Methods and compositions for preventing or treating eye diseases

Abstract

To provide methods for preventing or treating ophthalmic diseases or conditions in mammalian subjects.SOLUTION: The invention provides a method for preventing or treating an ophthalmic disease or a condition in a mammalian subject. The method comprises administration of a therapeutically effective amount of an aromatic-cationic peptide represented by a chemical formula D-Arg-2',6'-Dmt-Lys-Phe-NH(SS-31) or Phe-D-Arg-Phe-Lys-NH(SS-20). The invention provides a method for preventing or treating ophthalmic diseases or conditions such as diabetic retinopathy, cataract, retinitis pigmentosa, glaucoma, macular degeneration, choroidal neovascularization, retinal degeneration and oxygen-induced retinopathy.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 61 / 236,440, filed Aug. 24, 2009, U.S. Provisional Application No. 61 / 237,745, filed Aug. 28, 2009, and U.S. Provisional Patent No. 61 / 348,470, filed May 26, 2010, the entire contents of which are incorporated by reference. The present technology relates generally to compositions and methods for preventing or treating ocular diseases or conditions, and in particular to administering an effective amount of an aromatic-cationic peptide to prevent or treat an ocular disease or condition, such as diabetic retinopathy, cataracts, retinitis pigmentosa, glaucoma, choroidal neovascularization, and oxygen-induced retinopathy, in a mammalian subject. [Background technology]

[0002] The following explanation is provided to aid the understanding of the reader. None of the information provided or documents cited are admitted as prior art to the present invention.

[0003] Diseases and degenerative conditions of the optic nerve and retina are the leading causes of blindness worldwide. A notable degenerative condition of the retina is age-related macular degeneration (ARMD). ARMD is the most common cause of blindness in people over 50 years of age in the United States, and its prevalence increases with age. ARMD is classified as wet (neovascular) or dry (non-neovascular), with the dry form of the disease being more common. Macular degeneration occurs when the central retina becomes distorted and thinned, usually with age, but may also be characterized by endophthalmitis and neovascularization (wet ARMD only) and / or intraocular infection. Free radicals that cause oxidative tissue damage, local inflammation, and production of growth factors (such as VEGF and FGF), and subsequent production of inflammatory mediators, lead to inappropriate angiogenesis, as in the wet form of ARMD.

[0004] Retinopathy is the leading cause of blindness in type I diabetes and is also common in type II diabetes. The extent of retinopathy depends on the duration of diabetes, and typically begins to occur 10 years or more after the onset of diabetes. Diabetic retinopathy may be classified as nonproliferative, in which case the retinopathy is characterized by increased capillary permeability, edema and exudate, or proliferative, in which case the retinopathy is characterized by neovascularization extending from the retina to the vitreous, scarring, deposition of fibrous tissue, and possible retinal detachment. Diabetic retinopathy is thought to result from the formation of glycated proteins due to hyperglycemia. Some other less common retinopathies include choroidal neovascular membrane (CNVM), cystoid macular edema (CME), epiretinal membrane (ERM), and macular hole.

[0005] Glaucoma comprises a group of eye diseases that cause blindness through damage to the optic nerve. Elevated intraocular pressure (IOP) due to insufficient drainage of aqueous humor is the primary cause of glaucoma. Glaucoma often develops with ageing of the eye, or may occur as a result of ocular injury, inflammation, tumors, or in advanced cases of cataracts or diabetes. Elevated IOP caused by treatment with steroids may also be the cause. Medications with proven efficacy in glaucoma lower IOP by reducing vitreous humor production or by promoting drainage of aqueous humor. These drugs are often vasodilators, which themselves act on the sympathetic nervous system and include adrenergic antagonists. Summary of the Invention [Means for solving the problem]

[0006] The present technology relates generally to the treatment or prevention of ocular diseases or conditions in mammals by administration of a therapeutically effective amount of an aromatic-cationic peptide to a subject in need of such treatment or prevention.

[0007] In one aspect, the disclosure provides a method for treating or preventing an ocular disease in a mammalian subject in need thereof, comprising administering to the subject a therapeutically effective amount of the peptide D-Arg-2'6'-Dmt-Lys-Phe-NH2 or Phe-D-Arg-Phe-Lys-NH2. In one embodiment, the ocular disease is selected from the group consisting of diabetic retinopathy, cataracts, retinitis pigmentosa, glaucoma, macular degeneration, choroidal neovascularization, retinal degeneration, and oxygen-induced retinopathy.

[0008] In one aspect, the disclosure provides a method of treating or preventing an ocular disease in a mammalian subject comprising administering to the mammalian subject a therapeutically effective amount of an aromatic-cationic peptide. In some embodiments, the aromatic-cationic peptide is a peptide having: A net positive charge of at least 1; A minimum of 4 amino acids; up to about 20 amino acids; The minimum number of net positive charges (p m ) and the total number of amino acid residues (r), m is the maximum number less than or equal to r+1; the minimum number of aromatic groups (a) and the total number of net positive charges (p t ), 2a is p t +1, except when a is 1. t may be 1. In certain embodiments, the mammalian subject is a human.

[0009] In one embodiment, 2p m is a maximum number less than or equal to r+1 and may be equal to pt. The aromatic-cationic peptide may be a water-soluble peptide having a minimum of two or a minimum of three positive charges.

[0010] In one embodiment, the peptide comprises one or more non-naturally occurring amino acids, e.g., one or more D-amino acids. In some embodiments, the C-terminal carboxyl group of the amino acid at the C-terminus is amidated. In certain embodiments, the peptide has a minimum of four amino acids. The peptide may have a maximum of about six, a maximum of about nine, or a maximum of about twelve amino acids.

[0011] In one embodiment, the peptide may have the formula Phe-D-Arg-Phe-Lys-NH2 (SS-20) or 2',6'-Dmp-D-Arg-Phe-Lys-NH2. In a particular embodiment, the aromatic-cationic peptide has the formula D-Arg- ',6'-Dmt-Lys-Phe-NH2 (indistinguishably referred to as SS-31, MTP-131, or Bendavia™).

[0012] In one embodiment, the peptide is defined by Formula I: [ka] In the formula, R 1 and R 2 are each independently selected from the following: (i) hydrogen; (ii) linear or branched alkyl having 1 to 6 carbon atoms; (iii) [ka] (iv) [ka] (v) [ka] R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently selected from the following: (i) hydrogen; (ii) a linear or branched alkyl group having 1 to 6 carbon atoms; (iii) alkoxy having 1 to 6 carbon atoms; (iv) amino; (v) alkylamino having 1 to 4 carbon atoms; (vi) dialkylamino having 1 to 4 carbon atoms; (vii) nitro; (viii) hydroxyl; (ix) halogen, where “halogen” includes chloro, fluoro, bromo, and iodo, and n is an integer from 1 to 5.

[0013] In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are all hydrogen and n is 4. In another embodiment, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 11 are all hydrogen, R 8 and R 12 is methyl, R 10 is hydroxyl, and n is 4.

[0014] In one embodiment, the peptide is defined as follows: [ka] Here, R 1 and R 2 are each independently selected from the following: (i) hydrogen; (ii) a linear or branched alkyl group having 1 to 6 carbon atoms; (iii) [ka] (iv) [ka] (v) [ka] R 3 and R 4 are each independently selected from the following: (i) hydrogen; (ii) a linear or branched alkyl group having 1 to 6 carbon atoms; (iii) alkoxy having 1 to 6 carbon atoms; (iv) amino; (v) alkylamino having 1 to 4 carbon atoms; (vi) dialkylamino having 1 to 4 carbon atoms; (vii) nitro; (viii) hydroxyl; (ix) halogens, where “halogen” includes chloro, fluoro, bromo, and iodo; R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the following: (i) hydrogen; (ii) a linear or branched alkyl group having 1 to 6 carbon atoms; (iii) alkoxy having 1 to 6 carbon atoms; (iv) amino; (v) alkylamino having 1 to 4 carbon atoms; (vi) dialkylamino having 1 to 4 carbon atoms; (vii) nitro; (viii) hydroxyl; (ix) halogen, where “halogen” includes chloro, fluoro, bromo, and iodo, and n is an integer from 1 to 5.

[0015] The aromatic-cationic peptides can be administered in a variety of ways, hi some embodiments, the peptides can be administered intraocularly, orally, topically, intranasally, intravenously, subcutaneously, or transdermally (e.g., by iontophoresis).

[0016] In one aspect, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the peptide D-Arg-2'6'-Dmt-Lys-Phe-NH2 or Phe-D-Arg-Phe-Lys-NH2 formulated for topical, iontophoretic or intraocular administration.

[0017] In one embodiment, the present disclosure provides an ophthalmic preparation comprising a therapeutically effective amount of the peptide D-Arg-2'6'-Dmt-Lys-Phe-NH2 or Phe-D-Arg-Phe-Lys-NH2. In one embodiment, the formulation is soluble in the septum, aqueous humor, and lens. In one embodiment, the formulation further comprises a preservative. In one embodiment, the preservative is present at a concentration of less than 1%.

[0018] In one embodiment, the formulation further comprises an active agent selected from the group consisting of antioxidants, metal complexes, anti-inflammatory agents, antibiotics, and antihistamines, hi one embodiment, the antioxidant is vitamin A, vitamin C, vitamin E, lycopene, selenium, alpha-lipoic acid, coenzyme Q, glutathione, or a carotenoid.In one embodiment, the formulation comprises aceclidine, acetazolamide, anecortave, apraclonidine, atropine, azapentacene, azelastine, bacitracin, befunolol, betamethasone, betaxolol, bimatoprost, brimonidine, brinzolamide, carbachol, carteolol, celecoxib, chloramphenicol, chlortetracycline, ciprofloxacin, cromoglycate, cromolyn, cyclopentolate, cyclosporine, dapiprazole, demecarium, dexamethasone ... Samethasone, diclofenac, dichlorphenamide, dipivefrin, dorzolamide, echothiophate, emedastine, epinastine, epinephrine, erythromycin, ethoxzolamide, eucatropine, fludrocortisone, fluorometholone, flurbiprofen, fomivirzen, framycetin, ganciclovir, gatifloxacin, gentamicin, homatropine, hydrocortisone, idoxuridine, indomethacin, isoflurophate, ketorolac, ketotifen, latanovir, Prost, levobetaxolol, levobunolol, levocabastine, levofloxacin, lodoxamide, loteprednol, medrysone, methazolamide, metipranolol, moxifloxacin, naphazoline, natamycin, nedocromil, neomycin, norfloxacin, ofloxacin, olopatadine, oxymetazoline, pemirolast, pegaptanib, phenylephrine, physostigmine, pilocarpine, pindolol, pirenoxine, polymyxin B, prednisolone, proparacaine , ranibizumab, rimexolone, scopolamine, sezolamide, squalamine, sulfacetamide, suprofen, tetracaine, tetracycline, tetrahydrozoline, tetrizoline, timolol, tobramycin, travoprost, triamcinolone, trifluoromethazolamide, trifluridine, trimethoprim, tropicamide, unoprostone, vidarabine, xylometazoline, pharmaceutically acceptable salts thereof, and combinations thereof. [Brief description of the drawings]

[0019] [Figure 1]Figures 1A and 1B show the effect of different concentrations of SS-31 (10 nM, 100 nM, 1 μM, and 10 μM) used as a co-treatment with 30 mM glucose (HG). Figure 1A shows the analysis of apoptosis assessed by flow cytometry after Annexin V / PI staining, which showed that the viability (Q3) of HRECs 24 hours after treatment was 99.3%, 83.2%, 84.3%, 90.7%, 92.8%, and 94.3%, respectively. Figure 1B shows a graphical representation of the viability of HRECs. The data for 100 nM, 1 μM, and 10 μM concentrations of SS-31 were significantly higher than those seen in cells exposed to high glucose without co-treatment with SS-31. *p<0.05 vs. 30 mM high glucose treatment group. [Diagram 2] Figures 2A-2F are a series of micrographs showing that combined treatment with SS-31 reduced intracellular reactive oxygen species (ROS) in HRECs exposed to 30 mM glucose for 24 and 48 h. Intracellular ROS was measured using dihydroethidium. 2A, 2D were cultured in normal medium; 2B, 2E in 30 mM glucose; and 2C, 2F in 30 mM glucose + SS-31 (100 nM) for 24 and 48 h, respectively. [Diagram 3] Figures 3A and 3B show that SS-31 prevents the loss of mitochondrial membrane potential in HRECs treated with high glucose. Figure 3A: ΔΨm of HRECs was measured by flow cytometry after JC-1 fluorescent probe staining. High glucose (30 mM) treatment led to rapid loss of mitochondrial membrane potential in cultured HRECs at 24 and 48 h. Meanwhile, flow cytometry analysis showed that ΔΨm was increased in the 30 mM glucose group co-treated with SS-31 compared with the high glucose alone group. Figure 3B: Quantitative analysis of ΔΨm of high glucose HRECs co-treated with SS-31 for 24 and 48 h, where high glucose alone had a negative effect on ΔΨm. Meanwhile, SS-31 restored ΔΨm to the control concentration. Values ​​represent the mean ± standard deviation of six separate experiments performed in triplicate. *P<0.05. [Figure 4] Figures 4A and 4D are confocal microscopy images showing that cytochrome c staining and HSP60 staining were more accurately overlapped in HRECs treated with normal glucose and SS-31, indicating the colocalization of cytochrome c and mitochondria. After 24 and 48 h of treatment, cytochrome c was obviously increased in the cytoplasm of HRECs treated with 30 mM glucose. Figures 4B and 4E show the cytochrome c content in mitochondria and cytoplasm measured by Western blotting. Figures 4C and 4F show the quantitative analysis of the ratio of cytochrome c content in mitochondria and cytoplasm of HRECs treated with high glucose and SS-31 for 24 and 48 h. [Figure 5-1] 5A and 5B show by Western blotting that the increased expression of caspase 3 in HRECs treated with high glucose (HG) was reduced by co-treatment with SS-31. Caspase 3 expression was normalized to the expression of β-actin. [Figure 5-2] Figures 5C-E show that SS-31 increases the expression of Trx2 in high glucose treated HRECs. Figure 5C shows the mRNA levels of Trx2 in HRECs treated with SS-31 and exposed to 30 mM glucose for 24 and 48 h. Figure 5D shows the expression levels of Trx2 protein measured by Western blotting. Figure 5E shows the quantitative analysis of the protein concentration of Trx2 in HRECs 24 and 48 h after exposure to high glucose with and without concomitant treatment with SS-31. [Figure 6] Figure 6 shows photographs of the effect of SS-31 on diabetic rat lenses. Top row: lenses from diabetic rats; bottom row: lenses from diabetic rats treated with SS-31 or SS-20. [Figure 7] Figure 7 is a series of photographs showing the effects of SS-31 and SS-20 on the lenses of diabetic rats. Diabetes was induced with a high-fat diet and streptozotocin (HFD / STZ) (top row) or with streptozotocin (STZ) alone (bottom row). [Figure 8]8 is a series of photomicrographs showing lens epithelium from normal rats, diabetic rats, and diabetic rats treated with SS-31. Diabetes was induced by STZ. [Figure 9] 9 is a series of photomicrographs showing lens epithelium from normal, diabetic, and SS-31-treated diabetic rats. Diabetes was induced by HFD / STZ. [Figure 10] Figure 10 is a series of graphs showing blood-retinal barrier integrity in healthy rats (NRC), diabetic rats, and diabetic rats treated with SS-20 or SS-31, as analyzed by Evans blue extravasation: (A) STZ-induced diabetes; (B) HFD / STZ-induced diabetes. [Figure 11] FIG. 11 is a series of photomicrographs showing retinal microvasculature in healthy rats (NRC), diabetic rats (HFD / STZ), and diabetic rats treated with SS-31. [Figure 12] FIG. 12 is a series of photomicrographs showing retinal microvasculature in normal rats (NRC), diabetic rats (STZ), and diabetic rats treated with SS-31. [Figure 13] 13A-13D are a series of photomicrographs showing the distribution of the tight junction protein claudin 5 in retinal microvessels from healthy rats (A), STZ rats (B), STZ / SS-20-treated rats (C), or STZ / SS-31-treated rats (D). [Figure 14] FIG. 14 is a graph showing the lack of cytotoxicity of SS-31 on trabecular meshwork cells from unaffected individuals (HTM) and glaucoma affected individuals (GTM) treated with SS-31. [Figure 15] FIG. 15 is a series of confocal micrographs showing that combined treatment with SS-31 dose-dependently inhibited the decrease in mitochondrial membrane potential (ΔΨm) induced by 200 μM H2O2 in trabecular meshwork cells from glaucoma-affected individuals (GTM). [Figure 16]FIG. 16 is a series of graphs showing that co-treatment with SS-31 prevented the decrease in mitochondrial membrane potential (ΔΨm) as measured by TMRM and flow cytometry in trabecular meshwork cells from glaucoma-affected individuals (GTM) induced by 200 μM HO. [Figure 17] FIG. 17 is a graph comparing mitochondrial membrane potential (ΔΨm) in GTM and HTM cells. [Figure 18] FIG. 18 is a series of photomicrographs showing morphological changes in GTM cells in response to SS-31 treatment, as observed using inverse phase contrast microscopy. [Figure 19] FIG. 19 is a series of photomicrographs showing that co-treatment with SS-31 dose-dependently reduced the loss of mitochondrial membrane potential in GTM cells caused by 400 μM H2O2, as observed using confocal microscopy. [Figure 20] FIG. 20 is a series of photomicrographs showing that combined treatment with SS-31 reduced the loss of mitochondrial membrane potential (ΔΨm) in GTM cells caused by 400 μM HO, as observed using TMRM and confocal microscopy (200x magnification). [Figure 21] FIG. 21 is a series of photomicrographs showing morphological changes in GTM cells in response to SS-31 treatment, as observed using inverse phase contrast microscopy. [Figure 22] FIG. 22 is a graph showing that SS-31 has no effect on the viability of primary human retinal pigment epithelial (RPE) cells (as measured by the MTT assay). [Figure 23] Figure 23A is a graph showing the effect of various concentrations of tBHP on RPE cell viability (measured by MTT assay) and Figure 23B is a graph showing the effect of various concentrations of SS-31 on cell viability when exposed to high concentrations of tBHP. [Figure 24] Figures 24A-24C are a series of photomicrographs illustrating the pathological effects in a mouse model of choroidal neovascularization (CNV). Figure 24D is a graph showing the CNV area in treatment and control groups. [Diagram 25] Figure 25 is a series of photomicrographs illustrating various pathological effects in an oxygen-induced retinopathy (OIR) mouse model, showing areas of avascularity and neovascularization in P17 OIR mice compared to P17 healthy mice. [Figure 26] Figures 26A-26D are a series of photomicrographs showing the effect of SS-31 administration in an OIR mouse model. Figure 26E is a graph showing the neovascularized area in the control and treatment groups. SS-31 reduced the neovascularized area. [Figure 27] Figure 27A is a graph showing the effect of various doses of tBHP on cell viability of the 661W cone cell line derived from a mouse retinal tumor, and Figure 27B is a graph showing the effect of 1 μM SS-31 on reducing tBHP-induced 661W cell death. [Figure 28] FIG. 28 is a series of photomicrographs showing retinal outer nuclear layer (ONL) thickness in a mouse model of retinal degeneration in control and SS-31 treated mice. [Figure 29] FIG. 29 is a series of photomicrographs showing the density of cone cells in flat-mounted retinas stained with peanut agglutinin (PNA), which selectively stains cone inner and outer segments from control and SS-31-treated mice. [Diagram 30] FIG. 30 is a series of photomicrographs showing staining for acrolein, a marker of oxidative lipid damage, in a mouse model of retinal degeneration. [Diagram 31] Figure 31 is a series of graphs showing the fluorescence intensity of intracellular ROS production in three groups of RPE cells using FACS analysis. Figure 31A shows ROS production in control RPE cells; Figure 31B shows ROS production in RPE cells treated with 500 μM tBHP for 3 hours, and Figure 31C shows ROS production in RPE cells treated with 500 μM tBHP for 3 hours and 1 μM SS-31. [Diagram 32] Figure 32 is a series of graphs showing the analysis of MMPs labeled with JC-1 in a FACS assay. Three different concentrations of SS-31 groups were analyzed. [Diagram 33]Figures 33A-33C are a series of graphs showing the effect of 1 μM SS-31 on tBHP-induced MMP reduction. Figure 33A: control group; Figure 33B: group treated with 500 μM tBHP for 3 hours; Figure 33C: group treated with 1 μM SS-31 for 4 hours + 500 μM tBHP for 3 hours. Figure 33D is a graph comparing the fluorescence ratios of the various groups. *P<0.01, C vs. B. [Diagram 34] Figures 34A-34C are a series of graphs showing the effect of SS-31 on cell apoptosis induced by 250 μM tBHP for 24 hours. Figure 34A: control group; Figure 34B: group treated with 250 μM tBHP for 24 hours; Figure 34C: group treated with 1 μM SS-31 for 4 hours + 250 μM tBHP for 24 hours. Figure 34D is a graph comparing the fluorescence ratios of the various groups. *P<0.05, C vs. B. [Diagram 35] Figure 35 is a graph showing tBHP-induced MDA levels in three groups of RPE cells. *P<0.05, 1 μM SS-31 for 4 hrs + 250 μM tBHP for 24 hrs vs. 250 μM tBHP for 24 hrs. [Diagram 36] FIG. 36 shows a graph depicting the fluorescence intensity of TMRM in control and SS-31 treated GTM and HTM cells as measured using FACS analysis. [Figure 37] FIG. 37 shows a graph depicting the fluorescence intensity of ROS in control and SS-31 treated GTM and HTM cells measured using FACS analysis. [Figure 38] FIG. 38 is a series of graphs showing cell apoptosis in control and SS-31 treated groups as assessed by percentage of cells in the Q2+Q4 quadrant analysis. [Figure 39] FIG. 39 is a series of graphs showing that SS-31 reduced intracellular ROS production in GTM3 and iHTM cells treated with H2O2. [Diagram 40] FIG. 40 is a series of graphs showing that SS-31 prevented H2O2-induced mitochondrial depolarization in GTM3 and iHTM cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] It will be appreciated that certain forms, modes, embodiments, variations and features of the invention are described below in detail at all levels in order to provide a thorough understanding of the invention.

[0021] The practice of the present invention uses many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA. These techniques are well known and are described in the following references: Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. EnzymoL, (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth. EnzymoL, Vols. 154 and 155, Wu & Grossman, and Wu, Eds.

[0022] The following are definitions of terms used herein: Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0024] As used herein, "about" will be clear to those of skill in the art and may vary to some extent depending on the context in which it is used. If a term is used that is not clear to those of skill in the art, "about" may mean up to plus or minus 10% of the recited value, as determined by the context in which it is used.

[0025] As used herein, "administration" of a chemical, pharmaceutical, or peptide to a subject includes the route by which the compound is introduced or provided to the subject to perform its intended function. Administration can be by any suitable route, including oral, intraocular, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical. Administration includes self-administration and administration by another.

[0026] As used herein, the term "amino acid" includes naturally occurring and synthetic amino acids, as well as amino acid analogs or amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and o-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but maintain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that differs from the overall chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0027] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, such as an amount that results in the prevention or reduction of symptoms associated with an ocular disease. The amount of the composition administered to a subject will depend on the type and severity of the disease, and individual characteristics such as overall health, age, sex, weight, and drug tolerance. This will also depend on the stage, severity, and type of disease. The practitioner can determine the appropriate dose depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the aromatic-cationic peptide is administered to a subject having one or more signs or symptoms of an ocular disease. For example, a "therapeutically effective amount" of an aromatic-cationic peptide refers to a concentration that, at a minimum, improves the physiological effects of an ocular disease.

[0028] An "isolated" or "purified" polypeptide or peptide is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the chemical is extracted, or is substantially free of chemical precursors or other chemicals when chemically synthesized. For example, an isolated aromatic-cationic peptide will be free of substances that interfere with the diagnostic or therapeutic use of the chemical. Such interfering substances include enzymes, hormones, and other proteinaceous and non-proteinaceous solutes.

[0029] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers containing two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and to longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing, or by chemical modification techniques known in the art.

[0030] As used herein, the term "concurrent" therapeutic use means the administration of at least two active ingredients by the same route and at the same time or substantially the same time.

[0031] As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients by different routes, at the same time or substantially the same time.

[0032] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, by the same or different routes of administration. In particular, sequential use refers to the administration of one active ingredient prior to, or prior to, the administration of the other. Thus, one of the active ingredients can be administered minutes, hours, or days prior to the administration of the other active ingredient. In this case, there is no simultaneous treatment.

[0033] As used herein, the terms "treatment" or "treating" or "alleviating" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose being to prevent or inhibit (reduce) the targeted condition or disease. A subject is successfully "treated" for an ocular disease if, after administration of a therapeutic amount of an aromatic-cationic peptide according to the methods described herein, the subject exhibits a reduction or elimination of one or more observable and / or measurable signs or symptoms of the ocular disease. It should also be understood that the various modes of treating or preventing such conditions as described are intended to mean "substantial," including not only the entirety of the treatment or prevention, but less than the entirety, that some biologically or medically relevant result is achieved.

[0034] As used herein, "prevention" or "preventing" a disease or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disease or condition in treated subjects compared to untreated control subjects, or delays the onset of or reduces the severity of one or more symptoms of the disease or condition compared to untreated subjects. Aromatic Cationic Peptides

[0035] The present technology relates to the treatment or prevention of ocular diseases by administration of certain aromatic-cationic peptides. Without wishing to be limited by theory, the aromatic-cationic peptides can treat or prevent ocular diseases by reducing the severity or occurrence of intraocular oxidative damage. The aromatic-cationic peptides are water-soluble and highly polar. Despite these properties, the peptides can easily penetrate cell membranes. The aromatic-cationic peptides generally contain a minimum of three amino acids or a minimum of four amino acids covalently linked by peptide bonds. The maximum number of amino acids present in the aromatic-cationic peptide is about 20 amino acids covalently linked by peptide bonds. Preferably, the maximum number of amino acids is about 12, more preferably about 9, and most preferably about 6.

[0036] The amino acids of the aromatic-cationic peptides can be any amino acid. As used herein, "amino acid" is used to mean an organic molecule that contains at least one amino group and at least one carboxyl group. Generally, at least one amino group is located alpha to the carboxyl group. The amino acids may be naturally occurring. For example, natural amino acids include the 20 most common levorotatory (L) amino acids commonly found in mammalian proteins, namely alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Other natural amino acids include, for example, amino acids synthesized in metabolic processes unrelated to protein synthesis. For example, the amino acids ornithine and citrulline are synthesized in mammalian metabolism during ureogenesis. Another example of a natural amino acid is hydroxyproline (Hyp).

[0037] The peptide optionally includes one or more unnatural amino acids. Preferably, the peptide has no naturally occurring amino acids. The unnatural amino acids may be levorotatory (L-), dextrorotatory (D-), or mixtures thereof. Unnatural amino acids are those amino acids that are not generally synthesized by the normal metabolic processes of an organism and do not naturally occur in proteins. Furthermore, the unnatural amino acids are not recognized by common proteases. The unnatural amino acids can be present at any position in the peptide. For example, the unnatural amino acids can be present at the N-terminus, the C-terminus, or at a position between the N-terminus and the C-terminus.

[0038] For example, the unnatural amino acids may contain alkyl, aryl, or alkylaryl groups not found in natural amino acids. Some examples of unnatural alkyl amino acids include α-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, and ε-aminocaproic acid. Some examples of unnatural aryl amino acids include ortho-, meta-, and para-aminobenzoic acid. Some examples of unnatural alkylaryl amino acids include ortho-, meta-, and para-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid. Unnatural amino acids include derivatives of natural amino acids. Derivatives of the natural amino acids can include, for example, the addition of one or more chemical groups to a natural amino acid.

[0039] For example, one or more chemical groups can be added to one or more of the 2-, 3-, 4-, 5-, or 6-positions of the aromatic ring of a phenylalanine or tyrosine residue, or the 4-, 5-, 6-, or 7-positions of the benzo ring of a tryptophan residue. The groups can be any chemical group that can be added to an aromatic ring. Some examples of the groups include branched or unbranched C1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C1-4 alkyloxy (i.e., alkoxy), amino, C1-4 alkylamino, and C1-4 dialkylamino (e.g., methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro, chloro, bromo, or iodo). Some specific examples of non-natural derivatives of natural amino acids include norvaline (Nva) and norleucine (Nle).

[0040] Another example of modification of amino acids in peptides is derivatization of the carboxyl groups of aspartic acid or glutamic acid residues of the peptide. One example of derivatization is amidation with ammonia or with a primary or secondary amine, such as methylamine, ethylamine, dimethylamine, or diethylamine. Another example of derivatization is esterification, for example, with methyl or ethyl alcohol. Another such modification is derivatization of the amino groups of lysine, arginine, or histidine residues. For example, the amino groups can be acrylated. Some suitable acrylate groups include, for example, benzoyl or alkanoyl groups, including any of the alkyl groups having 1 to 4 carbon atoms mentioned above, such as acetyl or propionyl groups.

[0041] The unnatural amino acids are preferably resistant, and more preferably insensitive, to common proteases. Examples of unnatural amino acids that are resistant or insensitive to proteases include the dextrorotatory (D-) forms of any of the natural L-amino acids listed above, as well as L- and / or D-unnatural amino acids. D-amino acids do not normally occur in proteins, but are found in certain peptide antibiotics that are synthesized outside the normal ribosomal protein synthesis machinery of the cell. As used herein, D-amino acids are considered unnatural amino acids.

[0042] To minimize protease susceptibility, peptides should have less than 5, preferably less than 4, more preferably less than 3, and most preferably less than 2 adjacent L-amino acids recognized by common proteases, regardless of whether the amino acids are naturally occurring or not. Preferably, the peptides contain only D-amino acids and no L-amino acids. If the peptide contains a protease-susceptible sequence of amino acids, preferably at least one of the amino acids is a non-natural D-amino acid, thereby conferring protease resistance. Examples of protease-susceptible sequences include two or more adjacent basic amino acids that are easily cleaved by common proteases such as endopeptidases and trypsin. Examples of basic amino acids include arginine, lysine, and histidine.

[0043] The aromatic-cationic peptides must have a minimum number of net positive charges at physiological pH compared to the total number of amino acid residues in the peptide. The minimum number of net positive charges at physiological pH is given by: m ) The total number of amino acid residues in the peptide is hereinafter referred to as (r). All the minimum numbers of net positive charges mentioned below are at physiological pH. The term "physiological pH" as used herein means the normal pH in the cells of the tissues and organs of the mammalian body. For example, the physiological pH of a human is normally about 7.4, while the normal physiological pH of a mammal may be any pH between about 7.0 and about 7.8.

[0044] "Net charge" as used herein means the number of positive charges minus the number of negative charges carried by the amino acids present in the peptide. As used herein, it is understood that the net charge is measured at physiological pH. Natural amino acids that are positively charged at physiological pH include L-lysine, L-arginine, and L-histidine. Natural amino acids that are negatively charged at physiological pH include L-aspartic acid and L-glutamic acid.

[0045] Generally, peptides have a positively charged N-terminal amino group and a negatively charged C-terminal carboxyl group. The charges cancel each other out at physiological pH. As an example of net charge calculation, the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively charged amino acid (i.e., GLu) and four positively charged amino acids (i.e., two Arg residues, one Lys, and one His). Thus, the peptide has a net positive charge of three.

[0046] In one embodiment, the aromatic-cationic peptide is m The minimum number of net positive charges at physiological pH (p m ) and the total number of amino acid residues (r). In this embodiment, the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) are as follows: [Table 1]

[0047] In another embodiment, the aromatic-cationic peptide is m The minimum number of net positive charges (p m ) and the total number of amino acid residues (r). In this embodiment, the minimum number of net positive charges (p m ) and the total number of amino acids (r) is as follows: [Table 2]

[0048] In one embodiment, the minimum number of net positive charges (p m ) is equal to the total number of amino acid residues (r). In another embodiment, the peptide has 3 or 4 amino acid residues and a minimum of 1 net positive charge, preferably a minimum of 2 net positive charges, and more preferably a minimum of 3 net positive charges.

[0049] The aromatic-cationic peptide also has a total net positive charge (p t It is also important that the peptide has a minimum number of aromatic groups compared to the total number of aromatic groups in the peptide chain, which is referred to below as (a). Natural amino acids with aromatic groups include the amino acids histidine, tryptophan, tyrosine, and phenylalanine. For example, the hexapeptide Lys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributed by lysine and arginine residues) and three aromatic groups (contributed by tyrosine, phenylalanine, and tryptophan residues).

[0050] The aromatic cationic peptide is p t 3a is p unless is 1 and a is probably 1 too t The minimum number of aromatic groups (a) that is the maximum number less than or equal to +1 and the total number of net positive charges at physiological pH (p t In this embodiment, the minimum number of aromatic groups (a) and the total number of net positive charges (p t ) are as follows: [Table 3]

[0051] In another embodiment, the aromatic-cationic peptide comprises 2a, t The minimum number of aromatic groups (a) that is the maximum number less than or equal to +1 and the total number of net positive charges at physiological pH (p t In this embodiment, the relationship between the minimum number of aromatic amino acid residues (a) and the total number of net positive charges (p t) are as follows: [Table 4]

[0052] In another embodiment, the minimum number of aromatic groups (a) and the total number of net positive charges (p t ) are equal.

[0053] Carboxyl groups, particularly the terminal carboxyl group of the C-terminal amino acid, are suitably amidated, for example with ammonia to form the C-terminal amide. Alternatively, the terminal carboxyl group of the C-terminal amino acid may be amidated with a primary or secondary amine. The primary or secondary amine may be, for example, an alkyl, particularly a branched or unbranched alkyl or aryl amine having 1 to 4 carbon atoms. Thus, the amino acid at the C-terminus of the peptide may be converted to an amide group, an N-methylamide group, an N-ethylamide group, an N,N-dimethylamide group, an N,N-diethylamide group, an N-methyl-N-ethylamide group, or an N-phenyl-N-ethylamide group. Also, the free carboxylate groups of asparagine, glutamine, aspartic acid, and glutamic acid residues that do not occur at the C-terminus of the aromatic cationic peptide may be amidated wherever they occur within the peptide. Amidation of these internal positions may be with either ammonia or a primary or secondary amine as described above.

[0054] In one embodiment, the aromatic-cationic peptide is a tripeptide having two net positive charges and at least one aromatic amino acid. In a particular embodiment, the aromatic-cationic peptide is a tripeptide having two net positive charges and two aromatic amino acids.

[0055] Examples of aromatic-cationic peptides include, but are not limited to, the following peptide examples: Lys-D-Arg-Tyr-NH2 Phe-D-Arg-His D-Tyr-Trp-Lys-NH2 Trp-D-Lys-Tyr-Arg-NH2 Tyr-His-D-Gly-Met Phe-Arg-D-His-Asp Tyr-D-Arg-Phe-Lys-Glu-NH2 Met-Tyr-D-Lys-Phe-Arg D-His-Glu-Lys-Tyr-D-Phe-Arg Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH2 Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH2 Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH2 Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH2 Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH2 D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH2 Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH2 Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH2 Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH2 Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH2 Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH2

[0056] In one embodiment, the peptide having μ-opioid receptor agonist activity has the formula Tyr-D-Arg-Phe-Lys-NH2 (referred to herein as "SS-01"). SS-01 has a net positive charge of three contributed by the amino acids tyrosine, arginine, and lysine, and has two aromatic groups contributed by the amino acids phenylalanine and tyrosine. The tyrosine of SS-01 may be a modified derivative of tyrosine, such as 2'6'-dimethyltyrosine, which produces a compound having the formula 2',6'-Dmt-D-Arg-Phe-Lys-NH2 (referred to herein as "SS-02"). SS-02 has a molecular weight of 640 and carries a net positive charge at physiological pH. SS-02 readily penetrates the plasma membrane of multiple mammalian cell types in an energy-independent manner (Zhao et al., J. Pharmacol Exp Ther. 304:425-432, 2003).

[0057] Peptides that do not have μ-opioid receptor agonist activity generally do not have a tyrosine residue or a derivative of tyrosine at the N-terminus (i.e., amino acid position 1). The N-terminal amino acid may be a natural or unnatural amino acid other than tyrosine. In one embodiment, the N-terminal amino acid is phenylalanine or a derivative thereof. Exemplary derivatives of phenylalanine include 2′-methylphenylalanine (Mmp), 2′6′-dimethylphenylalanine (2′,6′-Dmp), N,2′,6′-trimethylphenylalanine (Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

[0058] An example of a peptide that does not have μ-opioid receptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH2 (referred to herein as "SS-20"). Alternatively, the N-terminal phenylalanine may be a derivative of phenylalanine, such as 2',6'-dimethylphenylalanine (2'6'-Dmp). SS-01, which contains 2',6'-dimethylphenylalanine at amino acid position 1, has the formula 2',6'-Dmp-D-Arg-Phe-Lys-NH2. In one embodiment, the amino acid sequence of SS-01 is rearranged so that Dmt is not at the N-terminus. An example of said aromatic-cationic peptide that does not have μ-opioid receptor agonist activity has the formula D-Arg-2'6'-Dmt-Lys-Phe-NH2 (SS-31).

[0059] SS-01, SS-20, SS-31 and their derivatives can further include functional analogs. A peptide is considered to be a functional analog of SS-01, SS-20 or SS-31 if the analog has the same function as SS-01, SS-20 or SS-31. For example, the analog can be a substitution isomer of SS-01, SS-20 or SS-31, where one or more amino acids are replaced with another amino acid.

[0060] Suitable substitution isomers of SS-01, SS-20, or SS-31 include conservative amino acid substitutions. Amino acids can be classified according to their physicochemical properties as follows: (a) Nonpolar amino acids: Ala (A) Ser (S) Thr (T) Pro (P) Gly (G) Cys (C); (b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q); (c) Basic amino acids: His(H) Arg(R) Lys(K); (d) hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and (e) Aromatic amino acids: Phe (F) Tyr (Y) Trp (W) His (H).

[0061] Substitution of an amino acid in a peptide with another amino acid in the same class is called a conservative substitution and can maintain the physical and chemical properties of the original peptide, whereas substitution of an amino acid in a peptide with another amino acid in a different group is generally more likely to change the properties of the original peptide.

[0062] In some embodiments, the aromatic-cationic peptide has a formula as shown in Table 5. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] Dab = diaminobutyric acid Dap = diaminopropionic acid Dmt = Dimethyltyrosine Mmt = 2'-methyltyrosine Tmt = N,2',6'-trimethyltyrosine Hmt = 2'-hydroxy, 6'-methyltyrosine dnsDap = β-dansyl-L-α, β-diaminopropionic acid atnDap = β-anthraniloyl-L-α, β-diaminopropionic acid Bio = Biotin

[0063] Other examples of aromatic-cationic peptides that do not activate the mu opioid receptor include, but are not limited to, the aromatic-cationic peptides shown in Table 6. [Table 6] Cha = cyclohexylalanine

[0064] The amino acids of the peptides shown in Tables 5 and 6 may be in the L- or D-configuration.

[0065] Peptides can be synthesized by any method known in the art. Suitable methods for chemically synthesizing proteins include, for example, those described by Stuart and Young in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984) and Methods Enzymol. 289, Academic Press, Inc, New York (1997). Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides - Patent application

[0066] The aromatic-cationic peptides described herein are useful for the prevention or treatment of diseases. In particular, the present disclosure provides both prophylactic and therapeutic methods for treating subjects at risk (or susceptible) to ocular diseases and conditions. Thus, the methods provide for the prevention and / or treatment of ocular diseases in a subject in need thereof by administering to the subject an effective amount of an aromatic-cationic peptide. For example, the subject can be administered an aromatic-cationic peptide composition for the purpose of ameliorating one or more factors that contribute to an ocular disease or condition.

[0067] One aspect of the technology includes a method for alleviating ocular disease in a subject for therapeutic purposes. In therapeutic applications, the composition or agent is administered to a subject suspected of or already suffering from the disease in an amount sufficient to treat or at least partially arrest the symptoms of the disease, including its complications and intermediate pathological phenotypes in the progression of the disease. Thus, the present disclosure provides a method for treating an individual suffering from an ocular disease. In some embodiments, the technology provides a method for treating or preventing certain ocular diseases, such as diabetic retinopathy, cataracts, retinitis pigmentosa, glaucoma, choroidal neovascularization, retinal degeneration, and oxygen-induced retinopathy, in a mammal by administering an aromatic-cationic peptide.

[0068] In one embodiment, an aromatic-cationic peptide is administered to a subject to treat or prevent diabetic retinopathy. Diabetic retinopathy is characterized by microaneurysms and pinpoint hemorrhages. Microvascular blockage then leads to the formation of cotton wool spots in the retina. In addition, retinal edema and / or hard white spots may form in individuals with diabetic retinopathy due to increased vascular permeability. Neovascularization then appears, and friction of connective tissue growth in the vitreous causes retinal detachment. Rubeosis iridis and neovascular glaucoma may also occur, which may ultimately lead to blindness. Symptoms of diabetic retinopathy include, but are not limited to, impaired reading ability, blurred vision, sudden blindness in one eye, seeing halos around lights, seeing dark spots, and / or seeing flashing lights.

[0069] In one embodiment, an aromatic-cationic peptide is administered to a subject to treat or prevent cataract. Cataract is a congenital or acquired disease characterized by a decrease in the transparency of the natural lens. An individual suffering from cataract may exhibit one or more symptoms, including but not limited to, clouding of the lens surface, clouding of the lens interior, and / or swelling of the lens. Typical examples of congenital cataract-related diseases are pseudocataract, intramembranous cataract, coronal cataract, lamellar cataract, punctate cataract, and filiform cataract. Typical examples of acquired cataract-related diseases are senile cataract, secondary cataract, browning cataract, combined cataract, diabetic cataract, and traumatic cataract. Acquired cataract can also be induced by electrical shock, radiation, ultrasound, drugs, systemic diseases, and nutritional disorders. Acquired cataract further includes postoperative cataract.

[0070] In one embodiment, an aromatic-cationic peptide is administered to a subject to treat or prevent retinitis pigmentosa. Retinitis pigmentosa is a disease characterized by damage to rod and / or cone cells. Dark lines in the retina are typical in individuals with retinitis pigmentosa. Individuals with retinitis pigmentosa also exhibit a variety of symptoms, including but not limited to headaches, numbness or tingling in the extremities, flashes of light, and / or visual disturbances. See, for example, the following reference: Heckenlively et al., Clinical findings and common symptoms in retinitis pigmentosa. Am J Ophthalmol. 105(5): 504-511 (1988).

[0071] In one embodiment, an aromatic-cationic peptide is administered to a subject to treat or prevent glaucoma. Glaucoma is a genetic disease characterized by elevated intraocular pressure, which leads to loss of vision. Glaucoma can result from a variety of ophthalmic conditions that are already present in the individual, such as trauma, surgery, and other structural deformities. Glaucoma can occur at any age, but it is more common in older individuals, leading to blindness. Patients with glaucoma typically have an intraocular pressure of 21 mmHg or higher. However, normal tension glaucoma, which shows glaucomatous deformations in the visual field and optic disc, can occur without the intraocular pressure being elevated, i.e., 21 mmHg or higher. Symptoms of glaucoma include, but are not limited to, blurred vision, severe eye pain, headache, seeing halos around lights, nausea, and / or vomiting.

[0072] In one embodiment, an aromatic-cationic peptide is administered to a subject to treat or prevent macular degeneration. Macular degeneration is typically an age-related disease. Common classifications of macular degeneration include wet, dry, and non-age-related macular degeneration. Dry macular degeneration, which accounts for approximately 80-90 percent of all cases, is also known as atrophic, non-exudative, or drusenoid macular degeneration. In dry macular degeneration, drusen typically accumulate under the retinal pigment epithelium tissue. When drusen interfere with the function of photoreceptors in the macula, blindness subsequently occurs. Symptoms of dry macular degeneration include, but are not limited to, astigmatism, distortion of central vision, light-dark distortion, and / or changes in color vision. Dry macular degeneration can cause a gradual loss of vision.

[0073] Wet macular degeneration is also known as neovascularization, subretinal neovascularization, exudation, or disciform degeneration. In wet macular degeneration, blood vessels grow abnormally under the macula. Blood leaks from the vessels into the macula, damaging the photoreceptors. Wet macular degeneration progresses quickly and can cause significant damage to central vision. Wet and dry macular degeneration have identical symptoms. However, non-age-related macular degeneration is rare and may be related to genetics, diabetes, poor nutrition, injury, infection, or other factors. Symptoms of non-age-related macular degeneration also include, but are not limited to, astigmatism, distortion of central vision, light and dark distortion, and / or changes in color vision.

[0074] In one embodiment, an aromatic-cationic peptide is administered to a subject to treat or prevent choroidal neovascularization. Choroidal neovascularization (CNV) is a disease characterized by the growth of new blood vessels in the choroidal layer of the eye. The newly formed blood vessels grow in the choroid through Bruch's membrane and invade the subretinal space. CNV can lead to impaired vision or complete blindness. Symptoms of CNV include, but are not limited to, flickering, flashing lights, or gray dots in the affected eye, blurred vision, astigmatism, and / or blindness.

[0075] In one embodiment, an aromatic-cationic peptide is administered to a subject to treat or prevent retinal degeneration. Retinal degeneration is a genetic disease associated with the destruction of the retina. Retinal tissue can degenerate for a variety of reasons, including arterial or venous blockage, diabetic retinopathy, retinopathy of prematurity, and / or retrolental fibroplasia. Retinal degeneration typically includes retinoschisis, lattice-like degeneration, and is associated with progressive macular degeneration. Symptoms of retinal degeneration include, but are not limited to, reduced vision, blindness, night blindness, constricted visual field, loss of peripheral vision, retinal detachment, and / or photosensitivity.

[0076] In one embodiment, an aromatic-cationic peptide is administered to a subject to treat or prevent oxygen-induced retinopathy. Oxygen-induced retinopathy (OIR) is a disease characterized by microvascular degeneration. OIR is an established model for studying retinopathy of prematurity. OIR is associated with vascular cell damage leading to abnormal angiogenesis. Microvascular degeneration leads to ischemia, which contributes to the physical changes associated with OIR. Oxidative stress also plays an important role in vascular occlusion in OIR, which predisposes endothelial cells to peroxidative injury. However, pericytes, smooth muscle cells, and perivascular astrocytes are generally resistant to peroxidative injury. See, for example, the following reference: Beauchamp et al., Role of thromboxane in retinal microvascular degeneration in oxygen-induced retinopathy, J Appl Physiol. 90: 2279-2288 (2001). OIR, including retinopathy of prematurity, is generally asymptomatic. However, abnormal eye movements, esotropia, severe myopia, and / or leukocoria may be signs of OIR or retinopathy of prematurity.

[0077] In one embodiment, the present invention provides a method for preventing ocular disease in a subject by administering to the subject an aromatic-cationic peptide that modulates one or more symptoms or markers of ocular disease. Subjects at risk for ocular disease can be identified, for example, by any or a combination of diagnostic or prognostic assays as described herein. In prophylactic applications, aromatic-cationic peptide pharmaceuticals or agents are administered to subjects suspected of or at risk for a disease or condition in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes in the progression of the disease. Administration of prophylactic aromatic-cationic agents can occur prior to the manifestation of symptoms characteristic of the abnormality, such that disease or injury is prevented or its progression is delayed. Depending on the type of abnormality, aromatic-cationic peptides that act, for example, to enhance or improve mitochondrial function or reduce oxidative damage can be used to treat the subject. Suitable compounds can be determined based on screening tests as described herein.

[0078] Determining the Biological Efficacy of Aromatic-Cationic Peptide-Based Therapies. In various embodiments, appropriate in vitro or in vivo testing is performed to determine the efficacy of a particular aromatic-cationic peptide-based therapy and whether its administration is therapeutic. In various embodiments, in vitro testing can be performed using cells representative of the type relevant to the subject's disorder to determine whether a particular aromatic-cationic peptide-based therapy has the desired effect on the cell type. Therapeutic compounds can be tested in suitable animal model systems, such as rats, mice, chickens, cows, monkeys, rabbits, etc., prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model systems known in the art can be used prior to administration to human subjects. In one embodiment, administration of an aromatic-cationic peptide to a subject exhibiting symptoms associated with an ocular disorder results in an improvement in one or more of those symptoms. Mode of Administration and Effective Dose

[0079] Any method known to one skilled in the art for contacting a cell, organ, or tissue with a peptide may be applied. Suitable modes include in vitro, ex vivo, or in vivo methods. Internal methods typically include administration of the aromatic-cationic peptides as described above to an animal, preferably a human. When used internally for therapy, the aromatic-cationic peptides are administered to the subject in an effective amount (i.e., an amount that has a desired therapeutic effect). The dosage and dosing regimen will depend on the extent of the subject's ocular disease, the characteristics of the particular aromatic-cationic peptide used, such as its therapeutic index, the subject, and the subject's medical history.

[0080] Effective amounts can be determined during preclinical and clinical trials by methods well known to physicians and clinicians. An effective amount of a peptide, preferably a pharmaceutical, useful in the methods of the invention can be administered to a mammal in need thereof by any of a number of known methods for administering pharmaceuticals. In some embodiments, the peptide can be administered systemically, topically, or intraocularly.

[0081] The aromatic-cationic peptides described herein can be incorporated into pharmaceuticals for administering to subjects alone or in combination for the treatment or prevention of disorders described herein.The compounds typically include active agents and pharmaceutically acceptable carriers.As used herein, the term "pharmaceutically acceptable carriers" includes physiological saline, solvents, dispersion media, coating agents, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, etc., suitable for pharmaceutical administration.Auxiliary active compounds can also be incorporated into the composition.

[0082] Pharmaceuticals are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal, or subcutaneous), oral, inhalation, transdermal (topical), ocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials. For the convenience of the patient or treating physician, the dosage formulations may be provided in a kit containing all the equipment needed for a course of treatment (eg, a vial of drug, a vial of diluent, a syringe, and needles).

[0083] Pharmaceuticals suitable for injection may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders so that sterile injectable solutions or dispersions can be prepared without prep. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, compositions for parenteral administration must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0084] The aromatic-cationic peptide composition can include a carrier, which may be a solvent or dispersion medium, for example, including water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomersal, and the like. Glutamine and other antioxidants can be introduced to prevent oxidation. In many cases, it may be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition a chemical that delays absorption, for example, aluminum monostearate or gelatin.

[0085] Sterile injectables can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above listed components, and then sterilizing by filtration if necessary. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle, which includes a basic dispersion medium and other required components listed above. In the case of sterile powders for preparing sterile injectables, typical methods of preparation include vacuum drying and freeze-drying, which can obtain the powder of the active ingredient plus additional required components from the solution thereof previously sterile-filtered.

[0086] For ophthalmic use, the therapeutic compounds are formulated into solutions, suspensions, and ointments suitable for use in the eye. For eye drop formulations, see generally Mitra (ed.), Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York, NY (1993), or see also Havener, WH, Ocular Pharmacology, CV Mosby Co., St. Louis (1983). Eye drop pharmaceuticals may be in the form of solutions, suspensions, ointments, creams, or solid inserts suitable for topical administration to the eye. For single doses, 0.1 ng to 5000 μg, 1 ng to 500 μg, or 10 ng to 100 μg of aromatic-cationic peptides can be instilled into the human eye.

[0087] Eye drop preparations may contain non-toxic substances such as antibacterial agents that are not harmful in use, for example, thimerosal, benzalkonium chloride, methyl and propyl parabens, benzyldodecinium bromide, benzyl alcohol, or phenylethanol; buffering ingredients such as sodium chloride, sodium borate, sodium acetate, sodium citrate, or gluconate; and other conventional ingredients such as sorbitan monolaurate, triethanolamine, polyoxyethylene sorbitan monopalmitate, ethylenediaminetetraacetic acid, and the like.

[0088] The eye drop solution or suspension is administered as often as necessary to maintain an acceptable concentration of aromatic-cationic peptide in the eye. Administration to the mammalian eye may be about once or twice daily.

[0089] Oral compositions generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated into excipients and can be used in the form of tablets, troches, or capsules, such as gelatin capsules. Pharmaceutically compatible binders and / or auxiliary substances can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, primogel, or corn starch; lubricants such as magnesium stearate or sterol; colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0090] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer, including those methods described in U.S. Patent No. 6,468,798.

[0091] Systemic administration of therapeutic compounds as described herein can also be carried out by transmucosal or transdermal methods. For transmucosal or transdermal administration, a penetrant that is compatible with the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays. For transdermal administration, the active compound is generally formulated into ointments, gels, or creams as known in the art. In one embodiment, transdermal administration can be carried out by iontophoresis.

[0092] The therapeutic protein or peptide can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic peptide is encapsulated in a liposome while maintaining the integrity of the peptide. As those skilled in the art will appreciate, there are various methods for preparing liposomes (see references: Lichtenberg et al., Methods Biochem. Anal, 33:337-462 (1988); Anselem et al, Liposome Technology, CRC Press (1993)). Liposomal formulations can slow clearance and increase cellular uptake (see Reddy, Ann., Pharmacother., 34 (7-8):915-923 (2000)). The active agent may also be incorporated within particles prepared from pharma- ceutically acceptable components, including, but not limited to, soluble, insoluble, permeable, non-permeable, biodegradable, or gastro-retentive polymers or liposomes, including, but not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles, and viral vector systems.

[0093] The carrier can also be a polymer, for example a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic peptide can be encapsulated in a polymer matrix while maintaining protein integrity. The polymer can be a natural polymer, such as a polypeptide, protein, or polysaccharide, or a synthetic polymer, such as poly-alpha-hydroxy acid. Examples include carriers made of collagen, fibronectin, elastin, cellulose acetate, nitrocellulose, polysaccharides, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is polylactic acid (PLA) or polylactic / glycolic acid (PGLA). The polymer matrix can be prepared and isolated in a variety of forms or sizes, including microspheres and nanospheres. Polymer formulations may provide a longer duration of therapeutic effect (see reference: Reddy, Ann. Pharmacother., 34 (7-8):915-923 (2000)). Polymeric formulations of human growth hormone (hGH) have been used in clinical trials (see reference: Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[0094] Examples of polymeric microsphere sustained release formulations are described in PCT Publication No. WO 99 / 15154 (Tracy et al.), U.S. Patent Nos. 5,674,534 and 5,716,644 (both Zale et al.), and PCT Publication No. WO 96 / 40073 (Zale et al.), and PCT Publication No. WO 00 / 38651 (Shah et al.). U.S. Patent Nos. 5,674,534 and 5,716,644 and PCT Publication No. WO 96 / 40073 describe a polymeric matrix containing particles of erythropoietin stabilized against aggregation with a salt.

[0095] In some embodiments, the therapeutic compound is prepared in a carrier that protects the therapeutic compound against elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using known techniques. Such materials are also commercially available, such as from Alza Corporation and Nova Pharmaceuticals, Inc. As pharmaceutically acceptable carriers, liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies against cell-specific antigens) can be used. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0096] The therapeutic compound can also be formulated to enhance intracellular delivery.For example, liposome delivery systems are known, see, for example, the following references: Chonn and Cullis, "Recent Advances in Liposome Drug Delivery Systems," Current Opinion in Biotechnology 6:698-708 (1995); Weiner, "Liposomes for Protein Delivery: Selecting Manufacturing and Development Processes," Immunomethods 4 (3) 201-9 (1994); and Gregoriadis, "Engineering Liposomes for Drug Delivery: Progress and Problems," Trends Biotechnol. 13 (12):527-37 (1995).Mizguchi et al., Cancer Lett, 100:63-69 (1996) describes the use of fusogenic liposomes to deliver proteins to cells both in vivo and in vitro.

[0097] The dosage, toxicity, and therapeutic efficacy of therapeutic drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutic in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are desirable. While compounds that exhibit toxic side effects may be used, care must be taken to design delivery systems that target the compound to the site of the affected tissue to minimize the potential for damage to non-infected cells. This will reduce side effects.

[0098] The data obtained from cell culture and animal studies can be used to formulate a range of doses for use in humans. The dose of the compound preferably falls within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dose may vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the method, a therapeutically effective amount can be estimated initially from cell culture studies. A dose can be formulated in an animal model that achieves a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Concentrations in plasma can be measured, for example, by high performance liquid chromatography.

[0099] Typically, an effective amount of the aromatic-cationic peptide sufficient to achieve a therapeutic or prophylactic effect ranges from about 0.000001 mg / kg body weight / day to about 10,000 mg / kg body weight / day. Suitably, the dose range is from about 0.0001 mg / kg body weight / day to about 100 mg / kg body weight / day. For example, the dose may range from 1 mg / kg body weight or 10 mg / kg body weight daily, every 2 days, or every 3 days, or from 1 to 10 mg / kg body weight weekly, every 2 weeks, or every 3 weeks. In one embodiment, a single dose of the peptide ranges from 0.1 to 10,000 μg / kg body weight. In one embodiment, the concentration of the aromatic-cationic peptide in the carrier ranges from 0.2 to 2000 μg / mL of the formulation delivered. Exemplary therapeutic dosing regimens call for administration once a day or once a week. Intervals may be more irregular than those indicated, with the subject's blood glucose or insulin levels being measured and the dosage or administration adjusted accordingly. In therapeutic applications, relatively high doses at relatively short intervals are often required until the progression of the disease is inhibited or halted, preferably until the subject shows partial or total improvement in the symptoms of the disease. The patient can then be placed on a prophylactic regimen.

[0100] In some embodiments, the therapeutically effective amount of the aromatic-cationic peptide is 10 -11 ~10 -6 Molar, e.g., about 10 -7 The therapeutic concentration can be defined as the concentration of peptide in a target tissue in millimolar doses. This concentration may be delivered at a systemic dose of 0.001-100 mg / kg or equivalent dose per body surface area. Dose schedules will be optimized to maintain therapeutic concentrations in the target tissue, most preferably once daily or weekly, but including continuous administration (e.g., intravenous infusion or transdermal application).

[0101] In some embodiments, the dose of aromatic-cationic peptide is defined as "low", "medium" or "high" dose level. In one embodiment, the low dose is about 0.0001 to about 0.5 mg / kg / h, preferably about 0.01 to about 0.1 mg / kg / h. In one embodiment, the medium dose is about 0.1 to about 1.0 mg / kg / h, preferably about 0.1 to about 0.5 mg / kg / h. In one embodiment, the high dose is about 0.5 to about 10 mg / kg / h, preferably about 0.5 to about 2 mg / kg / h.

[0102] Those skilled in the art will appreciate that certain factors may affect the dosage and timing required to effectively treat, including, but not limited to, the severity of the disease or disorder, the overall health and / or age of the subject, and the presence of other diseases. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[0103] Those skilled in the art will appreciate that certain factors may affect the dosage and timing required to effectively treat, including, but not limited to, the severity of the disease or disorder, the overall health and / or age of the subject, and the presence of other diseases. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[0104] The mammal treated according to the present method may be any mammal, including, for example, farm animals such as sheep, pigs, cows, and horses; pet animals such as dogs and cats; laboratory animals such as rats, mice, and rabbits. In a preferred embodiment, the mammal is a human. Combination treatment with aromatic-cationic peptides and other therapeutic agents

[0105] In some cases, it may be appropriate to administer at least one of the aromatic-cationic peptides (or pharma- ceutically acceptable salts, esters, amides, prodrugs, or solvates) described herein in combination with another therapeutic agent. By way of example only, if inflammation is one of the side effects experienced by a patient when administered one of the aromatic-cationic peptides herein, it may be appropriate to administer an anti-inflammatory agent in combination with the initial therapeutic agent. Alternatively, by way of example only, the therapeutic efficacy of one of the compounds described herein may be enhanced by administration of an adjunct (i.e., the adjunct alone has a minimal therapeutic effect, but in combination with another therapeutic agent, the overall therapeutic effect on the patient is enhanced). Alternatively, by way of example only, the effect experienced by the patient may be enhanced by administration of one of the compounds described herein with another therapeutic agent (including a treatment regimen) that also has a therapeutic effect in the prevention or treatment of an ocular disease. By way of example only, in the treatment of macular degeneration involving administration of one of the aromatic-cationic peptides, providing the patient with another therapeutic agent or treatment for macular degeneration may also provide an enhanced therapeutic effect. In any case, regardless of the ocular disease, disorder, or condition being treated, the overall effect experienced by the patient may simply be the additive effect of the two therapeutic agents, or the patient may experience a synergistic effect.

[0106] Specific non-limiting examples of possible combination therapies include using at least one aromatic-cationic peptide with nitric oxide (NO) inducers, statins, negatively charged phospholipids, antioxidants, minerals, anti-inflammatory agents, anti-angiogenic agents, matrix metalloproteinase inhibitors, and carotenoids. In some instances, suitable combination agents may fall into multiple categories (by way of example only, lutein is an antioxidant and a carotenoid). Additionally, aromatic-cationic peptides may be administered with additional agents that provide benefit to the patient, including, by way of example only, cyclosporin A.

[0107] Additionally, the aromatic-cationic peptides may be used in combination with procedures that provide synergistic benefits to the patient, including, by way of example only, the use of extracorporeal rheopheresis (also known as membrane differential filtration), the use of implantable miniature telescopes, laser photocoagulation of drusen, and microstimulation therapy.

[0108] The use of antioxidants has been proven to be beneficial for patients with macular degeneration and malnutrition.See, for example, the following references: Arch. Ophthalmol., 119: 1417-36(2001);Sparrow et al., J. Biol Chem., 278:18207-13(2003).Examples of suitable antioxidants that can be used in combination with at least one aromatic-cationic peptide include vitamin C, vitamin E, β-carotene and other carotenoids, coenzyme Q, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (also known as tempol), lutein, butylated hydroxytoluene, resveratrol, Trolox analog (PNU-83836-E), and cowberry extract.

[0109] The use of certain minerals has also been shown to be beneficial for patients with macular degeneration and malnutrition. See, for example, Arch. Ophthalmol., 119: 1417-36 (2001). Examples of suitable minerals that can be used in combination with at least one aromatic-cationic peptide include copper-containing minerals, such as cupric oxide; zinc-containing minerals, such as zinc oxide; and selenium-containing compounds.

[0110] The use of certain negatively charged phospholipids has also been shown to be beneficial for patients with macular degeneration and malnutrition. See, for example, the following references: Shaban & Richter, Biol. Chem., 383:537-45 (2002); Shaban, et al., 9 Exp. Eye Res., 75:99-108 (2002). Examples of suitable negatively charged phospholipids that can be used in combination with at least one aromatic-cationic peptide include cardiolipin and phosphatidylglycerol. Positively charged and / or neutral phospholipids can also be beneficial for patients with macular degeneration and malnutrition when used in combination with aromatic-cationic peptides.

[0111] The use of certain carotenoids has been linked to the maintenance of necessary photoprotection in photoreceptor cells. Carotenoids are natural yellow to red pigments of the terpenoid group that can be found in food, algae, bacteria, and certain animals such as birds and shellfish. Carotenoids are a collection of large molecules with over 600 naturally occurring carotenoids identified. Carotenoids include hydrocarbons (carotenes) and their oxygenated and alcohol derivatives (xanthophylls). These include actinioerythrol, astaxanthin, canthaxanthin, capsanthin, capsorubin, β-8'-apo-carotenal (apo-carotenal), β-12'-apo-carotenal, α-carotene, β-carotene "carotene" (a mixture of α- and β-carotene), γ-carotene, β-cryptoxanthin, lutein, lycopene, violeritrin, zeaxanthin, and esters of their hydroxyl- or carboxyl-containing moieties. Many of the carotenoids occur naturally as cis- and trans-isomers, while synthetic compounds are often racemic.

[0112] In humans, the retina selectively accumulates two carotenoids, primarily zeaxanthin and lutein. These two carotenoids are powerful antioxidants and absorb blue light, which may help protect the retina. Studies in quails have confirmed that animals fed a diet deficient in carotenoids had low retinal zeaxanthin concentrations and suffered severe light damage, as evidenced by a significant number of apoptotic photoreceptor cells, whereas animals with high zeaxanthin concentrations suffered minimal damage. Examples of carotenoids suitable for use in combination with at least one aromatic-cationic peptide include lutein and zeaxanthin, as well as the carotenoids mentioned above.

[0113] Suitable nitric oxide inducers include compounds that activate endogenous NO, or that increase the concentration of endogenous endothelium-derived relaxing factor (EDRF) in the body, or that are substrates for nitric oxide synthase. Such compounds include, for example, L-arginine, L-homoarginine, and N-hydroxy-L-arginine, including their nitrosated and nitrosylated analogs (e.g., nitrosated L-arginine, nitrosylated L-arginine, nitrosated N-hydroxy-L-arginine, nitrosylated N-hydroxy-L-arginine, nitrosated L-homoarginine, and nitrosylated L-homoarginine), and precursors of L-arginine and / or its pharma- ceutically acceptable salts, including, for example, citrulline, ornithine, glutamine, lysine, polypeptides comprising at least one of these amino acids, inhibitors of the enzyme arginase (e.g., N-hydroxy-L-arginine and 2(S)-amino-6-boronohexanoic acid) and substances for nitric oxide synthase, cytokines, adenosine, bradykinin, calreticulin, bisacodyl, and phenolphthalein. EDRF is a vasorelaxant factor secreted by endothelial cells and has been identified as nitric oxide or a closely related derivative thereof (Palmer et al, Nature, 327:524-526 (1987); Ignarro et al, Proc. Natl. Acad. Sci. USA, 84:9265-9269(1987)).

[0114] Statins act as lipid-lowering drugs and / or suitable nitric oxide inducers. Furthermore, a relationship between the use of statins and the onset or progression of macular degeneration has been demonstrated (G. McGwin, et al., British Journal of Ophthalmology, 87:1121-25(2003)). Thus, statins, when administered in combination with aromatic-cationic peptides, benefit patients suffering from ocular diseases, such as macular degeneration, malnutrition, and retinal dystrophies. Suitable statins include, by way of example only, rosuvastatin, pitavastatin, simvastatin, pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin, compactin, lovastatin, dalvastatin, fluidostatin, atorvastatin, atorvastatin calcium (which is the hemicalcium salt of atorvastatin), and dihydrocompactin.

[0115] Suitable anti-inflammatory drugs with which the aromatic-cationic peptides may be used include, by way of example only, aspirin and other salicylates, cromolyn, nedocromil, theophylline, zileuton, zafirlukast, montelukast, pranlukast, indomethacin, and lipooxygenase inhibitors; nonsteroidal anti-inflammatory drugs (NSAIDs) (such as ibuprofen and naproxen); prednisone, dexamethasone, cyclooxygenase inhibitors (i.e., naproxen® or cerebroside); COX-1 and / or COX-2 inhibitors such as Revlex™); statins (by way of example only, rosuvastatin, pitavastatin, simvastatin, pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin, compactin, lovastatin, dalvastatin, fluidostatin, atorvastatin, atorvastatin calcium (which is the hemicalcium salt of atorvastatin), and dihydrocompactin); and dissociative steroids.

[0116] To treat ocular diseases or conditions associated with macular or retinal degeneration, suitable matrix metalloproteinase (MMP) inhibitors may also be administered in combination with the aromatic-cationic peptides. MMPs are known to hydrolyze most components of the extracellular matrix. These proteinases play a central role in biological processes such as normal tissue remodeling, embryogenesis, wound healing, and angiogenesis. However, overexpression of MMPs has been observed in many disease states, including macular degeneration. Many MMPs have been identified, most of which are multidomain zinc endopeptidases. Many metalloproteinase inhibitors are known (see, for example, Whittacker M. et al., Review of MMP Inhibitors, Chemical Reviews 99(9):2735-2776 (1999)). Representative examples of MMP inhibitors include tissue inhibitors of metalloproteinases (TIMPs) (e.g., TIMP-1, TIMP-2, TIMP-3, or TIMP-4), alpha-2-macroglobulin, tetracyclines (e.g., tetracycline, minocycline, and doxycycline), hydroxamates (e.g., batimastat, marimistat, and trocade), chelating agents (e.g., EDTA, cysteine, acetylcysteine, D-penicillamine, and gold salts), synthetic MMP fragments, succinylmercaptopurine, phosphonamidates, and hydroxamic acids. Examples of MMP inhibitors that may be used in combination with the aromatic-cationic peptides include, by way of example only, any of the aforementioned inhibitors.

[0117] The use of anti-angiogenic or anti-VEGF drugs has also proven effective in patients with macular degeneration and malnutrition. Examples of suitable anti-angiogenic or anti-VEGF agents that may be used in combination with at least one aromatic-cationic peptide include Rhufab V2 (Lucentis™), tryptophanyl-tRNA synthetase (TrpRS), Eye001 (anti-VEGF pegylated aptamer), squalamine, Retaane™ 15 mg (anecortave acetate for depot suspension; Alcon, Inc.), combretastatin A4 prodrug (CA4P), Macugen™, Mifeprex™ (mifepristone-ru486), subtenon triamcinolone acetonide, intravitreal crystalline triamcinolone acetonide, prinomastat (AG3340 - a synthetic matrix metalloproteinase inhibitor, Pfizer), fluocinolone acetonide (fluocinolone intraocular implant, Bausch & Lomb / Control Delivery Systems), VEGFR inhibitors (Sugen), and VEGF traps (Regeneron / Aventis).

[0118] Other medications used to alleviate visual impairment can be used in combination with at least one aromatic-cationic peptide. Such therapies include Visudyne™ used with the use of a non-thermal laser, PKC 412, Endovion (NeuroSearch A / S), neurotrophic factors including, by way of example only, glial-derived neurotrophic factor and ciliary neurotrophic factor, diatazem, dorzolamide, photosynthetic products, 9-cis-retinal, phosphoryl iodide or echothiophate, or eye drops containing carbonic anhydrase inhibitors (including Echo Therapy), AE-941 (AEterna Laboratories, Inc), Sirna-027 (Sirna Therapeutics, Inc.), pegaptanib (NeXstar Pharmaceuticals / Gilead Sciences), neurotrophins (including, by way of example only, NT-4 / 5, Genentech), Cand5 (Acuity Pharmaceuticals), ranibizumab (Genentech), INS-37217 (Inspire Pharmaceuticals), integrin antagonists (Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (e.g., EntreMed, Inc.(Genentech), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan / Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus Biotech), microalgae compounds (Aquasearch / Albany, Mera Pharmaceuticals), D-9120 (Celltech Group pic), ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme / Celtrix), tyrosine kinase inhibitors (Allergan, SUGEN, Pfizer), NX-278-L (NeXstar Pharmaceuticals / Gilead Sciences), Opt-24 (OPTIS France SA), retinal ganglion neuroprotectant (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), and cyclosporine A.

[0119] In any case, the therapeutic agents can be administered in any order or even simultaneously. If simultaneously, the therapeutic agents can be provided in one combined dosage form or in several dosage forms (by way of example only, as one solution or two separate solutions). One of the therapeutic agents can be administered in multiple doses, or both can be administered in multiple doses. If not simultaneously, the interval between multiple administrations can vary from 0 weeks or more to about 4 weeks or less, about 6 weeks or less, about 2 months or less, about 4 months or less, about 6 months or less, or about 1 year or less. Furthermore, the combination methods, compositions, and formulations are not limited to the use of only two agents. By way of example only, an aromatic-cationic peptide is administered with at least one antioxidant and at least one negatively charged phospholipid; or an aromatic-cationic peptide is administered with at least one antioxidant and at least one inducer of nitric oxide production; or an aromatic-cationic peptide is administered with at least one inducer of nitric oxide and at least one negatively charged phospholipid, etc.

[0120] Additionally, the aromatic-cationic peptides may be used in combination with treatments that may provide additive or synergistic benefits to the patient. Treatments known, proposed, or believed to alleviate visual impairment include "limited retinal translocation," photodynamic therapy (including, by way of example only, receptor-targeted PDT, Bristol-Myers Squibb, Co.; injectable porfimer sodium with PDT; verteporfin, QLT Inc.; rostaporfin with PDT, Miravent Medical Technologies; talaporfin sodium with PDT, Nippon Petroleum; motexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides (including, by way of example only, products tested by Novagali Pharma SA, and ISIS-13650, Isis Pharmaceuticals), laser photocoagulation, drusen laser therapy, macular hole surgery, macular translocation, implantable miniature telescopes, file exercise angiography (also known as microlaser therapy and trophovascular coagulation therapy), proton beam therapy, microstimulation therapy, retinal detachment, and These include, but are not limited to, vitreous surgery, scleral buckle, submacular surgery, transpupillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal rheopheresis (also known as membrane differential filtration and rheotherapy), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including hypoxia responsive element gene therapy, Oxford Biomedica; Lentipak, Genetix; PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including retinal pigment epithelial cell Diacrin, Inc.; retinal cell transplants, Cell Genesys, Inc.), and acupuncture.

[0121] Further, combinations that may be used to provide benefits to individuals include using genetic testing to determine whether an individual is a carrier of a mutated gene known to be associated with a particular eye disease.By way of example only, defects in the human ABCA4 gene are believed to be associated with five distinct retinal phenotypes, including Stargardt's disease, cone-rod dystrophy, age-related macular degeneration, and retinitis pigmentosa.See, for example, the following references: Allikmets et al., Science, 277:1805-07 (1997); Lewis et al., Am. J. Hum. Genet., 64:422-34 (1999); Stone et al., Nature Genetics, 20:328-29 (1998); Allikmets, Am. JHum. Gen., 67:793-799 (2000); Klevering, et al., Ophthalmology, 11 1:546-553 (2004). Additionally, autosomal dominant Stargardt disease is caused by a mutated allele of the ELOV4 gene. See reference: Karan, et al., Proc. Natl. Acad. Sci. (2005). Patients carrying any of these mutated alleles are expected to benefit from the treatment and / or prevention methods described herein.

[0122] (Example) This invention is further illustrated by the following examples which should not be construed as limiting. EXAMPLES

[0123] Prevention of hyperglycemia induced damage in human retinal epithelial cells

[0124] The effect of aromatic-cationic peptides of the present invention in preventing hyperglycemia induced damage in human retinal epithelial cells (HRECs) was investigated in cultured HRECs.

[0125] The method of culturing HRECs in the present study is known. See generally the following references: Li B, Tang SB, Zhang G, Chen JH, Li BJ. Culture and characterization of human retinal capillary endothelial cell. Chin Ophthal Res 2005; 23: 20-2; Premanand C, Rema M, Sameer MZ, Sujatha M, Balasubramanyam M. Effect of curcumin on proliferation of human retinal endothelial cells under in vitro conditions. Invest Ophthalmol Vis Sci 2006; 47:2179-84.

[0126] Briefly, HREC cells were divided into three groups: normal control group, 30 mM glucose group, and 30 mM glucose + SS-31 group. The viability of HRECs co-treated with high glucose along with various concentrations of SS-31 (10 nM, 100 nM, 1 μM, 10 μM) was measured using Annexin V + PI test and flow cytometry.References: Koopman, G., Reutlingsperger, CP, Kuijten, GAM, Keehnen, RMJ, Pals, ST, and van Oers, MHJ 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415; Homburg, CH, de Haas, M., von dem Borne, AE, Verhoeven, AJ, Reutelingsperger, CP, and Roos, D. 1995. Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood 85: 532; Vermes , L , Haanen , C , Steffens-Nakken , H. , and Reutelingsperger , C. 1995. A novel assay for apoptosis -flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin VJ Immunol. Meth. 184: 39; Fadok, VA,Voelker, DR, Campbell, PA, Cohen, JJ, Bratton, DL, and Henson, PM 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148: 2207 .

[0127] The viability of HRECs treated with high glucose and SS-31 at 24 and 48 hours was examined. The results are shown in Figure 1. The administration of SS-31 significantly increased the viability of HRECs while decreasing the apoptotic and necrotic cells. SS-31 treatment also reduced the generation of ROS (Figure 2).

[0128] We investigated the evaluation of SS-31 as a protective agent against mitochondrial membrane potential loss in HRECs treated with high glucose. To determine whether mitochondrial-mediated pathways were important in the protective effect of SS-31 against high glucose-induced cell death, we measured ΔΨm by flow cytometry. After 24 or 48 h of treatment of HRECs with high glucose without SS-31, a rapid loss of mitochondrial membrane potential was detected by the JC-1 fluorescent probe, as indicated by a significant decrease in the ratio of red to green fluorescence observed in the high glucose group. In contrast, the ΔΨm of the 100 nM SS-31 co-treatment group remained virtually unchanged and was comparable to that of the normal glucose control group (Figure 3). These data suggest that SS-31 prevented the loss of mitochondrial membrane potential caused by exposure to a high glucose environment.

[0129] Glucose (30 mmol / L) induced the release of cytochrome c from mitochondria in HRECs. Fixed HRECs were immunolabeled with cytochrome c antibody and mitochondrial-specific antibody (HSP60). Confocal microscopy analysis showed that in HRECs cultured normally and co-treated with SS-31 together with glucose, cytochrome c staining and mitochondrial staining were overlapped, indicating that cytochrome c and mitochondria were colocalized (Figure 4). After 24 or 48 h of treatment with 30 mmol / L glucose, some cytochrome c was observed in the cytoplasm of HRECs, indicating that glucose induces the release of cytochrome c from mitochondria to the cytoplasm in HREC cells, but that SS-31 can reduce such translocation between mitochondria and cytoplasm.

[0130] Preventing cytochrome c release from mitochondria resulted in a decrease in the activity of caspase-3. As shown in Figure 5, SS-31 reduced the protein expression of caspase-3 in high glucose-treated HRECs. The cleaved caspase-3 protein expression level was measured by Western blotting (Figure 5A). When HRECs were exposed to 30 mM glucose for 24 and 48 h, the level of caspase-3 expression was dramatically increased. At the same time, the SS-31 co-treated group showed a significant decrease in caspase-3 protein level (*p<0.05). Figure 5B shows the quantitative analysis of caspase-3 expression level of HRECs in high glucose co-treated with SS-31 for 24 and 48 h.

[0131] SS-31 increased the expression of Trx2 in HRECs treated with high glucose. Figure 5C shows the mRNA levels of Trx2 in HRECs co-treated with SS-31 and exposed to 30 mM glucose for 24 and 48 h. The mRNA expression levels of Trx2 were measured by quantitative real-time PCR. The relative mRNA levels of Trx2 were normalized with 18S mRNA levels (*p<0.05 vs. normal glucose medium group and 30 mM high glucose treatment group). Three individual samples were used for each time. Figure 5D shows the expression levels of Trx2 protein measured by Western blotting. The protein expression of Trx in the high glucose group co-treated with SS-31 was significantly increased compared to the normal glucose group (*p<0.05). Figure 5E shows the quantitative analysis of the protein levels of Trx2 in HRECs after 24 and 48 h of exposure to high glucose without and with co-treatment with SS-31.

[0132] These results indicate that SS-31 enhances the survival rate of HREC cells in a high glucose environment.Similarly, SS-31 and other aromatic-cationic peptides may be useful as a method to prevent diabetic retinopathy. EXAMPLES

[0133] Prevention of diabetic retinopathy in rats fed a high-fat diet

[0134] The efficacy of the aromatic-cationic peptides of the present invention in preventing the progression of diabetic retinopathy was investigated in a Sprague-Dawley rat model. This Example describes the results of such an experiment.

[0135] A diabetic rat model was established in SD rats by a combination of a 6-week HFD and low-dose STZ (30 mg / kg) injection or a single high-dose STZ (65 mg / kg). See generally: K. Srinivasan, B. Viswanad, Lydia Asrat, CL Kaul and P. Ramarao, Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: A model for type 2 diabetes and pharmacological screening, Pharmacological Research, 52(4): 313-320, 2005. The same batch of rats fed a normal diet (NRC) was used as control. Tables 7-10 show the treatment schedule and experimental protocol. [Table 7] [Table 8] [Table 9] [Table 10]

[0136] Following such an experimental protocol, we demonstrated the efficacy of aromatic-cationic peptides in treating diabetes-related pathologies in an SD rat model. Administration of SS-20 and SS-31 led to the prevention or reversal of cataract formation in the lenses of diabetic rats (Figures 6 and 7, Tables 11 and 12). [Table 11] -: transparent; +: slightly turbid; ++: turbidity; +++: moderate turbidity; ++++: severe opacity; [Table 12]

[0137] The effects of the aromatic-cationic peptides on the lens epithelium in SD rat models were investigated. Administration of SS-31 reduced the changes in epithelial cells in both STZ rat models (Figure 8) and HFD / STZ rat models (Figure 9).

[0138] The effects of the aromatic-cationic peptides on the inner blood-retinal barrier function in an SD rat model were investigated. Administration of SS-20 and SS-31 improved the inner blood-retinal barrier function compared to rats fed a HFD without SS-20 or SS-31 (Figure 10).

[0139] The effects of the aromatic-cationic peptides on retinal microvessels in an SD rat model were investigated (FIGS. 11 and 12). Administration of SS-31 reduced the retinal microvascular changes observed in STZ or HFD / STZ rats.

[0140] The effect of the aromatic-cationic peptides on the distribution of tight junction protein claudin 5 in retinal microvessels in SD rat model was investigated. The distribution of tight junction protein claudin 5 was detected under confocal microscope (Figure 13). Claudin 5 was distributed smoothly, linearly, and uniformly along retinal vessels in normal rats (A), but the linear shape was disrupted in STZ rats (B). The distribution of claudin 5 in retinal vessels in STZ rats treated with SS-20 (10 mg / kg) or SS-31 (10 mg / kg) was similar to that in normal rats (panels C and D, respectively).

[0141] In summary, these findings collectively demonstrate that aromatic-cationic peptides prevent or correct the deleterious effects of diabetes in the eye, such as cataracts and the microvasculature, and thus administration of said aromatic-cationic peptides of the invention is useful in methods for preventing or treating diabetes-associated eye disease in human subjects. EXAMPLES

[0142] SS-31 prevents oxidative stress in glaucoma trabecular meshwork cells.

[0143] The effect of said aromatic-cationic peptides of the present invention in preventing or treating glaucoma was investigated by studying the effect of peptides on glaucoma trabecular meshwork cells. Glaucoma is the second most common cause of irreversible blindness worldwide. Primary open-angle glaucoma (POAG) is the main subtype of glaucoma. In POAG, there is no visible abnormality of the trabecular meshwork. However, it is believed that the ability of cells in the trabecular meshwork to perform normal functions is weakened.

[0144] In this example, the effects of the aromatic-cationic peptides of the invention were compared between trabecular meshwork cells from POAG patients (GTM) and non-diseased individuals (HTM). Methods useful in the study of the invention have been described. See generally the following references: He Y, Ge J, Tombran-Tink J., Mitochondrial defects and dysfunction in calcium regulation in glaucomatous trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2008, 49(11):4912-22; He Y, Leung KW, Zhang YH, Duan S, Zhong XF, Jiang RZ, Peng Z, Tombran-Tink J, Ge J. Mitochondrial complex I defect induces ROS release and degeneration in trabecular meshwork cells of POAG patients: protection by antioxidants. Invest Ophthalmol Vis Sci. 2008, 49(4): 1447-58. GTM cells show a significant impairment of mitochondrial membrane potential compared to HTM cells (Figure 18).

[0145] The cells were divided into three groups: "Group A" cells were exposed to hydrogen peroxide before administration of SS-31, "Group B" cells were exposed to SS-31 before administration of hydrogen peroxide, and "Group C" cells were simultaneously administered with SS-31 and hydrogen peroxide.

[0146] To assess whether SS-31 has a cytotoxic effect on HTM or GTM cells, cells were treated with various concentrations of SS-31 and cytotoxicity was measured using the LDH assay. The LDH cytotoxicity assay is a colorimetric method for testing cellular cytotoxicity. The assay quantitatively measures stable cytosolic lactate dehydrogenase (LDH) released from damaged cells. This released LDH is measured by a coupled enzymatic reaction resulting in the conversion of a tetrazolium salt (iodonitrotetrazolium (INT)) to a red formazan by diaphorase. Methods for detecting LDH from cells that are useful in the study of the present invention are known. See generally the following references: Haslam, G. et al. (2005) Anal. Biochem. 336: 187; Tarnawski, A. (2005) Biochem. Biophys. Res. Comm. 333: 207; Round, J. L et al (2005) J. Exp. Med. 201: 419; Bose, C et al (2005) Am. J. Physiol. Gastr. L. 289: G926; Chen, A. and Xu, J. (2005) Am. J. Physiol. Gastr. L. 288: G447. LDH activity is measured as NADH oxidation or INT reduction over a defined period of time. The results are shown in Figure 14 and show that SS-31 does not affect the viability of HTM and GTM cells.

[0147] Methods for measuring mitochondrial membrane potential using TMTR useful in the present study are described in the following references: Andrea Rasola and Massimo Geuna, A flow cytometry assay simultaneously detects independent apoptotic parameters, Cytometry 45:151-157, 2001; Mitoprobe JC-1 Kit for Flow Cytometry, Molecular Probes, Invitrogen, USA. Figure 16 shows the results in GTM cells. Taken together, these results demonstrate that SS-31 improves mitochondrial membrane potential in cells exposed to hydrogen peroxide prior to administration of SS-31.

[0148] Group A: We investigated the mitochondrial membrane potential (ΔΨm) of HTM and GTM cells when cells were exposed to hydrogen peroxide prior to administration of SS-31. First, we measured the mitochondrial membrane potential using confocal microscopy of cells labeled with tetramethylrhodamine methyl ester (TMRM, 500 nM x 30 min) (Figure 15). We also measured the mitochondrial membrane potential using flow cytometry by labeling cells with the mitochondria-selective probe tetramethylrhodamine methyl ester (TMRM, 500 nM x 30 min) (Figures 16, 17).

[0149] Group B: We investigated the morphology of GTM cells when the cells were exposed to SS-31 before administration of hydrogen peroxide. Figure 18 shows the results of inverted phase contrast microscopy of cells administered various concentrations of SS-31. The results show that SS-31 protects cells from hydrogen peroxide-mediated morphological changes in a concentration- and time-dependent manner. That is, hydrogen peroxide-mediated cell loss was reduced in cells exposed to the SS-31 peptide. We also investigated the mitochondrial membrane potential (ΔΨm) of HTM and GTM cells when the cells were exposed to SS-31 before administration of hydrogen peroxide. Mitochondrial membrane potential was measured using confocal microscopy of cells labeled with tetramethylrhodamine methyl ester (TMRM, 500 nM x 30 min) (Figures 19-21). These results show that SS-31 improves the mitochondrial membrane potential of cells exposed to hydrogen peroxide in a dose-dependent manner. Therefore, SS-31 provides a protective effect against oxidative stress in GTM cells.

[0150] The effect of SS-31 in alleviating acute oxidative damage in GTM and HTM cells was investigated. Figure 36 shows the fluorescence intensity of TMRM in GTM and HTM cells using FACS analysis. H2O2, SS-31 10 -6 M, SS-31 10 -7 M, SS-31 10 -8 The percentage of fluorescence intensity compared to the GTM control in M ​​was 35.2±2.12%, 56.2±4.04%, 50.3±4.46%, and 47.5±2.82%, respectively, n=4; and HTM group was 37.4±0.725%, 57.7±1.80%, 50.6±3.06%, and 49.4±2.27%, respectively, n=4; ** means P<0.01 compared to the GTM H2O2 group; * means P<0.05 compared to the GTM H2O2 group; ▲▲▲ means P<0.001 compared to the HTM H2O2 group.

[0151] Figure 37 shows the fluorescence intensity of ROS in GTM and HTM cells in the control and SS-31 treatment groups using FACS analysis. H2O2, SS-31 10 -6 M, SS-31 10 -7 M, SS-31 10 -8The percentage of intracellular ROS production compared to the GTM control in M ​​was 146.0±2.27%, 84.5±8.75%, 102.0±5.69%, 133.0±5.17%, respectively (n=3); HTM group was 153.0±3.46%, 79±2.39%, 91.8±3.49%, 129.0±8.24%, respectively (n=4), and H2O2 group was P<0.001 compared to the control GTM and HTM; *** means P<0.001 compared to the GTM H2O2 group; ▲▲▲ means P<0.001 compared to the HTM H2O2 group; ▲▲ means P<0.01 compared to the HTM H2O2 group. Figure 38 shows that SS-31 reduced the amount of cell apoptosis induced by H2O2.

[0152] We investigated the effect of SS-31 on persistent oxidative damage in GTM and HTM cells. To investigate the protective effect of SS-31, cells were cultured for 10 -6 , 10 -7 , 10 -8 The cells were pretreated with 200μM SS-31 for 1 hour, and then incubated with 200μM H2O2 for 1 hour. Figure 39 and Table 13 show the effect of SS-31 on ROS production from sustained oxidative damage in GTM and HTM cells. Figure 40 and Table 14 show the changes in MMP in GTM and HTM cells in each treatment group. [Table 13] [Table 14]

[0153] Taken together, these results suggest that SS-31 is -4 SS-31 (>10 M) was not cytotoxic to both GTM and HTM cells and reduced persistent and acute oxidative stress induced by hydrogen peroxide. -9 As such, the aromatic-cationic peptides of the present invention are useful in methods for preventing or treating glaucoma in human subjects. EXAMPLES

[0154] SS-31 prevents oxidative stress in primary retinal pigment epithelial cells.

[0155] To test the effect of the aromatic-cationic peptides of the present invention in preventing or reducing oxidative damage in primary retinal pigment epithelial (RPE) cells, these cells were cultured. Methods useful for testing primary retinal pigment epithelial cells have been described. See the following reference: Dunn l., ARPE-19, A Human Retinal Pigment Epithelial Cell Line with Differentiated Properties, Experimental Eye Research, 1996, 62(2): 155-170. First, it was found that SS-31 had no adverse effect on these cells. Primary human RPE cells were cultured with various concentrations of SS-31 alone, and cell viability was measured by MTT assay (Figure 22).

[0156] Next, we tested the viability of primary RPE cells in the presence of tBHP and various concentrations of SS-31. Cells were plated in 96-well plates at 10,000 cells per well and cultured for 24 hours, followed by starvation for 24 hours. Cells were then exposed to high concentrations of tBHP (Figure 23A) or pre-incubated with various concentrations of SS-31 for 4 hours and then stimulated with tBHP for 6 hours (Figure 23B). These results indicate that SS-31 enhanced cell viability in response to tBHP administration. Using FACS analysis, we also investigated intracellular ROS production in the three groups of RPE cells. Figure 31A shows ROS production in control RPE cells; Figure 31B shows ROS production in RPE cells treated with 500 μM tBHP for 3 hours; Figure 31C shows ROS production in RPE cells treated with 500 μM tBHP for 3 hours and 1 μM SS-31. Figure 32 shows MMPs labeled by JC-1 in FACS analysis. Three different concentrations of SS-31 were analyzed: 500 μM tBHP for 3 hours, green to red ratio of 1.08; 10 nM SS-31 for 4 hours + 500 μM tBHP for 3 hours, green to red ratio of 1.25; 100 nM SS-31 for 4 hours + 500 μM tBHP for 3 hours, green to red ratio of 1.4; 1 μM SS-31 for 4 hours + 500 μM tBHP for 3 hours, green to red ratio of 2.28. Figure 33 shows the effect of 1 μM SS-31 on the MMP reduction induced by tBHP. Figure 33A: control group, R / G is 3.63±0.24; Figure 33B: group treated with 500 μM tBHP for 3 hours, R / G is 1.08±0.11; Figure 33C: group treated with 1 μM SS-31 for 4 hours + 500 μM tBHP for 3 hours, R / G is 2.38±0.18. Figure 33D is a graph comparing the fluorescence ratios of the various groups. *P<0.01, C vs. B.

[0157] Figure 34 shows the effect of SS-31 on cell apoptosis induced by 250 μM tBHP for 24 hours. Figure 34A: Control group; (Q2+Q4)%=1.27±0.3%; Figure 34B: Treatment group with 250 μM tBHP for 24 hours; (Q2+Q4)%=15.7±0.6%; Figure 34C: Treatment group with 1 μM SS-31 for 4 hours + 250 μM tBHP for 24 hours; (Q2+Q4)%=8.4±0.8%. Figure 34D is a graph comparing the fluorescence ratios of various groups. *P<0.05, C vs. B. Figure 35 is a graph showing the MDA concentration induced by tBHP in three groups of RPE cells. (*P<0.05).

[0158] Taken together, these results demonstrate that SS-31 prevents oxidative stress in primary retinal pigment epithelial cells, making the aromatic-cationic peptides of the invention useful in methods for preventing or treating damage to retinal cells in human subjects. EXAMPLES

[0159] Prevention and treatment of choroidal neovascularization by the aromatic-cationic peptides of the present invention in a CNV mouse model

[0160] To further demonstrate the prevention of choroidal neovascularization (CNV) on the one hand and the treatment of CNV on the other hand, the aromatic-cationic peptides of the present invention were tested in a mouse model of CNV (Figure 24). CNV was induced in the eye by laser burn. For methods useful in this study, see the following reference: Reich, Mol Vis 2003; 9:210-216.

[0161] Briefly, male C57BL / 6 mice aged 5–6 weeks were anesthetized with chloral hydrate and pupils were dilated with tropicamide. Four laser spots (532 nm, 260 mw, 0.01 sec, 50 μm; Novus Spectra, Lumenis, USA) were applied to a circular fundus around the optic nerve head of the right eye with a coverslip used as a contact lens. Daily intraperitoneal injections of 1 mg / kg, 9 mg / kg SS-31, or vehicle were started the day before laser photocoagulation.

[0162] One week later, mice were deeply anesthetized and perfused through the left ventricle with 1 mL of PBS-buffered fluorescein dextran (50 mg / mL). Eyes were enucleated and fixed in 4% paraformaldehyde for 2 h. Eyes were dissected at the ocular equator, and the anterior half and retina were removed. The posterior segment, including the sclera and choroid, was cut into quarters by 4–5 radial cuts and mounted on slides. All flat-mounted specimens were examined under a fluorescent microscope (AxioCam MRC; Carl Zeiss). Measurement of the area of ​​each CNV lesion was performed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).

[0163] There were 48 neovascularization sites in each group. The area of ​​neovascularization was calculated using IMAGE-PROPLUS 6.0 software. The areas of neovascularization in the CNV model, 1 mg / kg SS-31, and 9 mg / kg SS-31 groups were 0.0130±0.0034, 0.0068±0.0025, and 0.0067±0, respectively. These results indicate that the two concentrations of SS-31 significantly reduced the area of ​​choroidal neovascularization (P<0.05) (Figure 24). EXAMPLES

[0164] Prevention and Treatment of Oxygen-Induced Retinopathy (OIR) by the Aromatic-Cationic Peptides of the Invention in an OIR Mouse Model

[0165] To further demonstrate prevention of oxygen-induced retinopathy (OIR), the aromatic-cationic peptides of the present invention were tested in a mouse model of OIR (Figure 25). In this model, 7-day-old mouse pups with partially developed retinal vasculature are exposed to hyperoxia (75% oxygen) for 5 days, which arrests retinal vascular growth and causes significant vascular occlusion. On postnatal day 12, the pups were returned to room air, and by postnatal day 17, a florid compensatory retinal neovascularization had developed. This model of pathological neovascularization is widely used as a surrogate for proliferative diabetic retinopathy (DR).

[0166] To investigate the effect of the aromatic-cationic peptides of the present invention on preventing OIR, OIR was induced in mouse pups and the mice were simultaneously administered an aromatic-cationic peptide (e.g., SS-20 or SS-30) for about 6 weeks. The results are shown in Figure 26, which shows that treatment with SS-31 prevented compensatory retinal neovascularization. Thus, the aromatic-cationic peptides of the present invention are useful in methods for preventing proliferative diabetic retinopathy in mammalian subjects. EXAMPLES

[0167] Antioxidants reduce photoreceptor cell death in a model of retinitis pigmentosa.

[0168] The cone cell specific line 661W was derived from a mouse retinal tumor. Methods useful in this study of 661W cells have been described previously. See generally the following reference: Gearoid Tuohy, Sophia Millington-Ward, Paul F. Kenna, Peter Humphries and G. Jane Farrar, Sensitivity of Photoreceptor-Derived Cell Line (661W) to Baculoviral p35, Z-VAD.FMK, and Fas-Associated Death Domain, Investigative Ophthalmology and Visual Science. 2002;43:3583-3589. These cells were cultured to test the effect of the aromatic-cationic peptides of the invention in preventing or reducing oxidative damage in cone cells (Figure 27). First, it was found that tBHP affects the viability of 661W cells (Figure 27A). Different doses of tBHP were administered to the cells for 3 hours. Next, we found that various doses of SS-1 reduced tBHP-induced 661W cell death (FIG. 27B).

[0169] The potential of SS-31 to prevent the decline in mitochondrial viability induced by tBHP, 100 nmol / L of SS-31, was administered to cultures of 661W cells. The results are shown in Figure 30 and show that SS-31 significantly increased mitochondrial viability as compared to cells that were not administered SS-1, as shown by the JC-1 assay. EXAMPLES

[0170] Effect of SS-31 in a mouse model of retinal degeneration.

[0171] To further demonstrate the prevention of retinal degeneration, the aromatic-cationic peptides of the present invention were tested in a mouse model of retinal degeneration. CNV is induced in the eye by laser burn (see Example 5). Mouse models of retinal degeneration have been investigated for many years in hopes of understanding the causes of photoreceptor cell death. The following naturally occurring mouse mutations have been found that express degeneration of photoreceptors in the retina while preserving all other retinal cell types: retinal degeneration (formerly rd, same as rodless retina r, now Pde6b rdl); Purkinje cell degeneration (pcd); neuronal (nr); retinal slow degeneration (rds, now Prph Rd2); retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinal degeneration 4 (rd4); retinal degeneration 5 (rd5); vitiligo (vit, now Mitfmi-vit); retinal degeneration 6 (rd6); retinal degeneration 7 (rd7); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8 (rd8); retinal degeneration 9 (rd9); retinal degeneration 10 (rd10); and cone photoreceptor loss of function (cpf11).

[0172] FIG. 28 is a series of photomicrographs showing the thickness of the outer retinal nuclear layer (ONL) in a mouse model of retinal degeneration in control and SS-31-treated mice. The results show that SS-31-treated mice maintained numerous rows of cells in the ONL compared to untreated mice. Flat-mounted retinas stained with peanut agglutinin (PNA), which selectively stains cone inner and outer segments, also show that cone cell density is higher in SS-31-treated mice (FIG. 29). These results indicate that treatment with SS-31 prevented compensatory damage to the outer retinal nuclear layer in a mouse model of retinal degeneration. Thus, the aromatic-cationic peptides of the present invention are useful in methods of preventing retinal degeneration in a mammalian subject.

[0173] (Corresponding patent) The present invention is not limited in terms of the specific embodiments described herein, which are intended to be only one illustration of individual aspects of the invention. As will be apparent to one skilled in the art, many modifications and variations of the present invention can be made without departing from its spirit and scope. In addition to what is recited herein, functionally equivalent methods and apparatuses within the scope of the present invention will be apparent to one skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is limited only by the appended claims, along with the full scope of equivalents to which the claims are entitled. It is to be understood that the present invention is not limited to the specific methods, reagents, compounds, compositions, or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0174] Further, when features or aspects of the disclosure are described in terms of the Markush Group, the parties thereby recognize that the disclosure is also described in terms of the individual members or subgroups of members of the Markush Group.

[0175] As will be apparent to those skilled in the art, for any and all purposes, particularly with respect to the provision of a written specification, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges thereof. Any described range can be easily recognized as fully describing and allowing for the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range disclosed herein can be easily divided into a lower third, middle third, upper third, etc. As will also be apparent to those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc. include the recited numbers, which are then ranges that can be divided into subranges as described above. Finally, as will be apparent to those skilled in the art, ranges include individual numbers. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0176] All patents, patent applications, provisional applications, and patent publications introduced or cited in this specification are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the express guidance of this specification.

[0177] Other embodiments are set forth in the following claims. Yet another aspect of the present invention may be as follows. [1] A method for treating or preventing an eye disease in a mammalian subject in need of such treatment or prevention, comprising: A therapeutically effective amount of a peptide having the formula D-Arg-2'6'-Dmt-Lys-Phe-NH2 or Phe-D-Arg-Phe-Lys-NH2 is administered to the subject. The method includes: [2] The method according to [1], The method, wherein the ocular disease is selected from the group consisting of diabetic retinopathy, cataracts, retinitis pigmentosa, glaucoma, macular degeneration, choroidal neovascularization, retinal degeneration, and oxygen-induced retinopathy. [3] The method according to [1], The method according to claim 1, wherein the peptide is D-Arg-2'6'-Dmt-Lys-Phe-NH2. [4] The method according to [1], The method of claim 2, wherein the peptide is Ala-D-Arg-Lys-NH2. [5] The method according to [1], The method, wherein the subject is a human. [6] The method according to [1], The method wherein the peptide is administered intraocularly, iontophoretically, orally, topically, systemically, intravenously, subcutaneously, or intramuscularly. [7] The method according to [1], The method further comprising administering separately, sequentially or simultaneously a second active agent. [8] The method according to [7], The method, wherein the second active agent is selected from the group consisting of antioxidants, metal complexes, anti-inflammatory agents, antibiotics, and antihistamines. [9] The method according to [8], The method, wherein the antioxidant is vitamin A, vitamin C, vitamin E, lycopene, selenium, alpha-lipoic acid, coenzyme Q, glutathione, or a carotenoid.

[10] The method according to [7], The second active agent may be aceclidine, acetazolamide, anecortave, apraclonidine, atropine, azapentacene, azelastine, bacitracin, befunolol, betamethasone, betaxolol, bimatoprost, brimonidine, brinzolamide, carbachol, carteolol, celecoxib, chloramphenicol, chlortetracycline, ciprofloxacin, cromoglycate, cromolyn, cyclopentolate, cyclosporine, dapiprazole, demecarium, demeclovir ... xamethasone, diclofenac, dichlorphenamide, dipivefrin, dorzolamide, echothiophate, emedastine, epinastine, epinephrine, erythromycin, ethoxzolamide, eucatropine, fludrocortisone, fluorometholone, flurbiprofen, fomivirzen, framycetin, ganciclovir, gatifloxacin, gentamicin, homatropine, hydrocortisone, idoxuridine, indomethacin, isoflurophate, ketorolac, ketotifen, Latanoprost, levobetaxolol, levobunolol, levocabastine, levofloxacin, lodoxamide, loteprednol, medrysone, methazolamide, metipranolol, moxifloxacin, naphazoline, natamycin, nedocromil, neomycin, norfloxacin, ofloxacin, olopatadine, oxymetazoline, pemirolast, pegaptanib, phenylephrine, physostigmine, pilocarpine, pindolol, pirenoxine, polymyxin B, prednisolone, promyelitis, wherein the active ingredient is selected from the group consisting of paracaine, ranibizumab, rimexolone, scopolamine, sezolamide, squalamine, sulfacetamide, suprofen, tetracaine, tetracycline, tetrahydrozoline, tetrizoline, timolol, tobramycin, travoprost, triamcinolone, trifluoromethazolamide, trifluridine, trimethoprim, tropicamide, unoprostone, vidarabine, xylometazoline, pharmaceutically acceptable salts thereof, and combinations thereof.

Claims

1. A pharmaceutical composition for treating retinal vascular occlusion in a mammalian subject in need thereof, comprising: Formula D-Arg-2'6'-Dmt-Lys-Phe-NH 2 A pharmaceutical composition comprising a peptide represented by the formula:

2. 2. The pharmaceutical composition of claim 1, The pharmaceutical composition, wherein the subject is a human.

3. The pharmaceutical composition according to any one of claims 1 to 2, The pharmaceutical composition is formulated for topical, subcutaneous, ocular, iontophoretic, oral, systemic, intravenous or intramuscular administration.

4. The pharmaceutical composition according to any one of claims 1 to 3, The pharmaceutical composition further comprises a second active agent.

5. 5. The pharmaceutical composition according to claim 4, A pharmaceutical composition wherein the second active agent is selected from the group consisting of antioxidants, metal complexes, anti-inflammatory agents, antibiotics, and antihistamines.

6. 6. The pharmaceutical composition according to claim 5, The pharmaceutical composition, wherein the antioxidant is vitamin A, vitamin C, vitamin E, lycopene, selenium, α-lipoic acid, coenzyme Q, glutathione, or a carotenoid.

7. 5. The pharmaceutical composition according to claim 4, The second active agent may be aceclidine, acetazolamide, anecortave, apraclonidine, atropine, azapentacene, azelastine, bacitracin, befunolol, betamethasone, betaxolol, bimatoprost, brimonidine, brinzolamide, carbachol, carteolol, celecoxib, chloramphenicol, chlortetracycline, ciprofloxacin, cromoglycate, cromolyn, cyclopentolate, cyclosporine, dapiprazole, demecarium, demetrium, cefotaxime ... xamethasone, diclofenac, dichlorphenamide, dipivefrin, dorzolamide, echothiophate, emedastine, epinastine, epinephrine, erythromycin, ethoxzolamide, eucatropine, fludrocortisone, fluorometholone, flurbiprofen, fomivirzen, framycetin, ganciclovir, gatifloxacin, gentamicin, homatropine, hydrocortisone, idoxuridine, indomethacin, isoflurophate, ketorolac, ketotifen, la Tanoprost, levobetaxolol, levobunolol, levocabastine, levofloxacin, lodoxamide, loteprednol, medrysone, methazolamide, metipranolol, moxifloxacin, naphazoline, natamycin, nedocromil, neomycin, norfloxacin, ofloxacin, olopatadine, oxymetazoline, pemirolast, pegaptanib, phenylephrine, physostigmine, pilocarpine, pindolol, pirenoxine, polymyxin B, prednisolone, propranolol, 1. A pharmaceutical composition comprising an active ingredient selected from the group consisting of caine, ranibizumab, rimexolone, scopolamine, sezolamide, squalamine, sulfacetamide, suprofen, tetracaine, tetracycline, tetrahydrozoline, tetrizoline, timolol, tobramycin, travoprost, triamcinolone, trifluoromethazolamide, trifluridine, trimethoprim, tropicamide, unoprostone, vidarabine, xylometazoline, pharmaceutically acceptable salts thereof, and combinations thereof.

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