Redox regulated organelle complex

Abstract

The contents disclosed herein include methods, compositions, and kits suitable for use in reducing or preventing oxidative stress and / or reductive stress. In some embodiments, the methods include contacting a redox sensitive composition with an effective amount of the disclosed redox modulating composition. In some embodiments, the methods comprise administering to a subject in need thereof an effective amount of the disclosed redox modulating composition. In some embodiments, the redox modulating composition comprises an isolated organelle complex. The organelle complex may include mitochondria and one or more of endoplasmic reticulum, peroxisome, lysosome, and Golgi. In some embodiments, the redox modulating compositions comprise isolated mitochondria. The redox modulating composition may be capable of reducing the level of ROS and / or RNS in a redox sensitive composition and / or cells.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 359,108, filed on July 7, 2022, under 35 U.S.C. §119(e), the contents of this related application being incorporated herein by reference in its entirety for all purposes. Background Art Technical Field

[0003] The present disclosure generally relates to methods of reducing and preventing oxidative stress and / or reductive stress.

[0004] Description of Related Technology

[0005] Mitochondria are intracellular organelles responsible for many metabolic conversions and regulatory functions. Mitochondria are highly dynamic organelles that move throughout the cell and undergo structural changes, changing length, morphology, shape and size. In addition, mitochondria are constantly eliminated and regenerated in a process called mitochondrial biogenesis. Although most mitochondrial genes have been transferred to the nuclear genome, the mitochondrial genome still encodes 13 subunits of rRNA, tRNA and electron transport chain (ETC). Therefore, functional communication between the nuclear genome and the mitochondrial genome is crucial for mitochondrial biogenesis, effective oxidative phosphorylation and normal health. Mitochondria are also the main source of free radicals and reactive oxygen species (ROS) that cause oxidative stress. In addition, mitochondria play a key role in the control of intracellular signal transduction and cell death (including apoptosis and necrosis). More and more evidence shows that mitochondrial dysfunction is associated with a variety of human diseases. Mitochondrial dysfunction (e.g., respiratory chain complex dysfunction) is the main cause of mitochondrial disease and aging.

[0006] Oxidative stress is caused by a disturbance in the normal redox state within cells. An imbalance between the regular production and detoxification of reactive oxygen species (such as peroxides and free radicals) can lead to oxidative damage to cellular structures and mechanisms. The most important source of reactive oxygen species under normal conditions for aerobic organisms is the leakage of reactive oxygen species from mitochondria during normal oxidative respiration. Damage associated with this process is suspected to contribute to mitochondrial diseases, neurodegenerative diseases, and diseases of aging.

[0007] Reductive stress can be caused by the reduction of important biological redox pairs such as NAD + / NADH、NADP + / NADPH and GSH / GSSG) redox balance is transformed to a more reducing state and occurs. Overexpression of the antioxidant enzyme system can lead to excessive reducing equivalents, which deplete reactive oxygen species and drive cells to produce reducing stress. Feedback regulation can be established, in which chronic reducing stress induces oxidative stress, which in turn stimulates reducing stress again. Excessive reducing equivalents can regulate cell signaling pathways, change transcriptional activity, induce changes in disulfide bond formation in proteins, reduce mitochondrial function, reduce cell metabolism, and therefore promote the progress of redox diseases and disorders (such as cardiomyopathy, pulmonary hypertension, stent stenosis, muscular dystrophy, nervous system diseases, Parkinson's disease, Alzheimer's disease, metabolic syndrome and insulin resistance, rheumatoid arthritis, kidney disease and cancer).

[0008] There is a need for redox modulating compositions and methods that can reduce or prevent oxidative stress and / or reductive stress. Summary of the invention

[0009] Disclosed herein include methods for reducing or preventing oxidative stress and / or reductive stress in a redox-sensitive composition. In some embodiments, the method comprises: contacting a 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 isolated organelle complexes. In some embodiments, the organelle complexes comprise mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. 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.

[0010] Disclosed herein include methods for reducing the level of reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) in a redox-sensitive composition. In some embodiments, the method comprises: contacting a 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. In some embodiments, reducing the level of ROS and / or RNS in the redox-sensitive composition thereby reduces or prevents oxidative stress and / or reductive stress in the redox-sensitive composition. In some embodiments, the redox-regulating composition comprises isolated organelle complexes. In some embodiments, the organelle complexes comprise mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. 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.

[0011] In some embodiments, the contacting step comprises incubating the mixture of the redox-sensitive composition and the redox-regulating composition for less than about 30 seconds. The redox-sensitive composition can be an oxidant-sensitive composition, a reductant-sensitive composition, or a combination thereof. In some embodiments, the effective amount comprises at least about 5ug / mL to about 5mg / mL of the redox-regulating composition. In some embodiments, the redox-sensitive composition is experiencing oxidative stress and / or reductive stress or is at risk of experiencing oxidative stress and / or reductive stress. In some embodiments, the redox-sensitive composition comprises one or more cells. The contacting step can include introducing the redox-regulating composition into the one or more cells. The one or more cells can be cells of a subject. The one or more cells may be experiencing hypoxia or are at risk of experiencing hypoxia. In some embodiments, the contacting is performed in vitro, in vitro, or in vivo.

[0012] In some embodiments, the redox-sensitive composition comprises a biological sample. In some embodiments, the biological sample is selected from the group consisting of soil samples, air samples, environmental samples, cell culture samples, bone marrow samples, rainfall samples, radioactive fallout samples, space samples, extraterrestrial samples, sewage samples, groundwater samples, wear samples, archaeological samples, food samples, blood samples, serum samples, plasma samples, urine samples, feces samples, semen samples, lymph samples, cerebrospinal fluid samples, nasopharyngeal wash samples, sputum samples, oral swab samples, throat swab samples, nasal swab samples, bronchoalveolar lavage samples, bronchial secretion samples, milk samples, amniotic fluid samples, biopsy samples, nail samples, hair samples, skin samples, cancer samples, tumor samples, tissue samples, cell samples, cell mitosis samples. Lysate samples, virus culture samples, forensic samples, infection samples, nosocomial infection samples, production samples, drug preparation samples, biomolecule production samples, protein preparation samples, lipid preparation samples, carbohydrate preparation samples, nucleotide solutions, polynucleotide solutions, nucleic acid solutions, peptide solutions, polypeptide solutions, amino acid solutions, protein solutions, synthetic polymer solutions, biochemical composition solutions, organic chemical composition solutions, inorganic chemical composition solutions, lipid solutions, carbohydrate solutions, combinatorial chemical product solutions, drug candidate molecule solutions, drug molecule solutions, drug metabolite solutions, cell suspensions, virus suspensions, microbial suspensions, metal suspensions, metal alloy suspensions, metal ion solutions, and any combination thereof.

[0013] Disclosed herein include methods for reducing or preventing oxidative stress and / or reductive 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 reductive stress in the subject. In some embodiments, the redox regulating composition comprises isolated organelle complexes. In some embodiments, the organelle complexes comprise mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. 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.

[0014] Disclosed herein include methods for treating or preventing a redox disease or disorder in a subject. 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. In some embodiments, the redox regulating composition comprises isolated organelle complexes. In some embodiments, the organelle complexes comprise mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a 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.

[0015] In some embodiments, the redox regulating composition comprises homogenized mitochondria, a first organelle complex, and / or a second organelle complex. In some embodiments, the first organelle complexes and the second organelle complexes comprise mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. In some embodiments, the first organelle complexes and the second organelle complexes are depleted of cytoplasmic macromolecules. In some embodiments, the first organelle complexes are derived from (i) frozen cells; (ii) floating cells; and / or (iii) cells contacted with a surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant. In some embodiments, the second organelle complexes are derived from (i) adherent cells; and / or (ii) cells contacted with a surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant. In some embodiments, the first organelle complexes and the second organelle complexes are derived from cells treated with a mitochondrial activator.

[0016] 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 ug to about 1 mg of the redox regulating composition. In some embodiments, the redox regulating composition does not comprise intact cells.

[0017] In some embodiments, the oxidative stress and / or the reductive stress include elevated levels of ROS, RNS and / or free radicals. In some embodiments, the oxidative stress and / or the reductive stress include altered cellular functions. In some embodiments, the oxidative stress and / or the reductive stress are associated with redox diseases or disorders. In some embodiments, 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%. In some embodiments, the redox regulating composition reduces the level of oxidative cell stress, and thereby restores or repairs these cellular functions. 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 generation and / or RNS generation in the subject. In some embodiments, the redox regulating composition has superoxide dismutase activity, catalase activity, peroxidase activity or any combination thereof.

[0018] In some embodiments, the redox regulating composition increases and / or decreases one or more of the following ratios in the subject or the redox sensitive composition: the oxidized form of nicotinamide adenine dinucleotide to the reduced form (NAD + / NADH), the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP + / NADPH), oxidized and reduced forms of glutathione (GSSG / GSH), and oxidized and reduced forms of thioredoxin (TrxSS / TrxSH 2 In some embodiments of the compositions and methods provided herein, the disclosed redox regulating compositions increase (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 any number or range between these values) one or more of the following ratios in the subject or the redox sensitive composition: the oxidized form of nicotinamide adenine dinucleotide to the reduced form (NAD + / NADH), the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP + / NADPH), oxidized and reduced forms of glutathione (GSSG / GSH), and oxidized and reduced forms of thioredoxin (TrxSS / TrxSH 2 ).

[0019] In some embodiments, the redox regulating composition has free radical scavenging activity and / or inhibits free radical formation. In some embodiments, the subject is experiencing oxidative stress and / or reductive stress or is at risk of experiencing 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).

[0020] In some embodiments, the reactive nitrogen species include nitric oxide (NO), nitrogen dioxide (NO 2 ), peroxynitrite anion (ONOO - ), peroxynitrite (ONOOH), nitrosoperoxycarbonate anion (ONOOCO 2 - ), nitronium cation (NO 2 + ), nitrosium cation (NO + ) or nitrogen trioxide (N 2 O 3 ) or any combination thereof. In some embodiments, the reactive oxygen species include superoxide (O 2 . - ), hydroperoxide (HO. 2 ), hydrogen peroxide (H 2 O 2 ), peroxynitrite (ONOO - ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO.), peroxyl radical (ROO.), alkoxyl radical (RO.), singlet oxygen ( 1 O 2 ), 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).

[0021] In some embodiments, the method comprises: identifying a subject who is experiencing oxidative stress and / or reductive stress or is at risk of experiencing oxidative stress and / or reductive stress. In some embodiments, the method comprises: measuring the level of RNS in the subject or the redox-sensitive composition with a probe that detects reactive nitrogen species; and / or measuring the level of ROS in the subject or the redox-sensitive composition with a probe that detects reactive oxygen species. In some embodiments, the probe that detects reactive nitrogen species is diaminonaphthalene, diaminofluorescein, diaminorhodamine, diaminocyanine, NiSPY, dichlorodiaminocalcein, 1,2-diaminoanthraquinone, or DAMBO-P HIn some embodiments, the probe for detecting reactive oxygen species is 2',7'-dichloro-fluorescein diacetate, dihydrorhodamine 123, 3'-(p-aminophenyl)fluorescein (API), 3'-(p-hydroxyphenyl)fluorescein (HPF), aminophenoxycalcein (APC), mitoAR, mitoHR, DPAX, DMAX, hydrogenocyanine or dihydroethidium.

[0022] In some embodiments, the subject has or is suspected of having a disease or disorder in which ROS are involved, the disease or disorder 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, Karns-Sayre syndrome, Reye's syndrome, Leber's hereditary optic neuropathy, mitochondrial myopathy, cardiomyopathy, deafness, mood disorders, movement disorders, dementia, amyotrophic lateral sclerosis, multiple sclerosis , tardive dyskinesia, brain injury, schizophrenia, epilepsy, AIDS dementia, endothelial nitroglycerin tolerance, doxorubicin toxicity, renal injury in type I diabetes, ex vivo renal 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 burns, cancer, inflammation and ischemia-reperfusion injury in stroke, heart attack, ultraviolet damage to the skin, organ transplantation, surgery, and any combination thereof.

[0023] In some embodiments, the redox regulating composition is administered to the subject to treat, alleviate or prevent ischemia-reperfusion injury in the subject. In some embodiments, the ischemia-reperfusion injury is caused by mitochondrial dysfunction, hypoxic injury, HMGB1 release or necrotic cell death. In some embodiments, the redox regulating composition inhibits ischemia-reperfusion injury, mitochondrial dysfunction, hypoxic injury, necrotic cell death or any combination thereof. In some embodiments, the redox disease or disorder includes ROS-mediated and / or RNS-mediated oxidative damage to one or more tissues of the subject. In some embodiments, the administration includes intravenous administration, intra-arterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration and / or intraocular administration.

[0024] In some embodiments, the redox disease or disorder is selected from the group consisting of: mitochondrial disorders; inherited mitochondrial diseases; Alpers disease; Barth syndrome; beta oxidation defects; 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 acidemia type II; Kearns-Sell syndrome (KSS); lactic acidosis; long-chain acyl-CoA dehydrogenase deficiency (LCAD); LCHAD; Leigh syndrome; Leigh-like syndrome; Leber's hereditary optic neuropathy (LHON); fatal infantile cardiomyopathy (LIC); Luft disease; multiple acyl-CoA dehydrogenase deficiency (MAD); medium-chain acyl-CoA dehydrogenase deficiency (MCAD); mitochondrial myoencephalopathy with lactic acidemia and stroke (MELAS); myoclonic epilepsy with red raggedness myofibrillar (MERRF); mitochondrial recessive ataxia syndrome (MIRAS); mitochondrial cytopathies, mitochondrial DNA depletion; mitochondrial encephalopathy; mitochondrial myopathy; myoneurogastrointestinal and encephalopathy (MNGIE); neuropathy, ataxia with 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 neurone disease; nervous system diseases; epilepsy; age-related diseases; macular degeneration; diabetes; metabolic syndrome; brain cancer; genetic diseases; Huntington's disease; mood disorders; schizophrenia; bipolar disorder; pervasive developmental disorder; autistic disorder; Asperger's syndrome; childhood disintegrative disorder (CDD); Rett's disorder; PDD-not otherwise specified (PDD-NOS); cerebrovascular accident; stroke; visual impairment; optic neuropathy; dominant juvenile optic atrophy; optic neuropathy caused by toxic agents; glaucoma; Stargardt's Macular dystrophy; diabetic retinopathy; diabetic maculopathy; retinopathy of prematurity; ischemia-reperfusion-related retinal damage; oxygen toxicity; hemoglobinopathies; thalassemia; sickle cell anemia; seizures; ischemia; renal tubular acidosis; attention-deficit / hyperactivity disorder (ADHD); neurodegenerative disorders resulting in hearing or balance impairment; dominant optic atrophy (DOA); maternally inherited diabetes and deafness (MIDD); chronic fatigue; contrast-induced renal injury; contrast-induced retinopathy injury; abetalipoproteinemia; retinitis pigmentosa; Wolfram's disease;Tourette syndrome; cobalamin C deficiency; methylmalonic acidemia; glioblastoma; Down syndrome; acute tubular necrosis; muscular dystrophy; leukodystrophy; progressive supranuclear palsy; spinal muscular atrophy; hearing loss; noise-induced hearing loss; traumatic brain injury; juvenile Huntington's disease; multiple sclerosis; NGLY1; multiple system atrophy; adrenoleukodystrophy; adrenomyeloneuropathy; and any combination thereof. ;

[0025] In some embodiments, the redox regulating composition improves one or more energy biomarkers in the subject or the redox sensitive composition, the one or more energy biomarkers selected from the group consisting of: lactate (lactate) level; pyruvate (pyruvate) level; lactate / pyruvate ratio; total glutathione level, reduced glutathione level, or oxidized glutathione level, or reduced glutathione / oxidized glutathione ratio; total cysteine ​​level, reduced cysteine ​​level, or oxidized cysteine ​​level, or reduced cysteine / oxidized cysteine ​​ratio; creatine phosphate level, NADH (NADH+H + ) level; NADPH (NADPH+H + ) level; 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 level (V02); carbon dioxide excretion level (VC02); respiratory quotient (VC02 / V02); exercise tolerance; anaerobic threshold; and any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1A to 1B Depicted are data relating to the effects of the second organelle complex (2nd OC) on ejection fraction (EF) in pigs with ischemia-reperfusion injury. Figure 1A Changes in EF (%) are depicted (control, N=5-3, two deaths before day 1; 250 μg of the second organelle complex, N=4). Figure 1B The percentage change 15 minutes after reperfusion relative to before ischemic treatment is depicted (control, N=5, 250 μg of the second organelle complex, N=4). Data are expressed as mean±SEM.

[0027] Figures 2A to 2BDepicts the experimental setup for comparing the anti-ROS activity between the second organelle complex (2OC) and homogenized mitochondria (H-mito) ( Figure 2A ) and the results of luminol determination ( Figure 2B ).

[0028] Figures 3A to 3B Depicted is the experimental setup associated with the NOC7-DAF-2 assay using 100 μg / mL of the indicated populations ( Figure 3A ) and data( Figure 3B ).

[0029] Figures 4A to 4B Depicted is an experimental setup associated with a cell-based assay to examine the in vivo redox modulating activity of the compositions provided herein ( Figure 4A ) and data( Figure 4B ). Figure 4B Shown are the results of an assay (performed in triplicate) using a second organelle complex (2nd OC) derived from HeLa cells.

[0030] Figures 5A to 5B Depicted is a method for examining the in vitro redox regulating activity of the compositions provided herein using HUEhT2 cells. 2.0 Assay-related experimental setup ( Figure 5A ) and data( Figure 5B ). Similar results were observed with Raw264.7 cells (not shown).

[0031] Figures 6A to 6B Depicts the experimental setup associated with a cell viability assay examining the redox regulating compositions provided herein ( Fig. 6A ) and data( Figure 6B The first organelle complex (1OC) derived from HeLa cells can prevent H 2 O 2 Treatment for 2 hours induced cell death. Cell viability results were compared with the control (without H 2 O 2 treatment) ratio.

[0032] Figures 7A to 7B Depicts the experimental setup associated with NAD+ / NADH assays examining the redox regulating compositions provided herein ( Fig. 7A ) and data( Figure 7B The first organelle complex (first OC) derived from HeLa cells is related to H 2 O 2 NAD 2 hours after exposure + / NADH ratio.

[0033] Figures 8A to 8BDepicted and compared the first organelle complex (1st OC) and the second organelle complex (2nd OC) derived from HeLa cells 2.0 Assay-related experimental setup ( Fig. 8A ) and data( Figure 8B ). The first and second organelle complexes prevent H 2 O 2 Cell death was observed at 2 hours (not shown), 4 hours (not shown), and 24 hours after exposure. Cell viability results were compared with those of PBS control (without H 2 O 2 treatment) ratio.

[0034] Fig. 9 Depicted are data related to the characteristics of the organelle complex populations provided herein.Intracellular structure / organelle Western blot protein analysis of the first organelle complex (1st OC) and the second organelle complex (2nd OC) prepared from HEK293T cells using the methods disclosed herein.

[0035] Figures 10A to 10C Depicted are data relating to the role of GSH in the anti-ROS activity of the first organelle complex. Fig. 10A Depicted are GSH concentrations in the first organelle complex derived from HEK293 cells that have been contacted with buthionine sulfoxide (BSO) (BSO-1st OC) or the first organelle complex derived from HEK293 cells that have not been contacted therewith (293-1st OC). Figures 10B to 10C Depicts the process of incubating HEK293 cells with H 2 O 2 (100 μM) and 293-1OC or BSO-1OC for 4 h ( Fig. 10B ) or 20 hours ( Fig. 10C ) after CellTiter-Glo cell viability results.

[0036] Figures 11A to 11C Depicted are data relating to the role of catalase in the anti-ROS activity of the first organelle complex. Fig.11A Depicted are data related to knockdown of catalase (CAT) in HEK293 cells 24 or 48 hours after transfection with catalase siRNA (catalase knockdown is highlighted by a box; vinculin is a loading control). Fig. 11B Catalase levels in untreated first organelle complex (1st OC) or catalase-depleted first organelle complex (1st OC-siRNA; knockdown is highlighted by a box) are depicted. Fig. 11CDepicted are CellTiter-Glo cell viability results for HEK293 cells (n=3) pre-incubated with either the first organelle complex (293-1OC) or catalase-depleted first organelle complex (293-1OC-siRNA) for 6 hours followed by H 2 O 2 (300 μM) for 18 hours. DETAILED DESCRIPTION

[0037] In the following detailed description, the accompanying drawings are mentioned, which form a part of this paper. Unless the context indicates otherwise, in the accompanying drawings, similar symbols generally represent similar components. The illustrative embodiments described in the detailed description, the accompanying drawings and the claims are not intended to be restrictive. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter described herein. It will be easily understood that aspects of the present disclosure (as generally described herein and shown in the accompanying drawings) may be arranged, substituted, combined, separated and designed in various different configurations, all of which are clearly considered herein and become part of the disclosure herein.

[0038] With respect to the related art, all patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety.

[0039] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition, 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 this disclosure, the following terms are defined as follows.

[0040] In the following detailed description, the accompanying drawings are mentioned, which form a part of this paper. Unless the context indicates otherwise, in the accompanying drawings, similar symbols generally represent similar components. The illustrative embodiments described in the detailed description, the accompanying drawings and the claims are not intended to be restrictive. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter described herein. It will be easily understood that aspects of the present disclosure (as generally described herein and shown in the accompanying drawings) may be arranged, substituted, combined, separated and designed in various different configurations, all of which are clearly considered herein and become part of the disclosure herein.

[0041] With respect to the related art, all patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety.

[0042] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition, 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 this disclosure, the following terms are defined as follows.

[0043] As used herein, "isolated" shall be given its ordinary meaning and shall also refer to a substance or entity that has been (1) separated from at least some of the components with which it was originally produced (whether in nature or in an experimental setting), and / or (2) produced, prepared and / or manufactured by the hand of man. In some embodiments, the isolated mitochondria or isolated population of organelle complexes have been processed to be obtained from a cellular environment via the methods provided herein.

[0044] As used herein, the term "cell" shall be given its ordinary meaning and shall also refer to eukaryotic cells, i.e., cells containing mitochondria in the cytoplasm, such as animal cells, such as mammalian cells, preferably human cells. As used herein, the term "cell" is used to include cells present in tissues, cells separated from tissues (e.g., single cells), and cells within cell populations (e.g., cell populations obtained from tissues of subjects and / or cell populations obtained from cell lines).

[0045] As used herein, the term "mitochondria" should be given its common meaning and should also refer to the organelle present in eukaryotic cells, which have a double lipid membrane, an inner membrane and an outer membrane, and a matrix surrounded by cristae and inner membranes. Mitochondria (more than one mitochondria) have enzymes on their inner membranes, such as respiratory chain complexes involved in oxidative phosphorylation. The inner membrane has a membrane potential due to the internal-external proton gradient formed by the effects of respiratory chain complexes, etc. When the inner membrane is destroyed, mitochondria are considered to be unable to maintain membrane potential. Known mitochondria have their own genome (mitochondrial genome), which is different from the genome in the nucleus.

[0046] As used herein, the term "organelle complex" should be given its usual meaning, and should also refer to a complex of mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. Organelle complexes can be depleted of cytoplasmic macromolecules (e.g., cytoplasmic proteins). In some embodiments, organelle complexes do not contain cytoplasmic macromolecules. In some embodiments, organelle complex populations include homogenized mitochondria. As used herein, the term "population" should be given its usual meaning, and should also refer to a group with multiple identical or different substances. For example, a "organelle complex population" is a group with at least multiple identical or different organelle complexes. A population may not always be homogenous, and may have a physical distribution, a chemical distribution, and / or a 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). Organelle complex populations can include a first organelle complex, a second organelle complex, homogenized mitochondria, or any combination thereof. As used herein, the term "homogenized mitochondria" shall be given its ordinary meaning, and shall also refer to mitochondria isolated via a method comprising one or more homogenization steps.

[0047] As used herein, the term "surfactant" shall be given its ordinary meaning and shall also refer to a molecule having a hydrophilic portion and a hydrophobic portion in one molecule. Surfactants have the effect of reducing interfacial surface tension or mixing polar and non-polar substances by forming micelles. Surfactants are roughly classified into nonionic surfactants and ionic surfactants. Nonionic surfactants are those in which the hydrophilic portion is not ionized, while ionic surfactants are those in which the hydrophilic portion contains cations or anions or contains both cations and anions.

[0048] As used herein, the term "critical micelle concentration" (CMC) shall be given its ordinary meaning and shall also refer to the concentration at which the surfactant forms micelles and further addition of surfactant to the system contributes to micelle formation, particularly bulk concentrations. When the concentration is above the critical micelle concentration, adding surfactant to the system may desirably increase the amount of micelles, particularly the number of micelles.

[0049] As used herein, "subject" refers to an animal that is the object of treatment, observation or experiment. "Animal" includes cold-blooded vertebrates and warm-blooded vertebrates and invertebrates, such as fish, shellfish, reptiles, and particularly mammals. As used herein, "mammal" refers to an individual belonging to the class mammalia, and includes, but is not limited to, humans, livestock and farm animals, zoo animals, sports animals, and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cattle; horses; primates, such as monkeys, chimpanzees, and apes, and particularly humans. In some embodiments, the mammal is a human. However, in some embodiments, the mammal is not a human.

[0050] As used herein, the term "treatment" refers to an intervention in response to a disease, illness or physiological condition exhibited by a patient. The purpose of treatment may include, but is not limited to, one or more of the following: relieving or preventing symptoms, slowing or stopping the progression or deterioration of a disease, illness or condition, and the alleviation of a disease, illness or condition. The terms "treat" and "treatment" include, for example, therapeutic treatments, preventive treatments, and applications in which the risk of a subject suffering from a disease or other risk factors is reduced. Treatment does not require a complete cure of the disease and encompasses embodiments in which symptoms or potential risk factors are alleviated. In some embodiments, "treatment" refers to both therapeutic treatments and preventive measures or preventive measures. Those that require treatment include those that have been affected by a disease or illness or an undesirable physiological condition and those in which a disease or illness or an undesirable physiological condition is to be prevented. As used herein, the term "prevention" refers to any activity that reduces the burden on individuals who subsequently exhibit these symptoms. This can occur at the primary, secondary and / or tertiary prevention levels, where: a) primary prevention avoids the progression of a symptom / disorder / condition; b) secondary prevention activities target the early stages of treatment of a symptom / disorder / symptom, thereby increasing the chances of intervention to prevent the progression of the symptom / disorder / symptom and the onset of symptoms; and c) tertiary prevention reduces the negative impact of an established symptom / disorder / symptom by, for example, restoring function and / or reducing any symptom / disorder / symptom or associated complications. The term "prevention" does not require 100% elimination of the likelihood of an event. Rather, it indicates that the likelihood of an event occurring has been reduced in the presence of a compound or method.

[0051] 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.

[0052] As used herein, the term "reductive stress" shall be given its ordinary meaning and shall also refer to the effects of stress on the metabolism of important biological redox pairs such as NAD + / NADH、NADP + In some embodiments, the reductive stress is a corresponding oxidative stress.

[0053] As used herein, the term "effective amount" refers to an amount sufficient to achieve beneficial or desired biological and / or clinical results.

[0054] In some embodiments, the methods, compositions, systems and kits provided herein can be used in conjunction 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 contents of which are incorporated herein by reference in their entirety).

[0055] Redox regulation of mitochondria

[0056] In some embodiments, a method of reducing or preventing oxidative stress and / or reductive stress in a redox-sensitive composition is provided. In some embodiments, the method comprises: contacting a 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.

[0057] In some embodiments, methods of reducing the level of reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) in a redox-sensitive composition are provided. In some embodiments, the method comprises: contacting a 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. In some embodiments, reducing the level of ROS and / or RNS in the redox-sensitive composition thereby reduces or prevents oxidative stress and / or reductive stress in the redox-sensitive composition.

[0058] The contacting step may include incubating the mixture of the redox-sensitive composition and the redox-regulating 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 may be experiencing oxidative stress and / or reductive stress or may be at risk of experiencing oxidative stress and / or reductive stress. The redox-sensitive composition may comprise one or more cells. The contacting step may include introducing the redox-regulating composition into the one or more cells. The one or more cells may be cells of a subject. The one or more cells may be experiencing hypoxia or at risk of experiencing hypoxia. The contacting may be performed ex vivo, in vitro, or in vivo.

[0059] The redox-sensitive composition may include a biological sample. The biological sample may be selected from the group consisting of soil samples, air samples, environmental samples, cell culture samples, bone marrow samples, rainfall samples, radioactive fallout samples, space samples, extraterrestrial samples, sewage samples, groundwater samples, wear samples, archaeological samples, food samples, blood samples, serum samples, plasma samples, urine samples, feces samples, semen samples, lymph samples, cerebrospinal fluid samples, nasopharyngeal wash samples, sputum samples, oral swab samples, throat swab samples, nasal swab samples, bronchoalveolar lavage samples, bronchial secretion samples, milk samples, amniotic fluid samples, biopsy samples, nail samples, hair samples, skin samples, cancer samples, tumor samples, tissue samples, cell samples, cell lysate samples samples, virus culture samples, forensic samples, infection samples, nosocomial infection samples, production samples, drug preparation samples, biomolecule production samples, protein preparation samples, lipid preparation samples, carbohydrate preparation samples, nucleotide solutions, polynucleotide solutions, nucleic acid solutions, peptide solutions, polypeptide solutions, amino acid solutions, protein solutions, synthetic polymer solutions, biochemical composition solutions, organic chemical composition solutions, inorganic chemical composition solutions, lipid solutions, carbohydrate solutions, combinatorial chemical product solutions, drug candidate molecule solutions, drug molecule solutions, drug metabolite solutions, cell suspensions, virus suspensions, microbial suspensions, metal suspensions, metal alloy suspensions, metal ion solutions, and any combination thereof.

[0060] In some embodiments, methods of reducing or preventing oxidative stress and / or reductive stress in a subject are 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.

[0061] In some embodiments, methods of treating or preventing a redox disease or disorder in a subject are provided. In some embodiments, the method comprises: administering to the subject an effective amount of a redox regulating composition, thereby treating or preventing the redox disease or disorder in the subject.

[0062] The redox regulating composition may include a population of organelle complexes. The population of organelle complexes may include a first organelle complex or a combination of a first organelle complex and a second organelle complex. The redox regulating composition may include an isolated organelle complex. The redox regulating composition may include homogenized mitochondria, a first organelle complex and / or a second organelle complex. These organelle complexes can include mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes and golgi bodies. The first organelle complex and / or the second organelle complex may be depleted of cytoplasmic macromolecules. The redox regulating composition may include isolated mitochondria. The redox regulating composition may include mitochondria isolated from intact cells and / or floating cells or frozen cells or a combination thereof. In certain 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) may include mitochondria and one, two, three or four of endoplasmic reticulum, peroxisomes, lysosomes and golgi bodies. The organelle complex (e.g., the first organelle complex, the second organelle complex) may 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; The ratio of mitochondria to other organelles (e.g., endoplasmic reticulum, peroxisome, lysosome, and Golgi) may vary in the population of organelle complexes.

[0063] Disclosed herein are methods for producing a first organelle complex population. In some embodiments, the method comprises: incubating cells in a first solution comprising a surfactant at a first temperature; removing the surfactant to form a second solution; and recovering the first organelle complex from the second solution. The first organelle complex may be derived from: (i) frozen cells; (ii) floating cells; and / or (iii) cells contacted with a surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant. In some embodiments, a second organelle complex is provided. In some embodiments, a method for separating a second organelle complex from a cell comprises treating cells in a first solution with a surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant, removing the surfactant to form a second solution, incubating cells in the second solution, and recovering the second organelle complex from the second solution. The second organelle complex may be derived from: (i) adherent cells; and / or (ii) cells contacted with a surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant. The organelle complex population may be derived from cells treated with a mitochondrial activator (e.g., resveratrol). Organelle complexes can be depleted of cytoplasmic macromolecules. Cytoplasmic macromolecules may not be present in the organelle complex population provided herein. Organelle complexes (e.g., the first organelle complex, the second organelle complex) population provided herein may include negligible and / or undetectable amounts of cytoplasmic macromolecules. Redox regulating compositions may include substantially pure organelle complex populations. Substantially pure organelle complex populations may include less than about 20% (e.g., less than about 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0% or any two of these values) cytoplasmic macromolecules. Cytoplasmic macromolecules may include cytoplasmic proteins (e.g., p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)). The first organelle complex and the second organelle complex may be derived from cells treated with mitochondrial activators. Homogenized mitochondria, the first organelle complex and / or the second organelle complex can be encapsulated in a lipid membrane-based vesicle. The method encapsulated in a lipid membrane-based vesicle is disclosed in PCT Patent Application Publication No. WO2021 / 132735, and its content is incorporated herein by reference in its entirety. Incubating cells and / or incubating the second solution in the first solution may include applying physical stimulation to the first solution and / or the second solution, such as pipetting, oscillation and / or stirring. Applying physical stimulation to the first solution and / or the second solution may include causing the first solution and / or the second solution to flow through a flow device (e.g., a flow reduction device). The flow device may include a fluid channel, which includes two or more sections with different cross-sectional diameters.Recovering the first organelle complexes from the second solution may include tangential flow filtration (TFF). In some embodiments, the systems, methods, compositions and kits provided herein may be used in conjunction with systems, methods, compositions and kits for generating first organelle complexes described in PCT patent application number PCT / US23 / 27014, entitled “ORGANELLECOMPLEXES”, filed on July 6, 2023 (the contents of which are incorporated herein by reference in their entirety).

[0064] The method may include at least about 5 ug / mL to about 5 mg / mL of a redox-sensitive composition. The effective amount may include at least about 1 ug to about 1 mg of a redox-regulating composition. The effective amount may include at least about 5 ug / mL to about 5 mg / mL of a redox-regulating composition. The amount of the redox-sensitive composition and / or the effective amount of the redox-regulating composition may be, may be about, may be at least, or may be at most 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, 401, 410, 420, 430, 440, 450 20, 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 0, 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, 2 000, 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, or a number or range in ug, mg, ug / mL and / or mg / mL between any two of these values.

[0065] Oxidative stress and / or reductive stress may include elevated levels of ROS, RNS and / or free radicals. Oxidative stress and / or reductive stress may include altered cellular function. Oxidative stress and / or reductive stress may be associated with redox diseases or disorders. In certain embodiments, the redox regulating composition reduces the level of one or more ROS and / or one or more RNS in a subject or a 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 higher level overlapping ranges therein). In certain embodiments, the redox regulating composition reduces the level of oxidative cell stress, and thus recovers or repairs cellular function.

[0066] The redox regulating composition may have ROS scavenging activity and / or RNS scavenging activity. In some embodiments, the redox regulating composition reduces or prevents ROS generation and / or RNS generation in the subject. The redox regulating composition may have superoxide dismutase activity, catalase activity, peroxidase activity, or any combination thereof. The redox regulating composition may have free radical scavenging activity and / or inhibit free radical formation.

[0067] In some embodiments, the redox modulating composition increases and / or decreases (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) one or more of the following ratios in a subject or a redox sensitive composition: oxidized to reduced forms of nicotinamide adenine dinucleotide (NAD+ / NADH), oxidized to reduced forms of nicotinamide adenine dinucleotide phosphate (NADP+ / NADPH), oxidized to reduced forms of glutathione (GSSG / GSH), and oxidized to reduced forms of thioredoxin (TrxSS / TrxSH 2 ).

[0068] Reactive nitrogen species can include nitric oxide (NO), nitrogen dioxide (NO 2 ), peroxynitrite anion (ONOO - ), peroxynitrite (ONOOH), nitrosoperoxycarbonate anion (ONOOCO 2 - ), nitronium cation (NO 2 + ), nitrosium cation (NO + ) or nitrogen trioxide (N 2 O3 ) or any combination thereof. The reactive oxygen species may include superoxide (O 2 . - ), hydroperoxide (HO. 2 ), hydrogen peroxide (H 2 O 2 ), peroxynitrite (ONOO - ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO.), peroxyl radical (ROO.), alkoxyl radical (RO.), singlet oxygen ( 1 O 2 ), lipid peroxides, lipid peroxyl radicals or lipid alkoxyl radicals or any combination thereof. ROS can be derived from neutrophils and / or xanthine oxidase (XO).

[0069] The redox regulating composition can prevent ROS-induced functional changes via anti-oxidation and / or reduction. The redox regulating composition can be an antioxidant composition, an anti-reductant 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 intracellular, extracellular, and / or in a cell-free environment. The redox regulating composition can improve (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 any number or range between these values) one or more energy biomarkers in a subject or redox sensitive composition, the 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 glutathione / oxidized glutathione ratio; total, reduced, or oxidized cysteine ​​level, or reduced cysteine / oxidized cysteine ​​ratio; phosphocreatine level, NADH (NADH+H + ) level; NADPH (NADPH+H +) level; 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 level (V02); carbon dioxide excretion level (VC02); respiratory quotient (VC02 / V02); exercise tolerance; anaerobic threshold; and any combination thereof. The redox regulating compositions provided herein can increase lactate and / or ATP production in a dose-dependent manner (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 any number or range between these values). In some embodiments, and without being bound by any particular theory, the redox regulating compositions regulate the conversion of reduced form nicotinamide adenine dinucleotide (NADH) to oxidized form nicotinamide adenine dinucleotide (NAD + In some embodiments, and without being bound by any particular theory, the redox regulating composition regulates the ratio of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP + ). NAD + / NADH can participate in the redox reaction of energy metabolism and mitochondrial function. NADH / NADPH can participate in ROS metabolism as an electron donor. Used to measure intracellular NAD +A variety of methods are known in the art and include LC-MS / MS, HPLC, NMR, MS imaging, in situ genetically encoded sensors, MRI assays, lysate-based methods and other methods, such as those provided in the following literature: Cambronne XA, Kraus WL. Trends Biochem Sci. October 2020; 45(10) / 858-873, the contents of which are hereby incorporated by reference in their entirety. The redox regulating composition can exert a reducing effect on the cells of the redox-sensitive composition and / or the subject. The redox regulating composition can reduce the generation of RNS and / or ROS in the redox-sensitive composition or the subject. The redox regulating composition can increase the removal of RNS and / or ROS in the redox-sensitive composition or the subject. In some embodiments, a redox regulating composition comprising a first organelle complex can reduce the generation of RNS and / or ROS in a redox sensitive composition or a subject and / or increase the removal of RNS and / or ROS in a redox sensitive composition or a subject by at least about 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or any number or range between these values) compared to a redox regulating composition comprising a second organelle complex. The redox regulating compositions provided herein can affect the redox system and thereby cause increased vitality (e.g., via antioxidant and / or antireductive effects). In some embodiments, excessive or lethal oxidative stress is reductive stress, and in some such embodiments, it requires not only a reduction effect through antioxidant effects, but also an oxidation effect through antireductive effects. The disclosed redox regulating compositions can have anti-oxidative stress effects and anti-reductive stress effects, such as redox enhancers. Reductive stress can include high lactate / pyruvate ratios. The redox regulating compositions provided herein can act by reducing excess reducing equivalent levels. In some embodiments, and without being bound by any particular theory, the redox regulating compositions disclosed herein provide removal of reductive stress and can reduce reductive stress by oxidizing NADH to NAD+, which can result in antioxidant activity, ATP production capacity, and / or cell proliferation capacity. In some embodiments, and without being bound by any particular theory, the redox regulating compositions provided herein can regulate NAD + / NADH、NADP +In some embodiments, and without being bound by any particular theory, oxidized NADPH and GSH / GSSG are increased or decreased by at least 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or any number or range between these values), thereby alleviating reductive stress. In some embodiments, and without being bound by any particular theory, oxidized NAD + Regulate the redox system connected by NADPH and / or act as a signaling molecule for steady state. The redox regulating compositions (e.g., organelle complexes) provided herein can be associated with redox systems (including those redox systems involved in ROS generation and / or ROS removal), such as peroxisomes, mitochondria, endosomes (e.g., plasma membrane endosomes), and endoplasmic reticulum. In some embodiments, the organelle complex contains more NAD + In some embodiments, NAD + is an active ingredient of a redox regulating composition provided herein. In some embodiments, the first organelle complex contains at least about 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or any number or range between these values) of NAD compared to the second organelle complex. + In some embodiments, NAD is removed from the redox regulating compositions provided herein. + This will reduce at least a portion of the effect performed by the redox regulating composition.

[0070] In an embodiment, the organelle complex population can treat, prevent, alleviate and / or improve clinical conditions caused by ischemia-reperfusion injury. In an embodiment, the organelle complex population can improve ejection fraction (EF), inhibit cardiac hypertrophy, and / or treat, prevent, improve and / or improve fibrosis after ischemia-reperfusion injury. The redox regulating composition can improve one or more cardiac function indicators in the subject (at least about 1.1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or any number or range between these values), and the one or more cardiac function indicators are selected from the group consisting of: cardiac output, ejection fraction, volume, stroke volume, pressure, end-diastolic volume (EDV) and end-systolic volume (ESV).

[0071] Pharmaceutically acceptable compositions and methods of administration

[0072] In some embodiments, the redox regulating 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 otherwise benefit from supplementing healthy, functional mitochondria. The present disclosure also provides the use of the redox regulating composition in the manufacture of a medicament for treating the diseases and disorders provided herein.

[0073] Administration can include intravenous administration, intraarterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and / or intraocular administration.The redox modulating composition can be administered orally or systemically.

[0074] As used herein, the phrase "local administration" or "topic administration" refers to any route of administration that brings a redox-modulating composition into contact with the body of an individual such that the resulting location of the redox-modulating composition within the body is local (limited to the specific tissue, organ, or other body site to be imaged). Exemplary routes of topical administration include injection by needle into a specific tissue, tube feeding into the gastrointestinal tract, and application of a solution containing the redox-modulating composition onto the surface of the skin.

[0075] As used herein, the term "systemic administration" refers to any route of administration that contacts the redox-modulating composition with the body of an individual such that the location of the resulting organelle complex population in vivo is systemic (i.e., not limited to the specific tissue, organ, or other body part that requires imaging). Systemic administration includes enteral administration and parenteral administration. Enteral administration is a systemic administration route in which a substance is administered via the digestive tract, and includes, but is not limited to, oral administration, administration through a gastric feeding tube, administration through a duodenal feeding tube, gastrostomy, enteral nutrition, and rectal administration. Parenteral administration is a systemic administration route in which a substance is administered by a route other than the digestive tract, and includes, but is not limited to, intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intravesical infusion.

[0076] In another aspect, the present disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of a redox regulating composition disclosed herein. As described in detail below, the pharmaceutical compositions of the present disclosure can be specifically formulated for administration in solid or liquid form, including those suitable for the following: (1) oral administration, such as an immersion (aqueous or non-aqueous solution or suspension), tablet, pill, powder, granule, paste; (2) parenteral administration, such as by subcutaneous, intramuscular or intravenous injection, such as as a sterile solution or suspension: (3) topical administration, such as as a cream, ointment or spray applied to the skin; (4) intravaginal or intrarectal, such as as a pessary, cream or foam; or (5) an aerosol containing a redox regulating composition, such as as an aqueous aerosol, liposomal formulation or solid particles. The pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers. As used herein, the phrase "therapeutically effective amount" can refer to an amount of a redox modulating composition disclosed herein that is effective to produce some desired therapeutic effect, such as cancer treatment, at a reasonable benefit / risk ratio.

[0077] The phrase "pharmaceutically acceptable / pharmaceutically acceptable" as used herein refers to those agents, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0078] 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, that is involved in carrying or transporting the subject chemical 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 causing harm to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch, potato starch, etc.; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate; (4) astragalus powder: (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; (1) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0079] Preparations that can be used for the methods of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, spray and / or parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient (e.g., redox regulating composition) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the redox regulating composition that produces a therapeutic effect. In general, in one hundred percent, this amount will be in the range of 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%.

[0080] Suspensions, in addition to the active agents, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0081] Dosage forms for topical or transdermal administration of the redox regulating 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 that may be required.

[0082] Ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0083] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also contemplated as being within the scope of this disclosure.

[0084] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present 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). Appropriate fluidity can be maintained, for example, by the use of coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[0085] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can ensure that prevention of microbial action is achieved by including various antibacterial and antifungal agents (e.g. parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents (such as sugar, sodium chloride, etc.) in the composition. In addition, the absorption of injectable drug forms can be extended by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0086] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be determined by the methods of the present disclosure so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without causing toxicity to the subject.

[0087] The subject may be experiencing oxidative stress and / or reductive stress or may be at risk of experiencing 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). In some embodiments, the method includes: identifying a subject who is experiencing oxidative stress and / or reductive stress or is at risk of experiencing 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 with a probe that detects reactive nitrogen species; and / or measuring the level of ROS in the subject or the redox-sensitive composition with a probe that detects reactive oxygen species. The probe for detecting reactive nitrogen species can be diaminonaphthalene, diaminofluorescein, diaminorhodamine, diaminocyanine, NiSPY, dichlorodiaminocalcein, 1,2-diaminoanthraquinone or DAMBO-P HThe probe for detecting reactive oxygen species may be 2',7'-dichloro-fluorescein diacetate, dihydrorhodamine 123, 3'-(p-aminophenyl)fluorescein (API), 3'-(p-hydroxyphenyl)fluorescein (HPF), aminophenoxycalcein (APC), mitoAR, mitoHR, DPAX, DMAX, hydrogenocyanine or dihydroethidium.

[0088] The subject may have or may be suspected of having a disease or condition in which ROS are involved, the disease or condition being 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, Reye's syndrome, Leber's hereditary optic neuropathy, mitochondrial myopathy, cardiomyopathy, deafness, mood disorders, movement disorders, dementia, amyotrophic lateral sclerosis, multiple sclerosis, late onset Movement disorders, brain injury, schizophrenia, epilepsy, AIDS dementia, endothelial nitroglycerin tolerance, doxorubicin toxicity, renal injury in type I diabetes, ex vivo renal 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 burns, cancer, inflammation and ischemia-reperfusion injury in stroke, heart attack, ultraviolet damage to the skin, organ transplantation, surgery, and any combination thereof.

[0089] In some embodiments, administering the redox regulating composition to the subject treats, reduces or prevents ischemia-reperfusion injury in the subject. Ischemia-reperfusion injury may be caused by mitochondrial dysfunction, hypoxic injury, HMGB1 release or necrotic cell death. In some embodiments, the redox regulating composition inhibits ischemia-reperfusion injury, mitochondrial dysfunction, hypoxic injury, necrotic cell death or any combination thereof.

[0090] The redox disease or disorder may include ROS-mediated and / or RNS-mediated oxidative damage to one or more tissues of the subject. The redox disease or disorder may be selected from the group consisting of: mitochondrial disorders; inherited mitochondrial diseases; Alpers disease; Barth syndrome; beta oxidation defects; 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; CPTII deficiency; Friedreich's ataxia (FA); glutaric acidemia type II; Kearns-Sell syndrome (KSS); lactic acidosis; long-chain acyl-CoA dehydrogenase deficiency (LCAD); LCHAD; Ley syndrome; Ley-like syndrome; Leber's hereditary optic neuropathy (LHON); fatal infantile cardiomyopathy (LIC); Luft disease; multiple acyl-CoA dehydrogenase deficiency (MAD); medium-chain acyl-CoA dehydrogenase deficiency (MCAD); mitochondrial myopathy-encephalopathy with hyperlactatemia and stroke (M ELAS); myoclonic epilepsy with red ragged fibers (MERRF); mitochondrial recessive ataxia syndrome (MIRAS); mitochondrial cytopathies, mitochondrial DNA depletion; mitochondrial encephalopathy; mitochondrial myopathy; myoneurogastrointestinal disorders and encephalopathy (MNGIE); neuropathy, ataxia with 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 (VL CAD); myopathy; cardiomyopathy; encephalomyopathy; neurodegenerative diseases; Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis (ALS); motor neuron disease; nervous system diseases; epilepsy; age-related diseases; macular degeneration; diabetes; metabolic syndrome; brain cancer; genetic diseases; Huntington's disease; mood disorders; schizophrenia; bipolar disorder; pervasive developmental disorders; autistic disorders; Asperger's syndrome; childhood disintegrative disorder (CDD); Rett's disorder; PDD-not otherwise specified (PDD-NOS); cerebrovascular accident; stroke; visual impairment; optic neuropathy optic neuropathy caused by toxic agents; glaucoma; Stargardt macular dystrophy; diabetic retinopathy; diabetic maculopathy; retinopathy of prematurity; ischemia-reperfusion-related retinal damage; oxygen toxicity; hemoglobinopathies; thalassemia; sickle cell anemia; seizures; ischemia; renal tubular acidosis; attention-deficit / hyperactivity disorder (ADHD); neurodegenerative disorders causing hearing or balance impairment; dominant optic atrophy (DOA); maternally inherited diabetes and deafness (MIDD); chronic fatigue;Contrast-induced renal injury; contrast-induced retinopathy injury; abetalipoproteinemia; retinitis pigmentosa; Wolfram's disease; Tourette syndrome; cobalamin C deficiency; methylmalonic acidemia; glioblastoma; Down syndrome; acute tubular necrosis; muscular dystrophy; leukodystrophy; progressive supranuclear palsy; spinal muscular atrophy; hearing loss; noise-induced hearing loss; traumatic brain injury; juvenile Huntington's disease; multiple sclerosis; NGLY1; multiple system atrophy; adrenoleukodystrophy; adrenomyoneuropathy; and any combination thereof. ;

[0091] Also provided herein are kits comprising one or more compositions described herein (e.g., formulations comprising redox regulating compositions) in suitable packaging, and may further comprise written materials, which may include instructions for use, discussions of clinical studies, lists of side effects, and the like. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and / or summaries of these, etc., which indicate or establish the activity and / or advantages of the compositions, and / or describe dosing, administration, side effects, drug interactions, or other information useful to healthcare providers. Such information may be based on the results of various studies, such as studies using experimental animals involving in vivo models and studies based on human clinical trials. The kit may comprise one or more unit doses described herein.

[0092] Examples

[0093] Some aspects of the above-described embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the present disclosure in any way.

[0094] Example 1

[0095] Effect of organelle complex II on ejection fraction in pigs with ischemia-reperfusion injury

[0096] The effect of the second organelle complex on ejection fraction in pigs with ischemia-reperfusion injury was studied. Ischemia was induced by inflation of a balloon placed at the D1 level in the LAD. The second organelle complex was administered around the LAD inflation site immediately before reperfusion. Figures 1A to 1B Depicted are data relating to the effects of the second organelle complex (2nd OC) on ejection fraction (EF) in pigs with ischemia-reperfusion injury. Figure 1A Changes in EF (%) are depicted (control, N=5-3, two deaths before day 1; 250 μg of the second organelle complex, N=4). Figure 1BThe percentage change 15 minutes after reperfusion relative to before ischemia treatment is depicted (control, N=5, 250 μg second organelle complex, N=4). Data are expressed as mean ± SEM. ROS and RNS production are involved in ischemia-reperfusion injury (see, Int. J. Mol. Sci. 2018, 19, 417; doi: 10.3390 / ijms19020417, incorporated herein by reference). These data indicate the redox regulation of the second organelle complex disclosed herein.

[0097] Example 2

[0098] Anti-ROS activity of redox modulating compositions

[0099] The luminol assay was used to detect H by chemiluminescence (CL). 2 O 2 (See React Oxyg Species (Apex). 2016 May; 1(3): 216–227. doi: 10.20455 / ros.2016.841, incorporated herein by reference.) In the presence of horseradish peroxidase (HRP), luminol can react with hydrogen peroxide to ultimately form an excited state of 3-aminophthalate (3-APA*). The decay of 3-APA* to a lower energy level results in photon emission, which can be measured as a chemiluminescent (CL) response for luminescence. Figure 2A The experimental setup for comparing the anti-ROS activity between the second organelle complex (2OC) and homogenized mitochondria (H-mito) is depicted, and Figure 2B Depicted are the results of the luminol assay for this comparison. The calculated IC50 values ​​are as follows: HeLa derived second organelle complex (2nd OC), 153.4 μg / ml; first organelle complex (1st OC), 166.9 μg / ml; homogenized mitochondria, 167.4 μg / ml. These data demonstrate the anti-ROS effects of various redox regulating compositions provided herein.

[0100] Example 3

[0101] Anti-RNS activity of redox-modulating compositions

[0102] The nitric oxide detection assay was performed using the NOC7-DAF-2 system. NOC7 was used as the nitric oxide donor and DAF-2 was used as the nitric oxide detector. Figure 3A The experimental setup associated with the 0.5 μM NOC7 and DAF-2 (1 μM) assay is depicted, and Figure 3B Data related to this assay are depicted. Figure 3BDepicted are the results of a NOC7-DAF-2 assay using 0.5M NOC7 with increasing doses of DAF-2. Anti-RNS activity was demonstrated using three different populations containing mitochondria (second organelle complex (2ndOC), first organelle complex (1stOC), and homogenized mitochondria (H-mito)) relative to control (Tris / Suc). 100 μg / mL ( Figure 3B ), 25 μg / mL (not shown), and 6.25 μg / mL (not shown), these data indicate dose-dependent anti-RNS effects of various redox regulating compositions provided herein.

[0103] Example 4

[0104] Cell-based assays for redox-regulating compositions

[0105] Cell-based assays are performed to examine the effects of the redox modulating compositions described herein on cells. Figures 4A to 4B Depicts the experimental setup associated with a cell-based assay for examining the in vitro redox regulating activity of the compositions provided herein ( Figure 4A ) and data( Figure 4B ). Figure 4B Shown are the results of an assay (performed in triplicate) using the second organelle complex (2nd OC) derived from HeLa cells. The second organelle complex (derived from HeLa cells) was found to inhibit tBHP-induced ROS in a dose-dependent manner.

[0106] Next, proceed 2.0 assays were performed to examine the effects of the redox regulating compositions described herein on HUEhT2 and Raw264.7 (data not shown) cells. The 2.0 assay can determine the number of viable cells in culture by quantifying ATP, which indicates the presence of metabolically active cells. The luminescence result can be directly proportional to the number of viable cells in culture. Figures 5A to 5B Describes methods for examining the in vitro redox regulating activity of the compositions provided herein 2.0 Assay-related experimental setup ( Figure 5A ) and data( Figure 5B ). Figure 5B Depicted are the results of an assay showing rescue of HUEhT2 cells by the second organelle complex (2nd OC, derived from 293T cells).These data demonstrate the in vitro redox modulating effects of the redox modulating compositions provided herein.

[0107] Example 5

[0108] Cell viability assay

[0109] Cell viability assays were performed to evaluate the effects of the disclosed redox regulating compositions. Fig. 6A Depicts the experimental setup associated with cell viability assays examining the redox regulating compositions provided herein, and Figure 6B Depicted are data related to this assay. The first organelle complex (1st OC) derived from HeLa cells was expressed in the presence of H 2 O 2 The cell viability ratio was improved 2 hours and 4 hours after contact (not shown). The first organelle complex (derived from HeLa cells) was found to improve cell viability in a dose-dependent manner at both time points.

[0110] Example 6

[0111] NAD + / NADH assay

[0112] Conduct NAD + / NADH assay to examine the effect of the first organelle complex on cells. Fig. 7A Depicted and examined NAD redox regulating compositions provided herein + / NADH assay-related experimental setup, and Figure 7B Depicted are data related to this assay. The first organelle complex (1st OC) derived from HeLa cells was expressed in the presence of H 2 O 2 Changes in NAD 2 and 4 hours after exposure (not shown) + / NADH ratio. The first organelle complex (derived from HeLa cells) increased NAD in a dose-dependent manner at both time points + In some embodiments, and without being bound by any particular theory, the redox regulating compositions disclosed herein provide relief of reductive stress and can be oxidized by NADH to NAD + to reduce reductive stress, which can lead to antioxidant activity, ATP production capacity and / or cell proliferation capacity.

[0113] Example 7

[0114] Comparison of the first and second organelle complexes

[0115] A cell-based assay was performed to compare the effects of the first organelle complex with the second organelle complex on viability. Fig. 8A Depicted and compared the first organelle complex (1st OC) and the second organelle complex (2nd OC) derived from HeLa cells 2.0 Determination of relevant experimental settings, and Figure 8BData related to this assay are depicted. Results of the first and second organelle complexes on cell viability ( Figure 8B ) are shown as a ratio relative to control cells treated with PBS. It was found that the first organelle complex (derived from HeLa cells) had a stronger anti-reductive stress effect (in a dose-dependent manner) than the second organelle complex. In view of these differences, the composition of the first organelle complex and the second organelle complex was compared to illustrate the active ingredients of the organelle complex provided herein for anti-reductive stress indications. Fig. 9 Depict non-limiting exemplary data related to the characteristics of the organelle complex population provided herein. It shows the intracellular structure / organelle Western blot protein analysis of the first organelle complex (1st OC) and the second organelle complex (2nd OC) prepared from HEK293T cells using the method disclosed herein.

[0116] Example 8

[0117] Elucidating the role of GSH and catalase in the anti-ROS activity of the first organelle complex

[0118] A cell-based assay was performed to examine the role of glutathione (GSH) and catalase in the anti-ROS activity of organelle complex I. Buthionine sulfoxide (BSO) was used to reduce GSH levels in organelle complex I. Fig. 10A Depicted is the concentration of GSH in the first organelle complex derived from HEK293 cells that have been contacted with BSO (BSO-1st OC) or the first organelle complex of HEK293 cells that have not been contacted with it (293-1st OC). HEK293 cells in culture were passaged and incubated with H 2 O 2 (100 μM) and 293-1OC or BSO-1OC were incubated together. CellTiter-Glo (CTG2.0) was used to determine the 2 O 2 Cell viability 4 and 20 hours after treatment. The results showed that GSH in the first organelle complex is not an important factor in its anti-ROS activity ( Figures 10B to 10C ). Next, the ROS scavenging activity of the first organelle complex after catalase depletion was evaluated. HEK293 cells were transfected with catalase siRNA (h) (Santa cruz Biotechnology (SCB); sc-45330; lot number B2423) using a common transfection reagent for 24 or 48 hours, and the expression level of catalase in total cell lysates was evaluated by Western blot analysis ( Fig.11A ). Fig. 11BCatalase levels in untreated first organelle complex (1st OC) or catalase-depleted first organelle complex (1st OC-siRNA) are depicted. After confirming the successful knockdown of catalase in the first organelle complex, the ROS scavenging activity of catalase-depleted first organelle complex (1st OC-siRNA) was evaluated. HEK293 cells were pretreated with the first organelle complex (293-1st OC) or catalase-depleted first organelle complex (293-1st OC-siRNA) for 6 hours, followed by H 2 O 2 (300 μM) for 18 hours. Cell viability was measured using CellTiter-Glo (CTG2.0). It was found that knockdown of catalase in the first organelle complex reduced its ROS scavenging activity with statistical significance (P<0.005) ( Fig. 11C ). These results provided in this example indicate that GSH depletion in the first organelle complex by BSO does not affect its anti-ROS activity, while the presence of catalase in the organelle complex (e.g., the first organelle complex, the second organelle complex) is an important factor in its ROS scavenging activity.

[0119] Unless such substitution is technically infeasible, in at least some of the previously described embodiments, one or more elements used in an embodiment may be interchangeably used in another embodiment. It should be understood by those skilled in the art 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 fall within the scope of the subject matter defined by the appended claims.

[0120] Regarding the use of any plural and / or singular terms herein, those skilled in the art can translate plural into singular and / or singular into plural according to context and / or application. For the sake of clarity, various singular / plural arrangements can be explicitly set forth herein. Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "one / a kind (a)", "one / a kind (an)" and "the / said (the)" include plural references. Unless otherwise indicated, any reference to "or" herein is intended to encompass "and / or".

[0121] Those skilled in the art will understand that, in general, the terms used herein and particularly in the appended claims (e.g., the bodies 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 "comprising" should be interpreted as "including but not limited to," etc.). Those skilled in the art will also understand that if a specific number of claim recitations is intended to be introduced, such intent will be explicitly recited in the claim, and in the absence of such a recitation, no such intent is present. For example, to aid understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that the introduction of claim recitations by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitations to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article, such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"); the same is true for the use of definite articles to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, one skilled in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., reciting "two recitations" alone without mentioning other modifiers means at least two recitations, or two or more recitations). Furthermore, in those cases where a convention similar to “at least one of A, B, and C, etc.” is used, generally speaking, such construction is intended to be in the sense that one skilled in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include, but is not limited to, systems having: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to “at least one of A, B, or C, etc.” is used, generally speaking, such construction is intended to be in the sense that one skilled in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include, but is not limited to, systems having: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will also appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms (whether in the specification, claims or drawings) should be understood to contemplate the possibility of including one of the terms, any one of the terms, or both of the terms.

[0122] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0123] As will be understood by those skilled in the art, for any and all purposes, such as in providing written specifications, all scopes disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof.Any listed scope can be easily considered to fully describe and enable the same scope to be decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each scope discussed herein can be lower third, middle third, and upper third, etc. It can also be understood by those skilled in the art that all languages ​​(such as "until", "at least", "greater than", "less than", etc.) include quoted numbers and refer to the scope of the sub-ranges that can be decomposed into as described above subsequently.Finally, as will be understood by those skilled in the art, scope includes each individual member.Therefore, for example, a group with 1 to 3 articles refers to a group with 1, 2 or 3 articles.Similarly, a group with 1 to 5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0124] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A method for reducing or preventing oxidative stress and / or reductive stress in a redox-sensitive composition, comprising: contacting a redox-sensitive composition with an effective amount of a redox modulating 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, wherein the organelle complex comprises mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus.

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 a 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, wherein the redox regulating composition comprises an isolated organelle complex, wherein the organelle complex comprises mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus.

3. The method of any one of claims 1 to 2, wherein the contacting step comprises incubating the mixture of the redox sensitive composition and the redox modulating composition for less than about 30 seconds.

4. The method of any one of claims 1 to 3, wherein the effective amount comprises at least about 5 ug / mL to about 5 mg / mL of the redox regulating composition.

5. The method according to any one of claims 1 to 4, wherein the redox-sensitive composition is experiencing oxidative stress and / or reductive stress or is at risk of experiencing oxidative stress and / or reductive stress.

6. The method of any one of claims 1 to 5, wherein 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, further optionally the one or more cells are cells of a subject, further optionally the one or more cells are experiencing or are at risk of experiencing hypoxia.

7. The method according to any one of claims 1 to 6, wherein the contacting is performed ex vivo, in vitro or in vivo.

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 of any one of claims 1 to 8, wherein the redox-sensitive composition comprises a biological sample.

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

11. A method for reducing or preventing oxidative stress and / or reductive 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 reductive stress in the subject, wherein the redox regulating composition comprises an isolated organelle complex, wherein the organelle complex comprises mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a 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 modulating composition, thereby treating or preventing a redox disease or disorder in the subject, wherein the redox regulating composition comprises an isolated organelle complex, wherein the organelle complex comprises mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus.

13. The method according to any one of claims 1 to 12, wherein the redox regulating composition comprises homogenized mitochondria, a first organelle complex and / or a second organelle complex, wherein the first organelle complex and the second organelle complex comprise mitochondria and one or more of endoplasmic reticulum, peroxisome, lysosome and Golgi apparatus, wherein said first organelle complex and said second organelle complex are depleted of cytoplasmic macromolecules, wherein the first organelle complex is derived from (i) frozen cells; (ii) floating cells; and / or (iii) cells contacted with a surfactant at a concentration equal to or greater than the critical micelle concentration (CMC) of the surfactant, and wherein the second organelle complex is derived from (i) adherent cells; and / or (ii) cells contacted with a surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant, Optionally, said first organelle complex and said 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.

14. The method of any one of claims 1 to 13, wherein the effective amount comprises at least about 1 ug to about 1 mg of the redox modulating composition.

15. The method of any one of claims 1 to 14, wherein the redox regulating composition does not comprise intact cells.

16. The method according to any one of claims 1 to 15, wherein the oxidative stress and / or the reductive stress comprises elevated levels of ROS, RNS and / or free radicals.

17. The method according to any one of claims 1 to 16, wherein the oxidative stress and / or the reductive stress comprises altered cellular function.

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

19. The method of 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 of any one of claims 1 to 19, wherein the redox regulating composition reduces the level of oxidative cellular stress and thereby restores or repairs the cellular function.

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

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

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

24. The method of any one of claims 1 to 23, wherein the redox modulating composition increases and / or decreases one or more of the following ratios in the subject or in the redox sensitive composition: the oxidized form of nicotinamide adenine dinucleotide to the reduced form (NAD + / NADH), the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP + / NADPH), oxidized and reduced forms of glutathione (GSSG / GSH), and oxidized and reduced forms of thioredoxin (TrxSS / TrxSH2).

25. The method according to any one of claims 1 to 24, wherein the reactive nitrogen species include nitric oxide (NO), nitrogen dioxide radicals (.NO2), peroxynitrite anions (ONOO - ), peroxynitrite (ONOOH), nitrosoperoxycarbonate anion (ONOOCO2 - ), nitronium cation (NO2 + ), nitrosium cation (NO + ) or nitrogen trioxide (N2O3) or any combination thereof.

26. The method according to any one of claims 1 to 25, wherein the reactive oxygen species comprises superoxide (O2. - ), hydroperoxide (HO.2), hydrogen peroxide (H2O2), peroxynitrite (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.

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

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

29. The method according to any one of claims 1 to 28, wherein the method comprises identifying a subject who is experiencing or is at risk of experiencing oxidative and / or reductive stress.

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

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

32. The method according to any one of claims 1 to 31, wherein the probe for detecting reactive oxygen species is 2',7'-dichloro-fluorescein diacetate, dihydrorhodamine 123, 3'-(p-aminophenyl)fluorescein (API), 3'-(p-hydroxyphenyl)fluorescein (HPF), aminophenoxycalcein (APC), mitoAR, mitoHR, DPAX, DMAX, hydrogenocyanine or dihydroethidine.

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

34. The method of any one of claims 1 to 33, wherein the subject has or is suspected of having a disease or condition in which ROS are implicated, the disease or condition 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, Reye's syndrome, Leber's hereditary optic neuropathy, mitochondrial myopathy, cardiomyopathy, deafness, mood disorders, movement disorders, dementia, amyotrophic lateral sclerosis sclerosis, multiple sclerosis, tardive dyskinesia, brain injury, schizophrenia, epilepsy, AIDS dementia, endothelial nitroglycerin tolerance, doxorubicin toxicity, renal injury in type I diabetes, ex vivo renal 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 burns, cancer, inflammation and ischemia-reperfusion injury in stroke, heart attack, ultraviolet damage to the skin, organ transplantation, surgery, and any combination thereof.

35. The method of any one of claims 1 to 34, wherein administering the redox regulating composition to the subject treats, reduces, or prevents ischemia-reperfusion injury in the subject.

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

37. The method of any one of claims 1 to 36, wherein the redox regulating composition inhibits ischemia-reperfusion injury, mitochondrial dysfunction, hypoxic injury, necrotic cell death, or any combination thereof.

38. The method of any one of claims 1 to 37, wherein the redox disease or disorder comprises ROS-mediated and / or RNS-mediated oxidative damage to one or more tissues of the subject.

39. The method of any one of claims 1 to 38, wherein the redox disease or disorder is selected from the group consisting of: mitochondrial disorders; inherited mitochondrial diseases; Alpers disease; Barth syndrome; beta oxidation defects; 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); CPTI deficiency; CPTII deficiency; Friedreich's ataxia (FA); glutaric acidemia type II; Kearns-Sell syndrome (KSS); lactic acidosis; long chain acyl-CoA dehydrogenase deficiency (LCA D); LCHAD; Leigh syndrome; Leigh-like syndrome; Leber's hereditary optic neuropathy (LHON); fatal infantile cardiomyopathy (LIC); Luft disease; multiple acyl-CoA dehydrogenase deficiency (MAD); medium-chain acyl-CoA dehydrogenase deficiency (MCAD); mitochondrial myopathy-encephalopathy with lactic acidosis and stroke (MELAS); myoclonic epilepsy with red ragged fibers (MERRF); mitochondrial recessive ataxia syndrome (MIRAS); mitochondrial cytopathic disease, mitochondrial DNA depletion; mitochondrial encephalopathy; mitochondrial myopathy; myoneurogastrointestinal encephalopathy and encephalopathy (MNGIE); neuropathy, ataxia with 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; Encephalomyopathies; Neurodegenerative diseases; Parkinson's disease; Alzheimer's disease; Amyotrophic lateral sclerosis (ALS); Motor neuron disease; Nervous system diseases; Epilepsy; Age-related diseases; Macular degeneration; Diabetes; Metabolic syndrome; Brain cancer; Genetic diseases; Huntington's disease; Mood disorders; Schizophrenia; Bipolar disorder; Pervasive developmental disorders ; autistic disorder; Asperger's syndrome; childhood disintegrative disorder (CDD); Rett's disorder; PDD-not otherwise specified (PDD-NOS); cerebrovascular accident; stroke; visual impairment; optic neuropathy; dominantly inherited juvenile optic atrophy; optic neuropathy caused by toxic agents; glaucoma; Stargardt's macular dystrophy; diabetic retinopathy; diabetic maculopathy; retinopathy of prematurity; ischemia-reperfusion-related retinal damage; oxygen toxicity; hemoglobinopathies; thalassemia; sickle cell anemia; seizures; ischemia; renal tubular acidosis; Attention-deficit / hyperactivity disorder (ADHD); neurodegenerative disorders resulting in hearing or balance impairment; dominant optic atrophy (DOA); maternally inherited diabetes and deafness (MIDD); chronic fatigue; contrast-induced renal injury; contrast-induced retinopathy injury; abetalipoproteinemia; retinitis pigmentosa; Wolfram's disease; Tourette syndrome; cobalamin c deficiency; methylmalonic acidemia; glioblastoma; Down syndrome; acute tubular necrosis; muscular dystrophy; leukodystrophy; progressive supranuclear palsy; spinal muscular atrophy; hearing loss; noise-induced hearing loss; traumatic brain injury; juvenile Huntington's disease; multiple sclerosis; NGLY1; multiple system atrophy; adrenoleukodystrophy; adrenomyoeloneuropathy; and any combination thereof.

40. The method of any one of claims 1 to 39, wherein the redox regulating composition improves one or more energy biomarkers in the subject or the redox sensitive composition, the 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 + ) level; NADPH (NADPH+H + ) level; 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 level (V02); carbon dioxide excretion level (VC02); respiratory quotient (VC02 / V02); exercise tolerance; anaerobic threshold; and any combination thereof.

41. The method of any one of claims 1 to 40, wherein the administration comprises intravenous administration, intra-arterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and / or intraocular administration.

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