Redox-regulated organelle complex

Abstract

This specification discloses methods, compositions, and kits suitable for use in reducing or preventing oxidative stress and / or redox stress. In some embodiments, the method comprises contacting a redox-sensitive composition with an effective amount of the disclosed redox-regulating composition. In some embodiments, the method comprises administering an effective amount of the disclosed redox-regulating composition to a subject in need thereof. In some embodiments, the redox-regulating composition comprises an isolated organelle complex. The organelle complex can comprise a mitochondrion and one or more of an endoplasmic reticulum, peroxisome, lysosome, and Golgi apparatus. In some embodiments, the redox-regulating composition comprises isolated mitochondria. The redox-regulating composition can reduce the levels of ROS and / or RNS in a redox-sensitive composition and / or in cells.

Description

Technical Field

[0001] Reference to Related Applications This application claims the benefit of 35 U.S.C.§119(e) of U.S. Provisional Patent Application No. 63 / 359,108, filed on July 7, 2022, and the content of this related application is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] The present disclosure generally relates to methods for reducing and preventing oxidative stress and / or reductive stress.

[0003] Mitochondria are intracellular organelles that undertake multiple metabolic conversion and regulatory functions. Mitochondria are highly dynamic organelles that move across cells, undergo structural transitions, and change in length, morphology, shape, and size. Furthermore, mitochondria are continuously removed and regenerated in a process known as mitochondrial biogenesis. Although most mitochondrial genes have been transferred to the nuclear genome, the mitochondrial genome still encodes the rRNA, tRNA, and 13 subunits of the electron transport system (ETC). Thus, functional communication between the nuclear genome and the mitochondrial genome is essential for mitochondrial biogenesis, efficient oxidative phosphorylation, and normal health. Mitochondria are also a major source of free radicals and reactive oxygen species (ROS) that cause oxidative stress. Furthermore, mitochondria play an important role in intracellular signaling and the control of cell death, including apoptosis and necrosis. Evidence is increasing that mitochondrial dysfunction is associated with a wide range of human diseases. Mitochondrial dysfunction, such as respiratory chain complex dysfunction, is a major cause involved in mitochondrial diseases and aging.

[0004] Oxidative stress is caused by a disturbance in the normal redox state within cells. An imbalance between the daily production and detoxification of reactive oxygen species such as peroxides and free radicals can lead to oxidative damage to cellular structures and machinery. The most important source of reactive oxygen species under normal conditions in aerobic organisms is the leakage of activated oxygen from mitochondria during normal oxidative respiration. Disorders associated with this process are suspected to contribute to mitochondrial diseases, neurodegenerative diseases, and diseases of aging.

[0005] Reductive stress can occur in response to a state that shifts the redox balance of important biological redox pairs such as NAD + / NADH, NADP + / NADPH, and GSH / GSSG towards a more reduced state. The overexpression of antioxidant enzyme systems can deplete reactive oxygen species and lead to excessive reducing equivalents that can drive cells into reductive stress. Chronic reductive stress can establish a feedback regulation that induces oxidative stress and then stimulates reductive stress again. Excessive reducing equivalents can regulate cell signaling pathways, modify transcriptional activity, induce changes in the formation of disulfide bonds in proteins, reduce mitochondrial function, decrease cell metabolism, and thus contribute to the development of redox diseases and disorders such as cardiomyopathy, pulmonary hypertension, stent stenosis, muscular dystrophy, neuropathy, Parkinson's disease, Alzheimer's disease, metabolic syndrome and insulin resistance, rheumatoid arthritis, kidney diseases, and cancer.

[0006] There is a need for redox regulatory compositions and methods that can reduce or prevent oxidative stress and / or reductive stress.

Summary of the Invention

[0007] This specification discloses a method for reducing or preventing oxidative stress and / or reductive stress in a redox-sensitive composition. In some embodiments, the method comprises contacting the redox-sensitive composition with an effective amount of a redox-regulating composition, thereby reducing or preventing oxidative stress and / or reductive stress in the redox-sensitive composition. In some embodiments, the redox-regulating composition comprises an isolated organelle complex. In some embodiments, the organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the redox-regulating composition comprises isolated mitochondria. In some embodiments, the redox-regulating composition comprises mitochondria isolated from intact cells and / or floating cells or frozen cells, or a combination thereof.

[0008] This specification discloses a method for reducing the levels of reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) in a redox-sensitive composition. In some embodiments, the method comprises contacting the redox-sensitive composition with an effective amount of a redox-regulating composition, thereby reducing the levels of ROS and / or RNS in the redox-sensitive composition. In some embodiments, the levels of ROS and / or RNS in the redox-sensitive composition are reduced, thereby reducing or preventing oxidative stress and / or reductive stress in the redox-sensitive composition. In some embodiments, the redox-regulating composition comprises an isolated organelle complex. In some embodiments, the organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the redox-regulating composition comprises isolated mitochondria. In some embodiments, the redox-regulating composition comprises mitochondria isolated from intact cells and / or floating cells or frozen cells, or a combination thereof.

[0009] In some embodiments, the contacting step comprises incubating a mixture of the redox-sensitive composition and the redox-regulating composition for less than about 30 seconds. The redox-sensitive composition can be an oxidizing agent-sensitive composition, a reducing agent-sensitive composition, or a combination thereof. In some embodiments, the effective amount comprises at least about 5 μg / mL to about 5 mg / mL of the redox-regulating composition. In some embodiments, the redox-sensitive composition is undergoing or at risk of undergoing oxidative stress and / or reductive stress. In some embodiments, the redox-sensitive composition comprises one or more cells. The contacting step can comprise introducing the redox-regulating composition into one or more cells. The one or more cells can be cells of a subject. The one or more cells can be undergoing or at risk of undergoing hypoxia. In some embodiments, the contacting is performed ex vivo, in vitro, or in vivo.

[0010] In some embodiments, the redox-sensitive composition comprises a biological sample. In some embodiments, the biological sample is a soil sample, an air sample, an environmental sample, a cell culture sample, a bone marrow sample, a rainfall sample, a fallout sample, a cosmic sample, an extraterrestrial sample, a sewage sample, a groundwater sample, a wear sample, an archaeological sample, a food sample, a blood sample, a serum sample, a plasma sample, a urine sample, a fecal sample, a semen sample, a lymph fluid sample, a cerebrospinal fluid sample, a nasopharyngeal wash sample, a sputum sample, an oral swab sample, a pharyngeal swab sample, a nasal swab fluid sample, a bronchoalveolar lavage sample, a bronchial secretion sample, a milk sample, an amniotic fluid sample, a biopsy sample, a nail sample, a hair sample, a skin sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a cell lysate sample, a virus culture sample, a forensic sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biomolecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, a nucleotide solution, a polynucleotide solution, a nucleic acid solution, a peptide solution, a polypeptide solution, an amino acid solution, a protein solution, a synthetic polymer solution, a biochemical composition solution, an organic chemical composition solution, an inorganic chemical composition solution, a lipid solution, a carbohydrate solution, a combinatorial chemistry product solution, a drug candidate molecule solution, a drug molecule solution, a drug metabolite solution, a cell suspension, a virus suspension, a microbial suspension, a metal suspension, a metal alloy suspension, a metal ion solution, and any combination thereof.

[0011] Disclosed herein is a method for reducing or preventing oxidative stress and / or reducing stress in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a redox-regulating composition, thereby reducing or preventing oxidative stress and / or reducing stress in the subject. In some embodiments, the redox-regulating composition comprises an isolated organelle complex. In some embodiments, the organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the redox-regulating composition comprises isolated mitochondria. In some embodiments, the redox-regulating composition comprises mitochondria isolated from intact cells and / or floating cells or frozen cells, or a combination thereof.

[0012] This specification discloses methods for treating or preventing redox diseases or disorders in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a redox modulating composition, thereby treating or preventing a redox disease or disorder in the subject. In some embodiments, the redox modulating composition comprises an isolated organelle complex. In some embodiments, the organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the redox modulating composition comprises isolated mitochondria. In some embodiments, the redox modulating composition comprises mitochondria isolated from intact cells and / or floating cells or frozen cells, or combinations thereof.

[0013] In some embodiments, the redox modulating composition comprises homogenized mitochondria, a first organelle complex, and / or a second organelle complex. In some embodiments, the first organelle complex and the second organelle complex each comprise mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the first organelle complex and the second organelle complex are depleted of cytoplasmic macromolecules. In some embodiments, the first organelle complex is derived from (i) frozen cells, (ii) floating cells, and / or (iii) cells contacted with the surfactant at a concentration above the critical micelle concentration (CMC) of the surfactant. In some embodiments, the second organelle complex is derived from (i) adherent cells and / or (ii) cells contacted with the surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant. In some embodiments, the first organelle complex and the second organelle complex are derived from cells treated with a mitochondrial activator.

[0014] In some embodiments, the homogenized mitochondria, the first organelle complex, and / or the second organelle complex are encapsulated in lipid membrane-based vesicles. In some embodiments, the effective amount comprises at least about 1 μg to about 1 mg of the redox-regulating composition. In some embodiments, the redox-regulating composition does not contain intact cells.

[0015] In some embodiments, the oxidative stress and / or the reductive stress include an increase in the levels of ROS, RNS, and / or free radicals. In some embodiments, the oxidative stress and / or the reductive stress include a change in cell function. In some embodiments, the oxidative stress and / or the reductive stress are associated with a redox disease or disorder. In some embodiments, the redox-regulating composition reduces the levels of one or more ROS and / or one or more RNS in the subject or the redox-sensitive composition by at least about 5%. In some embodiments, the redox-regulating composition reduces the level of oxidative cellular stress, thereby restoring or recovering the cell function. In some embodiments, the redox-regulating composition has ROS-scavenging activity and / or RNS-scavenging activity. In some embodiments, the redox-regulating composition reduces or prevents ROS production and / or RNS production in the subject. In some embodiments, the redox-regulating composition has superoxide dismutase activity, catalase activity, peroxidase activity, or any combination thereof.

[0016] In some embodiments, the redox-regulating composition is the oxidized form versus the reduced form of nicotinamide adenine dinucleotide (NAD + / NADH), the oxidized form versus the reduced form of nicotinamide adenine dinucleotide phosphate (NADP +(NADPH), the ratio of oxidized glutathione to reduced glutathione (GSSG / GSH), and the ratio of oxidized thioredoxin to reduced thioredoxin (TrxSS / TrxSH2) by increasing and / or decreasing one or more of them. In some embodiments of the compositions and methods provided herein, the disclosed redox modulating compositions are the oxidized to reduced forms of nicotinamide adenine dinucleotide (NAD + / NADH), the oxidized to reduced forms of nicotinamide adenine dinucleotide phosphate (NADP + / NADPH), the oxidized to reduced forms of glutathione (GSSG / GSH), and the oxidized to reduced forms of thioredoxin (TrxSS / TrxSH2) by (at least about 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) increasing one or more of them.

[0017] In some embodiments, the redox modulating composition has free radical scavenging activity and / or inhibits free radical formation. In some embodiments, the subject is undergoing oxidative stress and / or reductive stress or is at risk of undergoing oxidative stress and / or reductive stress (e.g., oxidative stress and / or reductive stress caused by ischemia-reperfusion injury or a redox disease or disorder).

[0018] In some embodiments, the reactive nitrogen species include nitric oxide (NO), nitrogen dioxide radical (NO2), peroxynitrite anion (ONOO - ), peroxynitrous acid (ONOOH), nitrosoperoxycarbonate anion (ONOOCO2 - ), nitronium cation (NO2 + ), nitrosyl cation (NO + ), or dinitrogen trioxide (N2O3), or any combination thereof. In some embodiments, the reactive oxygen species are superoxide (O2. -) Hydroperoxy (HO•), hydrogen peroxide (H2O2), peroxynitrous acid (ONOO - ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO•), peroxyl radical (ROO•), alkoxyl radical (RO•), singlet oxygen 1 O2), lipid peroxides, lipid peroxyl radicals or lipid alkoxyl radicals, or any combination thereof. In some embodiments, the ROS are derived from neutrophils and / or xanthine oxidase (XO).

[0019] In some embodiments, the method includes identifying a subject that is undergoing or at risk of undergoing oxidative stress and / or reductive stress. In some embodiments, the method includes measuring the level of RNS in the subject or the redox-sensitive composition using a probe that detects reactive nitrogen species, and / or measuring the level of ROS in the subject or the redox-sensitive composition using a probe that detects reactive oxygen species. In some embodiments, the probe that detects reactive nitrogen species is diaminonaphthalene, diaminofluorescein, diaminorhodamine, diaminocyanine, NiSPY, dichlorodiaminocarboxyfluorescein, 1,2-diaminoanthraquinone, or DAMBO-P H . In some embodiments, the probe that detects reactive oxygen species is 2’,7’-dichlorofluorescein diacetate, dihydrorhodamine 123, 3’-(p-aminophenyl) fluorescein (API), 3’-(p-hydroxyphenyl) fluorescein (HPF), aminophenoxycarboxyfluorescein (APC), mitoAR, mitoHR, DPAX, DMAX, hydrocyanine, or dihydroethidium.

[0020] In some embodiments, the subject has, or is suspected of having, a disease or condition involving ROS selected from the group consisting of atherosclerosis, heart disease, heart failure, hypertension, sepsis, diabetes, Alzheimer's disease, Parkinson's disease, toxin-induced parkinsonism, Huntington's disease, Wilson's disease, Friedreich's ataxia, Kearns-Sayre syndrome, Leigh syndrome, Leber's hereditary optic neuropathy, mitochondrial myopathy, cardiomyopathy, hearing loss, mood disorder, movement disorder, dementia, amyotrophic lateral sclerosis, multiple sclerosis, tardive dyskinesia, brain injury, schizophrenia, epilepsy, AIDS dementia, endothelial nitroglycerin resistance, adriamycin toxicity, kidney injury in type I diabetes, ex vivo kidney preservation, stroke, ischemia-reperfusion injury, ischemia-reperfusion injury, chronic inflammation, cocaine toxicity, alcoholic fatty liver disease, fatty liver disease, liver inflammation in hepatitis C virus patients, neuroprotection, immobilization-induced muscle atrophy, skeletal muscle burn, cancer, inflammation and ischemia-reperfusion injury in stroke, heart attack, UV damage to the skin, organ transplantation, surgery, and any combination thereof.

[0021] In some embodiments, administering the redox modulating composition to the subject treats, reduces, or prevents ischemia-reperfusion injury in the subject. In some embodiments, the ischemia-reperfusion injury is caused by mitochondrial dysfunction, hypoxia injury, HMGB1 release, or necrotic cell death. In some embodiments, the redox modulating composition suppresses ischemia-reperfusion injury, mitochondrial dysfunction, hypoxia injury, necrotic cell death, or any combination thereof. In some embodiments, the redox disease or disorder comprises ROS-mediated oxidative damage and / or RNS-mediated oxidative damage to one or more tissues of the subject. In some embodiments, the administration comprises intravenous administration, intraarterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and / or intraocular administration.

[0022] In some embodiments, the redox disease or disorder is mitochondrial disorder, hereditary mitochondrial disease, Alpers' disease, Barth syndrome, beta-oxidation deficiency, carnitine-acyl-carnitine deficiency, carnitine deficiency, creatine deficiency syndrome, coenzyme Q10 deficiency, complex I deficiency, complex II deficiency, complex III deficiency, complex IV deficiency, complex V deficiency, COX deficiency, chronic progressive external ophthalmoplegia (CPEO), CPT I deficiency, CPT II deficiency, Friedreich's ataxia (FA), glutaric aciduria type II, Kearns-Sayre syndrome (KSS), lactic acidosis, long-chain acyl-CoA dehydrogenase deficiency (LCAD), LCHAD, Leigh syndrome, Leigh-like syndrome, Leber hereditary optic neuropathy (LHON), lethal infantile cardiomyopathy (LIC), Luft disease, multiple acyl-CoA dehydrogenase deficiency (MAD), medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial myopathy, encephalopathy, lactic acidosis, stroke (MELAS), myoclonic epilepsy with ragged-red fibers (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, myoneurogastrointestinal encephalopathy (MNGIE), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, respiratory chain disorder, short-chain acyl-CoA dehydrogenase deficiency (SCAD), SCHAD, very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), myopathy, cardiomyopathy, encephalomyopathy, neurodegenerative disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), motor neuron disease, nerve disease, epilepsy, age-related disease, macular degeneration, diabetes, metabolic syndrome, brain tumor, genetic disease, Huntington's disease, mood disorder, schizophrenia, bipolar disorder, pervasive developmental disorder, autism, Asperger syndrome, childhood disintegrative disorder (CDD), Rett syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), cerebrovascular disorder, stroke, visual disorder, optic neuropathy, dominant hereditary juvenile optic atrophy, optic neuropathy due to toxic substances, glaucoma, Stargardt macular dystrophy, diabetic retinopathy, diabetic maculopathy, retinopathy of prematurity, ischemia-reperfusion-related retinal disorder, oxygen toxicity, hemoglobinopathy, thalassemia, sickle cell anemia, epileptic seizure,Selected from the group consisting of ischemic blood, renal tubular acidosis, attention deficit / hyperactivity disorder (ADHD), neurodegenerative diseases causing auditory or balance disorders, dominant optic atrophy (DOA), maternally inherited diabetes and deafness (MIDD), chronic fatigue, contrast-induced nephropathy, contrast-induced retinopathy disorder, abetalipoproteinemia, retinitis pigmentosa, Wolfram disease, Tourette syndrome, cobalamin c deficiency, methylmalonic aciduria, glioblastoma, Down syndrome, acute tubular necrosis, muscular dystrophy, white matter dystrophy, progressive supranuclear palsy, spinal muscular atrophy, deafness, noise-induced deafness, traumatic brain injury, juvenile Huntington's disease, multiple sclerosis, NGLY1, multiple system atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, and any combination thereof.

[0023] In some embodiments, the redox-regulating composition improves one or more energy biomarkers selected from the group consisting of lactate (lactate) levels, pyruvate (pyruvate) levels, lactate / pyruvate ratios, total, reduced or oxidized glutathione levels or reduced / oxidized glutathione ratios, total, reduced or oxidized cysteine levels or reduced / oxidized cysteine ratios, phosphocreatine levels, NADH (NADH+H + ) levels, NADPH (NADPH+H + ) levels, NAD levels, NADP levels, ATP levels, reduced coenzyme Q (CoQred) levels, oxidized coenzyme Q (CoQox) levels, total coenzyme Q (CoQtot) levels, oxidized cytochrome C levels, reduced cytochrome C levels, oxidized cytochrome C / reduced cytochrome C ratios, acetoacetate levels, b-hydroxybutyrate levels, acetoacetate / b-hydroxybutyrate ratios, 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, reactive oxygen species levels, oxygen consumption (V02) levels, carbon dioxide release (VC02) levels, respiratory quotient (VC02 / V02), exercise tolerance, anaerobic threshold, and any combination thereof in the subject or the redox-sensitive composition.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 10-2

Figure 11

Figure 11-2

Mode for Carrying Out the Invention

[0025] In the following detailed description, reference is made to the accompanying drawings which form a part hereof. In the drawings, like reference symbols typically identify like components unless the context dictates otherwise. The illustrative embodiments described in the mode for carrying out the invention, the drawings, and the claims are not meant to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure generally described and illustrated in the figures herein can be arranged, substituted, combined, separated, and designed in a variety of different configurations all of which are explicitly contemplated herein and form part of the disclosure herein.

[0026] All patents, published patent applications, other publications, and sequences from GenBank, as well as other databases, referred to herein are hereby incorporated by reference in their entirety for the relevant art.

[0027] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of the present disclosure, the following terms are defined below.

[0028] In the following detailed description, reference is made to the accompanying drawings which form a part hereof. In the drawings, like symbols typically identify like components unless the context dictates otherwise. The illustrative embodiments described in the embodiments of the invention, the drawings, and the claims are not meant to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure generally described herein and illustrated in the figures can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations all of which are explicitly contemplated herein and form a part of the disclosure herein.

[0029] All patents, published patent applications, other publications, and sequences from GenBank, as well as other databases, referred to herein are hereby incorporated by reference in their entirety for the relevant art.

[0030] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, see Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of the present disclosure, the following terms are defined below.

[0031] As used herein, "isolated" is given its ordinary meaning and also refers to (1) being isolated from at least a portion of the components associated therewith when first produced (in nature or in an experimental setting), and / or (2) a substance or entity that has been produced, prepared, and / or manufactured by human hand. In some embodiments, an isolated population of mitochondria or an isolated organelle complex is processed to be obtained from a cellular environment via the methods provided herein.

[0032] As used herein, the term "cell" is given its ordinary meaning and also refers to a eukaryotic cell, i.e., a cell that contains mitochondria in the cytoplasm, such as an animal cell, such as a mammalian cell, preferably a human cell. As used herein, the term "cell" is used in the sense of including cells present within a tissue, and cells separated from a tissue (e.g., a single cell), as well as cells within a population of cells (e.g., a population of cells obtained from a subject's tissue and / or a population of cells obtained from a cell line).

[0033] As used herein, the term "mitochondria" is given its ordinary meaning and refers to an organelle present within a eukaryotic cell that has a double lipid membrane, an inner and outer membrane, and a matrix surrounded by cristae membranes and the inner membrane. Mitochondria (plural) have enzymes such as respiratory chain complexes involved in oxidative phosphorylation on their inner membranes. The inner membrane has a membrane potential due to an inner and outer proton gradient formed by the action of the respiratory chain complexes. Mitochondria are thought to be unable to maintain the membrane potential when the inner membrane disintegrates. Mitochondria are known to have their own unique genomes (mitochondrial genomes) that are different from the genome in the cell nucleus.

[0034] As used herein, the term "organelle complex" is given its ordinary meaning and refers to a complex of mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. Organelle complexes can deplete cytoplasmic macromolecules (e.g., cytoplasmic proteins). In some embodiments, the organelle complex does not contain cytoplasmic macromolecules. In some embodiments, the organelle complex population contains homogenized mitochondria. As used herein, the term "population" is given its ordinary meaning and refers to a group of multiple same or different substances. For example, an "organelle complex population" is a group of at least multiple same or different organelle complexes. A population may not always be homogeneous and may have a physical, chemical, and / or physiological distribution. Physical distribution includes, for example, particle size and polydispersity index. Chemical distribution includes, for example, zeta potential distribution and lipid composition distribution. Physiological distribution includes, for example, differences in physiological functions (e.g., respiratory activity). An organelle complex population can contain a first organelle complex, a second organelle complex, homogenized mitochondria, or any combination thereof. As used herein, the term "homogenized mitochondria" is given its ordinary meaning and refers to mitochondria isolated via a method that includes one or more homogenization steps.

[0035] As used herein, the term "surfactant" is given its ordinary meaning and refers to a molecule having a hydrophilic portion and a hydrophobic portion in one molecule. Surfactants have the role of reducing the surface tension at the interface or mixing polar and nonpolar substances by forming micelles. Surfactants are broadly classified into nonionic surfactants and ionic surfactants. Nonionic surfactants are those in which the hydrophilic portion is not ionized, and ionic surfactants are those in which the hydrophilic portion contains a cation or an anion or both a cation and an anion.

[0036] As used herein, the term "critical micelle concentration" (CMC) is given its ordinary meaning and refers to the concentration at which surfactants form micelles and the surfactants added further to the system contribute to micelle formation, particularly the concentration in the bulk. At concentrations above the critical micelle concentration, when surfactants are added to the system, the amount of micelles, particularly the number of micelles, ideally increases.

[0037] As used herein, "subject" refers to an animal that is the subject of treatment, observation, or experiment. "Animal" includes cold-blooded vertebrates, warm-blooded vertebrates, and invertebrates such as fish, shellfish, reptiles, particularly mammals. "Mammal" as used herein refers to an individual belonging to the class of mammals and includes, but is not limited to, humans, domestic animals and livestock, zoo animals, sports and pet animals. Examples of mammals include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, as well as primates such as monkeys, chimpanzees, and apes, particularly humans. In some embodiments, the mammal is a human. However, in some embodiments, the mammal is not a human.

[0038] As used herein, the term "treatment" refers to an intervention carried out in response to a disease, disorder or physiological condition manifested by a patient. The purposes of treatment include, but are not limited to, one or more of alleviation of symptoms or prevention, delay or arrest of the progression or worsening of a disease, disorder or condition, and remission of a disease, disorder or condition. The terms "treating" and "treatment" include, for example, therapeutic treatment, prophylactic treatment, and uses for reducing the risk that a subject will develop a disorder or other risk factor. Treatment does not require complete cure of the disorder and encompasses embodiments that reduce symptoms or underlying risk factors. In some embodiments, "treatment" refers to both therapeutic and prophylactic means or prophylactic means. Subjects in need of treatment include those already affected by a disease or disorder or undesirable physiological condition, as well as those in whom a disease or disorder or undesirable physiological condition is to be prevented. As used herein, the term "prevention" refers to any activity that reduces the burden on an individual in whom those symptoms will later develop. This can be done at primary, secondary, and / or tertiary prevention levels, where a) primary prevention avoids the onset of symptoms / disorders / conditions, b) secondary prevention activities target the initial stages of state / disorder / symptom treatment, thereby increasing the opportunity for intervention to prevent the progression of the state / disorder / symptom and the appearance of symptoms, and c) tertiary prevention reduces the adverse effects of an already established state / disorder / symptom, for example, by restoring function and / or reducing any state / disorder / symptom or associated complications. The term "prevent" does not require the elimination of 100% of the likelihood of an event. Rather, it indicates that the likelihood of the occurrence of an event has been reduced in the presence of a compound or method.

[0039] As used herein, the term "oxidative stress" shall be given its ordinary meaning and shall also refer to an imbalance between the generation of reactive oxygen species, reactive nitrogen species, and / or free radicals and the antioxidant capacity of a biological system.

[0040] As used herein, the term "reductive stress" shall be given its ordinary meaning and shall also refer to NAD + / NADH, NADP + It shall refer to the response to a state that shifts the redox balance of important biological redox pairs such as / NADPH and GSH / GSSG to a more reduced state. In some embodiments, the reduction stress is equivalent oxidation stress.

[0041] As used herein, the term "effective amount" refers to an amount sufficient to produce a beneficial or desirable biological and / or clinical result.

[0042] The methods, compositions, systems, and kits provided herein can be used in combination with the methods, compositions, systems, and kits described in PCT Patent Application Publication Nos. WO2018 / 092839, WO2017 / 090763, WO2020 / 230601, WO2019 / 164003, WO2020 / 054824, WO2020 / 203961, WO2020 / 054829, WO2021 / 015298, and WO2021 / 132735, the entire contents of which are incorporated herein by reference in some embodiments. Redox-regulated mitochondria

[0043] In some embodiments, a method for reducing or preventing oxidation stress and / or reduction stress in a redox-sensitive composition is provided. In some embodiments, the method includes contacting the redox-sensitive composition with an effective amount of a redox-regulating composition, thereby reducing or preventing oxidation stress and / or reduction stress in the redox-sensitive composition.

[0044] In some embodiments, a method for reducing the level of reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) in a redox-sensitive composition is provided. In some embodiments, the method comprises contacting the redox-sensitive composition with an effective amount of a redox-modulating composition, thereby reducing the level of ROS and / or RNS in the redox-sensitive composition. In some embodiments, the level of ROS and / or RNS in the redox-sensitive composition is reduced, thereby reducing or preventing oxidative stress and / or reductive stress in the redox-sensitive composition.

[0045] The contacting step can include incubating a mixture of the redox-sensitive composition and the redox-modulating composition for less than about 30 seconds (e.g., 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 1 second, 1 millisecond, or a number or range between any of these values). The redox-sensitive composition can be one that is undergoing oxidative stress and / or reductive stress or is at risk of undergoing oxidative stress and / or reductive stress. The redox-sensitive composition can include one or more cells. The contacting step can include introducing the redox-modulating composition into the one or more cells. The one or more cells can be cells of a subject. The one or more cells can be undergoing hypoxia or be at risk of undergoing hypoxia. The contacting can be performed ex vivo, in vitro, or in vivo.

[0046] The redox-sensitive composition can contain a biological sample. The biological sample can be selected from the group consisting of a soil sample, an air sample, an environmental sample, a cell culture sample, a bone marrow sample, a rainfall sample, a fallout sample, a cosmic sample, an extraterrestrial sample, a sewage sample, a groundwater sample, a wear sample, an archaeological sample, a food sample, a blood sample, a serum sample, a plasma sample, a urine sample, a fecal sample, a semen sample, a lymph fluid sample, a cerebrospinal fluid sample, a nasopharyngeal wash sample, a sputum sample, an oral swab sample, a pharyngeal swab sample, a nasal swab fluid sample, a bronchoalveolar lavage sample, a bronchial secretion sample, a milk sample, an amniotic fluid sample, a biopsy sample, a nail sample, a hair sample, a skin sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a cell lysate sample, a virus culture sample, a forensic sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biomolecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, a nucleotide solution, a polynucleotide solution, a nucleic acid solution, a peptide solution, a polypeptide solution, an amino acid solution, a protein solution, a synthetic polymer solution, a biochemical composition solution, an organic chemical composition solution, an inorganic chemical composition solution, a lipid solution, a carbohydrate solution, a combinatorial chemistry product solution, a drug candidate molecule solution, a drug molecule solution, a drug metabolite solution, a cell suspension, a virus suspension, a microorganism suspension, a metal suspension, a metal alloy suspension, a metal ion solution, and any combination thereof.

[0047] In some embodiments, a method of reducing or preventing oxidative stress and / or reductive stress in a subject is provided. In some embodiments, the method comprises administering to the subject an effective amount of a redox-regulating composition, thereby reducing or preventing oxidative stress and / or reductive stress in the subject.

[0048] In some embodiments, a method of treating or preventing a redox disease or disorder in a subject is provided. In some embodiments, the method comprises administering to the subject an effective amount of a redox-regulating composition, thereby treating or preventing a redox disease or disorder in the subject.

[0049] The redox-regulating composition can include a population of organelle complexes. The population of organelle complexes can include a first organelle complex, or a combination of a first organelle complex and a second organelle complex. The redox-regulating composition can include an isolated organelle complex. The redox-regulating composition can include homogenized mitochondria, a first organelle complex, and / or a second organelle complex. The organelle complex can include mitochondria and one or more of the endoplasmic reticulum, peroxisome, lysosome, and Golgi apparatus. The first organelle complex and / or the second organelle complex can be depleted of cytoplasmic macromolecules. The redox-regulating composition can include isolated mitochondria. The redox-regulating composition can include mitochondria isolated from intact cells and / or floating or frozen cells, or a combination thereof. In some embodiments, the redox-regulating composition does not include intact cells. The organelle complexes provided herein (e.g., the first organelle complex, the second organelle complex) can include mitochondria and one, two, three, or four of the endoplasmic reticulum, peroxisome, lysosome, and Golgi apparatus.Organelle complexes (e.g., a first organelle complex, a second organelle complex) can include (i) mitochondria and endoplasmic reticulum, (ii) mitochondria and peroxisomes, (iii) mitochondria and lysosomes, (iv) mitochondria and Golgi apparatus, (v) mitochondria, endoplasmic reticulum, and peroxisomes, (vi) mitochondria, endoplasmic reticulum, and lysosomes, (vii) mitochondria, endoplasmic reticulum, and Golgi apparatus, (viii) mitochondria, endoplasmic reticulum, peroxisomes, and lysosomes, (ix) mitochondria, endoplasmic reticulum, peroxisomes, and Golgi apparatus, (x) mitochondria, endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus, (xi) mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus, (xii) mitochondria, peroxisomes, and lysosomes, (xiii) mitochondria, peroxisomes, and Golgi apparatus, (xiv) mitochondria, peroxisomes, lysosomes, and Golgi apparatus, and / or (xv) mitochondria, lysosomes, and Golgi apparatus. The ratio of mitochondria to additional organelles (e.g., endoplasmic reticulum, peroxisomes, lysosomes, and / or Golgi apparatus) within a population of organelle complexes can vary.

[0050] This specification discloses a method for generating a first population of organellar complexes. In some embodiments, the method includes incubating cells in a first solution containing a surfactant at a first temperature, removing the surfactant to form a second solution, and recovering the first organellar complex from the second solution. The first organellar complex can be derived from (i) frozen cells, (ii) floating cells, and / or (iii) cells that have been contacted with a surfactant at a concentration above the critical micelle concentration (CMC) of the surfactant. In some embodiments, a second organellar complex is provided. In some embodiments, the method for isolating a second organellar complex from cells includes treating the cells in a first solution having a surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant, removing the surfactant to form a second solution, incubating the cells in the second solution, and recovering the second organellar complex from the second solution. The second organellar complex can be derived from (i) adherent cells and / or (ii) cells that have been contacted with the surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant. The population of organellar complexes can be derived from cells treated with a mitochondrial activator (e.g., resveratrol). The organellar complex can deplete cytoplasmic macromolecules. The cytoplasmic macromolecules can be absent from the population of organellar complexes provided herein. The population of organellar complexes provided herein (e.g., the first organellar complex, the second organellar complex) can contain a negligible amount and / or an undetectable amount of cytoplasmic macromolecules. The redox-regulating composition can contain a substantially pure population of organellar complexes. The substantially pure population of organellar complexes can contain less than about 20% (e.g., about 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0%, or a range less than between any two of the values) of cytoplasmic macromolecules. The cytoplasmic macromolecules can include cytoplasmic proteins (e.g., p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)).The first organelle complex and the second organelle complex can be derived from cells treated with a mitochondrial activator. The homogenized mitochondria, the first organelle complex, and / or the second organelle complex can be encapsulated in lipid membrane-based vesicles. The method of encapsulating in lipid membrane-based vesicles is disclosed in PCT Patent Application Publication No. WO2021 / 132735, the entire content of which is incorporated herein by reference. Incubating the cells in the first solution and / or incubating the second solution can include applying physical stimuli, such as pipetting, shaking, and / or stirring, to the first solution and / or the second solution, respectively. Applying physical stimuli to the first solution and / or the second solution can include flowing the first solution and / or the second solution through a flow device (e.g., a reducer flow device). The flow device can include a fluid channel including two or more segments of various cross-sectional diameters. Recovering the first organelle complex from the second solution can include tangential flow filtration (TFF). The systems, methods, compositions, and kits provided herein can, in some embodiments, be used in combination with the systems, methods, compositions, and kits for generating the first organelle complex described in PCT Patent Application No. PCT / US23 / 27014, entitled "ORGANELLE COMPLEXES", filed on July 6, 2023, the entire content of which is incorporated herein by reference.

[0051] The method can include a redox-sensitive composition of at least about 5 μg / mL to about 5 mg / mL. The effective amount can include a redox-regulating composition of at least about 1 μg to about 1 mg. The effective amount can include a redox-regulating composition of at least about 5 μg / mL to about 5 mg / mL. The amount of the redox-sensitive composition and / or the effective amount of the redox-regulating composition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000 μg, mg, μg / mL, and / or mg / mL, or a number or range between any two of these values, and can be of the order of those numbers, or can be up to those numbers.

[0052] Oxidative stress and / or reductive stress can include an increase in the levels of ROS, RNS, and / or free radicals. Oxidative stress and / or reductive stress can include changes in cellular function. Oxidative stress and / or reductive stress can be associated with redox diseases or disorders. In some embodiments, the redox modulating composition reduces the levels of one or more ROS and / or one or more RNS in the subject or the redox-sensitive composition by at least about 5% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, or more and overlapping ranges therein). In some embodiments, the redox modulating composition reduces the level of oxidative cellular stress, thereby restoring or recovering cellular function.

[0053] The redox modulating composition can have ROS scavenging activity and / or RNS scavenging activity. In some embodiments, the redox modulating composition reduces or prevents ROS production and / or RNS production in a subject. The redox modulating composition can have superoxide dismutase activity, catalase activity, peroxidase activity, or any combination thereof. The redox modulating composition can have free radical scavenging activity and / or can inhibit free radical formation.

[0054] In some embodiments, the redox modulating composition increases and / or reduces in a subject or a redox-sensitive composition, one or more of the ratios of oxidized to reduced nicotinamide adenine dinucleotide (NAD+ / NADH), oxidized to reduced nicotinamide adenine dinucleotide phosphate (NADP+ / NADPH), oxidized to reduced glutathione (GSSG / GSH), and oxidized to reduced thioredoxin (TrxSS / TrxSH2) (by at least about 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values).

[0055] Reactive nitrogen species can include nitric oxide (NO), nitrogen dioxide radical (.NO2), peroxynitrite anion (ONOO - ), peroxynitrous acid (ONOOH), nitrosoperoxycarbonate anion (ONOOCO2 - ), nitronium cation (NO2 + ), nitrosyl cation (NO + ), or dinitrogen trioxide (N2O3), or any combination thereof. Reactive oxygen species can include superoxide (O2. - ), hydroperoxy (HO.2), hydrogen peroxide (H2O2), peroxynitrous acid (ONOO - ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO.), peroxyl radical (ROO.), alkoxyl radical (RO.), singlet oxygen ( 1 O2), lipid peroxides, lipid peroxyl radicals or lipid alkoxyl radicals, or any combination thereof. ROS can be derived from neutrophils and / or xanthine oxidase (XO).

[0056] The redox-regulating composition can protect against ROS-induced functional changes through antioxidant and / or reducing effects. The redox-regulating composition can be an antioxidant composition, a reducing composition, or a combination thereof. The redox-sensitive composition can be an oxidant-sensitive composition, a reductant-sensitive composition, or a combination thereof. The protection mediated by the redox-regulating composition provided herein can be in an intracellular, extracellular, and / or cell-free environment. The redox-regulating composition can be used to adjust the lactate (lactate) level, pyruvate (pyruvate) level, lactate / pyruvate ratio, total, reduced or oxidized glutathione level or reduced / oxidized glutathione ratio, total, reduced or oxidized cysteine level or reduced / oxidized cysteine ratio, phosphocreatine level, NADH (NADH+H + ) level, NADPH (NADPH+H +)One or more energy biomarkers selected from the group consisting of levels of lactate, NAD level, NADP level, ATP level, reduced coenzyme Q (CoQred) level, oxidized coenzyme Q (CoQox) level, total coenzyme Q (CoQtot) level, oxidized cytochrome C level, reduced cytochrome C level, oxidized cytochrome C / reduced cytochrome C ratio, acetoacetate level, b-hydroxybutyrate level, acetoacetate / b-hydroxybutyrate ratio, 8-hydroxy-2'-deoxyguanosine (8-OHdG) level, reactive oxygen species level, oxygen consumption (V02) level, carbon dioxide release (VC02) level, respiratory quotient (VC02 / V02), exercise tolerance, anaerobic threshold, and any combination thereof can be improved (by at least about 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values). The redox regulatory compositions provided herein can increase lactate and / or ATP production in a dose-dependent manner (by at least about 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values). In some embodiments, without being bound by any particular theory, the redox regulatory composition regulates the ratio of reduced (NADH) nicotinamide adenine dinucleotide to oxidized (NAD + ) nicotinamide adenine dinucleotide. In some embodiments, without being bound by any particular theory, the redox regulatory composition regulates the ratio of reduced (NADPH) nicotinamide adenine dinucleotide phosphate to oxidized (NADP + ) nicotinamide adenine dinucleotide phosphate. NAD + / NADH can be involved in redox reactions in energy metabolism and mitochondrial function. NADH / NADPH can be involved in ROS metabolism as an electron donor. Intracellular NAD +Various methods for measuring are known in the art and include LC-MS / MS, HPLC, NMR, MS imaging, in situ genetically encoded sensors, MRI assays, lysate-based approaches, and other methods such as those provided in Cambronne XA, Kraus WL. Trends Biochem Sci. 2020 Oct;45(10):858-873, the entire contents of which are incorporated herein by reference. The redox regulatory composition can exert a reducing effect on the redox-sensitive composition and / or the cells of the subject. The redox regulatory composition can reduce the production of RNS and / or ROS in the redox-sensitive composition or the subject. The redox regulatory composition can increase the removal of RNS and / or ROS in the redox-sensitive composition or the subject. In some embodiments, the redox regulatory composition comprising the first organelle complex reduces and / or increases the production of RNS and / or ROS in the redox-sensitive composition or the subject by at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) compared to the redox regulatory composition comprising the second organelle complex. The redox regulatory composition provided herein can affect the redox system and thereby result in an increase in survival rate (e.g., via antioxidant and / or anti-reducing effects). In some embodiments, the excessive or lethal oxidative stress is a reducing stress, and in some such embodiments, not only the reducing action by the antioxidant action but also the oxidative action by the anti-reducing action is required. The disclosed redox regulatory composition can be an antioxidant stress agent and an anti-reducing stress agent, e.g., a REDOX enhancer. The reducing stress can include a high lactate / pyruvate ratio. The redox regulatory composition provided herein can act by reducing the level of excessive reducing equivalents.In some embodiments, without being bound by any particular theory, the redox regulatory compositions disclosed herein provide for the elimination of redox stress and can reduce redox stress by oxidizing NADH to NAD+, which can result in antioxidant activity, ATP-generating capacity, and / or cell proliferation capacity. In some embodiments, without being bound by any particular theory, the redox regulatory compositions provided herein are NAD. + / NADH, NADP + / NADPH, and at least one of the ratios of GSH / GSSG by at least 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) to regulate (e.g., increase and / or decrease), thereby reducing redox stress. In some embodiments, without being bound by any particular theory, oxidized NAD + regulates the NADPH-linked redox system and / or acts as a signaling molecule for homeostasis. The redox regulatory compositions provided herein (e.g., organellar complexes) can be associated with redox systems (including those involved in ROS generation and / or ROS clearance) such as, for example, peroxisomes, mitochondria, endosomes (e.g., plasma membrane endosomes), and the endoplasmic reticulum. In some embodiments, the organellar complex contains more NAD + . In some embodiments, NAD + is the active ingredient of the redox regulatory compositions provided herein. In some embodiments, the first organellar complex contains at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) more NAD + than the second organellar complex. In some embodiments, NAD +Removing it reduces at least a portion of the effects exerted by the redox modulating composition.

[0057] In embodiments, the organelle complex population may treat, prevent, ameliorate, and / or improve the clinical condition due to ischemia-reperfusion injury. In embodiments, the organelle complex population may improve the ejection fraction (EF), inhibit cardiac hypertrophy, and / or treat, prevent, ameliorate, and / or improve fibrosis after ischemia-reperfusion injury. The redox modulating composition can improve one or more cardiac function indices in a subject selected from the group consisting of cardiac output, ejection fraction, volume, stroke volume, pressure, end-diastolic volume (EDV), and end-systolic volume (ESV) (by at least about 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values). Pharmaceutically acceptable compositions and methods of administration

[0058] In some embodiments, the redox modulating composition is administered to a subject in combination with one or more additional agents and / or additional therapies designed to treat a disease or disorder. In some embodiments, the present disclosure provides methods for treating diseases and disorders associated with mitochondrial dysfunction, or diseases or disorders that would benefit from replenishment of healthy, functional mitochondria. The present disclosure also provides the use of a redox modulating composition in the manufacture of a medicament for treating the diseases and disorders provided herein.

[0059] Administration can include intravenous administration, intra-arterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and / or intraocular administration. The redox modulating composition can be administered locally or systemically.

[0060] As used herein, the terms “local administration” or “topical administration” refer to any route of administration by which a redox modulating composition contacts an individual's body such that the location of the resulting redox modulating composition in the body is topical (i.e., limited to a particular tissue, organ, or other body part where imaging is desired). Exemplary local administration routes include injection into a particular tissue using a needle, enteral feeding into the gastrointestinal tract, and spreading a solution containing the redox modulating composition onto the skin surface.

[0061] As used herein, the term “systemic administration” refers to any route of administration by which a redox modulating composition contacts an individual's body such that the location of the resulting redox modulating composition in the body is systemic (i.e., not limited to a particular tissue, organ, or other body part where imaging is desired). Systemic administration includes enteral and parenteral administration. Enteral administration is a systemic route of administration by which a substance is administered via the gastrointestinal tract and includes, but is not limited to, oral administration, administration by gastrostomy tube, administration by duodenal tube, gastric fistula, enteral nutrition, and rectal administration. Parenteral administration is a systemic route of administration by which a substance is administered via a route other than the gastrointestinal tract and includes, but is not limited to, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intravesical instillation.

[0062] In another aspect, this disclosure provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of a redox modulating composition disclosed herein. As described in detail below, the pharmaceutical compositions of this disclosure can be administered orally, for example, as a drip (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste, (2) parenterally, for example, as a sterile solution or suspension, for example, by subcutaneous, intramuscular or intravenous injection, (3) topically, for example, as a cream, ointment or spray applied to the skin, (4) intravaginally or rectally, for example, as a pessary, cream or foam, or (5) for administration in solid or liquid form, including dosage forms suitable for, for example, aqueous aerosols, liposome preparations or aerosols as solid particles containing the redox modulating composition. The pharmaceutical composition can include one or more pharmaceutically acceptable carriers. As used herein, the phrase "therapeutically effective amount" can refer to an amount of the redox modulating composition disclosed herein that is effective, at a reasonable benefit / risk ratio, to produce some desired therapeutic effect, for example, cancer treatment.

[0063] As used herein, the phrase "pharmaceutically acceptable" refers to those agents, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, in contact with human and animal tissue, are suitable for use without excessive toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0064] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a subject chemical substance from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can act as pharmaceutically acceptable carriers are: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0065] Formulations useful in the methods of this disclosure include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral administration. The formulations may, for convenience, be in unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient (e.g., redox modulating composition) that can be combined with carrier materials to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of redox modulating composition that achieves a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0066] Suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof, in addition to the active agent.

[0067] Dosage forms for topical or transdermal administration of redox modulating compositions include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants as may be required.

[0068] Ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0069] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also considered to be within the scope of this disclosure.

[0070] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0071] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenolsorbic acid, etc. It may also be desirable to include in the composition isotonic agents such as sugars, sodium chloride, etc. Furthermore, the prolonged absorption of injectable pharmaceutical forms may be caused by including agents that delay absorption such as aluminum monostearate and gelatin.

[0072] The actual dosage level of the active ingredient in the pharmaceutical compositions of this disclosure can be determined by the methods of this disclosure in order to obtain an amount of the active ingredient that is non-toxic to the subject and effective to achieve the desired treatment response for a particular subject, composition, and mode of administration.

[0073] The subject can be or can have a risk of being subjected to oxidative stress and / or reductive stress, such as oxidative stress and / or reductive stress caused by ischemia-reperfusion injury or redox diseases or disorders. In some embodiments, the method includes identifying a subject that is or has a risk of being subjected to oxidative stress and / or reductive stress. In some embodiments, the method includes measuring the level of RNS in the subject or a redox-sensitive composition using a probe that detects reactive nitrogen species, and / or measuring the level of ROS in the subject or a redox-sensitive composition using a probe that detects reactive oxygen species. Probes that detect reactive nitrogen species can be diaminonaphthalene, diaminofluorescein, diaminorhodamine, diaminocyanine, NiSPY, dichlorodiaminocarboxyfluorescein, 1,2-diaminoanthraquinone, or DAMBO-P H and the like. Probes that detect reactive oxygen species can be 2’,7’-dichlorofluorescein diacetate, dihydrorhodamine 123, 3’-(p-aminophenyl) fluorescein (API), 3’-(p-hydroxyphenyl) fluorescein (HPF), aminophenoxycarboxyfluorescein (APC), mitoAR, mitoHR, DPAX, DMAX, hydrocyanine, or dihydroethidium

[0074] The subject can have, or be suspected of having, a disease or condition involving ROS selected from the group consisting of atherosclerosis, heart disease, heart failure, hypertension, sepsis, diabetes, Alzheimer's disease, Parkinson's disease, toxin-induced parkinsonism, Huntington's disease, Wilson's disease, Friedreich's ataxia, Kearns-Sayre syndrome, Leigh syndrome, Leber's hereditary optic neuropathy, mitochondrial myopathy, cardiomyopathy, hearing loss, mood disorder, movement disorder, dementia, amyotrophic lateral sclerosis, multiple sclerosis, tardive dyskinesia, brain injury, schizophrenia, epilepsy, AIDS dementia, endothelial nitroglycerin resistance, adriamycin toxicity, kidney injury in type I diabetes, ex vivo kidney preservation, stroke, ischemia-reperfusion injury, ischemia-reperfusion injury, chronic inflammation, cocaine toxicity, alcoholic fatty liver disease, fatty liver disease, liver inflammation in hepatitis C virus patients, neuroprotection, immobilization-induced muscle atrophy, skeletal muscle burn, cancer, inflammation and ischemia-reperfusion injury in stroke, heart attack, UV damage to the skin, organ transplantation, surgery, and any combination thereof.

[0075] In some embodiments, administering the redox modulating composition to the subject treats, reduces, or prevents ischemia-reperfusion injury in the subject. Ischemia-reperfusion injury can be caused by mitochondrial dysfunction, hypoxia injury, HMGB1 release, or necrotic cell death. In some embodiments, the redox modulating composition suppresses ischemia-reperfusion injury, mitochondrial dysfunction, hypoxia injury, necrotic cell death, or any combination thereof.

[0076] An oxidative-reductive disease or disorder can include ROS-mediated oxidative damage and / or RNS-mediated oxidative damage to one or more tissues of a subject. Oxidative-reductive diseases or disorders include mitochondrial disorders, hereditary mitochondrial diseases, Alpers' disease, Barth syndrome, beta-oxidation deficiency, carnitine-acyl-carnitine deficiency, carnitine deficiency, creatine deficiency syndrome, coenzyme Q10 deficiency, complex I deficiency, complex II deficiency, complex III deficiency, complex IV deficiency, complex V deficiency, COX deficiency, chronic progressive external ophthalmoplegia (CPEO), CPT I deficiency, CPT II deficiency, Friedreich's ataxia (FA), glutaric aciduria type II, Kearns-Sayre syndrome (KSS), lactic acidosis, long-chain acyl-CoA dehydrogenase deficiency (LCAD), LCHAD, Leigh syndrome, Leigh-like syndrome, Leber's hereditary optic neuropathy (LHON), lethal infantile cardiomyopathy (LIC), Luft disease, multiple acyl-CoA dehydrogenase deficiency (MAD), medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial myopathy, encephalopathy, lactic acidosis, stroke (MELAS), myoclonic epilepsy with ragged-red fibers (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, myoneurogastrointestinal encephalopathy (MNGIE), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, respiratory chain disorders, short-chain acyl-CoA dehydrogenase deficiency (SCAD), SCHAD, very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), myopathy, cardiomyopathy, encephalomyopathy, neurodegenerative diseases, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), motor neuron diseases, nerve diseases, epilepsy, age-related diseases, macular degeneration, diabetes, metabolic syndrome, brain tumors, genetic diseases, Huntington's disease, mood disorders, schizophrenia, bipolar disorder, pervasive developmental disorders, autism, Asperger's syndrome, childhood disintegrative disorder (CDD), Rett syndrome, PDD-NOS, cerebrovascular disorders, stroke, visual disorders, optic neuropathy, dominant hereditary juvenile optic atrophy, optic neuropathy due to toxic substances, glaucoma, Stargardt macular dystrophy, diabetic retinopathy, diabetic maculopathy,Retinopathy of prematurity, ischemia-reperfusion related retinal disorders, oxygen toxicity, hemoglobinopathy, thalassemia, sickle cell anemia, epileptic seizures, ischemia, renal tubular acidosis, attention deficit / hyperactivity disorder (ADHD), neurodegenerative diseases causing hearing or balance disorders, dominant optic atrophy (DOA), maternally inherited diabetes and deafness (MIDD), chronic fatigue, contrast-induced nephropathy, contrast-induced retinopathy disorder, abetalipoproteinemia, retinitis pigmentosa, Wolfram disease, Tourette syndrome, cobalamin c deficiency, methylmalonic aciduria, glioblastoma, Down syndrome, acute tubular necrosis, muscular dystrophy, leukodystrophy, progressive supranuclear palsy, spinal muscular atrophy, deafness, noise-induced deafness, traumatic brain injury, juvenile Huntington disease, multiple sclerosis, NGLY1, multiple system atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, and any combination thereof.

[0077] This specification also provides a kit comprising one or more of the compositions described herein (e.g., a formulation comprising a redox-regulating composition) in suitable packaging, and may further comprise written materials that can include instructions for use, conclusions of clinical studies, lists of side effects, etc. Such kits may also include information such as references to scientific literature, package insert materials, clinical trial results, and / or summaries thereof, that indicate and / or establish the activity and / or advantages of the composition, and / or describe dosage, administration, side effects, drug interactions, or other information useful to a healthcare provider. Such information may be based on the results of various studies, for example, studies using experimental animals including in vivo models and studies based on human clinical trials. The kit may include one or more unit doses as described herein.

Examples

[0078] Some aspects of the above-described embodiments are disclosed in more detail in the following examples and are in no way intended to limit the scope of the present disclosure. Example 1 Effect of the second organelle complex on the expulsion rate in pigs with ischemia-reperfusion injury

[0079] The effect of the second organelle complex on the ejection fraction in pigs with ischemia-reperfusion injury was examined. Ischemia was induced by balloon inflation placed at the D1 level in the LAD. The second organelle complex was given from the LAD periphery to the inflation site immediately before reperfusion. Figures 1A-1B show data related to the effect of the second organelle complex (2 nd OC) on the ejection fraction (EF) in pigs with ischemia-reperfusion injury. Figure 1A shows the change in EF (%) (control, N = 5-3, 2 died 1 day before, 250 μg of the second organelle complex, N = 4). Figure 1B shows the rate of change from pre-ischemic pretreatment for 15 minutes after reperfusion (control, N = 5, 250 μg of the second organelle complex, N = 4). The data are represented as mean ± SEM. The generation of ROS and RNS was involved in ischemia-reperfusion injury (see Int. J. Mol. Sci. 2018, 19, 417; doi:10.3390 / ijms19020417, which is incorporated herein by reference). These data demonstrate the redox regulatory effect of the second organelle complex disclosed herein. Example 2 Anti-ROS activity of the redox regulatory composition

[0080] The luminol assay was used for the detection of H2O2 by chemiluminescence (CL) (see React Oxyg Species (Apex). 2016 May;1(3):216-227.doi:10.20455 / ros.2016.841, which is incorporated herein by reference). In the presence of horseradish peroxidase (HRP), luminol can react with hydrogen peroxide and finally form an excited state of 3-aminophthalate (3-APA * ). The decay to a lower energy level results in photon emission that can be photometrically measured as a chemiluminescence (CL) response. Figure 2A shows the experimental setup, and Figure 2B shows the second organelle complex (2 * OC). ndThe results of a luminol assay comparing the anti-ROS activity between the second organellar complex (2 nd OC) derived from HeLa and homogenized mitochondria (H-mito) are shown. The calculated IC50 values are 153.4 μg / ml for the second organellar complex (2 st OC), 166.9 μg / ml for the first organellar complex (1 st OC), and 167.4 μg / ml for homogenized mitochondria. These data demonstrate the anti-ROS effects of the various redox-modulating compositions provided herein. Example 3 Anti-RNS activity of redox-modulating compositions

[0081] A nitric oxide detection assay was performed using the NOC7-DAF-2 system. NOC7 was used as a nitric oxide donor and DAF-2 was used as a nitric oxide detector. Figure 3A shows the experimental setup, and Figure 3B shows data related to 0.5 μM NOC7 by a DAF-2 (1 μM) assay. Figure 3B shows the results of an NOC7-DAF-2 assay using 0.5 M NOC7 and increasing the dose of DAF-2. Anti-RNS activity was demonstrated in three different populations including mitochondria (the second organellar complex (2 nd OC), the first organellar complex (1 st OC), and homogenized mitochondria (H-mito)) against the control (Tris / Suc). The populations shown were 100 μg / mL (Figure 3B), 25 μg / mL (not shown), and 6.25 μg / mL (not shown). These data demonstrate the dose-dependent anti-RNS effects of the various redox-modulating compositions provided herein. Example 4 Cell-based assays of redox-modulating compositions

[0082] Cell-based assays were performed to examine the effect of the redox-modulating compositions described herein on cells. Figures 4A-4B show the experimental setup (Figure 4A) and data related to a cell-based assay examining the in vitro redox-modulating activity of the compositions provided herein (Figure 4B). Figure 4B shows the second organellar complex (2nd The results of the assay (performed 3 times) using OC are shown. The second organellar complex (derived from HeLa cells) was found to suppress tBHP-induced OS in a dose-dependent manner.

[0083] Next, the CellTiter-Glo® 2.0 assay was performed to examine the effect of the redox-regulating composition described herein on HUEhT2 and Raw264.7 cells (data not shown). The CellTiter-Glo® 2.0 assay can determine the number of viable cells in culture by quantifying ATP, which can indicate the presence of metabolically active cells. The luminescence readout can be directly proportional to the number of viable cells in culture. Figures 5A - 5B show the experimental setup (Figure 5A) and data (Figure 5B) related to the CellTiter-Glo® 2.0 assay examining the in vitro redox-regulating activity of the composition provided herein. Figure 5B shows the rescue of HUEhT2 cells by the second organellar complex (2 nd OC, derived from 293T cells). These data demonstrate the in vitro redox-regulating effect of the redox-regulating composition provided herein. Example 5 Cell viability assay

[0084] A cell viability assay was performed to evaluate the effect of the disclosed redox-regulating composition. Figure 6A shows the experimental setup, and Figure 6B shows data related to the cell viability assay examining the redox-regulating composition provided herein. The first organellar complex (1 st OC) derived from HeLa cells improves cell viability at 2 and 4 hours (not shown) after contact with H2O2. The first organellar complex (derived from HeLa cells) was found to improve cell viability in a dose-dependent manner at both time points. Example 6 NAD + / NADH assay

[0085] NAD + / The NADH assay was performed to examine the effect of the first organellar complex on cells. Figure 7A shows the experimental setup, and Figure 7B shows the data related to the NAD + / NADH assay. The first organellar complex (1 st OC) derived from HeLa cells changes the ratio of NAD + / NADH at 2 and 4 hours (not shown) after contact with H2O2. The first organellar complex (derived from HeLa cells) was found to increase the ratio of NAD + / NADH in a dose-dependent manner at both time points. In some embodiments, without being bound by any particular theory, the redox-regulating compositions disclosed herein provide elimination of redox stress and can reduce redox stress by oxidizing NADH to NAD + , which can result in antioxidant activity, ATP-generating capacity, and / or cell proliferation capacity. Example 7 Comparison of the First Organellar Complex and the Second Organellar Complex

[0086] A cell-based assay was performed to compare the effects of the first organellar complex and the second organellar complex on viability. Figure 8A shows the experimental setup, and Figure 8B shows the first organellar complex (1 st OC) and the second organellar complex (2 ndData related to the CellTiter-Glo® 2.0 assay comparing (OC) are shown. Results of the first and second organellar complexes on cell viability (Figure 8B) are shown as ratios to PBS-treated control cells. The first organellar complex (derived from HeLa cells) was found to have a stronger (dose-dependently) anti-reducing stress effect than the second organellar complex. Compositions of the first and second organellar complexes were compared taking these differences into account to elucidate the active ingredients of the organellar complexes provided herein with respect to anti-reducing stress indication. Figure 9 shows non-limiting exemplary data related to the characterization of the organellar complex population provided herein. This is the first organellar complex (1 st OC) and the second organellar complex (2 nd OC) intracellular structure / organellar western blot protein analysis. Example 8 Elucidation of the role of GSH and catalase in the anti-ROS activity of the first organellar complex

[0087] Cell-based assays were performed to examine the role of glutathione (GSH) and catalase in the anti-ROS activity of the first organellar complex. Buthionine sulfoximine (BSO) was used to reduce GSH levels in the first organellar complex. Figure 10A shows the GSH concentration in the first organellar complex derived from HEK293 cells that were either in contact with BSO (BSO-1 st OC) or not in contact (293-1 st OC). ON cultures of HEK293 cells were incubated with H2O2 (100 μM) and 293-1 st OC or BSO-1 stIncubated with either OC. Cell viability at 4 hours and 20 hours after H2O2 treatment was determined by CellTiter-Glo (CTG2.0). The results showed that GSH in the first organellar complex is not an important factor in anti-ROS activity (Figures 10B - 10C). Next, the ROS scavenging activity of the first organellar complex with catalase depletion was evaluated. HEK293 cells were transfected with catalase siRNA (h) (Santa cruz Biotechnology (SCB); sc-45330; lot# B2423) using a common transfection reagent for either 24 hours or 48 hours, and the expression level of catalase in the whole cell lysate was evaluated by Western blotting analysis (Figure 11A). Figure 11B shows the catalase levels in untreated first organellar complex (1 st OC) or first organellar complex with catalase depletion (1 st OC-siRNA). It was confirmed that catalase in the first organellar complex was successfully knocked down, and the ROS scavenging activity of the first organellar complex with catalase depletion (1 st OC-siRNA) was evaluated. HEK293 cells were pretreated with either the first organellar complex (293-1 st OC) or the first organellar complex with catalase depletion (293-1 st OC-siRNA) for 6 hours, followed by treatment with H2O2 (300 μM) for 18 hours. Cell viability was measured by CellTiter-Glo (CTG2.0). It was found that knockdown of catalase in the first organellar complex significantly decreased its ROS scavenging activity statistically (P < 0.005) (Figure 11C). These results provided in this example show that depletion of GSH in the first organellar complex by BSO has no effect on its anti-ROS activity, while the presence of catalase in the organellar complex (e.g., the first organellar complex, the second organellar complex) is an important factor for its ROS scavenging activity.

[0088] In at least some of the foregoing embodiments, one or more of the elements used in the embodiments can also be used interchangeably in other embodiments, provided that such substitution is technically feasible. Those skilled in the art will understand that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to be within the scope of the subject matter defined by the appended claims.

[0089] Regarding the use of substantially any plural and / or singular terms herein, those skilled in the art can convert from plural to singular and / or from singular to plural as appropriate for the context and / or application. Various singular / plural substitutions may also be explicitly described herein for clarity. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any mention of "or" in this specification shall, unless otherwise specified, include "and / or."

[0090] One of ordinary skill in the art will generally understand that terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be open terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "including but not limited to", etc.). One of ordinary skill in the art will further understand that if a specific number is intended in the recitation of an introduced claim, such intent is explicitly recited in the claim, and if there is no such recitation, there is no such intent. For example, for purposes of illustration, the following appended claims may include the use of introductory phrases "at least one" and "one or more" to introduce the recitation of the claim. However, the use of such phrases should not be construed to limit a particular claim that includes such a recitation to embodiments that include only one such recitation, even if the same claim includes an introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"). Also, even if a specific number of an introduced claim recitation is explicitly recited, one of ordinary skill in the art will recognize that such recitation should be interpreted as meaning at least the recited number (e.g., a mere recitation of "two recitations" without other modifiers means at least two recitations, or two or more recitations). Further, when a convention similar to "at least one of A, B, and C, etc." is used, generally such an interpretation is intended in the sense that one of ordinary skill in the art understands the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together).When a convention similar to "at least one of A, B, or C" is used, generally, such an interpretation is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, a system having only A, only B, only C, both A and B together, both A and C together, both B and C together, and / or all of A, B, and C together). One of ordinary skill in the art can further understand that, in any of the specification, claims, and drawings, substantially any disjunctive and / or phrase presenting two or more alternative terms should be understood to contemplate the possibility of including one of the terms, any of the terms, or both terms.

[0091] Further, when a feature or aspect of the present disclosure is described in terms of a Markush group, one of ordinary skill in the art will recognize that the present disclosure is also described with respect to any individual member or subgroup of members of the Markush group.

[0092] As will be understood by one of ordinary skill in the art, for any and all purposes, such as for providing a written description, all ranges disclosed herein also inherently include any and all possible subranges and combinations of those subranges. It is readily recognized and described in sufficient detail to enable any recited range to be divided at least in half, into thirds, into quarters, into fifths, into tenths, etc. of the same range. By way of non-limiting example, each range described herein can be readily subdivided into lower thirds, middle thirds, upper thirds, etc. Also, as one of ordinary skill in the art will understand, all words such as "up to," "at least," "greater than," "less than," etc. include the recited numbers and refer to ranges that can later be subdivided into subranges as described above. Finally, as one of ordinary skill in the art will understand, ranges include each individual member. Thus, for example, a group having from 1 to 3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group having from 1 to 5 articles refers to a group having 1, 2, 3, 4, or 5 articles, etc.

[0093] Although various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are illustrative only and not intended to be limiting, while the true scope and spirit are indicated by the following claims.

Claims

**Claim 1** A method for reducing or preventing oxidative stress and / or reductive stress in a redox-sensitive composition, comprising: contacting the redox-sensitive composition with an effective amount of a redox-regulating composition, thereby reducing or preventing oxidative stress and / or reductive stress in the redox-sensitive composition, wherein the redox-regulating composition comprises an isolated organelle complex, and the organelle complex comprises mitochondria and one or more of endoplasmic reticulum, peroxisome, lysosome, and Golgi apparatus. **Claim 2** A method for reducing the level of reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) in a redox-sensitive composition, comprising: contacting the redox-sensitive composition with an effective amount of a redox-regulating composition, thereby reducing the level of ROS and / or RNS in the redox-sensitive composition, wherein the redox-regulating composition comprises an isolated organelle complex, and the organelle complex comprises mitochondria and one or more of endoplasmic reticulum, peroxisome, lysosome, and Golgi apparatus. **Claim 3** The method according to any one of claims 1 to 2, wherein the contacting step comprises incubating a mixture of the redox-sensitive composition and the redox-regulating composition for less than about 30 seconds. **Claim 4** The method according to any one of claims 1 to 3, wherein the effective amount comprises at least about 5 μg / mL to about 5 mg / mL of the redox-regulating composition. **Claim 5** The method according to any one of claims 1 to 4, wherein the redox-sensitive composition is under oxidative stress and / or reductive stress or at risk of undergoing oxidative stress and / or reductive stress. **Claim 6** The redox-sensitive composition comprises one or more cells. Optionally, the contacting step comprises introducing the redox-regulating composition into the one or more cells. Optionally, the one or more cells are cells of a subject. Optionally, the one or more cells are under hypoxia or at risk of hypoxia. The method according to any one of claims 1 to 5. **Claim 7** The method according to any one of claims 1 to 6, wherein the contacting is performed ex vivo, in vitro, or in vivo. **Claim 8** The method according to any one of claims 1 to 7, wherein the redox-sensitive composition is an oxidant-sensitive composition, a reductant-sensitive composition, or a combination thereof.

9. The method according to any one of claims 1 to 8, wherein the redox-sensitive composition contains a biological sample.

10. The biological sample is a soil sample, an air sample, an environmental sample, a cell culture sample, a bone marrow sample, a rainfall sample, a fallout sample, a cosmic sample, an extraterrestrial sample, a sewage sample, a groundwater sample, a wear sample, an archaeological sample, a food sample, a blood sample, a serum sample, a plasma sample, a urine sample, a fecal sample, a semen sample, a lymph fluid sample, a cerebrospinal fluid sample, a nasopharyngeal wash sample, a sputum sample, an oral swab sample, a pharyngeal swab sample, a nasal swab fluid sample, a bronchoalveolar lavage sample, a bronchial secretion sample, a milk sample, an amniotic fluid sample, a biopsy sample, a nail sample, a hair sample, a skin sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a cell lysate sample, a virus culture sample, a forensic sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biomolecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, a nucleotide solution, a polynucleotide solution, a nucleic acid solution, a peptide solution, a polypeptide solution, an amino acid solution, a protein solution, a synthetic polymer solution, a biochemical composition solution, an organic chemical composition solution, an inorganic chemical composition solution, a lipid solution, a carbohydrate solution, a combinatorial chemistry product solution, a drug candidate molecule solution, a drug molecule solution, a drug metabolite solution, a cell suspension, a virus suspension, a microbial suspension, a metal suspension, a metal alloy suspension, a metal ion solution, and any combination thereof, and is selected from the group consisting of the method according to any one of claims 1 to 9.

11. A method for reducing or preventing oxidative stress and / or reducing stress in a subject, comprising administering to the subject an effective amount of a redox-regulating composition, thereby reducing or preventing oxidative stress and / or reducing stress in the subject, wherein the redox-regulating composition contains an isolated organelle complex, and the organelle complex contains mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus.

12. A method for treating or preventing a redox disease or disorder in a subject, comprising administering to the subject an effective amount of a redox-regulating composition, thereby treating or preventing a redox disease or disorder in the subject. The redox regulation composition includes an isolated organelle complex, and the organelle complex includes mitochondria and one or more of the endoplasmic reticulum, peroxisome, lysosome, and Golgi apparatus.

13. The redox regulation composition includes homogenized mitochondria, a first organelle complex, and / or a second organelle complex. The first organelle complex and the second organelle complex include mitochondria and one or more of the endoplasmic reticulum, peroxisome, lysosome, and Golgi apparatus. The first organelle complex and the second organelle complex have depleted cytoplasmic macromolecules. The first organelle complex is derived from (i) frozen cells, (ii) floating cells, and / or (iii) cells contacted with the surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant. The second organelle complex is derived from (i) adherent cells and / or (ii) cells contacted with the surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant. Optionally, the first organelle complex and the second organelle complex are derived from cells treated with a mitochondrial activator. Further optionally, the homogenized mitochondria, the first organelle complex, and / or the second organelle complex are encapsulated in lipid membrane-based vesicles. The method according to any one of claims 1 to 12.

14. The effective amount includes at least about 1 μg to about 1 mg of the redox regulation composition. The method according to any one of claims 1 to 13.

15. The redox regulation composition does not include intact cells. The method according to any one of claims 1 to 14.

16. The oxidative stress and / or reduction stress includes an increase in the levels of ROS, RNS, and / or free radicals. The method according to any one of claims 1 to 15.

17. The oxidative stress and / or reduction stress includes a change in cell function. The method according to any one of claims 1 to 16.

18. The oxidative stress and / or reduction stress is associated with the redox disease or disorder. The method according to any one of claims 1 to 17.

19. The method according to any one of claims 1 to 18, wherein the redox-regulating composition reduces the level of one or more ROS and / or one or more RNS in the subject or the redox-sensitive composition by at least about 5%.

20. The method according to any one of claims 1 to 19, wherein the redox-regulating composition reduces the level of oxidative cellular stress, thereby restoring or recovering the cellular function.

21. The method according to any one of claims 1 to 20, wherein the redox-regulating composition has ROS scavenging activity and / or RNS scavenging activity.

22. The method according to any one of claims 1 to 21, wherein the redox-regulating composition reduces or prevents ROS production and / or RNS production in the subject.

23. The method according to any one of claims 1 to 22, wherein the redox-regulating composition has superoxide dismutase activity, catalase activity, peroxidase activity, or any combination thereof.

24. The redox regulation composition increases and / or decreases one or more of the ratios of the oxidized form to the reduced form of nicotinamide adenine dinucleotide (NAD ＋ / NADH), the oxidized form to the reduced form of nicotinamide adenine dinucleotide phosphate (NADP ＋ / NADPH), the oxidized form to the reduced form of glutathione (GSSG / GSH), and the oxidized form to the reduced form of thioredoxin (TrxSS / TrxSH ２ ) in the subject or the redox-sensitive composition, according to any one of claims 1 to 23.

25. The reactive nitrogen species is nitric oxide (NO), nitrogen dioxide radical (NO ２ ), peroxynitrite anion (ONOO － ), peroxynitrous acid (ONOOH), nitrosoperoxycarbonate anion (ONOOCO ２ － ), nitronium cation (NO ２ ＋ ), nitrosyl cation (NO ＋ ), or dinitrogen trioxide (N ２ O ３ ), or any combination thereof, according to any one of claims 1 to 24.

26. The reactive oxygen species are superoxide (O ２ . － ), hydroperoxy (HO.), ２ ), hydrogen peroxide (H ２ O ２ ), peroxynitrous acid (ONOO － ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO.), peroxyl radical (ROO.), alkoxyl radical (RO.), singlet oxygen ( １ O ２ ), lipid peroxide, lipid peroxyl radical or lipid alkoxyl radical, or any combination thereof, according to any one of claims 1 to 25.

27. The method according to any one of claims 1 to 26, wherein the redox-regulating composition has free radical scavenging activity and / or inhibits free radical formation.

28. The subject optionally has or is at risk of having oxidative stress and / or reductive stress caused by ischemia-reperfusion injury or the redox disease or disorder, according to any one of claims 1 to 27.

29. The method according to any one of claims 1 to 28, comprising identifying a subject having or at risk of having oxidative stress and / or reductive stress.

30. Measuring the level of RNS in the subject or the redox-sensitive composition using a probe that detects reactive nitrogen species, and / or Measuring the level of ROS in the subject or the redox-sensitive composition using a probe that detects reactive oxygen species, the method according to any one of claims 1 to 29.

31. The probe for detecting reactive nitrogen species is diaminonaphthalene, diaminofluorescein, diaminorhodamine, diaminocyanine, NiSPY, dichlorodiaminocalcein, 1,2-diaminoanthraquinone, or DAMBO-P Ｈ The method according to any one of claims 1 to 30, which is such.

32. The probe for detecting reactive oxygen species is 2′,7′-dichlorofluorescein diacetate, dihydrorhodamine 123, 3′-(p-aminophenyl) fluorescein (APF), 3′-(p-hydroxyphenyl) fluorescein (HPF), aminophenoxychalcein (APC), mitoAR, mitoHR, DPAX, DMX, hydrocyanine, or dihydroethidium, according to the method of any one of claims 1 to 31.

33. The ROS is derived from neutrophils and / or xanthine oxidase (XO), according to the method of any one of claims 1 to 32.

34. The subject has, or is suspected of having, a disease or condition involving ROS selected from the group consisting of atherosclerosis, heart disease, heart failure, hypertension, sepsis, diabetes, Alzheimer's disease, Parkinson's disease, toxin-induced parkinsonism, Huntington's disease, Wilson's disease, Friedreich's ataxia, Kearns-Sayre syndrome, Leigh syndrome, Leber's hereditary optic neuropathy, mitochondrial myopathy, cardiomyopathy, hearing loss, mood disorder, movement disorder, dementia, amyotrophic lateral sclerosis, multiple sclerosis, tardive dyskinesia, brain injury, schizophrenia, epilepsy, AIDS dementia, endothelial nitroglycerin resistance, adriamycin toxicity, kidney injury in type I diabetes, ex vivo kidney preservation, stroke, ischemia-reperfusion injury, ischemia-reperfusion injury, chronic inflammation, cocaine toxicity, alcoholic fatty liver disease, fatty liver disease, liver inflammation in patients with hepatitis C virus, neuroprotection, immobilization-induced muscle atrophy, skeletal muscle burns, cancer, inflammation and ischemia-reperfusion injury in stroke, heart attack, UV damage to the skin, organ transplantation, surgery, and any combination thereof, according to the method of any one of claims 1 to 33.

35. Administering the redox regulatory composition to the subject treats, reduces, or prevents ischemia-reperfusion injury in the subject, according to the method of any one of claims 1 to 34.

36. The ischemia-reperfusion injury is caused by mitochondrial dysfunction, hypoxia injury, HMGB1 release, or necrotic cell death, according to the method of any one of claims 1 to 35.

37. The method according to any one of claims 1 to 36, wherein the redox regulation composition suppresses ischemia-reperfusion injury, mitochondrial dysfunction, hypoxia injury, necrotic cell death, or any combination thereof. **Claim 38** The method according to any one of claims 1 to 37, wherein the redox disease or disorder comprises ROS-mediated oxidative damage and / or RNS-mediated oxidative damage to one or more tissues of the subject. **Claim 39** The redox disease or disorder is mitochondrial disorder, hereditary mitochondrial disease, Alpers' disease, Barth syndrome, beta-oxidation deficiency, carnitine-acyl-carnitine deficiency, carnitine deficiency, creatine deficiency syndrome, coenzyme Q10 deficiency, complex I deficiency, complex II deficiency, complex III deficiency, complex IV deficiency, complex V deficiency, COX deficiency, chronic progressive external ophthalmoplegia (CPEO), CPT I deficiency, CPT II deficiency, Friedreich's ataxia (FA), glutaric aciduria type II, Kearns-Sayre syndrome (KSS), lactic acidosis, long-chain acyl-CoA dehydrogenase deficiency (LCAD), LCHAD, Leigh syndrome, Leigh-like syndrome, Leber's hereditary optic neuropathy (LHON), lethal infantile cardiomyopathy (LIC), Luft disease, multiple acyl-CoA dehydrogenase deficiency (MAD), medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial myopathy, encephalopathy, lactic acidosis, stroke (MELAS), ragged red fiber myoclonic epilepsy (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, myoneurogastrointestinal encephalopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, respiratory chain disorder, short-chain acyl-CoA dehydrogenase deficiency (SCAD), SCHAD, very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), myopathy, cardiomyopathy, encephalomyopathy, neurodegenerative disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), motor neuron disease, nerve disease, epilepsy, age-related disease, macular degeneration, diabetes, metabolic syndrome, brain tumor, hereditary disease, Huntington's disease, mood disorder, schizophrenia, bipolar disorder, pervasive developmental disorder, autism, Asperger's syndrome, childhood disintegrative disorder (CDD), Rett syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), cerebrovascular disorder, stroke, visual disorder, optic neuropathy, dominant hereditary juvenile optic atrophy, optic neuropathy due to toxic substances, glaucoma, Stargardt macular dystrophy, diabetic retinopathy, diabetic maculopathy, retinopathy of prematurity, ischemia-reperfusion-related retinal disorder, oxygen toxicity, hemoglobinopathy, thalassemia, sickle cell anemia, epileptic seizure, ischemia,The method according to any one of claims 1 to 38, selected from the group consisting of renal tubular acidosis, attention deficit / hyperactivity disorder (ADHD), neurodegenerative diseases causing auditory or balance disorders, dominant optic atrophy (DOA), maternally inherited diabetes and deafness (MIDD), chronic fatigue, contrast agent-induced kidney injury, contrast agent-induced retinopathy disorder, abetalipoproteinemia, retinitis pigmentosa, Wolfram disease, Tourette syndrome, cobalamin c deficiency, methylmalonic aciduria, glioblastoma, Down syndrome, acute tubular necrosis, muscular dystrophy, leukodystrophy, progressive supranuclear palsy, spinal muscular atrophy, deafness, noise-induced deafness, traumatic brain injury, juvenile Huntington's disease, multiple sclerosis, NGLY1, multiple system atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, and any combination thereof. **Claim 40** The redox regulation composition improves one or more energy biomarkers selected from the group consisting of lactate (lactate) level, pyruvate (pyruvate) level, lactate / pyruvate ratio, total, reduced or oxidized glutathione level or reduced / oxidized glutathione ratio, total, reduced or oxidized cysteine level or reduced / oxidized cysteine ratio, phosphocreatine level, NADH (NADH+H ＋ ), NADPH (NADPH+H ＋ ), NAD level, NADP level, ATP level, reduced coenzyme Q (CoQred) level, oxidized coenzyme Q (CoQox) level, total coenzyme Q (CoQtot) level, oxidized cytochrome C level, reduced cytochrome C level, oxidized cytochrome C / reduced cytochrome C ratio, acetoacetate level, b-hydroxybutyrate level, acetoacetate / b-hydroxybutyrate ratio, 8-hydroxy-2'-deoxyguanosine (8-OHdG) level, reactive oxygen species level, oxygen consumption (V02) level, carbon dioxide release (VC02) level, respiratory quotient (VC02 / V02), exercise tolerance, anaerobic threshold, and any combination thereof, according to any one of claims 1 to 39. **Claim 41** The method according to any one of claims 1 to 40, wherein the administration comprises intravenous administration, intraarterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and / or intraocular administration.

Images