilled in the art will readily appreciate and understand that as described herein a retinal cell that exhibits reduced viability means that the length of time that a retinal cell survives in the cell culture system is reduced or decreased (decreased lifespan) and/or that the retinal cell exhibits a decrease, inhibition, or adverse effect of a biological or biochemical function (e.g., decreased or abnormal metabolism; initiation of apoptosis; etc.) compared with a retinal cell cultured in an appropriate control cell system (e.g., the cell culture system described herein in the absence of the cell stressor). Reduced viability of a retinal cell may be indicated by cell death; an alteration or change in cell structure or morphology; induction and/or progression of apoptosis; initiation, enhancement, and/or acceleration of retinal neuronal cell neurodegeneration (or neuronal cell injury).

Methods and techniques for determining cell viability are described in detail herein and are those with which skilled artisans are familiar. These methods and techniques for determining cell viability may be used for monitoring the health and status of retinal cells in the cell culture system and for determining the capability of the spin-trap antioxidant compounds described herein to alter (preferably increase, prolong, enhance, improve) retinal cell viability or retinal cell survival, and/or inhibit (i.e., decrease, slow the progression, reduce, prevent) degeneration of a retinal cell.

The addition of a cell stressor to the cell culture system is useful for determining the capability of spin-trap antioxidant compounds, and derivatives thereof, to abrogate, inhibit, eliminate, or lessen the effect of the stressor. The retinal neuronal cell culture system may include a cell stressor that is chemical (e.g., A2E, cigarette smoke concentrate); biological (for example, toxin exposure; beta-amyloid; lipopolysaccharides); or non-chemical, such as a physical stressor, environmental stressor, or a mechanical force (e.g., increased pressure or light exposure).

The retinal cell stressor model system may also include a cell stressor such as, but not limited to, a stressor that may be a risk factor in a disease or disorder or that may contribute to the development or progression of a disease or disorder, including but not limited to, light of varying wavelengths and intensities; cigarette smoke condensate exposure; glucose oxygen deprivation; oxidative stress (e.g., stress related to the presence of or exposure to hydrogen peroxide, nitroprusside, Zn++, or Fe++); increased pressure (e.g., atmospheric pressure or hydrostatic pressure), glutamate or glutamate agonist (e.g., N-methyl-D-aspartate (NMDA); alpha-amino-3- hydroxy-5-methylisoxazole-4-proprionate (AMPA); kainic acid; quisqualic acid; ibotenic acid; quinolinic acid; aspartate; trans-l-aminocyclopentyl-l^-dicarboxylate (ACPD)); amino acids (e.g., aspartate, L-cysteine; beta-N-methylamine-L-alanine); heavy metals (such as lead); various toxins (for example, mitochondrial toxins (e.g., malonate, 3-nitroproprionic acid; rotenone, cyanide); MPTP (l-methyl-4-phenyl- 1,2,3,6,-tetrahydropyridine), which metabolizes to its active, toxic metabolite MPP+ (1- methyl-4-phenylpryidine)); 6-hydroxydopamine; alpha-synuclein; protein kinase C activators (e.g., phorbol myristate acetate); biogenic amino stimulants (for example, methamphetamine, MDMA (3-4 methylenedioxymethamphetamine)); or a combination of one or more stressors. Useful retinal cell stressors include those that mimic a neurodegenerative disease that affects any one or more of the mature retinal cells described herein. A chronic disease model is of particular importance because most neurodegenerative diseases are chronic. Through use of this in vitro cell culture system, the earliest events in long-term disease development processes may be identified because an extended period of time is available for cellular analysis.

A retinal cell stressor may alter (i.e., increase or decrease in a statistically or biologically significant manner) viability of retinal cells such as by altering survival of retinal cells, including retinal neuronal cells, or by altering neurodegeneration of retinal neuronal cells. Preferably, a retinal cell stressor adversely affects a retinal neuronal cell such that survival of a retinal neuronal cell is decreased or adversely affected (i.e., the length of time during which the cells are viable is decreased in the presence of the stressor) or neurodegeneration (or neuron cell injury) of the cell is increased or enhanced. The stressor may affect only a single retinal cell type in the retinal cell culture, or the stressor may affect two, three, four, or more of the different cell types. For example, a stressor may alter viability and survival of photoreceptor cells but not affect all the other major cell types (e.g., ganglion cells, amacrine cells, horizontal cells, bipolar cells, RPE, and Mϋller glia). Stressors may shorten the survival time of a retinal cell (in vivo or in vitro), increase the rapidity or extent of neurodegeneration of a retinal cell, or in some other manner adversely affect the viability, morphology, maturity, or lifespan of the retinal cell.

The effect of a cell stressor on the viability of retinal cells in the cell culture system may be determined for one or more of the different retinal cell types. Determination of cell viability may include evaluating structure and/or a function of a retinal cell continually at intervals over a length of time or at a particular time point after the retinal cell culture is prepared. Viability or long term survival of one or more different retinal cell types or one or more different retinal neuronal cell types may be examined according to one or more biochemical or biological parameters that are indicative of reduced viability, such as apoptosis or a decrease in a metabolic function, prior to observation of a morphological or structural alteration.

A chemical, biological, or physical cell stressor may reduce viability of one or more of the retinal cell types present in the cell culture system when the stressor is added to the cell culture under conditions described herein for maintaining the long- term cell culture. Alternatively, one or more culture conditions may be adjusted so that the effect of the stressor on the retinal cells can be more readily observed. For example, the concentration or percent of fetal bovine serum may be reduced or eliminated from the cell culture when cells are exposed to a particular cell stressor. When a serum-free media is desired for a particular purpose, cells may be gradually weaned (i.e., the concentration of the serum is progressively and often systematically decreased) from an animal source of serum into a media that is free of serum or that contains a non-serum substitute. The decrease in serum concentration and the time period of culture at each decreased concentration of serum may be continually evaluated and adjusted to ensure that cell survival is maintained. When the retinal cell culture system described herein is exposed to a cell stressor, the serum concentration may be adjusted concomitantly with the application of the stressor (which may also be titrated (if chemical or biological) or adjusted (if a physical stressor)) to achieve conditions such that the stress model is useful for evaluating the effect of the stressor on a retinal cell type and/or for identifying an agent that inhibits, reduces, or abrogates the adverse effect(s) of a stressor on the retinal cell. Alternatively, retinal cells cultured in media containing serum at a particular concentration for maintenance of the cells may be abruptly exposed to media that does not contain any level of serum. In another embodiment, serum may be decreased in a retinal cell culture to less than 5%, 2%, 1%, 0.5%, less than 0.25%, less than 0.1 %, or less than 0.05% in a single step.

The retinal cell culture may be exposed to a cell stressor for a period of time that is determined to reduce the viability of one or more retinal cell types in the retinal cell culture system. The length of time that the culture is exposed to a cell stressor may be 3 hours, 6 hours, 9 hours, 12, hours, 18 hours, 24 hours, or 2 days, 3 days, 4 days, 5 days, 6 days, or a week, at least two weeks, and at least one month, or longer, or for any period of time between the time periods enumerated. The cells may be exposed to a cell stressor immediately upon plating of the retinal cells after isolation from retinal tissue. Alternatively, the retinal cell culture may be exposed to a stressor after the culture is established, or any time thereafter (e.g., one day, two days, 3-5 days, 6-10 days, 2 weeks, 3 weeks, or 4 weeks). When two or more cell stressors are included in the retinal cell culture system, each stressor may be added to the cell culture system concurrently and for the same length of time or may be added separately at different time points for the same length of time or for differing lengths of time during the culturing of the retinal cell system. Viability of the retinal cells in the cell culture system may be determined by any one or more of several methods and techniques described herein and practiced by skilled artisans (see also, e.g., methods and techniques described herein regarding determining viability in the presence of the spin-trap antioxidant compounds and derivatives thereof described herein. The effect of a stressor may be determined by comparing structure or morphology of a retinal cell, including a retinal neuronal cell, in the cell culture system in the presence of the stressor with structure or morphology of the same cell type of the cell culture system in the absence of the stressor, and therefrom identifying a stressor that is capable of altering neurodegeneration of the neuronal cell. The effect of the stressor on viability can also be evaluated by methods known in the art and described herein, for example by comparing survival of a neuronal cell of the cell culture system in the presence of the stressor with survival of a neuronal cell of the cell culture system in the absence of the stressor.

Survival of retinal cells may be determined according to methods described in detail herein and known in the art that identify and characterize retinal cells, for example, immunocytochemical methods. Antibodies that specifically bind to cell markers for a specific retinal or retinal neuronal cell type as well as antibodies that bind to cytoskeletal proteins common to more than one cell type are commercially available. Alternatively, such antibodies can be prepared according to standard methods and techniques known in the art {see, e.g., Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976) and improvements thereto; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Antibody Engineering, Methods and Protocols, Lo, ed., (Human Press 2004); U.S. Patent Nos. 5,693,762; 5,585,089; 4,816,567; 5,225,539; 5,530,101; U.S. Patent No. 5,223,409; Schlebusch et al., Hybridoma 16:47 (1997); and references cited therein; see also Andris-Widhopf et al., J Immunol. Methods 242 : 159-81 (2000)).

Photoreceptors may be identified using antibodies that specifically bind to photoreceptor-specific proteins such as opsins, peripherins, and the like. Photoreceptors in cell culture may also be identified as a morphologic subset of immunocytochemically labeled cells by using a pan-neuronal marker or may be identified morphologically in enhanced contrast images of live cultures. Outer segments can be detected morphologically as attachments to photoreceptors.

Retinal cells including photoreceptors can also be detected by functional analysis. For example, electrophysiology methods and techniques may be used for measuring the response of photoreceptors to light. Photoreceptors exhibit specific kinetics in a graded response to light. Calcium-sensitive dyes may also be used to detect graded responses to light within cultures containing active photoreceptors. For analyzing stress-inducing compounds or potential neurotherapeutics, retinal cell cultures can be processed for immunocytochemistry, and photoreceptors and/or other retinal cells can be counted manually or by computer software using photomicroscopy and imaging techniques. Other immunoassays known in the art {e.g., ELISA, immunoblotting, flow cytometry) may also be useful for identifying and characterizing the retinal cells and retinal neuronal cells of the cell culture model system described herein.

The retinal cell culture stress models may also be useful for identification of both direct and indirect pharmacologic agent effects by the bioactive agent of interest, such as spin-trap antioxidant compounds and derivatives thereof. For example, a bioactive agent added to the cell culture system in the presence of one or more retinal cell stressors may stimulate one cell type in a manner that enhances or decreases the survival of other cell types. Cell/cell interactions and cell/extracellular component interactions may be important in understanding mechanisms of disease and drug function. For example, one neuronal cell type may secrete trophic factors that affect growth or survival of another neuronal cell type (see, e.g., International Patent Publication No. WO 99/29279).

The methods described herein provide for culturing retinal neurons in vitro for extended periods of time, which may be longer than 2 weeks or 4 weeks, longer than 2 months, or longer than 3 months. Retinal cells from post-natal non- human primates and post-natal chickens may be used, but any adult or post-natal retinal tissue may be suitable for use within the described cell culture methods. The source of the retinal cells or tissue may be mammalian (e.g., human, non-human primate, rodent, canine, porcine, bovine, or other mammalian source), avian, or from other genera.

Light Stressor

Light is believed to cause or contribute to retinal cell death, particularly photoreceptor cell death. Exposure to cumulative amounts of light is considered a risk factor for onset of macular degeneration. The results from animal studies have indicated that mice exposed to high intensity light develop similar pathophysiological effects as observed in humans with macular degeneration (see, e.g., Dithmar et al., Arch. Ophthalmol. 119:1643-49 (2001); Gottsch et al., Arch. Ophthalmol. 111 :126-29 (1993)). For culture of retinal cells exposed to a light stressor, the light may be emitted from at least one fluorescent light, incandescent light, or at least one light- emitting diode. The exposure may be intermittent or constant, and the duration of exposure may be varied. Alternatively, light stress may be applied as a light shock whereby cells at some point prior to or during cell culture may be protected from exposure to any light source and then exposed to a light stress.

The intensity of the light stress may be measured in lux, which is a measure of light output at a surface. The retinal cell culture described herein is preferably exposed to light (white or blue light) at any intensity or at any range of intensities from about 1 to 20,000 lux, at any intensity or any range of intensities between about 1000-15,000 lux, between about 1000-8000 lux, between about 250- 8000 lux, 250-1000 lux, 250-2000 lux, 250-4000 lux, between about 4000-8000 lux, between about 1000-6000 lux, between about 1000-4000, between about 2000-6000, between about 2000-4000, between about 4000-6000 lux, or between about 1000-2000 lux. By way of example, cells are exposed to moderate intensity, for example, about 4000-6000 lux over a short period of time, for example, less than one week, between 18-96 hours, or between 18-48 hours. In another embodiment, the retinal cells are exposed to lower intensity of light (for example, between about 500-4000 lux, or between about 500-2000 lux, between about 250-1000, or between about 500-1000 lux) over a longer period of time (such as, longer than one week, at least two weeks, or at least one month). The latter set of conditions (lower intensity of light over a longer period of time) may provide a stress model for evaluating the effect of stress in chronic neurodegenerative retinal diseases and thus for determining the capability of a spin-trap antioxidant compound, or derivative thereof, described herein to treat chronic neurodegenerative retinal diseases. In a particular embodiment, the light stressor is a blue light. As described herein A2E is phototoxic and initiates blue light-induced apoptosis in RPE cells {see, e.g., Sparrow et al., Invest. Ophthalmol. Vis. Set 43:1222- 27 (2002)). Upon exposure to blue light, photooxidative products of A2E are formed (e.g., epoxides) that damage cellular macromolecules, including DNA (Sparrow et al., J Biol. Chem. 278(20): 18207-13 (2003)). The light stress may comprise ultraviolet or visible light at any wavelength varying from between 100 to 700 nm. The light stress may be visible light and include light at any wavelength from approximately 400 nm (violet light) to approximately 700 nm (red light) of the electromagnetic spectrum. In certain embodiments, the light stress is blue light in the visible spectrum from approximately 425 nm to 500 nm, for example, 470 nm. The ultraviolet part of the spectrum (up to approximately 300-400 nm) is divided into three regions: the near ultraviolet, the far ultraviolet, and the extreme ultraviolet. The three regions are distinguished by how energetic the ultraviolet radiation is and by the wavelength of the ultraviolet light, which is related to energy. The near ultraviolet is the light closest to optical or visible light. The extreme ultraviolet is the ultraviolet light closest to X-rays, and is the most energetic of the three types. The far ultraviolet lies between the near and extreme ultraviolet regions.

The source of light may be a fluorescent light, incandescent light, or a light-emitting diode (LED); the light source may be inserted into a tissue culture incubator to provide continuous exposure or to regulate exposure during the time that the retinal cells are cultured. High intensity light sources are useful, providing the capability to apply light at variable intensity levels. LED fixtures can be designed to provide light stress to the cell cultures from above the cell culture plate (which may be any cell culture dish, flask, or multi-well plate) from one LED and below the cell culture plate from a second separate LED. Each LED may emit light of the same intensity or of different intensities, which may be controlled for example by different potentiometers to independently control the current flowing through each LED. The emitted light may be constant, that is, having the same wavelength and intensity over a period of time, or may be cyclical, varying the wavelength or intensity. For example, emitted light that is cyclical may be controlled such that the light stress mimics or matches a circadian rhythm. Light sources that are mounted in a tissue culture incubator can be appropriately placed to ensure proper ventilation such that exposure of the cells to the light source does not result in exposure of the cells or a portion of the cells to changes in temperature. In another cell culture system, the source of light is a fluorescent light fixture, for example, a set of linear bulbs that provide ambient light to an entire plate, flask, or dish of cells. The bulb may also be large enough to permit exposure of multiple cell culture plates, dishes, or flasks. The effect of light on retinal cell viability, survival, or neurodegeneration of a retinal neuronal cell in the cell culture may be determined according to methods described herein and practiced in the art. The retinal cell culture light stress model described herein may be used as a model for diseases that affect photoreceptor cells, for example, macular degeneration. For example, the retinal cell culture is exposed to light, particularly blue light, which decreases the survival or kills photoreceptor cells without killing any of the other major retinal cell types that are present in the cell culture system described herein. By way of example, the retinal cell culture system prepared as described herein, when exposed to 6000 lux of white light for 48 hours results in death of photoreceptor cells (over 95%); however, survival of ganglion cells was not reduced or adversely affected.

This model may be also used for studying cellular processes that underlie the pathology of a neurodegenerative diseases or disorders, particularly retinal diseases and disorders. By way of example, light stress affects retinal cells by inducing inappropriate activation of apoptosis (programmed cell death), which can contribute to a variety of pathological disease states. Apoptosis can be determined by a variety of methods known in the art and disclosed herein.

The light stress model may also be useful in a method for determining whether a spin-trap antioxidant compound or derivative thereof, blocks light from harming the eye. As described in more detail herein, the model may be used in methods for determining that a spin-trap antioxidant compound, or derivative thereof, has the capability to block, inhibit, or prevent light from decreasing survival of retinal cells (e.g., photoreceptor cells) or to decrease or inhibit the progression of or reverse neurodegeneration. The spin trap antioxidant compound thus acts like a filter at the cellular level to block out harmful light such as ultraviolet or blue light. By way of example, light output applied only above a retinal cell culture and measured below cells that were maintained in culture media containing phenol red (which acts as an acid-base indicator and tints the media red) was 25% less luminous (decreased intensity) than the level of light output measured above the cells. Thus, the red media had a filtering effect that protected photoreceptor cells from the light stress.

Cigarette Smoke Condensate as a Cell Stressor

The retinal cell stressor may be tobacco smoke, one or more compounds found in tobacco smoke, or cigarette smoke condensate. Smoking is believed to be a risk factor for developing macular degeneration (Delcourt et al., Arch. Ophthalmol. 116:1031-35 (1998)). Tobacco smoke contains numerous mutagenic and carcinogenic compounds such as polyaromatic hydrocarbons (PAHs)₅ tobacco-specific nitrosamines (TSNAs), carbazole, phenol, and catechol. PAHs are a group of chemicals in which constituent atoms of carbon and hydrogen are linked by chemical bonds that form two or more rings. Thus PAHs are sometimes called poly cyclic hydrocarbons or polynuclear aromatics. Examples of such chemical arrangements are anthracene (3 rings), pyrene (4 rings), benzo(a)pyrene (5 rings), and similar polycyclic compounds. Exposure of bovine retinal pigment epithelial cells to benzo(a)pyrene appeared to inhibit growth and replication of the cells (Patton et al., Exp. Eye Res. 74:513-522 (2002)).

Tobacco specific nitrosamines (TSNAs) are electrophilic alkylating agents that are potent carcinogens. TSNAs are formed by reactions involving free nitrate during processing and storage of tobacco and by combustion of tobacco that contains the alkaloids, nicotine and nomicotine, in a nitrate rich environment. Fresh- cut, green tobacco contains virtually no tobacco specific nitrosamines (see, e.g., U.S. Pat. Nos. 6,202,649 and 6,135,121). In contrast, cured tobacco products obtained according to conventional methods contain a number of nitrosamines, including N'- nitrosonornicotine (NNN) and 4-(N-nitrosomethylamino)-l-(3-ρyridyl)-l-butanone (NNK).

Additional toxic compounds produced in cigarette smoke include carbazole, phenol, and catechol. Carbazole is a heterocyclic aromatic compound containing a dibenzopyrrole system and is a suspected carcinogen. The phenolic compounds present in cigarette smoke occur as a result of pyrolysis of the polyphenols chlorogenic acid and rutin. Phenolic compounds in tobacco smoke include catechol, phenol, hydroquinone, resorcinol, o-cresol, m-cresol, and p-cresol. Catechol is the most abundant phenol in tobacco smoke (80-400 μg/cigarette) and has been identified as a co-carcinogen with benzo[a]pyrene.

Cigarette smoke condensate (CSC) may be prepared according to methods described herein and known in the art or may be purchased from a vendor such as Murty Pharmaceuticals (Lexington, KY). A mechanical device such as an FTC Smoke Machine or Phipps-Bird 20-channel smoking machine may be used for generating tobacco smoke. Examples of cigarettes used for preparing CSC include 1R4F or 1R3F research cigarettes or the like {see, e.g., Meckley et al., Food Chem. Toxicol. 42:851-63 (2004); Putnam et al., Toxicol. In Vitro 16:599-607 (2002)). To prepare CSC, for example, particulate constituents of tobacco smoke that is generated by one or more cigarettes may be deposited or collected on a filter, such as a glass fiber filter or another filter that is inert during the extraction process. Compounds are extracted from the filters using a solvent, for example, dimethyl sulfoxide (DMSO). The extraction procedure may also include a mechanical force such as sonication that is useful for aiding the removal of the particulate matter from the filters. The effect of tobacco smoke on survival of retinal cells, particularly retinal neuronal cells, or on neurodegeneration of the retinal neuronal cells, in the presence and absence of a spin-trap antioxidant, or derivative thereof, may be determined using the retinal cell culture system described herein. A retinal cell culture may be exposed to cigarette smoke condensate, tobacco smoke, or to one or more constituent compounds of tobacco smoke, including but not limited to the compounds discussed herein. The retinal cells may be exposed to a CSC cell stressor prior to culture of the retinal cells or for a period of time during the culture of the cells. Cells may be exposed to CSC for at least about 3 hours, 6 hours, 9 hours, 12, hours, 18 hours, 24 hours, or 2 days, 3 days, 4 days, 5 days, 6 days, or a week, 2 weeks, 4 weeks, 2 months, 4 months, or longer, or for any period of time between the time periods enumerated. The effect of the cell stressor on cell viability, survival, or alternatively on neurodegeneration, of the retinal cells in the cell culture may be determined according to methods described herein and known in the art.

Cigarette Smoke Condensate Plus Light as a Stressor A retinal neuronal cell culture may be exposed to more than one cell stressor, for example, the culture may be exposed to at least two retinal cell stressors. For example, one retinal cell stressor may be cigarette smoke condensate and a second cell stressor may be light as described herein.

A retinal neuronal cell culture may be exposed to two cell stressors such as cigarette smoke condensate and a light source, separately or together, and then cultured. Alternatively, the retinal cell culture may be exposed to two cell stressors such as cigarette smoke condensate and a light source, separately or together, during the culture of the retinal neuronal cells. The retinal neuronal cells may be exposed to either one or both of the cell stressors prior to culturing the cells, or the cells may be exposed to one cell stressor prior to culture and then exposed to either one or both of the cell stressors during culture of the cells. The effect of a spin-trap antioxidant compound, or derivative thereof, in the presence of the cell stressors, on the survival or alternatively neurodegeneration of the retinal cells in the cell culture may be determined according to methods described herein and known in the art. The time of exposure of the retinal neuronal cell culture to each cell stressor may differ. Cells may be exposed to CSC and/or light for at least about 3 hours, 6 hours, 9 hours, 12, hours, 18 hours, 24 hours, or 2 days, 3 days, 4 days, 5 days, 6 days, or a week, at least two weeks, and at least one month, or longer, or for any period of time between the time periods enumerated.

As described herein for culture of retinal cells exposed to a light stressor, the light may be emitted from at least one fluorescent light, incandescent light, or at least one light-emitting diode. The exposure may be intermittent or constant, and the duration of exposure may be varied. Alternatively, light stress may be applied as a light shock whereby cells at some point prior to or during cell culture may be protected from exposure to any light source and then exposed to a light stress. The light source may be inserted into a tissue culture incubator to provide continuous exposure or to regulate exposure during the time that the retinal cells are cultured.

The retinal cell culture system described herein may be used as model for diseases that affect photoreceptor cells, for example, macular degeneration. When a light stressor is combined with a CSC stressor, the number of photoreceptor cells that survive is reduced compared to the number of photoreceptor cells that survive when exposed to CSC alone.

The retinal cell culture system comprising a CSC stressor and a light stressor may be also used for studying the effect of a spin-trap antioxidant compound, or derivative thereof, on cellular processes that underlie the pathology of a neurodegenerative disease or disorder, particularly a retinal disease or disorder. For instance, such stressors may affect retinal cells by inducing inappropriate activation of apoptosis (programmed cell death), which can contribute to a variety of pathological disease states. Apoptosis can be determined by a variety of methods known in the art and described herein.

Physical Stressor: Increased Hydrostatic Pressure

The retinal cell stressor may be a physical cell stressor such as elevated hydrostatic pressure (pressure exerted by a liquid, which may be applied by methods described herein and practiced in the art such as, for example, increasing atmospheric pressure). Elevated intraocular pressure (IOP) is known in the art to correlate with glaucoma in patients. Ocular cells exposed to a hydrostatic pressure of 50 mm mercury (Hg) did not appear to have decreased viability, but morphological changes were observed as well as changes in distribution of actin stress fibers in certain cells (see Wax et al., Br. J. Ophthalmol. 84:423-28 (2000)). In one embodiment, the retinal cell culture system comprises isolated mature retinal cells, including retinal neuronal cells, and increased or elevated hydrostatic pressure (or atmospheric pressure) as a cell stressor. Cells may be exposed to a pressure that is 40, 45, 50, 55, 60, 70, 75, 80, 100, 110, 120, or 130 mm Hg (or at any pressure between the mm Hg enumerated). Increased pressure may be applied using methods described herein and known to a skilled artisan, for example, by using a pressure incubator (see, e.g., Healey et al., J Vase. Surg. 38:1099-105 (2003)) or by placing a pressure chamber within a tissue culture incubator (see, e.g., Wax et al., supra; see also Vouyouka et al., J Surg. Res. 110:344-51 (2003)). The retinal neuronal cell culture system may be exposed to increased atmospheric pressure for at least 6 hours, 9 hours, 12, hours, 18 hours, 24 hours, or 2 days, 3 days, 4 days, 5 days, 6 days, or a week, at least two weeks, and at least one month (4 weeks), or longer, or for any period of time between the time periods enumerated.

One or more culture conditions may be adjusted so that the effect of the physical stressor, such as increased hydrostatic pressure, on the retinal cells can be more readily observed. For example, the concentration or percent of fetal bovine serum may be reduced or eliminated from the cell culture when cells are exposed to increased pressure.

In another embodiment, the retinal cell culture system comprises increased hydrostatic pressure (or increased atmospheric pressure) as one cell stressor and a second cell stressor. The retinal neuronal cells may be exposed to increased pressure concomitantly with the second stressor or the cells may be exposed first to one cell stressor and then to the second stressor. In alternative embodiments, the retinal neuronal cells may be exposed to either one or both of the cell stressors prior to culturing the cells; alternatively, the cells may be exposed to one cell stressor prior to culture and then exposed to either one or both of the cell stressors during culture of the cells. The effect of a spin-trap antioxidant compound, or derivative thereof, described herein in the presence of the cell stressors on retinal cell viability, survival, or neurodegeneration of a retinal neuronal cell, may be determined according to methods described herein and known in the art.

Chemical Stressors: Retinoid N-retinylidene-N-retinyl-ethanolamine (A2E) Cell Stressor

Alternatively, the stressor may be a chemical. For example, the chemical stressor may be a vitamin A derivative, such as retinoid N-retinylidene-N- retinyl-ethanolamine (A2E), or a derivative of A2E. A2E stress may include any one or more of A2E isomers including, such as iso- A2Ε (13-Z photo-isomer of A2E {see, e.g., Parish et al., Proc. Natl. Acad. Sd. USA 95:14609-13 (1998); Ben-Shabat et al., Angew. Chem. Int. Ed. 41:814-17 (2002)), or the stress may include all isoforms of A2E. A2E is a component of retinal lipofuscin, which according to non-limiting theory is formed from retinal, digested rhodopsin, and ethanolamine (a cell membrane component), in retinal pigment epithelial cells that line the photoreceptor rods and cones during processing of cellular debris {see, e.g., Parish et al., supra; Mata et al., Proc. Natl. Acad. Sd. USA 97:7154-59 (2000)). Accumulation of A2E has been hypothesized to contribute to development of age-related neurodegeneration of retinal cells, particularly macular degeneration. Exposure of the retinal neuronal cell culture system to A2E results in selective killing of certain cells, particularly photoreceptor cells, that are present in the retinal cell culture system.

The photoreceptors in the retina, designed to initiate the cascade of events that link the incoming light to the sensation of "vision," are susceptible to damage by light, particularly blue light. The damage can lead to cell death and diseases, particularly the dry form of macular degeneration. The turnover of retinal, an essential element of the visual process, is the basis of the events that lead to damage. Free retinal, absorbing in the blue region of the visible spectrum, is phototoxic and is a precursor of the (photo)toxic compound A2E, which specifically targets cytochrome oxidase and thereby induces cell death by apoptosis.

The retinal cell culture system may be exposed to A2E at any concentration between 1 pM and 200 μM {e.g., 1 pM, 10 pM, 100 pM, 250 pM, 500 pM, 750 pM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 2 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 40 μM, 50 μM, 75 μM, 100 μM, 120 μM, 200 μM); or 250 μM, 500 μM, or 750 μM), between 1 μM and 40 μM, or between 10 μM and 20 μM, for a period of time, for example, between 2 and 48 hours or between 12 and 36 hours. In another embodiment, the cell culture may be exposed to lower concentrations of A2E (for example, between 1 pM and 10 μM or between 1 nM and 1 μM) for longer times (such as about one week, about two weeks, or about one month (4 weeks)). By way of example, the retinal cell culture system prepared as described herein when exposed to 20 μM A2E for 48 hours results in death of photoreceptor cells (more than 90% of photoreceptor cells die compared to photoreceptor cells not exposed to A2E); survival of ganglion cells is not adversely affected (i.e., ganglion cell viability is not reduced).

As described herein, more than one stressor may be applied to the retinal cell culture system. For example, a culture may be exposed to a light stressor and a chemical stressor such as A2E according to methods and techniques described herein. Additional stressors that are known in the art and described herein, including but not limited to glucose oxygen deprivation, pressure, and neurotoxins, may be combined with either a light stressor or a chemical stressor or both stressors.

Chemical Cell Stressor: Glutamate

A retinal cell culture system may include glutamate as a cell stressor. In the mammalian central nervous system (CNS), the transmission of nerve impulses is controlled by the interaction between a neurotransmitter, which is released by a sending neuron, and a surface receptor on a receiving neuron, which causes excitation of this receiving neuron. Excitatory amino acids (EAAs), principally glutamic acid (the primary excitatory neurotransmitter) and aspartic acid, mediate the major excitatory pathway in the mammalian central nervous system. Thus, glutamic acid can bring about changes in the postsynaptic neuron that reflect the strength of the incoming neural signals. The receptors that respond to glutamate are called excitatory amino acid receptors (EAA receptors) (see, e.g., Watkins et al., Trans. Pharm. Sci. 11:25 (1990);

Monaghan et al., Annu. Rev. Pharmacol. Toxicol. 29:365 (1989); Watkins et al., Annu.

Rev. Pharmacol Toxicol. 21:165 (1981)). The excitatory amino acids play a role in a variety of physiological processes, such as long-term potentiation (learning and memory), the development of synaptic plasticity, motor control, respiration, cardiovascular regulation, and sensory perception.

Excitatory amino acid receptors are classified into two general types: ionotropic and metabotropic. The ionotropic receptors contain ligand-gated ion channels and mediate ion fluxes for signaling, while the metabotropic receptors use G- proteins for signaling. Both types of receptors appear not only to mediate normal synaptic transmission along excitatory pathways, but also to participate in the modification of synaptic connections during development and throughout life (see, e.g., Schoepp et al., Trends in Pharmacol. Sci. 11:508 (1990); McDonald et al., Brain Res. Rev. 15:41 (1990)).

Further sub-classification of the ionotropic EAA glutamate receptors is based upon the agonists (stimulating agents) other than glutamic and aspartic acid that selectively activate the receptors. The at least three subtypes of the ionotropic receptors are defined by the depolarizing actions of allosteric modulators: a receptor responsive to N-methyl-D-aspartate (NMDA); a receptor responsive to alpha-amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA); and a receptor responsive to kainic acid (KA). The NMDA receptor controls the flow of both divalent (Ca⁺⁺) and monovalent (Na , K ) ions into the postsynaptic neural cell. The AMPA and KA receptors also regulate the flow into postsynaptic cells of monovalent K⁺ and Na⁺, and occasionally divalent calcium (Ca⁺⁺). Other glutamate agonists in addition to NMDA, AMPA, and KA include aspartate, ACPD, quisqualic acid, ibotenic acid, and quinolinic acid. A glutamate agonist may be included as a retinal cell stressor in the mature retinal cell culture system at concentrations and for a duration and at times as described herein for the inclusion of glutamate as a cell stressor.

The G-protein excitatory amino acid receptor is coupled to multiple second messenger systems that lead to enhanced phosphoinositide hydrolysis, activation of phospholipase D, increased or decreased c-AMP formation, and/or changes in ion channel function (see, e.g., Schoepp et al., Trends in Pharmacol. Sci. 14:13 (1993)). The metabotropic EAA receptors are divided into three sub-groups, which are unrelated to ionotropic receptors, and are coupled via G-proteins to intracellular second messengers. These metabotropic EAA receptors are classified based on receptor homology and second messenger linkages. EAA receptors have been implicated during development in specifying neuronal architecture and synaptic connectivity and may be involved in experience-dependent synaptic modifications. These receptors appear to be involved in a broad spectrum of CNS disorders. For example, during brain ischemia caused by stroke or traumatic injury, excessive amounts of the EAA glutamic acid are released from damaged or oxygen- deprived neurons. Binding of this excess glutamic acid to the postsynaptic glutamate receptors opens their ligand-gated ion channels, thereby allowing an ion influx that in turn activates a biochemical cascade resulting in protein, nucleic acid, and lipid degradation, and cell death. This phenomenon, known as excitotoxicity, may also be responsible for the neurological damage associated with other disorders ranging from hypoglycemia, ischemia, and epilepsy to chronic neurodegeneration that occurs in Huntington's, Parkinson's, and Alzheimer's diseases (see, e.g., Kannurpatti et al., Neurochem. Int. 44:361-69 (2004); Curr. Top. Med. Chem. 4:149-77 (2004); Swanson et al., Curr. MoI. Med. 4:193-205 (2004)). Excessive activation of ionotropic receptors and group I metabotropic receptors may result in neuronal death. Many neurodegenerative conditions, including Parkinson's disease, Alzheimer's disease, cerebral ischemia, epilepsy, Huntington's chorea and amyotrophic lateral sclerosis (ALS), have been linked to disturbed glutamate homeostasis (Tortarolo et al., J Neurochem. 88:481-93 (2004); Lipton et al, New Eng. J. Med. 330:613-22 (1994); Gegelashvili et al., MoI. Pharmacol. 52:6-15 (1997); Robinson et al., Adv. Pharmacol. 37:69-115 (1997)). In glaucoma, the increased release of glutamate is a major cause of retinal ganglion cell death (see, e.g., El-Remessy et al., Am. J. Pathol. 163:1997-2008 (2003)). Extracellular glutamate concentrations are maintained within physiological levels exclusively by glutamate transporters, permitting normal excitatory transmission as well as protecting against excitotoxicity (Robinson et al., Adv. Pharmacol. 37:69-115 (1997)). Nerve damage may be caused by abnormal accumulation of glutamate that leads to overexcitation of the receiving nerve cell or may be caused by oversensitive glutamate receptors on the receiving nerve cell.

The cell culture system described herein comprising mature retinal cells including retinal neuronal cells for determining the effect of a spin-trap antioxidant compound, or derivative thereof, on retinal cell survival or cell viability may comprise glutamate or a derivative thereof {see, e.g., U.S. Patent Application No. 2002/0115688) or a glutamate agonist as a cell stressor (see Luo et al., supra). The concentration of glutamate added to a retinal cell culture may be between 0.5 nM-100 μM, such as about 0.5 nM, 1 nM, 2 nM, 4 nM, 5 nM, 7.5 nM, 10 nM, 20 nM, 40 nM, 50 nM, 75 nM, 100 nM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 20 μM, 25 μM, 40 μM, 50 μM, 60 μM, 75 μM, or 100 μM, or between 100 μM and 1 mM, such as about 150 μM, 200 μM, 250 μM, 300 μM, 400 μM, 500 μM, 600 μM, 750 μM, 800 μM, 900 μM, and 1000 μM (1 mM). Glutamate acting as a cell stressor may be added to a retinal cell culture at the time the freshly harvested (isolated) retinal cells are prepared and plated for tissue culture. Alternatively, glutamate may be added at a time subsequent to plating and establishment of the retinal cells in culture. Glutamate may be added one day after plating the retinal cells, two days, three days, four days, five days, six days, or 7 days (one week), 2 weeks, 3 weeks, 4 weeks, or 6 weeks, or longer, after plating of the cells. Glutamate may also be combined with one or more other cell stressors described herein, for example, light stress, CSC, A2E stress, or increased hydrostatic pressure. As described herein when a retinal cell culture is exposed to two or more cell stressors, glutamate and one or more other cell stressors may be applied or added to the cell culture together at the same time or may be applied or added to the cell culture separately at different times and in any order. The time of exposure to each cell stressor may be different or may be the same.

A glutamate stress retinal cell culture model with or without additional cell stressors may be used. Neurodegeneration may be affected any one of a number of different pathways and receptors that are affected by excitotoxic mechanisms. For example, activation of glutamate receptors can trigger death of neurons and some types of glial cells, particularly when cells are also subjected to adverse conditions such as reduced levels of oxygen or glucose, increased levels of oxidative stress, exposure to toxins, or a genetic mutation. Excitotoxic death that occurs as a result of one or more of these adverse conditions may involve excessive calcium influx, release of calcium from internal cell organelles, radical oxygen species production, and engagement of apoptotic cascades. See, e.g., Mattson, Neuromolecular Med. 3:65-94 (2003); Atlante et al., FEBS Lett. 497:1-5 (2001).

Assay Methods In one embodiment, the cell culture system and the in vitro retinal cell culture stress model described herein are used for biological testing of compounds, such as spin-trap antioxidant compounds, such as PBN and derivatives thereof, as described herein, that may be suitable for treatment of neurological diseases or disorders in general, and for treatment of degenerative diseases of the eye and brain in particular. As described herein, a method is provided for enhancing survival or viability of a retinal cell and/or inhibiting degeneration of a retinal cell (which may be a retinal neuronal cell such as a photoreceptor cell, amacrine cell, ganglion cell, horizontal cell, and/or a bipolar cell) wherein the method comprises contacting (i.e., combining, mixing, or otherwise permitting interaction of) a spin-trap antioxidant compound (such as PBN), or a derivative thereof, with the mature retinal cells present in a retinal cell culture system (in the absence or presence of one or more cell stressors) under conditions and for a time sufficient to permit interaction between the spin-trap antioxidant and the cell culture system, and then comparing survival of a plurality of mature retinal cells in the presence of the candidate agent with the survival of a plurality of mature retinal cells in the absence of the candidate agent. As described herein, the plurality of retinal cells that are not exposed to the spin-trap antioxidant may be prepared simultaneously from the same retinal tissue as the retinal cells that are exposed to the compound. Alternatively, survival of retinal cells in the presence of the spin-trap antioxidant may be quantified and compared to survival of a standard retinal cell culture (i.e., a retinal cell culture system as described herein that provides repeatedly consistent, reliable, and precise determinations of retinal cell survival and viability).

The conditions under which a compound and a retinal cell are permitted to interact include those with which a skilled artisan will be familiar, such as temperature, media components, buffers, appropriate diluent and testing concentrations of the compound, etc. A person skilled in the art may readily and routinely determine conditions and time sufficient for performing these methods on the basis of the description provided herein and on the basis of procedures with which a skilled artisan is familiar. Methods for altering (increasing or decreasing in a statistically or biologically significant manner), preferably increasing in a statistically significant or biologically significant manner, or maintaining the viability of a mature retinal cell comprise contacting (i.e., combining, mixing, or otherwise permitting interaction of) a spin-trap antioxidant compound, such as PBN, with the mature retinal cells present in a retinal cell culture system (in the absence or presence of one or more cell stressors) under conditions and for a time sufficient to permit interaction between the spin-trap antioxidant compounds and the cell culture system, and then comparing the viability of a plurality of mature retinal cells in the presence of the compound with the viability of a plurality of mature retinal cells in the absence of the candidate agent. The plurality of retinal cells that are not exposed to the spin-trap antioxidant compound may be prepared simultaneously from the same retinal tissue as the retinal cells that are exposed to the compound. Alternatively, the viability of retinal cells in the presence of the spin- trap antioxidant compound may be quantified and compared to viability of a standard retinal cell culture (i.e., a retinal cell culture system as described herein that provides repeatedly consistent, reliable, and precise determinations of retinal cell viability).

In another embodiment, a spin-trap antioxidant (such as PBN), or a derivative thereof, is incorporated into screening assays comprising the retinal cell culture stress model system to determine whether the compound is capable of altering (i.e., impairing, inhibiting, preventing, abrogating, reducing, slowing the progression of, or accelerating in a statistically significant manner) degeneration of a retinal cell, including whether the compound is capable of altering neurodegeneration of a retinal neuronal cell. A compound useful for treating a retinal disease or disorder preferably inhibits (i.e., reduces, abrogates, slows the progression of, or impairs) degeneration of a retinal cell, particularly a retinal neuronal cell; is capable of regenerating a retinal cell; and/or is capable of enhancing or prolonging survival (i.e., promoting, improving, or increasing survival or increasing cell viability, thus delaying injury and/or death) of a retinal cell. A compound that inhibits degeneration of a retinal cell may be identified by contacting (i.e., mixing, combining, or otherwise permitting interaction between the compound and retinal cells of the cell culture system) with the cell culture system under conditions and for a time sufficient to permit interaction between the compound and the retinal cells, particularly the mature retinal neuronal cells of the cell culture system described herein.

A bioactive spin-trap antioxidant agent or a derivative thereof as described herein that effectively alters (preferably inhibits, impairs, slows the progression of, prevents, decreases, or reverses) degeneration of a retinal cell, including neurodegeneration or neuronal cell injury of a retinal neuronal cell, and/or that enhances (prolongs) retinal cell survival may be identified and/or evaluated for such capability by techniques known in the art and described herein. For instance, such techniques and methods include, but are not limited to, determining (a) the effects of the compound on neuronal cell structure or morphology; (b) expression of neuronal cell markers (e.g., β3-tubulin, rhodopsin, recoverin, visinin, calretinin, calbindin, Thy-1, tau, microtubule-associated protein 2, neuron-specific enolase, protein gene product 95, and the like (see, e.g., Espanel et al., Int. J. Dev. Biol. 41:469-76 (1997); Ehrlich et al., Exp. Neurol. 167:215-26 (2001); Kosik et al., J Neurosci. 7:3142-53 (1987); Luo et al., supra); and (c) cell survival (i.e., cell viability or length of time until cell death). Antibodies that may be used for determining the aforementioned properties and characteristics include antibodies that specifically bind to a protein that is expressed by specific cell types (e.g., opsins expressed by photoreceptor cells, for example, rhodopsin expressed by rods; β3-tubulin expressed by interneurons and ganglion cells; and NFM expressed by ganglion cells), and include antibodies that specifically identify a cell marker expressed by a retinal cell that is from a specific animal source. Preferably, a spin-trap antioxidant compound enhances survival of retinal cells including retinal neuronal cells, and more particularly photoreceptor cells. That is, the spin-trap antioxidant compound promotes survival or prolongs survival such that the time period in which neuronal cells are viable is extended (i.e., increases cell viability). The methods described herein may be used for identifying or determining that a spin-trap antioxidant (such as PBN or a derivative thereof) alters viability (i.e., alters survival and/or neurodegeneration and/or neuronal cell injury) of one, two, three, or more, or all retinal cell types and may also be used to identify and/or determine that the compound alters viability of one, two, three, four, or all retinal cells and retinal neuronal cell types (amacrine cell, a photoreceptor cell, a ganglion cell, horizontal cell, and bipolar cell). In certain other embodiments, the screening methods may be used to identify and/or determine that a spin-trap antioxidant alters viability (preferably enhances or promotes survival and/or inhibits degeneration or cell injury) of one retinal cell type, such as an amacrine cell, a photoreceptor cell, or a ganglion cell, horizontal cell, or bipolar cell.

In another embodiment, a spin-trap antioxidant (for example, PBN or a derivative thereof) is incorporated into screening assays comprising the retinal cell culture stress model system described herein to determine whether the compound increases viability (i.e., increases in a statistically significant or biologically significant manner) of a plurality of retinal cells. A person skilled in the art would readily appreciate and understand that as described herein a retinal cell that exhibits increased viability means that cell survival is enhanced or promoted and that the length of time that a retinal cell survives in the cell culture system is increased (increased lifespan) and/or that the retinal cell maintains a biological or biochemical function (normal metabolism and organelle function; lack of apoptosis; etc.) compared with a retinal cell cultured in an appropriate control cell system (e.g., the cell culture system described herein in the absence of the spin-trap antioxidant compound). Increased viability of a retinal cell may be indicated by delayed cell death or a reduced number of dead or dying cells; maintenance of structure and/or morphology; lack of or delayed initiation of apoptosis; delay, inhibition, slowed progression, and/or abrogation of retinal neuronal cell neurodegeneration or delaying or abrogating or preventing the effects of neuronal cell injury. Methods and techniques for determining viability of a retinal cell and thus whether a retinal cell exhibits increased viability are described in greater detail herein and are routinely practiced by persons skilled in the art. In one embodiment, a method is provided for determining whether a spin-trap antioxidant compound (e.g., PBN, or a derivative thereof) enhances (promotes) survival (or increases cell viability or decreases neurodegeneration) of photoreceptor cells. One method comprises contacting a retinal cell culture system as described herein with the spin-trap antioxidant compound under conditions and for a time sufficient to permit interaction between the retinal cells and the compound. Enhanced survival (i.e., prolonged survival) may be measured according to methods described herein and known in the art, including detecting expression of rhodopsin. Rhodopsin, which is composed of the protein opsin and retinal (a vitamin A form), is located in the membrane of the photoreceptor cell in the retina of the eye and catalyzes the only light sensitive step in vision. The 11 -cis-retinal chromophore lies in a pocket of the protein and is isomerised to α//-£røns-retinal when light is absorbed. The isomerisation of retinal leads to a change of the shape of rhodopsin, which triggers a cascade of reactions that lead to a nerve impulse that is transmitted to the brain by the optical nerve.

The capability of a spin-trap antioxidant (for example, PBN or a derivative thereof) to increase retinal cell viability (or in certain embodiments, to maintain cell viability) and/or to enhance (promote or prolong) cell survival (that is, to extend the time period in which retinal cells are viable), and/or inhibit (impair, slow the progression of, retard, or impede) degeneration, in the presence or absence of a herein described stress or as a direct or indirect result of same, may be determined by any one of several methods practiced by those skilled in the art. For example, changes in cell morphology in the absence and presence of a spin-trap antioxidant may be determined by visual inspection such as by light microscopy, confocal microscopy, or other microscopy methods known in the art. Survival of cells can also be determined by counting viable and/or nonviable cells, for instance. Immunochemical or immunohistological techniques (such as fixed cell staining or flow cytometry) may be used to identify and evaluate cytoskeletal structure (e.g., by using antibodies specific for cytoskeletal proteins such as glial fibrillary acidic protein, fibronectin, actin, vimentin, tubulin, or the like) or to evaluate expression of cell markers as described herein.

The effect of a compound of interest on cell integrity, morphology, and/or survival may also be determined by measuring the phosphorylation state of neuronal cell polypeptides, for example, cytoskeletal polypeptides (see, e.g., Sharma et al., J. Biol. Chem. 274:9600-06 (1999); Li et al., J Neurosci. 20:6055-62 (2000)). Viability, cell survival or, alternatively cell death, may also be determined according to methods described herein and known in the art for measuring apoptosis (for example, annexin V binding, DNA fragmentation assays (such as terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL)); caspase activation; mitochondrial membrane potential breakdown; marker analysis, e.g., poly(ADP-ribose) polymerase (PARP); detection with antibodies specific for enzymes or polypeptides expressed during apoptosis (e.g., an anti-caspase-3 antibody; etc.)).

Viability (or survival) of one or more retinal cell types that are present in the cell culture system described herein may be determined according to methods described herein and with which a skilled artisan is familiar. For example, viable cells may be differentiated from non- viable cells by uptake of particular dyes, such as trypan blue. Alternatively, cell death and cell lysis may be quantified by measuring cellular metabolites or enzymes, such as alkaline and acid phosphatase, glutamate-oxalacetate transaminase, glutamate pryuvate transaminase, argininosuccinate lyase, and lactate dehydrogenase, that are released into cell culture media supernatant from the damaged cells (e.g., via damaged or compromised plasma membranes) or upon cell expiration. For example, viability assays may be employed that use esterase substrates, stain nucleic acids, or that measure oxidation or reduction (see Molecular Probes, Eugene, OR, Invitrogen Life Sciences, Carlsbad, CA). Viability of living cells that are not actively dividing, such as retinal neuronal cells, may be determined by evaluating one or more metabolic processes. Such methods incorporate reagents that may be detected by colorimetric or fluorimetric analyses. Companies that provide assay kits for determining cell viability/vitality or cytotoxicity include Roche Applied Science, Indianapolis, IN and Molecular Probes. Viability of one or more retinal cell types in the cell culture system may be determined by assessing survival of the one, two, three, or more retinal cell types. Viability or survival of retinal cells in the cell culture system in the absence or presence of one or more cell stressors may be determined, as well as viability (survival) in the absence or presence of a spin-trap antioxidant (such as PBN or a derivative thereof). In one embodiment, the spin-trap antioxidant enhances (i.e., prolongs) survival of one or more retinal cell types. Survival may be determined by comparing the number (or percent) of retinal cells exposed to the spin-trap antioxidant thereof that are viable over a defined period of time relative to the number (or percent) of retinal cells not exposed to the compound that are viable over the same defined time period. Survival of retinal cells in the cell culture system may be compared during the time the cells are exposed to the spin-trap antioxidant or may be compared for a period(s) of time after the compound is removed from the cell culture system. The time period may be 1 day, 2-3 days, 4-7 days, 7-14 days, or 14-28 days, 2 months, 4, months, or longer. A compound such as a spin-trap antioxidant may act directly upon a retinal cell, such as a retinal neuronal cell, in a manner that affects survival or degeneration (or neuronal cell injury) of the cell. Alternatively, the compound may act indirectly by interacting with one retinal cell type that consequently, via a biological response to the compound, affects viability, that is survival and/or degeneration, of another retinal cell. Not wishing to be bound by theory, glial cells such as Mϋller glial cells, which are associated with retinal neurons and interact with retinal neurons such that the Mϋller glial cells support the metabolic function of the neurons, may be acted upon by the spin-trap antioxidant. The effect of the compound on the biological or biochemical function of a Mϋller glial cell may in turn affect the metabolism, viability, and/or survival of the associated retinal neuron(s). For instance, viability, survival, or neurodegeneration of a retinal neuronal cell may be indirectly affected or altered in a statistically or biologically significant manner by a compound that maintains viability or enhances survival of a Mϋller glial cell.

The disclosed methods and cell culture model systems permit precise measurements of specific interactions occurring between retinal neurons, as well as enabling detailed analysis of subtleties in retinal neuron structure. For instance, the methods and cultured cells described herein are compatible with neurochips, cell-based biosensors, and other multielectrode or electrophysiologic devices for stimulating and recording data from cultured neurons {see, for instance, M.P. Maher et al., J Neurosci. Meth. 87:45-56 (1999); K.H. Gilchrist et al., Biosensors & Bioelectronics 16:557-64, (2001)).

Also provided herein are methods for identifying agents and measuring the activity of bioactive agents such as spin-trap antioxidant compounds (for example, PNB), or derivatives thereof, that may be useful for treating neurodegenerative diseases. Such neurodegenerative disease include but are not limited to retinal diseases (also referred to as ocular or ophthalmic diseases) such as glaucoma, macular degeneration, including dry form macular degeneration, diabetic retinopathy, retinal detachment, retinal blood vessel (artery or vein) occlusion, retinitis pigmentosa, hemorrhagic retinopathy, retinopathy of prematurity, inflammatory retinal diseases, optic neuropathy, proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusion related retinal injury, hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, a retinal disorder associated with viral infection, a retinal disorder related to light overexposure, and retinal disorders associated with other degenerative diseases such as Alzheimer's disease, multiple sclerosis, and Parkinson's disease, or associated with AIDS or other diseases of the brain. The cultured mature retinal neurons provided herein are particularly useful for screening compounds and determining the biological activity of compounds such as spin-trap antioxidant compounds that enable or effect regeneration of retinal tissue that has been damaged by disease. For example, the presence of photoreceptors with an intact outer segment is relevant in such an assay to identify and analyze compounds useful for treating neurodegenerative eye diseases.

Bioactive agents or compounds of interest as described herein may be incorporated into screening assays comprising the cell culture system described herein to determine whether the compound is capable of altering neurodegeneration of neuronal cells (impairing, inhibiting, preventing, or accelerating in a statistically or biologically significant manner). A preferred compound is one that enhances cell survival (i.e., prolongs survival and cell viability), such as enhancing or prolonging survival of photoreceptor cells, or that inhibits or impairs degeneration of a retinal cell, including inhibits or impairs neurodegeneration of a retinal neuronal cell, or that is capable of regenerating a neuronal cell. A compound that inhibits degeneration of a retinal cell may be identified by contacting a candidate compound, for example, a compound from a library of a spin-trap antioxidant compounds such as a library of alpha-phenyl-N-tert-butyl nitrone (PBN) derivatives, with the cell culture system, under conditions and for a time sufficient to permit interaction between a candidate agent and the retinal neuronal cells. A compound may act directly upon a retinal cell to affect survival or degeneration of the cell. Alternatively, a bioactive agent may act indirectly by interacting with one cell that consequently responds to the agent by affecting survival or degeneration of another retinal cell. Such bioactive agents that enhance survival of retinal cells {e.g., a retinal neuronal cell, particularly a photoreceptor cell) may be used in methods for enhancing or prolonging neuronal cell survival in a subject who has a disease or disorder of the retina as described herein.

The methods described herein for identifying and evaluating spin-trap antioxidant compound compounds that enhance or prolong survival of retinal cells and/or that inhibit degeneration of retinal cells may also include an additional stressor as described herein, for example, light of varying wavelengths and intensities, compounds such as A2E, cigarette smoke concentrate, glucose oxygen deprivation, pressure, various toxins, or a combination of one or more stressors. By way of example, the photoreceptors in the retina, designed to initiate the cascade of events that link the incoming light to the sensation of vision, are susceptible to damage by light, particularly blue light. The damage can lead to cell death and diseases, particularly dry form of macular degeneration. The turnover of retinal, an essential element of the visual process, is the basis of the events that lead to damage. Free retinal, absorbing in the blue region of the visible spectrum, is phototoxic, and is a precursor of the (photo)toxic compound A2E, which specifically targets cytochrome oxidase and thereby induces cell death by apoptosis. As described herein, A2E is a component of retinal lipofuscin, which according to non-limiting theory is formed from retinal, digested rhodopsin, and ethanolamine (a cell membrane component), in retinal pigment epithelial cells that line the photoreceptor rods and cones during processing of cellular debris {see, e.g., Parish et al., supra; Mata et al., Proc. Natl. Acad. Sci. USA 97:7154-59 (2000)). Accumulation of A2E may play some role in development of age-related degeneration of retinal cells, particularly macular degeneration. Exposure of the retinal cell culture system described herein to A2E results in selective killing of certain cells, particularly photoreceptor cells, that are present in the retinal cell culture.

Treatment of Neurodegenerative Diseases of the Eye

As described herein methods are provided for enhancing (promoting) retinal cell survival, increasing (promoting, enhancing) retinal cell viability, and/or inhibiting (impairing, preventing, abrogating, reducing, slowing the progression of) retinal cell degeneration. In particular embodiments, methods are provided for treating neurodegenerative diseases and disorders particularly neurodegenerative retinal diseases and ophthalmic diseases as described herein. In one embodiment, such methods comprise administering a spin-trap antioxidant compound to a subject having a neurodegenerative disease of the eye (i.e., a retinal disease) by administering to the subject a composition comprising a spin-trap antioxidant or derivative thereof and a pharmaceutically acceptable carrier (i.e., pharmaceutically (physiologically) acceptable excipient, diluent, etc. as described herein), hi one embodiment, the spin-trap antioxidant compound is a spin-trap antioxidant such as alpha-phenyl-N-tert-butyl nitrone (PBN), or a derivative thereof (e.g., S-PBN). These compounds may be suitable for treatment of neurological diseases or disorders in general, and for treatment of degenerative diseases of the eye and brain in particular (see also U.S. Patent Application No. 2003/0018044). These methods enhance survival, increase viability, and/or inhibit degeneration of a retinal cell including any one or more of the different types of retinal neuronal cells, such as a photoreceptor cell, amacrine cell, horizontal cell, ganglion cell, or bipolar cell. A person skilled in the art will readily appreciate and understand that as described herein a retinal cell present in the eye of a subject that exhibits increased viability means that the length of time that a retinal cell survives is prolonged or increased (increased lifespan) and/or that the retinal cell maintains or exhibits improvement of a biological or biochemical function (e.g., normal metabolism; lack of apoptosis or other cell death related events; etc.) compared with a retinal cell in a subject who is afflicted with a similar disease or disorder or compared with viability, survival, or degeneration of a retinal cell in the subject to be treated or being treated prior to treatment with a spin trap antioxidant. Reduced viability of a retinal cell may be indicated by cell death; an alteration or change in cell structure or morphology; induction and/or progression of apoptosis; initiation, enhancement, and/or acceleration of retinal neuronal cell neurodegeneration (or neuronal cell injury).

A neurodegenerative disease or disorder for which the compounds and methods described herein may be used for treating, curing, preventing, ameliorating the symptoms of, slowing, inhibiting, and/or stopping the progression of, is a disease or disorder that leads to or is characterized by retinal neuronal cell loss, which is the cause of visual impairment. Such a disease or disorder includes, but is not limited to, glaucoma, macular degeneration, including dry form macular degeneration, diabetic retinopathy, retinal detachment, retinal blood vessel (artery or vein) occlusion, retinitis pigmentosa, hemorrhagic retinopathy, retinopathy of prematurity, inflammatory retinal diseases, optic neuropathy, proliferative vitreoretinopathy, retinal dystrophy, ischemia- reperfusion related retinal injury (such as that caused by transplant, surgical trauma, hypotension, thrombosis or trauma injury), traumatic injury to the optic nerve (such as by physical injury, excessive light exposure, or laser light), hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, neuropathy due to a toxic agent or caused by adverse drug reactions or vitamin deficiency, a retinal disorder associated with viral infection (e.g., cytomegalovirus or herpes simplex virus), a retinal disorder related to light overexposure, and retinal disorders associated with other degenerative diseases such as Alzheimer's disease, multiple sclerosis, and Parkinson's disease, or associated with AIDS or other diseases of the brain or other forms of progressive retinal atrophy or degeneration. In another specific embodiment, the disease or disorder results from mechanical injury, chemical or drug-induced injury, thermal injury, radiation injury, light injury, laser injury. The spin trap antioxidant compounds described herein are also useful for preventing ophthalmic injury from environmental factors such as light- induced oxidative retinal damage, laser-induced retinal damage, etc.

Macular degeneration as described herein is a disorder that affects the macula (central region of the retina) and results in the decline and loss of central vision. Age-related macular degeneration occurs typically in individuals over the age of 55 years. The etiology of age-related macular degeneration may include both an environmental influence and a genetic component {see, e.g., Iyengar et al., Am. J. Hum. Genet. 74:20-39 (2004) (Epub 2003 December 19); Kenealy et al., MoI. Vis. 10:57-61 (2004); Gorin et al., MoI. Vis. 5:29 (1999)). More rarely, macular degeneration occurs in younger individuals, including children and infants, and generally the disorder results from a genetic mutation. Types of juvenile macular degeneration include Stargardt's disease {see, e.g., Glazer et al., Ophthalmol. Clin. North Am. 15:93-100, viii (2002); Weng et al., Cell 98:13-23 (1999)); Best's vitelliform macular dystrophy {see, e.g., Kramer et al., Hum. Mutat. 22:418 (2003); Sun et al., Proc. Natl. Acad. Sci. USA 99:4008-13 (2002)), Doyne's honeycomb retinal dystrophy {see, e.g., Kermani et al., Hum. Genet. 104:77-82 (1999)); Sorsby's fundus dystrophy, Malattia Levintinese, fundus flavimaculatus, and autosomal dominant hemorrhagic macular dystrophy {see also Seddon et al., Ophthalmology 108:2060-67 (2001); Yates et al., J Med. Genet. 37:83-7 (2000); Jaakson et al., Hum. Mutat. 22:395-403 (2003)).

In another embodiment, a subject may be treated for diabetic retinopathy or diabetic maculopathy. Diabetes increases the permeability of blood vessel walls beneath the retina, allowing fluids and fatty exudates to accumulate in the macula. This accumulation causes macular edema, destabilizes RPE membranes, and causes abnormal blood vessel function, leading to light-exacerbated vision loss. Preventing the accumulation of these exudates (or phototoxic constituents, such as A2E) could protect the diabetic retina from degeneration. As used herein, a patient (or subject) may be any mammal, including a human, that may have or be afflicted with a neurodegenerative disease or condition, including an ophthalmic disease or disorder, or that may be free of detectable disease. Accordingly, the treatment may be administered to a subject who has an existing disease, or the treatment may be prophylactic, administered to a subject who is at risk for developing the disease or condition. Treating or treatment refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.

The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination. Accordingly, the term "treating" includes the administration of the compounds or agents described herein to treat pain, hyperalgesia, allodynia, or nociceptive events and to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with pain, hyperalgesia, allodynia, nociceptive events, or other disorders. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or sequelae of the disease in the subject. Treatment also includes restoring or improving retinal neuronal cell functions (including photoreceptor function) in a vertebrate visual system, for example, such as visual acuity and visual field testing etc., as measured over time (e.g., as measured in weeks or months). Treatment also includes stabilizing disease progression (i.e., slowing, minimizing, or halting the progression of an ophthalmic disease and associated symptoms) and minimizing additional degeneration of a vertebrate visual system. Treatment also includes prophylaxis and refers to the administration of a spin-trap antioxidant compound to a subject in need thereof to prevent degeneration or further degeneration or deterioration or further deterioration of a retinal cell and the vertebrate visual system of the subject and to prevent or inhibit development of the disease and/or related symptoms and sequelae. A subject in need of such treatment may be a human or may be a non- human primate or other animal (i.e., veterinary use) who has developed symptoms of a retinal disease or disorder or who is at risk for developing a retinal disease or disorder. Examples of non-human primates and other animals include but are not limited to farm animals, pets, and zoo animals (e.g., horses, cows, buffalo, llamas, goats, rabbits, cats, dogs, chimpanzees, orangutans, gorillas, monkeys, elephants, bears, large cats, etc.). Subjects in need of treatment using the compounds and methods described herein may be identified according to accepted screening methods in the medical art that are employed to determine risk factors or symptoms associated with an ophthalmic disease or condition described herein or to determine the status of an existing ophthalmic disease or condition in a subject. These and other routine methods allow the clinician to select patients in need of therapy using the methods and compositions described herein.

Administering a spin-trap antioxidant such as alpha-phenyl-N-tert-butyl nitrone (PBN) or a derivative thereof (e.g., S-PBN) to a subject or patient for enhancing survival of photoreceptor cells may be particularly useful for treating retinal diseases that include photoreceptor neurodegeneration as a sequelae of the disease, including but not limited to the dry form of macular degeneration. As described herein, dry or atrophic macular degeneration results in the loss of RPE cells and photoreceptors and is characterized by diminished retinal function due to an overall atrophy of the cells. By contrast, the wet form or neovascular form of macular degeneration involves proliferation of abnormal choroidal vessels, which penetrate the Bruch's membrane and RPE layer into the subretinal space, thereby forming extensive clots and/or scars (see, e.g., Hamdi et al., Front. Biosci. 8:e305-14 (2003)). Macugen® (pegaptanib sodium injection) is a pegylated single-stranded nucleic acid that specifically inhibits VEGF, which was very recently approved by the FDA for the treatment of neovascular (wet) ARMD; the nucleic acid is administered to patients by intravitreal injection every six weeks. The dry form often precedes development of the wet form; therefore, a patient or subject at risk for developing the wet form of macular degeneration is a subject who has or presents symptoms of the dry form of macular degeneration. A composition, including a pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion, which comprises a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient, which is also referred to as a physiologically or pharmaceutically suitable carrier, excipient, or diluent) in addition to the spin-trap antioxidant compound. Such compositions may be in the form of a solid, liquid, or gas (aerosol). Alternatively, compositions described herein may be formulated as a lyophilizate, or compounds may be encapsulated within liposomes using technology known in the art. Pharmaceutical compositions within the scope of the present invention may also contain other components, which may be biologically active or inactive. Such components include, but are not limited to, buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, stabilizers, dyes, flavoring agents, and suspending agents and/or preservatives.

Any pharmaceutically acceptable (pharmaceutically suitable) carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions useful for practicing the present invention. A pharmaceutically acceptable or suitable carrier includes an ophthalmologically suitable or acceptable carrier. Carriers for therapeutic use are well known, and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. 1985). In general, the type of carrier is selected based on the mode of administration. Modes of local administration can include, for example, eye drops, intraocular injection or periocular injection. Pharmaceutical compositions may be formulated for any appropriate manner of administration, including, for example, intraocular, subconjunctival, topical, oral, nasal, intrathecal, rectal, vaginal, sublingual or parenteral administration, including subcutaneous, intravenous, intramuscular, intrasternal, intracavernous, intrameatal or intraurethral injection or infusion. For parenteral administration, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. Suitable nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose, ethyl cellulose, cellulose, glucose, sucrose, magnesium carbonate, and the like. (See, e.g., Gennaro, Ed., Remington "Pharmaceutical Sciences", 17 Ed., Mack Publishing Co., Easton, Pennsylvania, 1985.

A pharmaceutical composition (e.g., for oral administration or delivery by injection or for ocular administration) may be in the form of a liquid. A liquid pharmaceutical composition may include, for example, one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils that may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents; antioxidants; chelating agents; buffers and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The use of physiological saline is preferred, and an injectable pharmaceutical composition or a composition that is delivered ocularly is preferably sterile.

The compositions described herein may be formulated for sustained or slow release. Such compositions may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain an agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

Pharmaceutical compositions may be administered in a manner appropriate to the disease to be treated (or prevented) as determined by those skilled in the medical arts. Systemic drug absorption of a drug or composition administered via an ocular route is known to those skilled in the art {see, e.g., Lee et al., Int. J. Pharm. 233:1-18 (2002)), which may be, for example, intraocular or periocular. Periocular injection typically comprises an injection into the conjunctiva or into the tennon, which is the fibrous tissue overlying the eye. Injection by an intraocular route typically refers to injection of the therapeutic agent into the vitreous of the eye. Alternatively, a spin- trap antioxidant (e.g., PNB) or derivative (e.g., S-PBN) thereof may be delivered by a topical ocular delivery method (see, e.g., Curr. Drug Metab. 4:213-22 (2003)).

The composition may be in the form of an eye drop, salve, or ointment or the like, such as, aqueous eye drops, aqueous ophthalmic suspensions, non-aqueous eye drops, and non-aqueous ophthalmic suspensions, gels, ophthalmic ointments, etc. For preparing a gel, for example, carboxyvinyl polymer, methyl cellulose, sodium alginate, hydroxypropyl cellulose, ethylene maleic anhydride polymer and the like can be used. The dose of the composition of the present invention may differ, depending upon the patient's (e.g., human) condition, that is, stage of the disease, general health status, age, and other factors that a person skilled in the medical art will use to determine dose. When the composition of the present invention is used as eye drops, for example, one to several drops per unit dose, preferably 1 or 2 drops (about 50 μl per 1 drop), may be applied about 1 to about 6 times daily. Suitable ophthalmological compositions include those that are administrable locally to the eye, such as by eye drops, injection or the like. In the case of eye drops, the formulation can also optionally include, for example, ophthalmologically compatible agents such as isotonizing agents such as sodium chloride, concentrated glycerin, and the like; buffering agents such as sodium phosphate, sodium acetate, and the like; surfactants such as polyoxyethylene sorbitan mono-oleate (also referred to as Polysorbate 80), polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil, and the like; stabilization agents such as sodium citrate, sodium edentate, and the like; preservatives such as benzalkonium chloride, parabens, and the like; and other ingredients. Preservatives can be employed, for example, at a level of from about 0.001 to about 1.0% weight/volume. The pH of the formulation is usually within the range acceptable to ophthalmologic formulations, such as within the range of about pH 4 to 8.

The PNB compound or derivative thereof may also be administered orally or intravenously, providing systemic distribution of the compound. For injection, the spin trap antioxidant compound, such as PNB, can be provided in an injection grade saline solution, in the form of an injectable liposome solution, or the like. Intraocular and periocular injections are known to those skilled in the art and are described in numerous publications including, for example, Spaeth, Ed., Ophthalmic Surgery: Principles of Practice, W. B. Sanders Co., Philadelphia, Pa., 85-87, 1990.

An appropriate dosage (an effective does) and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dosage and treatment regimen provides the spin-trap antioxidant compound in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of or diminish the severity of a disease associated with neurodegeneration of retinal neuronal cells. Optimal dosages may generally be determined using experimental models and/or clinical trials. The optimal dose may depend upon the body mass, weight, or blood volume of the patient. The doses of the spin trap antioxidant compounds can be suitably selected depending on the clinical status, condition and age of the subject, dosage form and the like, hi the case of eye drops, a compound can be administered, for example, from about 0.01 mg, about 0.1 mg, or about 1 mg, to about 25 mg, to about 50 mg, to about 90 mg per single dose. Eye drops can be administered one or more times per day, as needed. In the case of injections, suitable doses can be, for example, about 0.0001 mg, about 0.001 mg, about 0.01 mg, or about 0.1 mg to about 10 mg, to about 25 mg, to about 50 mg, or to about 90 mg of the compound, one to four times per week. In other embodiments, about 1.0 to about 30 mg of a spin trap antioxidant compound such as PNB can be administered one to three times per week.

Oral doses can typically range from about 1.0 to about 1000 mg, one to four times, or more, per day. An exemplary dosing range for oral administration is from about 10 to about 250 mg one to three times per day.

The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES

EXAMPLE 1

PNB ENHANCES PHOTORECEPTOR SURVIVAL

This Example describes the effect of a spin-trap antioxidant such as alpha-phenyl-N-tert-butyl nitrone (PBN) on photoreceptor cell survival in an extended retinal neuronal cell culture system.

All compounds and reagents were obtained from Sigma Aldrich Chemical Corporation (St. Louis, MO), except as noted.

Retinal Neuronal Cell Culture

Porcine eyes were obtained from Kapowsin Meats, Inc. (Graham, WA). Eyes were enucleated, and muscle and tissue were cleaned away from the orbit. Eyes were cut in half along their equator and the neural retina was dissected from the anterior part of the eye in buffered saline solution, according to standard methods used in the art. Briefly, the retina, ciliary body, and vitreous were dissected away from the anterior half of the eye in one piece, and the retina was gently detached from the clear vitreous. Each retina was dissociated with papain (Worthington Biochemical Corporation, Lakewood, NJ), followed by inactivation with fetal bovine serum (FBS) and addition of 134 Kunitz units/ml of DNasel. The enzymatically dissociated cells were triturated and collected by centrifugation, resuspended in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco BRL, Invitrogen Life Technologies, Carlsbad, CA) containing 25 μg/ml of insulin, 100 μg /ml of transferin, 60 μM putrescine, 30 nM selenium, 20 nM progesterone, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 0.05 M Hepes, and 10% FBS. Dissociated primary retinal cells were plated onto PoIy-D- lysine- and Matrigel (BD, Franklin Lakes, NJ)-coated glass coverslips that were placed in 24-well tissue culture plates (Falcon Tissue Culture Plates, Fisher Scientific, Pittsburgh, PA) and maintained in 0.5 ml of media at 37°C and 5% CO₂. PBN (Sigma, Dallas, TX) was diluted in dimethylsulfoxide (DMSO) and added to the culture wells at a final concentration of 100 nM for 24 hours at 37° C and

5% CO₂. The cells were stressed by changing to media that contained both 25 μM A2E

(obtained from Dr. Koji Nakanishi, Columbia University, New York City, NY; diluted in ethanol) and 100 nM PBN and incubated for 24 hours.

Immunohistochemistry Analysis

Immunohistochemistry analysis was performed according to standard methods known in the art. Rod photoreceptors were identified by labeling with a rhodopsin-specific antibody (mouse monoclonal, diluted 1:500; Chemicon, Temecula, CA). An antibody to mid-weight neurofilament (NFM rabbit polyclonal, diluted 1:10,000, Chemicon) was used to identify ganglion cells; an antibody to beta3-tubulin was used to generally identify interneurons, and antibodies to calbindin and calretinin were used to identify subpopulations of calbindin- and calretinin-expressing interneurons in the inner nuclear layer. Briefly, the retinal cell cultures were fixed with 4% paraformaldehyde (Polysciences, Inc, Warrington, PA) and/or ethanol, rinsed in Dulbecco's phosphate buffered saline (DPBS), and incubated with primary antibody for 1 hour at 37° C. The cells were then rinsed with DPBS, incubated with a secondary antibody (Alexa 488- or Alexa 568-conjugated secondary antibodies (Molecular Probes, Eugene, OR)), and rinsed with DPBS. Nuclei were stained with 4', 6-diamidino-2- phenylindole (DAPI, Molecular Probes), and the cultures were rinsed with DPBS before removing the glass coverslips and mounting them with Fluoromount-G (Southern Biotech, Birmingham, AL) on glass slides for viewing and analysis.

Cultures were analyzed by counting rhodopsin-labeled photoreceptors and NFM-labeled ganglion cells using an Olympus K81 or CZX41 microscope (Olympus, Tokyo, Japan). Twenty fields of view were counted per coverslip with a 2Ox objective lens. Six coverslips were analyzed by this method for each condition in each experiment. Cells that were not exposed to either PBN or to any stressor were counted, and cells exposed to a stressor with or without treatment with a PBN were normalized to the number of cells in the control. Figure 1 shows representative rhodopsin- expressing photoreceptors before stress. Figure 2 shows representative rhodopsin- expressing photoreceptors after stress (25 μM A2E for 24 hours). The small dots are debris; the total live cell count is much smaller than in Figure 1. Figure 3 shows rhodopsin-expressing photoreceptors under stress but with addition of PBN (100 nM) for the same duration. The live cell count is much greater than it is in Figure 2, indicating neuroprotection of photoreceptors.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Claims

CLAIMS We claim the following:

1. A method of enhancing retinal cell survival comprising contacting a retinal cell with a spin-trap antioxidant.

2. A method of inhibiting degeneration of a retinal cell comprising contacting a retinal cell with a spin-trap antioxidant.

3. A method of inhibiting degeneration of a retinal cell in a subject who has or who is at risk of developing a retinal disease or disorder comprising administering to the subject a composition that comprises a spin-trap antioxidant and a pharmaceutically acceptable carrier.

4. The method of claim 3 wherein the retinal disease or disorder is selected from macular degeneration, glaucoma, diabetic retinopathy, diabetic maculopathy, retinal detachment, retinal blood vessel occlusion, hemorrhagic retinopathy, retinitis pigmentosa, retinopathy of prematurity, optic neuropathy, inflammatory retinal disease, proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusion related retinal injury, hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, a retinal disorder associated with Alzheimer's disease, a retinal disorder associated with multiple sclerosis, a retinal disorder associated with Parkinson's disease, a retinal disorder associated with viral infection, a retinal disorder related to light overexposure, and a retinal disorder associated with AIDS.

5. The method according to any one of claims 1-3 wherein the retinal cell is a retinal neuronal cell.

6. The method according to claim 5 wherein the retinal neuronal cell is selected from a photoreceptor cell, a ganglion cell, an amacrine cell, a horizontal cell, and a bipolar cell.

7. The method according to any one of claims 1-3 wherein the retinal cell is a photoreceptor cell.

8. The method according to any one of claims 1-3 wherein the spin-trap antioxidant is selected from alpha-phenyl-N-tert-butyl nitrone (PBN); 5,5- dimethylpyrroline-N-oxide (DMPO); alpha-(4-pyridyl l-oxide)-N-tert-butyl nitrone (POBN); and 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO).

9. The method according to any one of claims 1-3, wherein the spin-trap antioxidant is alpha-phenyl-N-tert-butyl nitrone (PBN), or a derivative thereof.

10. The method according to claim 3, wherein the retinal disease or disorder is dry form macular degeneration.

11. The method according to claim 3, wherein the retinal disease or disorder is wet form macular degeneration.

12. The method according to claim 3, wherein the composition is administered topically to an eye of the subject, or is administered orally, intravenously, intraocularly, or periocularly.

13. A method of treating a retinal disease or disorder in a subject, comprising administering to the subject a composition that comprises a spin-trap antioxidant and pharmaceutically acceptable carrier.

14. The method of claim 13 wherein the retinal disease or disorder is selected from macular degeneration, glaucoma, diabetic retinopathy, diabetic maculopathy, retinal detachment, retinal blood vessel occlusion, hemorrhagic retinopathy, retinitis pigmentosa, retinopathy of prematurity, optic neuropathy, inflammatory retinal disease, proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusion related retinal injury, hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, a retinal disorder associated with Alzheimer's disease, a retinal disorder associated with multiple sclerosis, a retinal disorder associated with Parkinson's disease, a retinal disorder associated with viral infection, a retinal disorder related to light overexposure, and a retinal disorder associated with AIDS.

15. The method of claim 13 wherein the retinal disease is dry form macular degeneration.

16. The method of claim 13 wherein the retinal disease is wet form macular degeneration.

17. The method of claim 13 wherein the spin-trap antioxidant is selected from alpha-phenyl-N-tert-butyl nitrone (PBN); 5,5-dimethylpyrroline-N-oxide (DMPO); alpha-(4-pyridyl l-oxide)-N-tert-butyl nitrone (POBN); and 2,2,6,6- tetramethylpiperidine-1 -oxyl (TEMPO).

18. The method of claim 13 wherein the spin-trap antioxidant is alpha-phenyl-N-tert-butyl nitrone (PBN), or a derivative thereof.

19. A method for treating a retinal disease or disorder in a subject, comprising administering to the subject a composition that comprises alpha-phenyl-N- tert-butyl nitrone (PBN), or a derivative thereof, and pharmaceutically acceptable carrier.

20. The method of either claim 13 or claim 19 wherein the composition is administered topically to an eye of the subject, or is administered orally, intravenously, intraocularly, or periocularly.

21. Use of a spin-trap antioxidant for the manufacture of a medicament for treating a retinal disease or disorder.

22. The use according to claim 21 wherein the spin-trap antioxidant is selected from alpha-phenyl-N-tert-butyl nitrone (PBN); 5,5-dimethylpyrroline-N- oxide (DMPO); alpha-(4-pyridyl l-oxide)-N-tert-butyl nitrone (POBN); and 2,2,6,6- tetramethylpiperidine-1 -oxyl (TEMPO).

23. The use according to claim 21 wherein the spin-trap antioxidant is PBN.

24. The use according to claim 21 wherein the retinal disease or disorder is selected from macular degeneration, glaucoma, diabetic retinopathy, diabetic maculopathy, retinal detachment, retinal blood vessel occlusion, hemorrhagic retinopathy, retinitis pigmentosa, retinopathy of prematurity, optic neuropathy, inflammatory retinal disease, proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusion related retinal injury, hereditary optic neuropathy, metabolic optic neuropathy, Stargardt's macular dystrophy, Sorsby's fundus dystrophy, Best disease, a retinal injury, a retinal disorder associated with Alzheimer's disease, a retinal disorder associated with multiple sclerosis, a retinal disorder associated with Parkinson's disease, a retinal disorder associated with viral infection, a retinal disorder related to light overexposure, and a retinal disorder associated with AIDS.

25. The use according to claim 21 wherein the retinal disease or disorder is wet form or dry form macular degeneration.