Methods and compositions for preventing or treating ophthalmic conditions

Abstract

To provide methods of preventing or treating ophthalmic diseases or conditions in a mammalian subject.SOLUTION: The present invention provides methods of preventing or treating ophthalmic diseases or conditions in a mammalian subject. The methods comprise administering a therapeutically effective amount of an aromatic-cationic peptide represented by the chemical formula D-Arg-2',6'-Dmt-Lys-Phe-NH2 (SS-31) or Phe-D-Arg-Phe-Lys-NH2 (SS-20). The present invention provides methods of preventing or treating ophthalmic diseases or conditions such as diabetic retinopathy, cataracts, 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 August 24, 2009, U.S. Provisional Application No. 61 / 237,745, filed August 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 ocular diseases or conditions (e.g., 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 reader's understanding: None of the information provided or documents cited is admitted to be 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 the age of 50 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 typically occurs when the central retina becomes distorted and thinned with age, but it may also be characterized by endophthalmitis and neovascularization (wet ARMD only) and / or intraocular infection. Oxidative tissue damage, local inflammation, and the subsequent production of free radicals, which lead to the production of growth factors (e.g., VEGF and FGF), and inflammatory mediators, can lead to inappropriate angiogenesis, as in the wet form of ARMD.

[0004] Retinopathy is the leading cause of blindness in type 1 diabetes and is also common in type 2 diabetes. The extent of retinopathy depends on the duration of diabetes, typically beginning 10 years or more after the onset of diabetes. Diabetic retinopathy can be classified as nonproliferative, characterized by increased capillary permeability, edema, and exudate, or proliferative, characterized by neovascularization extending from the retina to the vitreous, scarring, fibrous tissue deposition, and possible retinal detachment. Diabetic retinopathy is thought to result from the formation of glycated proteins due to hyperglycemia. 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 inadequate aqueous humor drainage is the primary cause of glaucoma. Glaucoma often progresses with aging of the eye or can occur as a result of ocular injury, inflammation, or tumor, or in advanced cases of cataracts or diabetes. Elevated IOP caused by treatment with steroids can also be the cause. Effective drug treatments for glaucoma lower IOP by reducing vitreous humor production or by promoting aqueous humor drainage. These medications 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 generally relates to treating or preventing ocular diseases or conditions in mammals by administering to a subject in need thereof a therapeutically effective amount of an aromatic-cationic peptide.

[0007] In one aspect, the disclosure provides a method of treating or preventing an ocular disease in a mammalian subject in need thereof, the method 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 four 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), 3p m is the maximum number of aromatic groups (a) and the total number of net positive charges (p t ) in the relationship, 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 up to about six, up to about nine, or up to about 12 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-2',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 , R11 , and R 12 are each independently selected from the following: (i) Hydrogen; (ii) a linear or branched alkyl 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 by Formula II: [ka] where R 1 and R 2are each independently selected from the following: (i) Hydrogen; (ii) a linear or branched alkyl 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 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 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 present 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 aspect, the disclosure provides an ophthalmic formulation comprising a therapeutically effective amount of the peptide D-Arg-2'6'-Dmt-Lys-Phe-NH2 or Phe-D-Arg-Phe-Lys-NH2, which in one embodiment is soluble in the septum, aqueous humor, and lens. In one embodiment, the formulation further comprises a preservative, which in one embodiment 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 an antioxidant, a metal complex, an anti-inflammatory agent, an antibiotic, and an antihistamine, 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 contains aceclidine, acetazolamide, anecortave, apraclonidine, atropine, azapentacene, azelastine, bacitracin, befunolol, betamethasone, betaxolol, bimatoprost, brimonidine, brinzolamide, carbachol, carteolol, celecoxib, chloramphenicol, chlortetracycline, ciprofloxan, cromoglycate, cromolyn, cyclopentolate, cyclosporine, dapiprazole, demecarium, dextromethorphan, benzodiazepine, benzophenone, benzocaine, benzodiazepine, benzocaine ... 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, latanolide 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 explanation of the drawings]

[0019] [Figure 1]Figures 1A and 1B show the effects of various 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, revealing that the viability (Q3) of HRECs 24 h after treatment was 99.3%, 83.2%, 84.3%, 90.7%, 92.8%, and 94.3%, respectively. Figure 1B shows a graphical representation of HREC viability. Data from 100 nM, 1 μM, and 10 μM SS-31 were significantly higher than those seen in cells exposed to high glucose without co-treatment with SS-31. *p<0.05 vs. the 30 mM high glucose treatment group. [Figure 2] Figures 2A–2F are a series of photomicrographs 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. [Figure 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 resulted in rapid loss of mitochondrial membrane potential in cultured HRECs at 24 and 48 hours. Meanwhile, flow cytometry analysis revealed that ΔΨm was increased in the 30 mM glucose group treated with SS-31 compared with the high glucose group. Figure 3B: Quantitative analysis of ΔΨm of high glucose HRECs treated with SS-31 for 24 and 48 hours. High glucose alone had a negative effect on ΔΨm. Meanwhile, SS-31 restored ΔΨm to the control level. Values ​​represent the mean ± standard deviation of six independent experiments performed in triplicate. *P<0.05. [Figure 4] Figures 4A and 4D show confocal microscopy images showing that cytochrome c staining and HSP60 staining overlapped more precisely in HRECs treated with normal glucose and SS-31, demonstrating the colocalization of cytochrome c and mitochondria. Cytochrome c was significantly increased in the cytoplasm of HRECs treated with 30 mM glucose for 24 and 48 hours after treatment. Figures 4B and 4E show the mitochondrial and cytoplasmic cytochrome c content measured by Western blotting. Figures 4C and 4F show quantitative analysis of the ratio of mitochondrial to cytoplasmic cytochrome c content in HRECs treated with high glucose and SS-31 for 24 and 48 hours. [Figure 5-1] Figures 5A and 5B show by Western blotting that the increased expression of caspase 3 in HRECs treated with high glucose (HG) was attenuated by co-treatment with SS-31. Caspase 3 expression was normalized to β-actin expression. [Figure 5-2] Figures 5C-E show that SS-31 increases Trx2 expression in high-glucose-treated HRECs. Figure 5C shows Trx2 mRNA levels in HRECs treated with SS-31 and exposed to 30 mM glucose for 24 and 48 hours. Figure 5D shows Trx2 protein expression levels measured by Western blotting. Figure 5E shows quantitative analysis of Trx2 protein levels in HRECs 24 and 48 hours 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 the lenses of diabetic rats. Top row: lenses obtained from diabetic rats; bottom row: lenses obtained 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 lens 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, diabetic, 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 the integrity of the blood-retinal barrier 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 microvessels from normal rats (NRC), diabetic rats (HFD / STZ), and diabetic rats treated with SS-31. [Figure 12] FIG. 12 is a series of photomicrographs showing retinal microvessels from normal rats (NRC), diabetic rats (STZ), and diabetic rats treated with SS-31. [Figure 13] Figures 13A-13D are a series of photomicrographs showing the distribution of the tight junction protein claudin 5 in retinal microvessels of 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 to 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 HO 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 TMRM and mitochondrial membrane potential (ΔΨm) measured by 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 combined treatment with SS-31 dose-dependently reduced the loss of mitochondrial membrane potential in GTM cells caused by 400 μM HO, 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), and Figure 24D is a graph showing the area of ​​CNV in treatment and control groups. [Figure 25] Figure 25 is a series of photomicrographs illustrating various pathological effects in a mouse model of oxygen-induced retinopathy (OIR), 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 effects of SS-31 administration in an OIR mouse model. Figure 26E is a graph showing the neovascularization area in the control and treatment groups. SS-31 reduced the neovascularization 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] Figure 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 of control and SS-31-treated mice. [Figure 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. [Figure 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. [Figure 32] 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 were analyzed. [Figure 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. [Figure 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. [Figure 35] Figure 35 is a graph showing MDA levels induced by tBHP in three groups of RPE cells. *P<0.05, 1 μM SS-31 for 4 hours plus 250 μM tBHP for 24 hours vs. 250 μM tBHP for 24 hours. [Figure 36] FIG. 36 shows a graph depicting the fluorescence intensity of TMRM in GTM and HTM cells in control and SS-31-treated groups as measured using FACS analysis. [Figure 37] FIG. 37 shows a graph depicting the ROS fluorescence intensity of GTM and HTM cells in control and SS-31 treated groups 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 the 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 H 2 O 2 . [Figure 40] FIG. 40 is a series of graphs showing that SS-31 prevented H 2 O 2 -induced mitochondrial depolarization in GTM3 and iHTM cells. DETAILED DESCRIPTION OF THE INVENTION

[0020] It will be appreciated that certain forms, modes, embodiments, variations and features of the present invention are described below in detail at all levels in order to provide a thorough understanding of the present 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, 1986). 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 definitions of terms used herein are provided below: 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 the context clearly dictates 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 skilled 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 skilled in the art, "about" may mean up to plus or minus 10% of the recited value, depending on the context in which it is used.

[0025] As used herein, "administration" of a chemical, pharmaceutical, or peptide to a subject includes any 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 not only naturally occurring and synthetic amino acids, but also amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by genetic code as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and o-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but maintain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a compound that has a structure different from the overall chemical structure of an amino acid but 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 the 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 a 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. A practitioner can determine the appropriate dose based 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, an aromatic-cationic peptide is administered to a subject with 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, ameliorates 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 would 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 comprising 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 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 "simultaneous" 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 means 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 active ingredient can be administered minutes, hours, or days before 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, where the objective is 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 are intended to mean "substantial," including not only the totality of the treatment or prevention, but also less than the totality, that achieve some biologically or medically relevant result.

[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 through the 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 or 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" refers to an organic molecule containing 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 acid may be naturally occurring. For example, naturally occurring amino acids include the 20 most common levorotatory (L) amino acids commonly found in mammalian proteins: 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 naturally occurring 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 naturally occurring amino acid is hydroxyproline (Hyp).

[0037] The peptide optionally includes one or more unnatural amino acids. Preferably, the peptide does not have any naturally occurring amino acids. The unnatural amino acids may be levorotatory (L-), dextrorotatory (D-), or a mixture thereof. Unnatural amino acids are those amino acids that are not typically synthesized through 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, C-terminus, or between the N-terminus and C-terminus.

[0038] For example, the unnatural amino acid may contain an alkyl, aryl, or alkylaryl group 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, natural amino acids with one or more chemical groups added.

[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 such groups include branched or unbranched C1-C4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl; C1-C4 alkyloxy (i.e., alkoxy); amino; C1-C4 alkylamino; and C1-C4 dialkylamino (e.g., methylamino, dimethylamino); nitro; hydroxyl; halo (i.e., fluoro, chloro, bromo, or iodo); some specific examples of unnatural derivatives of natural amino acids include norvaline (Nva) and norleucine (Nle).

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

[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 naturally occurring 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 cells. As used herein, D-amino acids are considered unnatural amino acids.

[0042] To minimize protease susceptibility, peptides should have fewer than five, preferably fewer than four, more preferably fewer than three, and most preferably fewer than two adjacent L-amino acids recognized by common proteases, regardless of whether the amino acids are naturally occurring or non-occurring. Preferably, the peptide contains only D-amino acids and no L-amino acids. If the peptide contains a protease-sensitive 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-sensitive 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). The minimum number of net positive charges stated below is the value at physiological pH. As used herein, the term "physiological pH" refers to the normal pH in the cells of the tissues and organs of a mammalian body. For example, the physiological pH in humans is normally about 7.4, but the normal physiological pH in mammals can be anywhere between about 7.0 and about 7.8.

[0044] As used herein, "net charge" refers to the difference between the number of positive and negative charges carried by the amino acids present in a peptide. As used herein, it should be understood that net charge is measured at physiological pH. Naturally occurring amino acids that are positively charged at physiological pH include L-lysine, L-arginine, and L-histidine. Naturally occurring 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 calculating net charge, 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). Therefore, the peptide has a net positive charge of three.

[0046] In one embodiment, the aromatic-cationic peptide is a 3p 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 acid residues (r) are as follows: [Table 1]

[0047] In another embodiment, the aromatic-cationic peptide is 2p 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 number of net positive charges (p t It is also important to have a minimum number of aromatic groups relative to the total number of aromatic groups. The minimum number of aromatic groups 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 (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 is 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 (p t In this embodiment, 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 preferably amidated, for example, with ammonia to form a 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 C1-C4 alkyl or aryl amine. 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. Additionally, 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 also 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 peptides: 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, a 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 two aromatic groups, contributed by the amino acids phenylalanine and tyrosine. The tyrosine in 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 cell membranes 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 without 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 the Dmt is not at the N-terminus. An example of the aromatic-cationic peptide without 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 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, in which 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

[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 for (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 an effective amount of an aromatic-cationic peptide to the subject. For example, a subject can be administered an aromatic-cationic peptide composition to ameliorate one or more factors contributing to an ocular disease or condition.

[0067] One aspect of the present technology includes a method for alleviating ocular disease in a subject for therapeutic purposes. In therapeutic applications, a 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 prevent the progression of the disease, including its complications and intermediate pathological phenotypes in the progression of the disease. Thus, the present disclosure provides methods for treating individuals suffering from ocular diseases. 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 petechiae. Microvascular blockage then leads to the formation of cotton wool spots in the retina. Furthermore, retinal edema and / or hard exudates may form in individuals with diabetic retinopathy due to increased vascular permeability. Subsequently, neovascularization develops, and friction of connective tissue growth in the vitreous causes retinal detachment. Rubeosis iridis and neovascular glaucoma, which can ultimately lead to blindness, may also occur. 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 cataracts. Cataracts are congenital or acquired diseases characterized by a decrease in the transparency of the natural lens. Individuals suffering from cataracts may exhibit one or more symptoms, including, but not limited to, clouding of the lens surface, clouding of the lens interior, and / or lens swelling. Typical examples of congenital cataract-related diseases include pseudocataract, intramembranous cataract, coronal cataract, lamellar cataract, punctate cataract, and filiform cataract. Typical examples of acquired cataract-related diseases include senile cataract, secondary cataract, brown cataract, combined cataract, diabetic cataract, and traumatic cataract. Acquired cataracts can also be induced by electrical shock, radiation, ultrasound, drugs, systemic diseases, and nutritional disorders. Acquired cataracts also include postoperative cataracts.

[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 various symptoms, including, but not limited to, headaches, numbness or tingling in the extremities, flashing lights, 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, leading to vision loss. Glaucoma can result from various pre-existing ocular conditions, such as trauma, surgery, and other structural anomalies. Glaucoma can occur at any age, but it commonly develops in older individuals and leads to blindness. Patients with glaucoma typically have an intraocular pressure of 21 mmHg or higher. However, normal-tension glaucoma, characterized by glaucomatous changes in the visual field and optic disc, can occur even without elevated intraocular pressure, i.e., 21 mmHg or higher. Symptoms of glaucoma include, but are not limited to, blurred vision, severe eye pain, headache, 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. General 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 beneath the retinal pigment epithelium. 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, central vision distortion, light-dark distortion, and / or changes in color vision. Dry macular degeneration can result in 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 beneath the macula. These vessels leak blood into the macula, damaging photoreceptors. Wet macular degeneration progresses rapidly and can cause significant damage to central vision. Wet and dry macular degeneration share the same symptoms. However, non-age-related macular degeneration is rare and may be related to genetics, diabetes, malnutrition, injury, infection, or other factors. Symptoms of non-age-related macular degeneration also include, but are not limited to, astigmatism, distortion of central vision, distortion of light and dark, 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 choroid 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 visual impairment or complete blindness. Symptoms of CNV include, but are not limited to, seeing 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 and lattice-like degeneration, which are associated with progressive macular degeneration. Symptoms of retinal degeneration include, but are not limited to, decreased vision, blindness, night blindness, tunnel vision, loss of peripheral vision, retinal detachment, and / or light sensitivity.

[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 the vascular occlusion of OIR, predisposing 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 aspect, 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, such as those 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 onset 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 the screening tests described herein.

[0078] Determining the Biological Effect of Aromatic-Cationic Peptide-Based Therapies. In various embodiments, appropriate in vitro or in vivo testing is performed to determine the effect 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 appropriate animal model systems, such as rats, mice, chickens, cows, monkeys, rabbits, etc., before testing in human subjects. Similarly, for in vivo testing, any animal model system known in the art can be used before administration to human subjects. In one embodiment, administration of an aromatic-cationic peptide to a subject exhibiting symptoms associated with an ocular disease results in improvement in one or more of those symptoms. Mode of Administration and Effective Dose

[0079] Any method known to those skilled in the art for contacting cells, organs, or tissues with peptides may be applied. Suitable methods include in vitro, ex vivo, or in vivo methods. Internal methods typically involve administration of the aromatic-cationic peptides described above to animals, preferably humans. When used internally for therapy, the aromatic-cationic peptides are administered to the subject in an effective amount (i.e., an amount that has the 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 many 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 pharmaceutical preparations for administration to a subject, either alone or in combination, for the treatment or prevention of the disorders described herein. The compounds typically include an active agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and the like, all of which are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[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 glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and an agent for adjusting tonicity such as sodium chloride or dextrose. 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 unit of treatment (eg, drug vial, diluent vial, syringe, and needle).

[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 further preparation. 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 preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0084] The aromatic-cationic peptide composition can contain a carrier, which may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of a desired particle size in the case of dispersions, and 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 added to prevent oxidation. In many cases, it is desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and 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 injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the ingredients listed above, and then sterilizing by filtration, if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, typical methods of preparation include vacuum drying and freeze-drying, which can be used to obtain a powder of the active ingredient plus any other required ingredients from a previously sterile-filtered solution thereof.

[0086] For ophthalmic use, 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). Ophthalmic pharmaceuticals may be suitable for topical administration to the eye in the form of solutions, suspensions, ointments, creams, or solid inserts. 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, such as thimerosal, benzalkonium chloride, methyl and propylparaben, 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, polyoxyethylenesorbitan monopalmitate, ethylenediaminetetraacetic acid, etc.

[0088] The ophthalmic solution or suspension is administered as often as necessary to maintain an acceptable concentration of the 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 an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated into an excipient and 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, disintegrating agents such as alginic acid, Primogel, or cornstarch; 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, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. 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] Therapeutic proteins or peptides 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 the following 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 can also be incorporated into particles prepared from pharmaceutically acceptable components, including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable, or gastroretentive 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, e.g., 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 a poly-α-hydroxy acid. Examples include carriers made from 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 various forms or sizes, including microspheres and nanospheres. Polymeric formulations may provide a longer-lasting therapeutic effect (see Reddy, Ann. Pharmacother., 34 (7-8):915-923 (2000)). High molecular weight 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.), 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 can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. Such materials are commercially available from sources such as Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies against cell-specific antigens) can be used as pharmaceutically acceptable carriers. 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). Mizuguchi 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 agents 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 a population) and ED50 (the dose therapeutically effective in 50% of a 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 uninfected cells. This will reduce side effects.

[0098] Data obtained from cell culture and animal studies can be used to formulate a range of dosages for use in humans. The dosage of the compound preferably falls within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods, a therapeutically effective dose can be estimated initially from cell culture studies. A dose can be formulated in animal models that achieves a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms), as determined in cell culture. This information can be used to more accurately determine useful doses in humans. Plasma concentrations may 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. Preferably, the dose range is from about 0.0001 mg / kg body weight / day to about 100 mg / kg body weight / day. For example, doses may range from 1 mg / kg body weight or 10 mg / kg body weight daily, every two days, or every three days, or from 1 to 10 mg / kg body weight weekly, every two weeks, or every three 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 delivered formulation. Exemplary therapeutic dosing regimens call for daily or weekly administration. Intervals may be more irregular than those indicated by measuring the subject's glucose or insulin blood levels and adjusting the dose or administration 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 moles per 1000 mg of target peptide. This concentration may be delivered at a systemic dose of 0.001-100 mg / kg or equivalent dose per body surface area. The dosing schedule 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 the aromatic-cationic peptide is defined as a "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, 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, may affect the dosage and timing required for effective treatment. 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, 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, may affect the dosage and timing required for effective treatment. 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, cattle, and horses; pet animals such as dogs and cats; and 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 a pharmaceutically acceptable salt, ester, amide, prodrug, or solvate) 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 upon administration of one of the aromatic-cationic peptides described 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 effectiveness of one of the compounds described herein may be enhanced by administration of an ancillary agent (i.e., the ancillary agent alone may have a minimal therapeutic effect, but when combined 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 administering 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 treating macular degeneration with 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 addition of the effects of the two therapeutic agents, or the patient may experience a synergistic effect.

[0106] Specific, non-limiting examples of possible combination therapies include the use of 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 (e.g., lutein is an antioxidant and a carotenoid). Furthermore, 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, aromatic-cationic peptides may be used in combination with procedures that provide synergistic benefits to patients, 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 effective 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, beta-carotene and other carotenoids, coenzyme Q, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (also known as tempol), lutein, butylhydroxytoluene, resveratrol, Trolox analog (PNU-83836-E), and cowberry extract.

[0109] The use of certain minerals has also been shown to be effective 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 group 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 potent antioxidants and absorb blue light, which may help protect the retina. Studies in quail have confirmed that animals fed a carotenoid-deficient diet had low retinal zeaxanthin concentrations and suffered severe light damage, as evidenced by a significant number of apoptotic photoreceptor cells, while animals with high zeaxanthin concentrations experienced 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 pharmaceutically acceptable salts thereof, including, for example, citrulline, ornithine, glutamine, lysine, polypeptides containing 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)). Therefore, when administered in combination with aromatic-cationic peptides, statins can 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 (the hemicalcium salt of atorvastatin), and dihydrocompactin.

[0115] Suitable anti-inflammatory drugs with which the aromatic-cationic peptides can 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 cephalosporin); 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, an appropriate matrix metalloproteinase (MMP) inhibitor may also be administered in combination with the aromatic-cationic peptide. 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 pathologies, 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)). Typical 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 / Controlled 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 in conjunction 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, photosynthates, 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.(manufactured by Sigma-Aldrich), 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 multiple therapeutic agents can be administered in any order or simultaneously. If administered simultaneously, the multiple therapeutic agents can be provided in a single 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 administered simultaneously, the interval between multiple doses 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, combination methods, compositions, and formulations are not limited to the use of only two agents. By way of example only, an aromatic-cationic peptide can be administered with at least one antioxidant and at least one negatively charged phospholipid; or an aromatic-cationic peptide can be administered with at least one antioxidant and at least one inducer of nitric oxide production; or an aromatic-cationic peptide can be administered with at least one inducer of nitric oxide and at least one negatively charged phospholipid, etc.

[0120] Additionally, 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 vegetative vascular 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 response 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 benefit individuals include using genetic testing to determine whether an individual is a carrier of a mutant gene known to be associated with a particular eye disease. By way of example only, defects in the human ABCA4 gene are thought to be associated with five distinct retinal phenotypes, including Stargardt 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. J. Hum. Gen., 67:793-799 (2000); Klevering, et al., Ophthalmology, 11 1:546-553 (2004). Additionally, autosomal dominant Stargardt disease is caused by a mutant allele of the ELOV4 gene. See the following reference: Karan, et al., Proc. Natl. Acad. Sci. (2005). Patients carrying any of these mutant 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. [Example]

[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 for culturing HRECs used 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 cells. 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: a normal control group, a group treated with 30 mM glucose, and a group treated with 30 mM glucose plus SS-31. The viability of HRECs treated with high glucose and various concentrations of SS-31 (10 nM, 100 nM, 1 μM, and 10 μM) was measured using the Annexin V plus PI assay 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 SS-31 and high glucose was examined at 24 and 48 hours. The results are shown in Figure 1. SS-31 administration significantly increased the viability of HRECs while reducing apoptotic and necrotic cells. SS-31 treatment also reduced ROS production (Figure 2).

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

[0129] Glucose (30 mmol / L) induced the release of cytochrome c from mitochondria in HRECs. Fixed HRECs were immunolabeled with cytochrome c and mitochondrial-specific antibodies (HSP60). Confocal microscopy analysis revealed that cytochrome c and mitochondrial staining overlapped in HRECs cultured under normal conditions and those co-treated with glucose and SS-31, indicating colocalization of cytochrome c and mitochondria (Figure 4). After 24 or 48 hours 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 and that SS-31 can reduce such transfer between mitochondria and the cytoplasm.

[0130] Preventing cytochrome c release from mitochondria resulted in reduced caspase-3 activity. As shown in Figure 5, SS-31 reduced caspase-3 protein expression in high-glucose-treated HRECs. The protein expression level of cleaved caspase-3 was measured by Western blotting (Figure 5A). When HRECs were exposed to 30 mM glucose for 24 and 48 hours, the level of caspase-3 expression 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 levels in high-glucose HRECs co-treated with SS-31 for 24 and 48 hours.

[0131] SS-31 increased Trx2 expression in HRECs treated with high glucose. Figure 5C shows the Trx2 mRNA levels in HRECs co-treated with SS-31 and exposed to 30 mM glucose for 24 and 48 hours. Trx2 mRNA expression levels were measured by quantitative real-time PCR. Relative Trx2 mRNA levels were normalized to 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 point. Figure 5D shows the Trx2 protein expression levels measured by Western blotting. Trx protein expression in the SS-31 co-treated high glucose group was significantly increased compared to the normal glucose group (*p<0.05). Figure 5E shows quantitative analysis of Trx2 protein levels in HRECs after 24 and 48 hours of high glucose exposure, without and with SS-31 co-treatment.

[0132] These results suggest that SS-31 enhances the survival of HREC cells in a high-glucose environment. Similarly, SS-31 and other aromatic-cationic peptides may be useful as a method for preventing diabetic retinopathy. [Example]

[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 the above experiment.

[0135] A diabetic rat model was established in SD rats by combining a 6-week HFD with a low-dose STZ (30 mg / kg) injection or a single high-dose STZ injection (65 mg / kg). See generally the following reference: 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. Rats from the same batch fed a normal diet (NRC) were used as controls. Tables 7–10 show the treatment schedule and experimental protocol. [Table 7] [Table 8] [Table 9] [Table 10]

[0136] Following this 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 prevented or reversed cataract formation in the lenses of diabetic rats (Figures 6 and 7, Tables 11 and 12). [Table 11] -: transparent; +: slightly cloudy; ++: turbidity; +++: moderate turbidity; ++++: severe opacity; [Table 12]

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

[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 with rats fed a HFD without SS-20 or SS-31 (Figure 10).

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

[0140] The effects of the aromatic-cationic peptide on the distribution of the tight junction protein claudin-5 in retinal microvessels in an SD rat model were investigated. The distribution of the tight junction protein claudin-5 was detected under a confocal microscope (Figure 13). Claudin-5 was distributed smoothly, linearly, and uniformly along the 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 adverse effects of diabetes in the eye, such as cataracts and the microvasculature, and therefore administration of the aromatic-cationic peptides of the present invention is useful in methods for preventing or treating diabetes-related eye disease in human subjects. [Example]

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

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

[0144] In this example, the effects of the aromatic-cationic peptides of the present invention were compared between trabecular meshwork cells from POAG patients (GTM) and non-affected individuals (HTM). Methods useful in studying the present 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 exhibit 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 the cytotoxic effect of SS-31 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. This assay quantitatively measures stable cytosolic lactate dehydrogenase (LDH) released from damaged cells. This released LDH is measured by a coupled enzymatic reaction that results in the conversion of a tetrazolium salt (iodonitrotetrazolium (INT)) to red formazan by diaphorase. Methods for detecting LDH in 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 time period. The results are shown in Figure 14 and demonstrate 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 study of the present invention, 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 for GTM cells. Collectively, 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 the cells were exposed to hydrogen peroxide before 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 and 17).

[0149] Group B: We investigated the morphology of GTM cells when the cells were exposed to SS-31 before hydrogen peroxide administration. Figure 18 shows the results of inverse phase contrast microscopy of cells treated with various concentrations of SS-31. The results indicate that SS-31 protects cells from hydrogen peroxide-mediated morphological changes in a concentration- and time-dependent manner. Specifically, cells exposed to the SS-31 peptide exhibited reduced hydrogen peroxide-mediated cell loss. We also investigated the mitochondrial membrane potential (ΔΨm) of HTM and GTM cells when the cells were exposed to SS-31 before hydrogen peroxide administration. 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 indicate that SS-31 dose-dependently improves the mitochondrial membrane potential of cells exposed to hydrogen peroxide. Therefore, SS-31 provides a protective effect against oxidative stress in GTM cells.

[0150] The effect of SS-31 on 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 those in the HTM group were 37.4±0.725%, 57.7±1.80%, 50.6±3.06%, and 49.4±2.27%, respectively, n=4; ** indicates P<0.01 compared to the GTM H2O2 group; * indicates P<0.05 compared to the GTM H2O2 group; ▲▲▲ indicates 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 treated groups using FACS analysis. -6 M, SS-31 10 -7 M, SS-31 10 -8The percentages of intracellular ROS production in the GTM group compared to the control were 146.0 ± 2.27%, 84.5 ± 8.75%, 102.0 ± 5.69%, and 133.0 ± 5.17%, respectively (n = 3); and in the HTM group were 153.0 ± 3.46%, 79 ± 2.39%, 91.8 ± 3.49%, and 129.0 ± 8.24%, respectively (n = 4). P < 0.001 for the HO group compared to the control in GTM and HTM; *** indicates P < 0.001 compared to the GTM HO group; ▲▲▲ indicates P < 0.001 compared to the HTM HO group; ▲▲ indicates P < 0.01 compared to the HTM HO group. Figure 38 shows that SS-31 reduced the amount of HO-induced cell apoptosis.

[0152] The effects of SS-31 on persistent oxidative damage in GTM and HTM cells were investigated. To investigate the protective effect of SS-31, cells were cultured for 10 min. -6 , 10 -7 , 10 -8 The cells were pre-treated with 200 μM SS-31 for 1 hour, followed by incubation with 200 μM HO 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 -4 SS-31 (>10 M) was not cytotoxic to both GTM and HTM cells and inhibited the persistent and acute oxidative stress induced by hydrogen peroxide. -9 Therefore, the aromatic-cationic peptides of the present invention are useful in methods for preventing or treating glaucoma in human subjects. [Example]

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

[0155] To test the effectiveness 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 I., ARPE-19, A Human Retinal Pigment Epithelial Cell Line with Differentiated Properties, Experimental Eye Research, 1996, 62(2): 155-170. First, we found that SS-31 had no adverse effects on these cells. Primary human RPE cells were cultured with various concentrations of SS-31 alone, and cell viability was measured using the MTT assay (Figure 22).

[0156] Next, we examined 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 24 hours of starvation. Cells were then exposed to high concentrations of tBHP (Figure 23A) or preincubated 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 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; and 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. The group treated with 500 μM tBHP for 3 hours had a green-to-red ratio of 1.08; the group treated with 10 nM SS-31 for 4 hours plus 500 μM tBHP for 3 hours had a green-to-red ratio of 1.25; the group treated with 100 nM SS-31 for 4 hours plus 500 μM tBHP for 3 hours had a green-to-red ratio of 1.4; and the group treated with 1 μM SS-31 for 4 hours plus 500 μM tBHP for 3 hours had a green-to-red ratio of 2.28. Figure 33 shows the effect of 1 μM SS-31 on tBHP-induced MMP reduction. Figure 33A: Control group, R / G was 3.63±0.24; Figure 33B: Treatment with 500 μM tBHP for 3 hours, R / G was 1.08±0.11; Figure 33C: Treatment with 1 μM SS-31 for 4 hours + 500 μM tBHP for 3 hours, R / G was 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: Group treated with 250 μM tBHP for 24 hours; (Q2+Q4)% = 15.7 ± 0.6%; Figure 34C: Group treated 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 levels induced by tBHP in the three groups of RPE cells. (*P<0.05).

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

[0159] Prevention and Treatment of Choroidal Neovascularization with the Aromatic-Cationic Peptides of the 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 (FIG. 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 (5-6 weeks old) were anesthetized with chloral hydrate, and pupils were dilated with tropicamide. Using a coverslip used as a contact lens, four laser spots (532 nm, 260 mW, 0.01 s, 50 μm; Novus Spectra, Lumenis, USA) were applied to the fundus of the right eye in a circular pattern around the optic disc. Daily intraperitoneal injections of 1 mg / kg, 9 mg / kg SS-31, or vehicle were initiated the day before laser photocoagulation.

[0162] One week later, mice were deeply anesthetized and perfused via the left ventricle with 1 mL of PBS-buffered fluorescein dextran (50 mg / mL). Eyes were enucleated and fixed in 4% paraformaldehyde for 2 hours. Eyes were dissected at the equator, and the anterior half and retina were removed. The posterior segment, including the sclera and choroid, was cut into quarters using 4–5 radial cuts and mounted on slides. All flat-mount specimens were examined under a fluorescence microscope (AxioCam MRC; Carl Zeiss). The area of ​​each CNV lesion was measured 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 neovascularization areas 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 SS-31 at both concentrations significantly reduced the area of ​​choroidal neovascularization (P < 0.05) (Figure 24). [Example]

[0164] Prevention and treatment of oxygen-induced retinopathy (OIR) by aromatic-cationic peptides of the present 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 were exposed to hyperoxia (75% oxygen) for 5 days, which arrests retinal vascular growth and leads to significant vascular occlusion. On postnatal day 12, the pups were returned to room air, and by postnatal day 17, compensatory retinal neovascularization with a pale complexion 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 treated with an aromatic-cationic peptide (e.g., SS-20 or SS-30) for approximately 6 weeks. The results are shown in Figure 26, demonstrating that treatment with SS-31 prevented compensatory retinal neovascularization. Therefore, the aromatic-cationic peptides of the present invention are useful in methods for preventing proliferative diabetic retinopathy in mammalian subjects. [Example]

[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 for this study of 661W cells have been previously described. 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 efficacy of the aromatic-cationic peptides of the present invention in preventing or reducing oxidative damage in cone cells (Figure 27). tBHP was first found to affect the viability of 661W cells (Figure 27A). Various 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 tested in cultures of 661W cells. The results are shown in Figure 30 and show that SS-31 significantly increased mitochondrial viability compared to cells that were not treated with SS-1, as demonstrated by the JC-1 assay. [Example]

[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 retinal 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] Figure 28 is a series of photomicrographs showing the thickness of the retinal outer nuclear layer (ONL) in control and SS-31-treated mice in a mouse model of retinal degeneration. The results show that SS-31-treated mice maintained a higher number of cell rows 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 higher cone cell density in SS-31-treated mice compared to SS-31-treated mice (Figure 29). These results indicate that treatment with SS-31 prevented compensatory damage to the retinal outer nuclear layer in a mouse model of retinal degeneration. Therefore, the aromatic-cationic peptides of the present invention are useful in methods for preventing retinal degeneration in mammalian subjects.

[0173] (Corresponding patent) The present invention is not limited in terms of the specific embodiments described herein, which are intended to be merely illustrative of particular aspects of the invention. It will be apparent to those skilled in the art that many modifications and variations of the present invention can be made without departing from its spirit and scope. Functionally equivalent methods and apparatuses within the scope of the invention, in addition to those recited herein, will be apparent to those 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 such claims are entitled. It is to be understood that the present invention is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and not for the purpose of limitation.

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

[0175] Those skilled in the art will appreciate that, for all purposes, particularly with respect to the provision of a written specification, all ranges disclosed herein encompass all possible subranges and subrange combinations thereof. Any stated range can be readily recognized as fully delineated and allows 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 readily divided into a lower third, middle third, upper third, etc. Those skilled in the art will also appreciate that 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, those skilled in the art will appreciate that ranges include individual numbers. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 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 herein, including all figures and tables, are incorporated by reference in their entirety to the extent not inconsistent with the express guidance of this specification.

[0177] Other embodiments are set forth in the following claims. 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 thereof, comprising: A method comprising administering to a subject a therapeutically effective amount of a peptide prepared by the formula Dmt-Lys-Phe-NH2 or Phe-D-Arg-Lys-NH2. [2] The method according to [1], The method, wherein the ocular disease is selected from the group consisting of diabetic retinopathy, cataract, retinitis pigmentosa, glaucoma, macular degeneration, choroidal neovascularization, retinal degeneration, and oxygen-induced retinopathy. [3] The method according to [1], The method wherein the peptide is D-Arg-2'6'-Dmt-Lys-Phe-NH2. [4] The method according to [1], The method of claim 1, wherein the peptide is Phe-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, by iontophoresis, orally, topically, systemically, intravenously, subcutaneously, or intramuscularly. [7] The method according to [1], The method further comprising administering a second active agent separately, sequentially, or simultaneously. [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, ciprofloxan, cromoglycate, cromolyn, cyclopentolate, cyclosporine, dapiprazole, demecarium, dextromethorphan, benzodiazepine, benzophenone, benzocaine, benzodiazepine, benzocaine ... azamethasone, 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, progesterone 10. The method of claim 1, wherein the medicament 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. 1. A method for treating or preventing an ocular disease in a mammalian subject in need thereof, comprising: Chemical formula D-Arg-2'6'-Dmt-Lys-Phe-NH 2 or Phe-D-Arg-Phe-Lys-NH 2 administering to a subject a therapeutically effective amount of the peptide prepared in the method of claim 1.

2. 10. The method of claim 1, The method, wherein the ocular disease is selected from the group consisting of diabetic retinopathy, cataract, retinitis pigmentosa, glaucoma, macular degeneration, choroidal neovascularization, retinal degeneration, and oxygen-induced retinopathy.

3. 10. The method of claim 1, The peptide is D-Arg-2′6′-Dmt-Lys-Phe-NH 2 That's the method.

4. 10. The method of claim 1, the peptide is Phe-D-Arg-Phe-Lys-NH 2 That's the method.

5. 10. The method of claim 1, The method, wherein the subject is a human.

6. 10. The method of claim 1, The method wherein the peptide is administered intraocularly, by iontophoresis, orally, topically, systemically, intravenously, subcutaneously, or intramuscularly.

7. 10. The method of claim 1, The method further comprising administering a second active agent separately, sequentially, or simultaneously.

8. 8. The method of claim 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. 9. The method of claim 8, The method, wherein the antioxidant is vitamin A, vitamin C, vitamin E, lycopene, selenium, α-lipoic acid, coenzyme Q, glutathione, or a carotenoid.

10. 8. The method of claim 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, dextromethorphan, benzodiazepine, benzophenone, benzocaine, benzodiazepine, benzocaine ... azamethasone, 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, progesterone 10. The method of claim 1, wherein the medicament 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.

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