Treatment of diseases and disorders associated with oxidative stress

Abstract

Peptide compounds and their use in the treatment of diseases and disorders caused by or characterized by cellular oxidative stress. The disclosed compounds comprise or consist of an amino acid sequence having the formula Gly-X-Thr-Pro, where X is a sequence of 3 amino acids selected from Arg-Ala-Chs(acid). Further, the disclosed compounds comprise or consist of the amino acid sequence Gly-ArgGly-Cys(acid)-Gly-Gly-Gly-Asp-Gly.

Description

Technical Field

[0001] Related Applications This patent application claims priority to U.S. Provisional Patent Application No. 63 / 350,373, filed on June 8, 2022, entitled Treatments for Diseases and Disorders That Involve Oxidative Stress, the entire disclosure of which is hereby expressly incorporated by reference herein. Further, this patent application is a continuation of a portion of co-pending U.S. Patent Application No. 16 / 882,656, filed on May 25, 2020, entitled Peptide Compounds and Related Methods, which is a portion of U.S. Patent Application No. 16 / 012,706, filed on June 19, 2018, now abandoned, and claims priority to U.S. Provisional Patent Application No. 62 / 521,984, filed on June 19, 2017, entitled Peptide Compositions and Related Methods, the entire disclosure of each such application being hereby expressly incorporated by reference herein.

[0002] The present disclosure generally relates to the fields of chemistry, life sciences, pharmacy, and medicine, and more specifically to pharmaceutical formulations and their use in the treatment of diseases.

Background Art

[0003] In accordance with 37 CFR 1.71(e), this patent document contains material subject to copyright protection, and the owner of this patent document reserves all copyrights whatsoever. Risuteganib (sometimes referred to herein as RSG) is an unnatural peptide having the molecular formula C22-H39-N9-O11-S and the following structural formula.

[0004]

Chem.

[0005] Rustegravir (RSG) and / or formulations containing it as the main active ingredient are also referred to by other names, nomenclatures, and designations, including ALG-1001, Gly-Lys-Gly-Asp-Thr-Pro, glycyl-L-arginylglycyl-3-sulfo-L-alanyl-L-threonyl-L-proline, Arg-Gly-NH-CH(CH2-SO3H)COOH, and Luminate® (Allegro Ophthalmics, LLC, San Juan Capistrano, CA). Rustegravir has been shown to downregulate many integrins upstream of the oxidative stress pathway. Rustegravir acts broadly to downregulate multiple angiogenesis and inflammatory processes, including those associated with oxidative stress.

[0006] Additional descriptions of rustegravir and other compounds and information regarding them are provided in Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4, and Patent Document 5 and Patent Document 6, and the co-pending U.S. Provisional Patent Application No. 62836858 entitled Compositions And Methods Useable For Treatment Of Dry Eye filed on April 22, 2019 and the same No. 62 / 879281 entitled Peptides For Treating Dry Macular Degeneration And Other Disorders Of The Eye filed on July 26, 2019, and the entire disclosure content of each of such patents and patent applications is expressly incorporated herein by reference.

[0007] The excessive formation of reactive oxygen species (ROS) such as free radicals, and the dysfunction of defensive antioxidant systems can generally lead to conditions known as oxidative stress. Mitochondrial respiration is a major source of free radicals thought to cause oxidative stress in many diseases and disorders. Mitochondria are organelles found in many types of cells. Mitochondria function to produce energy by generating adenosine triphosphate (ATP), which serves as fuel for many of the body's functions. Since muscle and nerve cells have high energy demands, mitochondrial dysfunction frequently appears in the form of muscle or nerve disorders. Mitochondrial disorders that mainly affect muscle are sometimes referred to as mitochondrial myopathy. Mitochondrial disorders that mainly affect the nerves are sometimes referred to as mitochondrial neuropathy or mitochondrial encephalomyopathy. Mitochondrial dysfunction can contribute to a wide variety of disorders such as neurodegeneration, metabolic diseases, congestive heart failure, chronic heart failure with reduced ejection fraction, chronic heart failure with preserved ejection fraction, Barth syndrome, kidney diseases and renal failure resulting from percutaneous renal angiography for renal artery stenosis, skeletal muscle function in the elderly, primary muscle mitochondrial myopathy and neuropathy, ischemia-reperfusion injury, and protozoal infections. Non-Patent Document 1. Mitochondria enable adaptation to stress at the cellular level and provide both energy and signals for direct adaptation. See Non-Patent Document 2, therefore. Also, mitochondrial dysfunction has been identified as a factor in the development of autism spectrum disorder. See Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9, and Non-Patent Document 10.

[0008] There is a need to develop new therapies for diseases and disorders that cause, are caused by, or otherwise involve inflammation or oxidative stress and / or mitochondrial dysfunction and / or unwanted angiogenesis or neovascularization.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Document

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non - Patent Document 10

Summary of the Invention

[0011] As used herein, the terms "patient" or "subject" refer to a human or non - human animals such as humans, primates, mammals, and vertebrates. As used herein, the terms "treating" or "treatment" refer to preventing, excluding, curing, suppressing, reducing the severity of, or reducing at least one symptom of a condition, disease or disorder.

[0012] As used herein, the phrase "effective amount" or "amount effective for" refers to the amount of an agent that produces some desired effect with a reasonable benefit / risk ratio. In certain embodiments, this term refers to the amount necessary or sufficient to treat a specified condition or disorder. The effective amount can vary depending on factors such as the disease or condition being treated, the particular composition being administered, the route of administration, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular agent without undue experimentation.

[0013] Throughout this patent application, an amino acid or amino acid residue may be referred to interchangeably using the amino acid name, three - letter code, and / or one - letter code as shown in Table 1 below.

[0014]

Table 1

[0015] The present disclosure describes certain compounds and their use for treating diseases and disorders that cause, are caused by, or are otherwise associated with oxidative stress, mitochondrial dysfunction, and / or other actions and etiologies described herein. In some embodiments, the compounds can be oligopeptides.

[0016] The compounds disclosed herein can comprise, or consist of, either a) the amino acid sequence Gly-Arg-Gly-Cys(acid)-Gly-Gly-Gly-Asp-Gly, or b) an amino acid sequence according to the following general formula 1: General formula 1 Gly-X-Thr-Pro wherein X is one of three amino acid sequences selected from Arg-Ala-Cys(acid), Arg-Gly-Cys, Arg-Asp-Gly, Arg-Ala-Glu, Arg-Gly-Asn, Asp-Gly-Arg, Cys(acid)-Gly-Arg, and Lys-Gly-Asp.

[0017] In some embodiments, where practicable, the compounds disclosed herein can be modified by adding one or more additional amino acids / residues to either the N-terminus or C-terminus of the compound. In such embodiments, the resulting (modified) amino acid sequence can contain more than 6 but less than 11 amino acids / residues. In some such embodiments, the resulting (modified) amino acid sequence has 7 amino acids / residues. In some such embodiments, the resulting (modified) amino acid sequence has 8 amino acids / residues. In some such embodiments, the resulting (modified) amino acid sequence has 9 amino acids / residues. In some such embodiments, the resulting (modified) amino acid sequence has 10 amino acids / residues.

[0018] In some embodiments, the compound consisting of or comprising Gly-Arg-Gly-Cys(acid)-Gly-Gly-Gly-Asp-Gly or General Formula 1 (above) may be modified by replacing the N-terminal Gly with another amino acid / residue or other chemical substance selected from Arg, Asp, His, Lys, Trp, Phe, Tyr, Met, Ala, Leu, and guanidino.

[0019] In some embodiments, the compound comprising General Formula 1 (above) may be modified by replacing one or both of the C-terminal Thr-Pro amino acid / residues with one or two other amino acids / residues selected from Ser, Val, Thr, Phe, Tyr, Lys, and Asp.

[0020] In some embodiments, the compound comprising either Gly-Arg-Gly-Cys(acid)-Gly-Gly-Gly-Asp-Gly or General Formula 1 (above) may be a salt, such as a salt form selected from hydrochloride, acetate, and trifluoroacetate.

[0021] Also disclosed are pharmaceutical formulations comprising either Gly-Arg-Gly-Cys(acid)-Gly-Gly-Gly-Asp-Gly or General Formula 1 (above), and / or any of the described modifications thereof, in combination with at least one pharmaceutically acceptable carrier, diluent, solvent, or excipient.

[0022] The compounds disclosed herein include a series of test compounds referred to herein by the alphanumeric designations ALG-3001 to ALG-3009, or alternatively P1 to P9, having the amino acid sequences and structures specified in Table 2 below:

[0023]

Table 2-1

[0024]

Table 2-2

[0025]

Table 2-3

[0026] Some of ALLEG-3001 to ALG-3009 (also referred to as P1 to P9) and other compounds have previously demonstrated efficacy in inhibiting pathological angiogenesis, as described in incorporated U.S. Patent Application No. 16 / 882,656, entitled "PEPTIDE COMPOSITIONS AND RELATED METHODS" and U.S. Patent Application No. 16 / 882,660, entitled "PEPTIDE COMPOSITIONS AND RELATED METHODS".

[0027] The present disclosure includes uses and methods for using the disclosed compounds, and pharmaceutical formulations containing such compounds, for the treatment of diseases and disorders in human or animal subjects in need of treatment of diseases and disorders, including, for example, diseases or disorders that cause, are caused by, or are associated with oxidative stress and / or mitochondrial dysfunction. Such methods can include administering to the subject a therapeutically effective amount of at least one disclosed compound, or a pharmaceutically acceptable salt, ester or amide thereof, and / or a pharmaceutical formulation containing one or more of such compounds. In some uses, the subject may be suffering from a disorder that causes, contributes to, or is caused by angiogenesis, unwanted angiogenesis, inflammation, oxidative stress, and / or mitochondrial bioenergetic dysfunction. In such cases, the compound(s) may be in an amount that protects a particular cell or tissue from the effects of oxidative stress and / or at least some of the dysfunction of mitochondrial bioenergetics, such as chemical toxicity, hypoxic or ischemic injury, metabolic stress, heart failure, chronic heart failure with reduced ejection fraction, chronic heart failure with preserved ejection fraction, Barth syndrome, kidney disease, renal failure due to percutaneous renal angiography for renal artery stenosis, functional impairment of skeletal muscle function in the elderly, primary muscle mitochondrial myopathy or neuropathy, ischemia-reperfusion injury, protozoal infection, peripheral neuropathy, skin disorders, neurodegenerative diseases, retinal degeneration (e.g., dry age-related macular degeneration, retinitis pigmentosa, glaucoma, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), another disorder that causes progressive degeneration of the function and / or structure of nerve cells in the central nervous system (CNS), skin disorders, rash, abnormal pigmentation or acrocyanosis, itching, atopic dermatitis, psoriasis, and hemorrhoids, peripheral nerve disorders, peripheral neuralgia, or mitochondrial dysfunction, diabetes, abnormal glucose metabolism, oxidative stress or chemotherapy, nerve pain that causes, contributes to, or is caused by heart failure or reduced cardiac output, and may be administered in an amount to restore or prevent such nerve pain.

[0028] Auditory problems associated with tinnitus, or ringing in the ears that occurs in both ears, are thought to develop due to abnormal neural activity in the absence of sound stimuli and are sounds that cannot be heard from the outside. It is seen in 10 - 17% of the general population and 33% of the elderly population. Many factors, including auditory trauma, stress, ototoxic drugs, metabolic diseases, ear diseases, and neurological diseases, have been identified as causative factors, but most cases remain idiopathic. Oxidative stress has been strongly suggested as a fundamental cause of tinnitus. Other treatable ear disorders include other inflammations of the middle or inner ear, Meniere's disease, and other sensorineural hearing losses.

[0029] Generally, the test compounds identified as ALG - 3001 to ALG - 3009 (i.e., P1 to P9) have been tested in several experimental models, including an in vivo OIR / ROP mouse model that develops retinal neovascularization (NV) due to hypoxia, and in vitro donor retinal pigment epithelial (RPE) cells stressed with high and low levels of hydroquinone (HQ), i.e., toxins found in tobacco smoke and air pollutants. Table 3 below summarizes the results of this test.

[0030] [Table 3]

[0031] In some embodiments, in any of ALG-3001 to ALG-3005 and ALG-3007 to ALG-3009, the N-terminal arginine (G-) and the C-terminal threonine-proline (-T-P) can be modified or replaced by other amino acid(s) or amino acid group(s), and doing so will not render the compound ineffective for its intended use. For example, in some embodiments, the N-terminal Gly can be replaced by an amino acid such as Arg, Asp, His, Lys, Trp, Phe, Tyr, Met, Ala, Leu, or guanidine (guanidino group) or other entity. For example, in some embodiments, one or both of the C-terminal Thr-Pro can be replaced by other amino acid(s) / residue(s) selected from Ser, Val, Thr, Phe, Tyr, Lys, and Asp.

[0032] Further aspects and details of the present disclosure will be understood by reading the detailed description and examples set forth below in this specification.

Brief Description of the Drawings

[0033]

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DETAILED DESCRIPTION OF THE INVENTION

[0034] The following detailed description and the accompanying drawings referred to therein are not necessarily to describe all embodiments or implementations of the disclosed subject matter, but are intended to describe some embodiments or implementations. The embodiments or implementations described are considered to be illustrative in all respects and not restrictive. The content of this detailed description and the accompanying drawings does not limit the scope of the present disclosure in any way.

[0035] The treatments described in this patent application can be administered by any suitable route(s) of administration and in any suitable dosage form. Possible routes of administration known in the art include systemic, topical, regional, parenteral, enteral, inhalation, local, intramuscular, subcutaneous, intravenous, intravitreal, intraarterial, intrathecal, intravesical, oral, endoscopic (e.g., via endoscope, bronchoscope, colonoscope, sigmoidoscope, hysteroscope, laparoscope, arthroscope, gastroscope, cystoscope, etc.), transurethral, transtympanic, rectal, nasal, oral, tracheal, bronchial, esophageal, gastric, enteral, peritoneal, urethral, blister, urethral, vaginal, uterine, fallopian, buccal, sublingual, sublingual, and mucosal, but are not necessarily limited thereto. Possible dosage forms known in the art include liquid, biphasic liquid, solid, semi-solid, vapor, aerosol, solution, suspension, mixture, syrup, lozenge, gel, cream, paste, ointment, lotion, application, collodion, emulsion, transdermal delivery patch, suppository, capsule, tablet, powder, granule, edible, chewable, drop, spray, enema, drench, lozenge, etc., but are not limited thereto.

[0036] In Vivo OIR / ROP Mouse Model Study of Retinal Neovascularization In this study, each of the investigational agent (ALG-3001 to ALG-3009, alternatively referred to as P1 to P9), the investigational agent (ALG-3001 to ALG-3009, alternatively referred to as P1 to P9), and the test compounds (ALG-3001 to ALG-3009, alternatively referred to as P1 to P9) was tested for its effectiveness in reducing retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR), compared to a known inactive control peptide having the amino acid sequence Gly-Arg-Gly-Tyr-Pro (RGE) (e.g., negative control).

[0037] Method: OIR pups were exposed to 5 days of hyperoxia (75% O2) to regress developing retinal blood vessels. Upon return to room air, retinal angiogenesis develops due to the imbalance between oxygen supply and demand. At the time of return to room air, the eyes of OIR pups received a single intravitreal injection of either a control peptide (known to be inactive), a positive control (known to be active), or a test compound:

[0038] [Table 4]

[0039] Injectable solutions were prepared by dissolving either the control peptide, risteginterferon, or test compound in 0.9% NaCl saline at a concentration of 10 μg / μL. One microliter (1.0 μL) of each solution was injected intravitreally into each eye, thereby delivering a 10 μg dose of the indicated compound per eye. Eighteen days after injection, the mice were euthanized, the retinas were removed, and stained with fluorescein-labeled dextran for fluorescein microscopy. The prepared retinas were then examined microscopically, and the area of each retina showing angiogenesis was measured.

[0040] Results: The results of this study are summarized in Table 3 (top), Table 4 (bottom), and Figures 1A, 1B, 2, 3, 4, 5, 7, 8, 9, and 10.

[0041] [Table 5]

[0042] The positive control, risteginterferon (RSG), caused a 47 - 64% reduction in the area of neovascularization compared to the inactive control peptide (GRGETP). All test compounds (ALG-3001 (P1) - ALG-3009 (P9)) caused a reduction in the area of neovascularization equivalent to that caused by risteginterferon (RSG).

[0043] In Vitro Cell Culture Study of ALG-3001~ALG-3009 (P1~P9) in Retinal Pigment Epithelial (RPE) Cells under Stress In this study, ALG-3001~ALG-3009 (alternatively, P1~P9) were tested for their cytoprotective, mitochondrial stabilization, and other therapeutic properties in a human RPE cell culture model. The cells were stressed with tobacco smoke toxin hydroquinone, which is known to reduce cell viability, increase reactive oxygen species (ROS), and reduce mitochondrial function. This model replicates the disease scenarios of retinal degeneration and specifically dry age-related macular degeneration.

[0044] RPE cells form a single layer of highly specialized polarized epithelial cells that intervene between the choroid capillaries and photoreceptors. RPE cells play an important role in the homeostasis of the retina and are essential for the health of photoreceptor cells and visual function.

[0045] Dysfunction or death of RPE cells is thought to be an important contributor to age-related macular degeneration (AMD). RPE cells are constantly exposed to oxidants throughout life, and oxidative stress plays a major role in the etiology and progression of AMD. Tobacco smoke contains high concentrations of free oxidants and is involved as a major environmental risk factor for AMD. Hydroquinone (HQ), a major oxidant of both tobacco smoke and air pollutants, increases the production of reactive oxygen species (ROS) and promotes oxidative stress. ROS, a group of unstable oxygen-containing molecules that can readily react with other molecules within the cell, are produced during cell metabolism and in response to various stimuli. In cells, the major site of ROS production is the mitochondrial electron transport chain, where some electrons leak from the transport process and react spontaneously with molecular oxygen to produce superoxide anions. ROS have important physiological functions, but excessive ROS can cause oxidative damage to RPE cells.

[0046] Methods: Human donor eyes from a 62-year-old male donor were obtained from organ donors and eye banks in accordance with the Helsinki Declaration regulations for research involving human tissues. Cells were grown at 37 °C in a humidified environment containing 5% CO2 in Eagle's Minimum Essential Medium (MEM, Invitrogen) with 10% fetal bovine serum (Thermo Scientific) and 1× penicillin / streptomycin (Thermo Scientific). Human donor RPE cells were seeded onto collagen-coated plates. On the 6th day after seeding, the cells were washed twice with serum-free, phenol red-free MEM (SF-MEM) and treated with HQ for various times at 37 °C regardless of the presence or absence of peptide drugs in SF-MEM.

[0047] For the WST assay, RPE cells in triplicate wells of a 96-well plate were treated with different concentrations of HQ (in the range of 140 μM to 170 μM) for 2.5 hours in the presence of any of the following: no treatment (control); 400 μM of ridaforolimus (RSG) (positive control) or test compounds (ALG-3001 to ALG-3009) at concentrations of 100 μM, 400 μM, and 800 μM. One of the test compounds, ALG-3002, was further tested at concentrations of 12.5 μM, 25 μM, 50 μM, and 100 μM.

[0048] After an initial 2.5-hour incubation, the medium was removed and the cells were incubated with WST-1 solution for 30 minutes at 37 °C. A colorimetric assay was performed based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase in viable cells. The plates were read on a spectrophotometer at 440 nm and a reference wavelength of 690 nm.

[0049] RPE cells in the wells of a 96-well plate with a transparent bottom, in three replicates of the black plate, were washed with SF-MEM, loaded with 20 μM CM-H2DCFDA in SF-MEM at 37 °C for 30 minutes, and then washed twice. The cells were then treated with HQ (160 μM) in the presence or absence of the peptide drug. Fluorescence was measured at various times using a fluorescence plate reader (490 nm excitation, 522 nm emission).

[0050] For mitochondrial membrane potential measurement, RPE cells in the wells of a 96-well plate with a transparent bottom, in three replicates of the black plate, were washed with SF-MEM, loaded with 10 μM JC-1 dye in SF-MEM at 37 °C for 30 minutes, and then washed twice. The cells were then treated with HQ (160 μM) regardless of the presence or absence of RSG (0.4 mM). Fluorescence was measured at various times using a fluorescence plate reader, and the green JC-1 monomer (490 nm excitation, 522 nm emission) and red JC-1 aggregates (535 nm excitation, 590 nm emission) were quantified.

[0051] For the glutathione (GSH) assay, RPE cells in the wells of a 96-well plate, in three replicates, were washed with SF-MEM and treated with HQ regardless of the presence or absence of the peptide drug. After 1.5 hours of treatment, the cell culture medium was removed, and GSH-Glo was incubated at room temperature for 30 minutes. Next, the detection reagent was added for 15 minutes of incubation at room temperature. A plate reader was used to measure the relative light units, followed by an LDH assay by collecting the supernatant.

[0052] Data are presented as mean ± standard deviation. Two-way ANOVA with Tukey's multiple comparison correction was used to determine whether there were statistically significant differences between the treatment groups. The results were plotted using GraphPad Prism 9.0 with asterisks indicating the magnitude of the P-value (N.S. = not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001).

[0053] Results: The results showing the cytoprotective effect of the test compounds are summarized in Tables 5 and 6 (below).

[0054] [Table 6-1]

[0055] [Table 6-2]

[0056] · In the above experiment, HQ + 400 μM RSG increases the cell viability of cells above the level decreased by HQ exposure. · In the above experiment, HQ + 400 μM ALG - 3001 to ALG - 3009, and HQ + 800 μM ALG - 3001 to ALG - 3009 significantly increase the cell viability of cells above the level decreased by HQ exposure.

[0057] Table 6 (below) compares the amino acid sequences and the highest observed RPE cell viability in cells stressed with HQ for each test compound (ALLEG - 3001 to 3009 (P1 to P9)) and the positive control, lysitinib (RSG).

[0058] [Table 7]

[0059] These results are also summarized graphically in the following figures: Figure 1C and 1D - ALG - 3001 (P1); Figure 2A, 2B, 2C, and 2D - ALG - 3002 (P2); Figure 3A and 3B - ALG - 3003 (P3); Figure 4A and 4B - ALG - 3004 (P4); Figure 5A and 5B - (ALG - 3005 (P5); Figure 6A and 6B - (ALG - 3006 (P6); Figure 7A and 7B - ALG - 3007 (P7); Figure 8A and 8B - (ALG - 3008 (P8); and Figure 9A and 9B - ALG - 3009 (P9).

[0060] Furthermore, ALG-3002 (P2) was determined to be protective not only against HQ-induced stress but also against CCCP-induced stress. Table 7 (below) and Figure 10 compare the effects of 100 uM ALG 3002 (P2) in RPE cells stressed with either CCCP or HQ. ALG-3002 (P2) showed a cytoprotective effect in both cells stressed with CCCP and cells stressed with HQ.

[0061]

Table 8

[0062] · In cells exposed to HQ or CCCP alone, the cell viability is 41%. · The cell viability of HQ + 400 μM ALG-3002 (P2) is 108%. · The cell viability of CCCP + 400 μM ALG-3002 (P2) is 88%.

[0063] RPE cell degeneration is central to the etiology of age-related macular degeneration (AMD), a disease that leads to progressive loss of visual function and blindness. LISTEGANIB, along with these nine new peptides, showed in vitro signals indicating that they can maintain the viability of RPE cells, reduce ROS levels, and improve mitochondrial integrity in human RPE cells stressed by oxidants associated with macular degeneration. These peptides may be beneficial for the treatment of AMD and other degenerative eye diseases.

[0064] All nine of the test compounds protected cell survival, and ALG-3004 (P4), ALG-3006 (P6), and ALG-3007 (P7) provided a greater cytoprotective effect than RSG (positive control). Among the nine test compounds, ALG-3002 (P2) showed the greatest cytoprotective effect in this study, which appears to be dose-related.

[0065] Reactive oxygen species (ROS) The ROS level is a measure of the oxidative stress experienced by cells. The ROS level after treatment was measured using a CM-H2DCFDA-based ROS assay. ALG-3002 (P2), ALG-3004 (P4), and ALG-3007 (P7) were tested, plotted, and summarized in Table 8 (below), and as graphically summarized in Figures 11 (ALG-3002 (P2)), 12 (ALG-3004 (P4)), and 13 (ALG-3007 (P7)), it was found that they significantly reduced the ROS level in retinal pigment epithelium (RPE) cells co-treated with carbonyl cyanide phenylhydrazone (CCCP) or stressed retinal pigment epithelium (RPE) cells HQ.

[0066]

Table 9

[0067] · Reactive oxygen species increased by 230% with HQ while HQ+ALG-3002 decreased reactive oxygen species by 73%. · Reactive oxygen species increased by 47% with HQ while HQ+ALG-3004 decreased reactive oxygen species by 62%.

[0068] · Reactive oxygen species increased by 29% with HQ while HQ+ALG-3007 decreased reactive oxygen species by 79%. Mitochondrial membrane potential The mitochondrial membrane potential is an indicator of mitochondrial integrity, and a lower potential means that the mitochondria are deteriorating and the cell health is reduced. ALG-3002, ALG-3004, and ALG-3007 were tested, plotted, and also summarized in Table 9 (below), and as shown graphically in Figures 14 (ALG-3002 (P2)), 15 (ALG-3004 (P4)), and 16 (ALG-3007 (P7)), it was found that all three significantly improved the mitochondrial membrane potential in cells co-treated with HQ.

[0069]

Table 10

[0070] · The mitochondrial membrane potential was reduced by 25% by HQ while being increased by 91% by HQ+ALG-3002. · The mitochondrial membrane potential was reduced by 20% by HQ while being increased by 55% by HQ+ALG-3004.

[0071] · The mitochondrial membrane potential was reduced by 16% by HQ while being increased by 75% by HQ+ALG-3007. Level of glutathione (GSH) Glutathione (GSH) is a natural antioxidant used by cells to maintain redox homeostasis. GSH levels were measured using the GSH-Glo GSH assay after treatment. Cells stressed with HQ showed a reduction in GSH levels, while both RSG and ALG-3002 significantly improved GSH levels, as summarized in Table 10 (below) and shown graphically in Figure 17.

[0072] [Table 11]

[0073] · The untreated control level of glutathione (GSH) is 9064 RLU / LDH units. · The presence of the HQ toxin reduces the level of glutathione (GSH) to 597 RLU / LDH units.

[0074] · 800 μM of RSG has no effect on the normal level of glutathione (GSH) of 8995 RLU / LDH units. · The presence of 120 μM of HQ + 400 μM of RSG reduces the level of glutathione (GSH) to 3248 RLU / LDH units.

[0075] · 100 μM of ALG-3002 has no effect on the normal level of glutathione (GSH) at 8657 RLU / LDH units. · The presence of 120 μM of HQ + 400 μM of ALG-3002 increases the level of glutathione (GSH) up to 8060 RLU / LDH units.

[0076] Effect of RSG and ALG-3002 on mitochondrial ultrastructure in human RPE cells This study compared the effects of rimegepant (RSG) and ALG-3002 (P2) on the morphology of mitochondria in primary human RPE cells stressed with hydroquinone (HQ), a tobacco smoke toxin. HQ is known to induce intracellular oxidative stress and lead to cytotoxicity.

[0077] Methods: Primary human RPE cells cultured in 6-well plates were treated with HQ (180 μM) for 4 hours regardless of the presence or absence of RSG (800 μM) and ALG-3002 (100 μM). After treatment, the medium was removed, and then the cells were washed with warm PBS (2 mL / well). The cells were lifted with trypsin-EDTA (600 μL / well), followed by the addition of PBS to dilute the trypsin (1 mL / well). The cells were pelleted, washed with PBS (1 mL / well), and then fixed with 3% PFA and 2% glutaraldehyde in PBS at 4°C for 18 hours. The cell pellets were then washed with 1 mL of PBS and imaged under a transmission EM system for mitochondrial ultrastructure changes due to HQ and peptides.

[0078] Results / Discussion: Figures 18A - 18C are representative micrographs showing that normal mitochondrial morphology was observed in non-stressed control cells (Figure 25A), while exposure of cells to HQ (Figure 25B) caused changes in mitochondrial morphology including mitochondrial swelling, the presence of vacuoles, and loss of cristae. Both the positive control RSG (Figure 25C) and the test compound ALG-3002 (P2) (Figure 25D) appeared to significantly reduce the severity of HQ-induced morphological changes in mitochondria.

[0079] Conclusion: In these primary human RPE cell cultures, both RSG and ALG-3002 protected the mitochondria of RPE cells against morphologically evident HQ stress. Therapeutic use of ALG-3001 - ALG-3009 Mitochondria are intracellular organelles that contain the main respiratory machinery within the cell and are essential for energy production and cell homeostasis. Due to the high level of metabolic demand by photoreceptors, RPE cells are rich in numerous mitochondrial populations to meet high energy needs. As a result, RPE mitochondrial dysfunction can lead to tissue damage and is involved in the development of AMD. Damaged, fragmented, and ruptured mitochondria have been observed in RPE cells of eyes with AMD. The level of mtDNA mutations also increases in RPE cells of eyes with AMD.

[0080] Mitochondrial respiration plays an important role in cell survival. The role of RSG in the regulation of mitochondrial function was evaluated. Mitochondria are a major source of ROS production that contributes to oxidative stress-mediated cell death. ROS levels were measured to examine whether RSG reduces oxidative stress-mediated ROS production. As shown, ROS production was significantly increased in HQ-treated cells compared to the control, but co-treatment with RSG significantly decreased HQ-induced ROS production. RSG alone did not significantly change the ROS level compared to the control.

[0081] Primary mitochondrial disorders can cause skin symptoms (e.g., rash, abnormal pigmentation, digital cyanosis), and primary skin disorders can be associated with mitochondrial dysfunction. A number of skin disorders (e.g., pruritus, atopic dermatitis, psoriasis) can benefit from treatment as described herein to improve mitochondrial function. Mitochondrial dysfunction has not been the exception in skin diseases but has rather been characterized as a rule. Feichtinger, R.G., et al.; Mitochondrial dysfunction: a neglected component of skin diseases; Experimental Dermatology Vol. 23, Issue 9, September 2014 (607 - 614); https: / / doi.org / 10.1111 / exd.12484 Based on the above data, in addition to the anti - angiogenic effects described in incorporated U.S. Patent Application No. 16 / 882,656, titled "PEPTIDE COMPOSITIONS AND RELATED METHODS" and No. 16 / 882,660, titled "PEPTIDE COMPOSITIONS AND RELATED METHODS", the peptide therapies described herein can be used to treat a variety of disorders including, but not necessarily limited to: Eye: diabetic retinopathy, retinal angiogenesis, exudative or wet macular degeneration, non - exudative or dry macular degeneration, retinitis pigmentosa, glaucoma, other retinal degenerations, dry eye disease, retinal inflammation, scleritis, episcleritis, or corneal inflammation.

[0082] Neurological: multiple sclerosis, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), peripheral neuropathy, peripheral neuralgia, stroke, tinnitus, and other disorders that cause functional and / or structural degeneration in central neurons and / or peripheral neurons; Lung: chronic obstructive pulmonary disease, pulmonary fibrosis; Cardiovascular / Kidney: congestive heart failure, chronic heart failure with reduced ejection fraction, chronic heart failure with preserved ejection fraction, Barth syndrome, renal failure, kidney disease, renal failure due to percutaneous renal angiography for renal artery stenosis, vascular inflammation, vasculitis, hemorrhoids; Liver: non-alcoholic steatohepatitis (NASH) Muscle: impairment of skeletal muscle function in the elderly, primary mitochondrial myopathy or neuropathy, ischemia-reperfusion injury, myocardial or skeletal muscle dysfunction caused by protozoal or other microbial infections; Skin / Local: rash, abnormal pigmentation, acrocyanosis, atopic dermatitis, marasmus, pruritus, psoriasis, and hemorrhoids; Ear: inflammation of the middle ear or inner ear, Meniere's disease, sensorineural hearing loss, or tinnitus The dosage, dosing schedule, and / or route of administration may vary depending on the type and severity of the disease or disorder being treated. For example, in at least some uses where a compound comprising ALG-3001 to ALG-3009 is administered intravitreally or topically to the eye of interest, the dosage of the compound selected from ALG-3001 to ALG-3009 may be in the following range(s) or other ranges that may be clinically appropriate: a) Intravitreal injection or implantation: 0.01 mg / 50 uL to 10.0 mg / 50 uL, or 1.0 mg / 50 uL to 2.0 mg / 50 uL b) Topical application to the eye: -0.01 g / 100 mL to 40 g / 100 mL, or 0.60 g / 100 mL c) Intravenous injection or infusion: 0.01 mg / kg body weight to 50 mg / kg body weight, or 5.0 mg / kg body weight to 6.0 mg / kg body weight d) Topical application to the skin or mucosa: 0.01 g / 100 g to 40 g / 100 g, or 0.40 g / 100 g to 0.50 g / 100 g e) Intradermal, subcutaneous, intramuscular, intraperitoneal, or other injection or implantation: 0.01 mg / kg body weight to 50 mg / kg body weight, or 5.0 mg / kg body weight to 6.0 mg / kg body weight f) Intrathecal, epidural: 0.01 mg / kg body weight to 50 mg / kg body weight, or 2.0 mg / kg body weight to 6.0 mg / kg body weight g) Application to the ear (e.g., the ear, eardrum, inner eardrum, inner cochlea) for inflammation of the middle ear or inner ear, Meniere's disease, sensorineural hearing loss, or tinnitus: 0.01 g / 100 g to 40 g / 100 g, or 0.40 g / 100 g to 0.50 g / 100 g h) Rectal or transmucosal application (e.g., for the treatment of vascular inflammation, vasculitis, hemorrhoids, or for systemic transmucosal absorption, suppositories or topical application): 0.01 g / 100 g to 40 g / 100 g, or 0.40 g / 100 g to 0.50 g / 100 g The above description refers to examples or embodiments, but various additions, deletions, changes, and modifications can be made to the described examples and embodiments without departing from the intended spirit and scope of the invention disclosed herein. For example, unless otherwise specified or doing so would render the embodiment or example unsuitable for its intended use, any element, step, member, component, composition, reactant, part, or portion of an embodiment or example can be removed / excluded from, incorporated into, or used with another embodiment or example. Also, if the steps of a method or process are described or listed in a particular order, unless otherwise specified or doing so would render the method or process unsuitable for its intended use, the order of such steps can be changed. Further, any element, step, member, component, composition, reactant, part, or portion of any invention or example described herein can optionally be present, or can be utilized in the absence or substantial absence of any other element, step, member, component, composition, reactant, part, or portion, unless otherwise noted. All reasonable additions, deletions, modifications, and alterations are considered equivalents of the described examples and embodiments and are included within the scope of the following claims.

Claims

1. A compound comprising or consisting of an amino acid sequence having the following formula: Gly-X-Thr-Pro A compound in which X is a sequence of three amino acids selected from Arg-Ala-Cys(acid), Arg-Gly-Cys, Arg-Asp-Gly, Arg-Ala-Glu, Arg-Gly-Asn, Asp-Gly-Arg, Cys(acid)-Gly-Arg, and Lys-Gly-Asp.

2. The compound according to claim 1, wherein one or more additional amino acids are added to either or both ends of the compound such that the compound consists of more than six but no more than ten amino acids.

3. The compound according to claim 1, wherein the N-terminal Gly is replaced by another amino acid or group selected from Arg, Asp, His, Lys, Trp, Phe, Tyr, Met, Ala, Leu, and guanidino.

4. The compound according to claim 1, wherein one or both of the Thr-Pro groups at the C-terminus are replaced by one or two other amino acids selected from Ser, Val, Thr, Phe, Tyr, Lys, and Asp.

5. A compound according to any one of claims 1 to 4, prepared as a salt.

6. The compound according to claim 5, comprising a salt form selected from hydrochloride, acetate, and trifluoroacetate.

7. A pharmaceutical formulation comprising the compound according to any one of claims 1 to 4, in combination with at least one pharmaceutically acceptable carrier, diluent, solvent, or excipient.

8. A pharmaceutical formulation according to claim 7 for treating a disease or disorder in human or animal subjects that causes, is caused by, or is characterized by cellular oxidative stress.

9. The pharmaceutical preparation according to claim 7 for treating mitochondrial bioenergy dysfunction in humans or animals.

10. The pharmaceutical preparation according to claim 7 for treating eye diseases or disorders, non-exudative or dry macular degeneration, retinitis pigmentosa, glaucoma, other retinal degenerations, dry eye diseases, retinitis, scleritis, episcleritis, or corneal xerosis or inflammation.

11. The pharmaceutical preparation according to claim 7 for treating congestive heart failure, chronic heart failure with reduced ejection fraction, chronic heart failure with preserved ejection fraction, Barth syndrome, renal failure, kidney disease, or renal failure, vasculitis, vasculitis, or hemorrhoids as determined by percutaneous renal angiography for renal artery stenosis.

12. The pharmaceutical formulation according to claim 7 for treating skeletal muscle dysfunction, primary myocardial mitochondrial myopathy or neuropathy, ischemia-reperfusion injury, protozoan or other microbial infection-related dysfunction of myocardium or skeletal muscle in elderly patients.

13. The pharmaceutical formulation according to claim 7 for treating neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), stroke, and other disorders causing degeneration of the function and / or structure of neurons of the central nervous system (CNS).

14. The pharmaceutical preparation according to claim 7 for treating skin disorders, rashes, hyperpigmentation abnormalities, acrocyanosis of the limbs, atopic dermatitis, malasma, itching, or psoriasis.

15. The pharmaceutical formulation according to claim 7 for treating neurological disorders or nerve disorders, multiple sclerosis, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), peripheral neuropathy, peripheral neuralgia, stroke, or other disorders causing degeneration of function and / or structure in central and / or peripheral neurons.

16. The pharmaceutical preparation according to claim 7 for treating inflammation of the middle or inner ear, Meniere's disease, sensorineural hearing loss, tinnitus, or another hearing disorder.

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