WO2022178423A1 - Okn-007 as an agent to improve longevity - Google Patents

Abstract

The present invention includes compositions and methods for increasing longevity of a cell or organism comprising administering to the cell or organism a composition comprising a therapeutically effective amount of 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl α-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof to increase a longevity of a cell or organism.

Description

OKN-007 AS AN AGENT TO IMPROVE LONGEVITY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/152,163, filed February 22, 2021, the entire contents of which is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates in general to the field of a therapeutic agent for the improvement of longevity.

STATEMENT OF FEDERALLY FUNDED RESEARCH [0003] Not Applicable.

BACKGROUND OF THE INVENTION

[0004] Without limiting the scope of the invention, its background is described in connection with compositions and methods for improving longevity.

[0005] One such treatment is taught in U.S. Patent No. 10,772,938, issued to Niklason, et ak, entitled “Compositions and methods of increasing longevity or treating cellular stress”. These inventors are said to teach methods and compositions for increasing longevity of a cell and increasing cellular resistance to stress that includes a method to induce gene expression of a homolog of Pachytene Checkpoint 2 (pch-2) or bmk-1 gene and methods to treat oxidative stress or induce cellular death or apoptosis by administering a composition comprising a modulator of pch-2 or bmk-1 homolog gene expression.

[0006] However, a need remains for novel agents that can be used more broadly to treat improving longevity.

SUMMARY OF THE INVENTION

[0007] In one embodiment, the present invention includes a method for increasing longevity of a cell or organism comprising administering to the cell or organism a composition comprising a therapeutically effective amount of 2,4-Disulfonyl-N-Tert- Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof to increase a longevity of a cell or organism. In one aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or intraperitoneally. In another aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof are from about 40 to 1,200 mg/kg body weight/day, 100 to 450 mg/kg body weight/day, 200 to 400 mg/kg body weight/day, 300 to 800 mg/kg body weight/day, 350 to 1,000 mg/kg body weight/day, or 400 to 1,100 mg/kg body weight/day. In another aspect, the

2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone, or a pharmaceutically acceptable salt thereof, is provided in a sustained-release formulation. In another aspect, the composition at least one of: reduces free radicals in the brain, protects neurons, prevents microglial cell activation, or prevents a decrease in muscle mass. In another aspect, the composition inhibits the transforming growth factor bΐ (TGF-bI) pathway. In another aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is at least one of: (1) administered at least one of continuously, intermittently, systemically, or locally, (2) administered one or more times a day; (3) sequentially or concomitantly, with another pharmaceutical agent; or (4) as a single agent or in combination with another pharmaceutical agent.

[0008] In another embodiment, the present invention includes a method for increasing cellular resistance to stress, aging or DNA damage in a cell comprising contacting the cell with a composition that inhibits the transforming growth factor bΐ (TGF-bI) pathway. In one aspect, the composition comprises a therapeutically effective amount of

2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof to increase a longevity of a cell or organism. In another aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or intraperitoneally. In another aspect, the 2,4-Disulfonyl-N-Tert- Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof are from about 40 to 1,200 mg/kg body weight/day, 100 to 450 mg/kg body weight/day, 200 to 400 mg/kg body weight/day, 300 to 800 mg/kg body weight/day, 350 to 1,000 mg/kg body weight day, or 400 to 1,100 mg/kg body weight/day. In another aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a- phenyl tertiary butyl nitrone, or a pharmaceutically acceptable salt thereof, is provided in a sustained-release formulation. In another aspect, the composition at least one of: reduces free radicals in the brain, protects neurons, prevents microglial cell activation, or prevents a decrease in muscle mass. In another aspect, the 2,4-Disulfonyl-N-Tert- Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is at least one of: (1) administered at least one of continuously, intermittently, systemically, or locally, (2) administered one or more times a day; (3) sequentially or concomitantly, with another pharmaceutical agent; or (4) as a single agent or in combination with another pharmaceutical agent.

[0009] In another embodiment, the present invention includes a method for increasing cellular resistance to stress, aging or DNA damage in a cell comprising contacting the cell with a composition comprising a therapeutically effective amount of 2,4-Disulfonyl- N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof to increase a longevity of a cell or organism. In one aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or intraperitoneally. In another aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof are from about 40 to 1,200 mg/kg body weight/day, 100 to 450 mg/kg body weight/day, 200 to 400 mg/kg body weight/day, 300 to 800 mg/kg body weight/day, 350 to 1,000 mg/kg body weight/day, or 400 to 1,100 mg/kg body weight/day. In another aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone, or a pharmaceutically acceptable salt thereof, is provided in a sustained-release formulation. In another aspect, the composition at least one of: reduces free radicals in the brain, protects neurons, prevents microglial cell activation, or prevents a decrease in muscle mass. In another aspect, the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is at least one of: (1) administered at least one of continuously, intermittently, systemically, or locally, (2) administered one or more times a day; (3) sequentially or concomitantly, with another pharmaceutical agent; or (4) as a single agent or in combination with another pharmaceutical agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

[0011] FIGS. 1A to ID show that OKN-007 reduces free radical levels in LPS-exposed rat brains, an inflammaging model. At 1-week post-LPS there are increased free radical levels (p<0.0001) compared to saline. Representative Tl-weighted images overlaid with a post-minus a pre-contrast image following administration of the anti-DMPO probe that targets DMPO-trapped macromolecular radicals for (FIG. 1A) LPS-administered rat brains at 1-week post-LPS, and (FIG. IB) OKN-007 treatment following LPS-exposure after 1-week. (FIG. 1C) MRI signal intensity percent (%) differences normalized to muscle graphs of LPS-and saline-administered rat brains given anti-DMPO probes at 1- week post-LPS, saline or LPS + OKN-007. There was a significant decrease in detection of DMPO-trapped radicals in the LPS + OKN-007-treated rat brains at 1-week (p<0.001) post-LPS, compared to LPS alone. (FIG. ID) Illustration of the anti-DMPO probe, which consists of Gd-DTPA, anti-DMPO antibody bound to albumin, and biotin. n=5 for each group.

[0012] FIGS. 2A and 2B show: FIG. 2A shows a-MN counts; at 25 months of age the number of a-motor neurons is higher in treated mice (n = 10, mean SD = 15.45 2.79) comparing to untreated controls (n = 9, mean SD = 11.44 2.11; unpaired two-tailed t- test p = 0.0028 (denoted by #). Data from young control and older control mice from the inventrors’ previous publication (Piekarz et ak, 2020) are also graphed for comparison. Analysis by one way ANOVA for all four groups revealed differences as indicated on the graph). FIG. 2B - a representative image of a lumbar spinal cord ventral hom of an old untreated WT mouse (old control) and old OKN-007-treated WT mouse (old OKN- 007).

[0013] FIGS. 3A to 3E show that OKND007 decreases microglia proliferation and activation, without affecting astrocyte proliferation. FIG. 3A - a representative image of immunostaining for Ibal in lumbar spinal cord ventral hom of treated and untreated old wildtype mice; inset shows the morphology of a representative micro-glial cell; FIG. 3B - quantification of microglia proliferation determined as the number of Ibal -positive cells per ventral hom; the treated group has less microglia (treated: n = 10, mean ± SD = 91.05 ± 14.49; untreated: n = 7, mean ± SD = 103.9 ± 3.63 unpaired t-test with Welch’s correction p = 0.0216); FIG. 3C -microglia activation represented as the number of non-activated microglia (expressed as a percentage of all Ibal -positive cells per ventral hom). Microglia activation is higher in treated group (n = 10, mean ± SD = 26.72 ± 18.67) comparing to the control (n = 7, mean ± SD = 9.488 ± 5.08; unpaired t- test with Welch’s correction p = 0.0091); FIG. 3D - a representative image of anti- GFAP immunostaining to visualize astrocytes in ventral hom of treated and untreated wildtype mice; the inset shows a close-up of a representative astrocyte; FIG. 3E - quantification of GFAP proliferation. The treatment does not affect the number of GFAP-positive cells (treated: n = 10, mean ± SD = 81.36 ± 7.27; untreated: n = 9, mean ± SD = 76.46 ± 11.59; two-tailed unpaired.

[0014] FIGS. 4A and 4B shows that OKN-007 restores brain vascularity to normal in LPS-exposed rat brains (cerebral cortex and hippocampus) better than rapamycin. LPS- treated rat brains have significantly decreased relative cerebral blood flow (rCBF) at 1-6 weeks in both the cerebral cortex (A) and hippocampus (B) regions post-LPS exposure (LPS vs. saline: cerebral cortex (****p<0.0001 at 1, ***p<0.001 at 3 weeks, and **p<0.01 at 6 weeks; hippocampus (**p<0.05 at 1-week, **p<0.01 at 3 weeks, and ***p<0.001 at 6 weeks). rCBF was found to be significantly restored by OKN-007 treatment in LPS-exposed rat brains in the cerebral cortex (†††p<0.001 at 1-week post- LPS, ††p<0.01 at 3 weeks, and †p<0.05 at 6 weeks in the cerebral cortex; †p<0.05 at 1 and 3 weeks, and ††p<0.01 at 6 weeks in the hippocampus), when compared to LPS- exposure alone. Rapamycin was only effective at restoring rCBF at 1 and 3 weeks in the cerebral cortex (††††p<0.0001 and †p<0.05, respectively), compared to LPS- alone. n=20-28 for all groups, except n=5 for rapamycin groups.

[0015] FIG. 5 shows that OKN-007 restores brain metabolites to normal following LPS- exposure better than rapamycin. LPS-treated rat brains have decreased brain metabolites at various time-points following LPS exposure. Metabolite/choline (Cho) ratios for NAA/Cho (24 hours and 6 weeks post-LPS; *p<0.05), Cr/Cho (3 wks post-LPS; **p<0.01), and Myolns/Cho (1 wk (*p<0.05), 3 wks (****p<0.0001) and 6 wks (*p<0.05) post-LPS) are decreased when comparing LPS- and saline- administered rat brains. OKN-007 restores brain metabolites (NAA/Cho ratio at 24 hrs and 1 wk (††p<0.01 for both), as well as 3 and 6 wks post-LPS (†p<0.05 for both; (Cr/Cho ratio at 24 hrs (††p<0.01) and 3 wks (†p<0.05) post-LPS); and (Myo-Ins/Cho ratio at 24 hrs ((††††p<0.0001), 1 wk (†p<0.05), 3 wks (†††p<0.001), and 6 wks (††p<0.01) post-LPS), when compared to LPS alone. There was a significant increase in NAA between the LPS alone group vs. the LPS + RAPA group at 1, 3 and 6 wks post-LPS; a significant increase in Cr between the LPS alone group vs. the LPS + RAPA group at 24 hrs; and a significant decrease in Myo- Ins (M-I) between the LPS alone group vs. the LPS + RAPA group at 24 hrs. n=20-28 for all, except n=5 for RAPA.

[0016] FIG. 6 shows mouse body weights obtained at pre- treatment and post-treatment (OKN-007 vs. control) in aged mice. n=ll-13 for each group. Two-way ANOVA (p=0.3349 regarding treatment effect).

[0017] FIG. 7 shows the lean mass weights obtained at pre-treatment and post- treatment (OKN-007 vs. control) of aged mice. n=10-13 for each group. Two- way ANOVA (p=0.0045 regarding treatment effect).

[0018] FIG. 8 shows mouse fat weights obtained at pre-treatment and post-treatment (OKN-007 vs. control) in aged mice. n=ll-13 for each group. Two-way ANOVA (p=0.2675 regarding treatment effect).

[0019] FIGS. 9A to 9D show the 34 genes in intersection between old treated and untreated groups. The length of the bar indicates the t-test statistics resulting from a test for differential expression between the old treated and untreated groups. The t-test statistic is a normalized version of the fold change by the intergroup variation that compresses information on both fold change sign and p-value ordering.

[0020] FIG. 10 shows number of up-regulated and down-regulated genes when comparing young mice to old mice and when comparing untreated old (control) mice to OKN-007-treated old mice.

[0021] FIG. 11 shows the top upstream regulators with target molecules when comparing old-OKN-007-treated mice with old untreated (control) mice.

[0022] FIG. 12 shows the top canonical pathways when comparing old-OKN-007- treated mice with old untreated (control) mice.

[0023] FIG. 13 shows the top diseases by activation when comparing old-OKN-007- treated mice with old untreated (control) mice.

[0024] FIG. 14 the shows top diseases by p-value when comparing old-OKN-007- treated mice with old untreated (control) mice. [0025] FIG. 15 shows the top up-regulated molecules when comparing old-OKN-007- treated mice with old untreated (control) mice.

[0026] FIG. 16 shows the top down-regulated molecules when comparing old-OKN- 007-treated mice with old untreated (control) mice.

FIGS. 17A and 17B shows that OKN-007 decreases BSCB permeability. FIG. 17A - a representative image of a lumbar spinal cord ventral hom for OKN-007 treated and untreated old WT mice after HRP injection and visualization with DAB; FIG. 17B - there is a decrease in the number of HRP extravasation regions in the treated group (n = 5, mean ± SD = 1 ± 1.17) compared to untreated controls (n = 3, mean ± SD = 2.667

1.155, one-tailed unpaired t-test, p = 0.0491 denoted by #). Data from young control and older control mice from the inventors’ previous publication [11] are also graphed for comparison. Analysis by one way ANOVA for the four groups revealed differences as indicated on the graph.

[0027] Fig. 18 shows the Top 20 differentially expressed molecules (increased and decreased expression) in old treated and untreated groups. The length of the bar indicates the t-test statistics resulting from a test for differential expression between the old treated and untreated groups. The t-test statistic is a normalized version of the fold change by the intergroup variation that compresses information on both fold change sign and p-value ordering.

[0028] Fig. 19 shows the genes reversed in old mice by OKND007 treatment. A - Heatmap showing 131 genes that are differentially expressed in young vs old mice and which OKN-007 brings back to the “youthful” state; B - top upstream regulators pre dicted by IPA with names of affected molecules listed in the bars; C - top ten canonical pathways involved in OKN-007 aging reversal effect, ordered by p-value (with B-H correction) with molecules involved in each pathway listed inside the bars. D - top ten diseases related to OKN-007 aging reversal effect, sorted by p-value, identified by IPA analysis; the number in each bar represents the number of molecules affected in each disease category.

DETAILED DESCRIPTION OF THE INVENTION

[0029] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0030] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0031] The present invention includes various methods of treating patients with compound 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone, also known as: OKN-007, NXY-059, 2,4-DSPBN, 2,4-disulfonyl phenyl tert- butyl nitrone, 2,4-ds-PBN and Cerovive. It has been found that OKN-007 is a small molecule that can easily cross the blood brain barrier and has been demonstrated to have no adverse effects on normal cells, unlike other agents that have systemic toxicity. OKN-007 can be administered either orally in pill form (patient compliant), or intravenously.

[0032] Further, it was found that OKN-007 is a neuroprotective agent (both in brain and spinal cord). There are currently no pharmaceutical compounds or agents that protect motor neurons, as well as inhibit late-stage therapies that have these qualities. All other therapeutic approaches do not prevent late-stage disease progression.

[0033] The therapeutic methods of the present invention (which include prophylactic treatment) in general include administration of a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a neurodegenerative disease, or having a symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, marker (as defined herein), family history, and the like).

[0034] As used herein, a “pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

[0035] As used herein, the term “safe and effective amount” refers to the quantity of a component that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.

[0036] As used herein, the term “therapeutically effective amount” is meant an amount of a compound of the present invention effective to yield the desired therapeutic response. For example, an amount effective to delay or reverse a neurodegenerative disease. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

[0037] As used herein, a “pharmaceutical salt” is salt for making an acid or base salts of the compound. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. Preferably the salts are made using an organic or inorganic acid. These preferred acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. The preferred phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium.

[0038] As used herein, a “pharmaceutical carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the OKN-007 compound to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutical carrier.

[0039] OKN-007 is typically administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and/or carriers (collectively referred to herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and as consistent with conventional pharmaceutical practices. Depending on the best location for administration, the OKN-007 may be formulated to provide, e.g., maximum and/or consistent dosing for the particular form for oral, rectal, topical, intravenous injection or parenteral administration. While the OKN-007 may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier. The carrier may be solid or liquid, depending on the type and/or location of administration selected.

[0040] Techniques and compositions for making useful dosage forms using the present invention are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., HANDBOOK OF CLINICAL DRUG DATA, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., PRINCIPLES OF DRUG ACTION, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., BASIC AND CLINICAL PHARMACOLOGY, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Tenth Edition, McGraw Hill, 2001; REMINGTON’S PHARMACEUTICAL SCIENCES, 20th Ed., Lippincott Williams & Wilkins., 2000, and updates thereto; Martindale, THE EXTRA PHARMACOPOEIA, Thirty- Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference, and the like, relevant portions incorporated herein by reference.

[0041] For example, the OKN-007 may be included in a tablet. Tablets may contain, e.g., suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents and/or melting agents. For example, oral administration may be in a dosage unit form of a tablet, gel cap, caplet or capsule, the active drug component being combined with an non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, mixtures thereof, and the like. Suitable binders for use with the present invention include: starch, gelatin, natural sugars (e.g., glucose or beta-lactose), com sweeteners, natural and synthetic gums (e.g., acacia, tragacanth or sodium alginate), carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants for use with the invention may include: sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, mixtures thereof, and the like. Disintegrators may include: starch, methyl cellulose, agar, bentonite, xanthan gum, mixtures thereof, and the like.

[0042] OKN-007 may be administered in the form of liposome delivery systems, e.g., small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles, whether charged or uncharged. Liposomes may include one or more: phospholipids (e.g., cholesterol), stearylamine and/or phosphatidylcholines, mixtures thereof, and the like.

[0043] OKN-007 may also be coupled to one or more soluble, biodegradable, bioacceptable polymers as drug carriers or as a prodrug. Such polymers may include: polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, mixtures thereof, and the like. Furthermore, the OKN-007 may be coupled one or more biodegradable polymers to achieve controlled release of the OKN- 007, biodegradable polymers for use with the present invention include: polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels, mixtures thereof, and the like.

[0044] In one embodiment, gelatin capsules (gel caps) may include the OKN-007 and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Like diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as immediate-release, mixed-release or sustained-release formulations to provide for a range of release of medication over a period of minutes to hours. Compressed tablets may be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere. An enteric coating may be used to provide selective disintegration in, e.g., the gastrointestinal tract.

[0045] For oral administration of OKN-007 in a liquid dosage form, the oral drug components may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents, mixtures thereof, and the like. [0046] Liquid dosage forms for oral administration of the OKN-007 may also include coloring and flavoring agents that increase patient acceptance and therefore compliance with a dosing regimen. In general, water, a suitable oil, saline, aqueous dextrose (e.g., glucose, lactose and related sugar solutions) and glycols (e.g., propylene glycol or polyethylene glycols) may be used as suitable carriers for parenteral solutions. Solutions for parenteral administration include generally, a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffering salts. Antioxidizing agents such as sodium bisulfite, sodium sulfite and/or ascorbic acid, either alone or in combination, are suitable stabilizing agents. Citric acid and its salts and sodium EDTA may also be included to increase stability. In addition, parenteral solutions may include pharmaceutically acceptable preservatives, e.g., benzalkonium chloride, methyl-or propyl-paraben, and/or chlorobutanol. Suitable pharmaceutical carriers are described in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, a standard reference text in this field, relevant portions incorporated herein by reference.

[0047] Capsules. Capsules with OKN-007 may be prepared by filling standard two- piece hard gelatin capsules each with 10 to 500 milligrams of powdered active ingredient, 5 to 150 milligrams of lactose, 5 to 50 milligrams of cellulose and 6 milligrams magnesium stearate.

[0048] Soft Gelatin Capsules. A mixture of OKN-007 is dissolved in a digestible oil such as soybean oil, cottonseed oil or olive oil. The active ingredient is prepared and injected by using a positive displacement pump into gelatin to form soft gelatin capsules containing, e.g., 100-500 milligrams of the active ingredient. The capsules are washed and dried.

[0049] Tablets. A large number of tablets are prepared by conventional procedures so that the dosage unit of OKN-007, e.g., 1-500 milligrams of OKN-007, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 50-275 milligrams of microcrystalbne cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

[0050] To provide an effervescent tablet appropriate amounts of, e.g., monosodium citrate and sodium bicarbonate, are blended together and then roller compacted, in the absence of water, to form flakes that are then crushed to give granulates. The granulates are then combined with the active ingredient, drug and/or salt thereof, conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants.

[0051] Injectable solution. A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in deionized water and mixed with, e.g., up to 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized using, e.g., ultrafiltration.

[0052] Suspension. An aqueous suspension is prepared for oral administration so that each 5 ml contain 100 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 ml of vanillin.

[0053] It has been found that OKN-007 [OKlahoma Nitrone; disodium 4-(tert-butyl- imino) methyl) benzene- 1,3 -disulfonate N-oxide); also known as 2,4-disulfonyl-phenyl- N-tert-butyl nitrone; and also, NXY-059], is a nitrone compound that is anti inflammatory and neuroprotective in aging mice. OKN-007 is currently in phase II clinical trials as an Investigational New Drug (IND) for patients with recurrent glioblastoma (GBM) and has been found to have no adverse effects in these patients (~40 GBM patients), as well as over 3,000 human patients when this compound was assessed as a potential therapy for stroke (phase I-III clinical trials). The mechanism-of-action (MOA) for OKN-007 in GBM is via the inhibition of the tumorigenic transforming growth factor bΐ (TGF-bI) pathway, particularly through the downregulation of collagen and matrix metalloproteinase genes [1] In addition, in rodent models for GBM, OKN- 007 has been found to be a free radical scavenger and decrease the levels of oxidative proteins and lipids [2] It was found that OKN-007 protects motor neurons and prevents microglia from being activated in an aging mouse model. In addition, OKN-007 was able to restore brain vascularity and brain metabolites, including a neuron-specific metabolite, N-acetyl aspartate (NAA), in a lipopolysaccharide (LPS)-induced neuroinflammatory rat model [3], and was found to perform better than rapamycin, which is a well-studied agent for lifetime extension.

[0054] OKN-007 as an anti-inflammatory agent. Age-associated inflammation, also referred to as “inflammaging”, has been studied as a major player in shortened lifespan and chronic disease [4] Nitrone compounds are known to be anti-inflammatory in rodent models for encephalopathy, and the MOA is thought to be via NF-KB (nuclear factor kB) inhibition [5-7] In endotoxin (LPS)-induced rodents, PBN (phenyl N-tert- butyl nitrone; parent compound for OKN-007) was found to inhibit the induction of iNOS in mice [8] and also inhibited apoptosis-associated gene expression (e.g. Fas-A, Bax) [9] and multiple anti-inflammatory cytokines (tumor necrosis factor (TNF-a), interleukins (IL-la and IL-Ib), nuclear factor KB (NF-KB), activator protein- 1 (AP-1)) [7] in rats. PBN was also found to provide a neuroprotective effect by reducing nitric oxide production in LPS-induced meningitis within rats [10], and inhibiting hypoxia- ischemia (Hl)-induced up-regulation of IL-Ib, TNF-a and iNOS mRNA expression following HI [11] For the HI study, it was also found that PBN had free radical scavenging activity [11]

[0055] It is demonstrated herein that OKN-007 is a free radical scavenger in the rat LPS-induced neuroinflammatory model [3], as shown in FIGS. 1A to ID. A combination of immuno-spin trapping (1ST) and molecular-targeted MRI was used to initially trap free radicals with the spin trapping agent, DMPO (1,5-dimethyl-pyrroline- N-oxide), and then visualize the trapped radicals by attached an anti-DMPO antibody to an MRI contrast agent (anti-DMPO probe; anti-DMPO antibody-albumin-Gd-DTPA- biotin).

[0056] OKN-007 protects neurons and prevents microglia from being activated in aged mice. It was found that OKN-007 in an aged mouse model (25 months; OKN-007 administered orally in drinking water (150 mg/kg/day) starting at 16 months of age) contributes to increased motor neuron (MN) survival (see FIGS. 2 A and 2B), as well as decreases microglia activation (see FIGS. 3 A to 3E).

[0057] FIGS. 2A and 2B show: FIG. 2A shows a-MN counts; at 25 months of age the number of a-motor neurons is higher in treated mice (n = 10, mean SD = 15.45 2.79) comparing to untreated controls (n = 9, mean SD = 11.44 2.11; unpaired two-tailed t- test p = 0.0028 (denoted by #). Data from young control and older control mice from the inventors’ previous publication (Piekarz et ak, 2020) are also graphed for comparison. Analysis by one way ANOVA for all four groups revealed differences as indicated on the graph). FIG. 2B - a representative image of a lumbar spinal cord ventral hom of an old untreated WT mouse (old control) and old OKN-007-treated WT mouse (old OKN- 007). [0058] FIGS. 3A to 3E show that OKN-007 decreases microglia proliferation and activation, without affecting astrocyte proliferation. FIG. 3A - a representative image of immunostaining for Ibal in lumbar spinal cord ventral hom of treated and untreated old wildtype mice; inset shows the morphology of a representative micro-glial cell; FIG. 3B - quantification of microglia proliferation determined as the number of Ibal -positive cells per ventral hom; the treated group has less microglia (treated: n = 10, mean ± SD = 91.05 ± 14.49; untreated: n = 7, mean ± SD = 103.9 ± 3.63 unpaired t-test with Welch’s correction p = 0.0216); FIG. 3C -microglia activation represented as the number of non- activated microglia (expressed as a percentage of all Ibal -positive cells per ventral hom). Microglia activation is higher in treated group (n = 10, mean ± SD = 26.72 ± 18.67) comparing to the control (n = 7, mean ± SD = 9.488 ± 5.08; unpaired t-test with Welch’s correction p = 0.0091); FIG. 3D - a representative image of anti-GFAP immunostaining to visualize astrocytes in ventral hom of treated and untreated wildtype mice; the inset shows a close-up of a representative astrocyte; FIG. 3E - quantification of GFAP proliferation. The treatment does not affect the number of GFAP -positive cells (treated: n = 10, mean ± SD = 81.36 ± 7.27; untreated: n = 9, mean ± SD = 76.46 ± 11.59; two-tailed unpaired.

[0059] FIGS. 4A and 4B shows that OKN-007 restores brain vascularity to normal in LPS-exposed rat brains (cerebral cortex and hippocampus) better than rapamycin. LPS- treated rat brains have significantly decreased relative cerebral blood flow (rCBF) at 1-6 weeks in both the cerebral cortex (A) and hippocampus (B) regions post-LPS exposure (LPS vs. saline: cerebral cortex (****p<0.0001 at 1, ***p<0.001 at 3 weeks, and **p<0.01 at 6 weeks; hippocampus (**p<0.05 at 1-week, **p<0.01 at 3 weeks, and ***p<0.001 at 6 weeks). rCBF was found to be significantly restored by OKN-007 treatment in LPS-exposed rat brains in the cerebral cortex (†††p<0.001 at 1-week post- LPS, ††p<0.01 at 3 weeks, and †p<0.05 at 6 weeks in the cerebral cortex; †p<0.05 at 1 and 3 weeks, and ††p<0.01 at 6 weeks in the hippocampus), when compared to LPS- exposure alone. Rapamycin was only effective at restoring rCBF at 1 and 3 weeks in the cerebral cortex (††††p<0.0001 and †p<0.05, respectively), compared to LPS-alone. n=20-28 for all groups, except n=5 for rapamycin groups.

[0060] FIG. 5 shows that OKN-007 restores brain metabolites to normal following LPS- exposure better than rapamycin. LPS-treated rat brains have decreased brain metabolites at various time-points following LPS exposure. Metabolite/choline (Cho) ratios for NAA/Cho (24 hours and 6 weeks post-LPS; *p<0.05), Cr/Cho (3 wks post-LPS; **p<0.01), and Myo-Ins/Cho (1 wk (*p<0.05), 3 wks (****p<0.0001) and 6 wks (*p<0.05) post-LPS) are decreased when comparing LPS- and saline- administered rat brains. OKN-007 restores brain metabolites (NAA/Cho ratio at 24 hrs and 1 wk (††p<0.01 for both), as well as 3 and 6 wks post-LPS (†p<0.05 for both; (Cr/Cho ratio at 24 hrs (††p<0.01) and 3 wks (†p<0.05) post-LPS); and (Myo-Ins/Cho ratio at 24 hrs ((††††p<0.0001), 1 wk (†p<0.05), 3 wks (†††p<0.001), and 6 wks (††p<0.01) post-LPS), when compared to LPS alone. There was a significant increase in NAA between the LPS alone group vs. the LPS + RAPA group at 1, 3 and 6 wks post-LPS; a significant increase in Cr between the LPS alone group vs. the LPS + RAPA group at 24 hrs; and a significant decrease in Myo- Ins (M-I) between the LPS alone group vs. the LPS + RAPA group at 24 hrs. n=20-28 for all, except n=5 for RAPA.

[0061] FIG. 6 shows mouse body weights obtained at pre-treatment and post-treatment (OKN-007 vs. control) in aged mice. n=ll-13 for each group. Two-way ANOVA (p=0.3349 regarding treatment effect). FIG. 7 shows the lean mass weights obtained at pre-treatment and post-treatment (OKN-007 vs. control) of aged mice. n=10-13 for each group. Two-way ANOVA (p=0.0045 regarding treatment effect). FIG. 8 shows mouse fat weights obtained at pre-treatment and post-treatment (OKN-007 vs. control) in aged mice. n=ll-13 for each group. Two-way ANOVA (p=0.2675 regarding treatment effect).

[0062] In addition, in a pilot study it was found that OKN-007 treatment in an aged mouse model also prevented an age-related decrease in muscle mass (see FIG. 7).

[0063] Thus, OKN-007 has a beneficial effect on protecting neurons and preventing microglia from being activated in aged mice, as well as maintaining brain blood barrier (BBB) integrity, brain vasculature (rCBF), and brain metabolites in an inflammaging model. OKN-007 has been well tolerated and found to have no adverse effects in both pre-clinical and human clinical studies.

[0064] OKN-007 is able to restore brain vasculature and metabolites better than rapamycin in an inflammaging model. The data herein shows that OKN-007 is able to perform better than rapamycin in an LPS-induced inflammaging rat model regarding restoring brain blood vasculature (measured as rCBF) (see FIGS. 4 A and 4B) and brain metabolites (see FIG. 5). It known that RAPA treatment can increase lifespan and health span in mice [17,18]

[0065] TREATMENT PROTOCOL.

[0066] Based on these data in the aged mouse study, the calculated dose is 150 mg/kg body weight/day (administered orally via drinking water, prepared fresh every 2-3 days) for the initial longevity trial in the ITP.

[0067] Rationale for dosage. A dose of 150 mg/kg/day in drinking water is recommended, as this dose was well tolerated by the mice as evidenced by a lack of change in body weight (see FIG. 6) or fat weight (see FIG. 8) in aged mice following OKN-007 treatment, compared to controls.

[0068] Route of administration. Based on the mice (aged or GBM) and rat (LPS- induced inflammaging) models, the drinking water administration route was effective, well tolerated, and no adverse effects were observed. Alternatively, OKN-007 could also be administered in a pill or orally directly. OKN-007 remains stable at room temperature in a water solution for up to 3 days without loss of stability or activity. Generally, the water containers should be shielded from light (brown bottles or covered in aluminum foil) to maintain stability. OKN-007 powder is stored at 4°C, and remains stable for over a year.

[0069] Monitoring the effects of OKN-007. The inventors used body/fat/muscle weights, immunofluorescence markers for neurons (NeuN) and activated microglia (Ibal), and MRI assessments for BBB disruption, brain vascularity perfusion rates, brain metabolites, and free radical scavenging, to determine the effect of OKN-007 in rodent models for aging and inflammaging.

[0070] Age at which treatment begins, and duration of treatment. Weaned mice are placed on the OKN-007 supplemented drinking water immediately, or at some predetermined age (e.g. at six weeks of age; e.g., the inventors have used 16 weeks of age). Since oxidative stress is minimal in young animals, but progressively increases with aging, the age of starting the OKN-007 supplement could start after a pre determined age after weaning (6-10 weeks of age).

[0071] Safety and toxicity studies on OKN-007. Extensive safety and toxicity studies have been previously done on OKN-007 (NXY-059) when it was considered for stroke treatment. Based on initial clinical trials, it has been established that NXY-059 (OKN- 007) did not reveal any serious adverse events from initial pharmacokinetics Phase I and safety Phase II studies [12-16]

[0072] OKNIH007 treatment is associated with maintenance of a OH motor neuron numbers in old mice. The inventors measured a-motor neuron number in two cohorts of mice that were treated with or without OKN-007 in drinking water starting at 16 months of age. As shown in in FIGS. 2A and 2B, at 25 months of age, a significantly greater number of a-motor neurons was observed in OKN-007-treated mice compared to age matched untreated controls (15.45 ± 2.79 (SD) versus 11.44 ± 2.11 (SD) motor neurons/ ventral hom (p = 0.0028). For comparison, data from [11] is shown and the analysis of all four groups by one-way ANOVA. Values from old OKN-treated mice are similar to values from the young control mice supporting a protective effect of OKN -007 on motor neuron numbers.

[0073] It was found that OKNIH007 decreases microglia proliferation and activation, without affecting astrocyte proliferation. Immunostaining for Ibal was used to measure microglia proliferation and activation in the ventral hom of the lumbar spinal accord FIGS. 3A-3C). This analysis revealed a greater than 10% decrease (p =0.0216) in the number of microglia identified by Ibal staining (FIG. 3B), suggesting reduced proliferation and a 1.8-fold increase (p = 0.0091) in the number of ramified or basal resting microglia (FIG. 3C), suggesting reduced activation in the spinal cord of OKN- 007 treated compared to untreated old wildtype mice. In contrast, the treatment had no effect on the number of cells immunolabelled by GFAP (glial fibrillary acidic protein), a marker of reactive astrocytes, suggesting no change (p = 0.2803) in astrocyte proliferation (FIGS. 3D and 3E).

[0074] OKN-007 decreases blood-spinal cord barrier (BSCB) permeability. Because inflammation is known to be casual in increased BSCB permeability [37-39], the inventors determined whether the anti-inflammatory properties of OKN-007 could reduce age related BSCB permeability. BSCB permeability was measured following i.p. injection of horse radish peroxidase (HRP) and found that the number of HRP extravasation regions in the ventral spinal cord of untreated older mice was more than 2.5-fold higher (p = 0.0491) than in old mice treated with OKN-007 (FIGS. 17A and 17B) showing a reduction in BSCB permeability in response to OKN-007. For comparison, the data from [11] is also show and the analysis of all four groups by one way ANOVA. [0075] Treatment with OKNIH007 changes the spinal cord transcriptome RNA-seq analysis was performed using Ingenuity Pathway Analysis (IP A) in samples of spinal cord from young, old and old OKN-007-treated mice, and the results are summarized in FIGS. 9A-9D and 10 to 13. This analysis revealed 2370 differentially expressed genes (DEGs) among the three groups analyzed (young untreated, old untreated, old OKN-007 treated). As shown in FIG. 10, there were 970 DEGs in the young vs old comparison dataset (669 upregulated with age/301 downregulated with age) and 117 DEGs in old OKN-007-treated vs old untreated mice (50 upregulated/67 downregulated in old spinal cord in OKN-007-treated mice versus old untreated mice). There was an overlap of 34 genes between the young vs old and old treated versus untreated analyses. These 34 genes are listed in FIG. 18. Most of these genes (21/34) were decreased in expression as a function of age, and increased by OKN-007 treatment. Two genes (TEKT4 and GPD1) were decreased with age and decreased further by OKN-007. Of the 11/34 genes showing increased expression as a function of age, eight were reduced following OKN- 007 treatment and three were increased even further by OKN-007. The top 20 most differentially expressed genes in a comparison of old control versus old OKN-007- treated mice are listed in FIGS. 9A to 9D. Surprisingly, at least 16 out of 117 DEGs (13 out of top 20 genes; FIGS. 9 A to 9D) in treated vs untreated old groups are poorly described predicted genes. However, among the known genes identified as down regulated transcripts in OKN-007 treated vs untreated old mice are solute carrier family 22 organic cation transporter member 1 (Slc22al), an organic cation transporter and guanylate cyclase 1 soluble alpha 2 (Gucyla2) which is involved in guanylate synthase signaling pathways.

[0076] In contrast, among the genes that exhibit an increase in expression in the spinal cord from OKN-007 mice are the sodium coupled glucose transporter (Slc25al2) that also transports monocarboxylates such as lactate, pyruvate, nicotinate, propionate, butyrate and beta-D-hydroxy butyrate, and chemokine ligand 13 (Cxcll3), which acts to attract B lymphocytes. Among the top pathways altered by OKN-007 treatment are G- protein coupled receptor (GPCR) pathways, fibrosis related pathways and glucose-e- phosphate signaling. IPA analysis also shows that the top diseases affected by the treatment with OKN-007 include disease related to neurotransmission, nervous system and neuron structure/morphology, and disease that alter movement and locomotion (FIG. 13). [0077] Interestingly, most of the differentially expressed (DE) genes in old mice differed significantly from both young and OKN-treated mice (29 out of 34), shown treatment reversal effect of expression variation pattern toward similar expression levels similar between young and treated mice. The data from young and treated mice were then collapsed into a single group and contrasted it to old mice. Since the t-statistic considers the difference between group averages relative to within-group variation, a strong t-statistic would indicate both strong difference between old mice versus the other groups, and similar expression levels in the young and treated mice. The resulting 131 differentially expressed genes include the 29 genes mentioned above, providing an extended set of genes that differ in the young vs untreated old, and that are no longer different in the old OKN-007-treated mice, suggesting a shift back towards the “youthful” state in response to OKN-007. An upstream regulator analysis for this dataset revealed that most of the predicted upstream regulators for this shift in gene expression were predicted to be inhibited and the effected genes are listed in FIG. 10. The top affected canonical pathways (FIG. 12) for this analysis pointed to changes in the GPCR pathways, similar to the results outline in FIG. 11 in the Old Control vs Old OKN-007 analysis. In addition, pathways involved in CREB, endocannabinoid, synaptogenesis and nNOS signaling were implicated as well. Overall, the top identified likely disease pathways were very similar to the Old Control vs Old OKN-007 comparison (FIG. 13).

[0078] FIGS. 9A to 9D show the 34 genes in intersection between old treated and untreated groups. The length of the bar indicates the t-test statistics resulting from a test for differential expression between the old treated and untreated groups. The t-test statistic is a normalized version of the fold change by the intergroup variation that compresses information on both fold change sign and p-value ordering.

[0079] FIG. 10 shows number of up-regulated and down-regulated genes when comparing young mice to old mice and when comparing untreated old (control) mice to OKN-007-treated old mice.

[0080] FIG. 11 shows the top upstream regulators with target molecules when comparing old-OKN-007-treated mice with old untreated (control) mice. FIG. 12 shows the top canonical pathways when comparing old-OKN-007-treated mice with old untreated (control) mice. FIG. 13 shows the top diseases by activation when comparing old-OKN-007-treated mice with old untreated (control) mice. FIG. 14 the shows top diseases by p-value when comparing old-OKN-007-treated mice with old untreated (control) mice. FIG. 15 shows the top up-regulated molecules when comparing old- OKN-007-treated mice with old untreated (control) mice. FIG. 16 shows the top down- regulated molecules when comparing old-OKN-007-treated mice with old untreated (control) mice.

[0081] FIG. 18 shows the Top 20 differentially expressed molecules (increased and decreased expression) in old treated and untreated groups. The length of the bar indicates the t-test statistics resulting from a test for differential expression between the old treated and untreated groups. The t-test statistic is a normalized version of the fold change by the intergroup variation that compresses information on both fold change sign and p-value ordering.

[0082] FIG. 19 shows the genes reversed in old mice by OKND007 treatment. A - Heatmap showing 131 genes that are differentially expressed in young vs old mice and which OKN-007 brings back to the “youthful” state; B - top upstream regulators predicted by IPA with names of affected molecules listed in the bars; C - top ten canonical pathways involved in OKN-007 aging reversal effect, ordered by p-value (with B-H correction) with molecules involved in each pathway listed inside the bars. D - top ten diseases related to OKN-007 aging reversal effect, sorted by p-value, identified by IPA analysis; the number in each bar represents the number of molecules affected in each disease category.

[0083] Materials and Methods. Animals. The experimental protocols were approved by the Oklahoma Medical Research Foundation (OMRF) Institutional Animal Care and Use Committee. Male C57BL/6J from OMRF colony and NIA were treated with OKN-007 in drinking water (150 mg/kg/day) from 16 months of age until sacrifice at 25 months of age. OKN -007-treated and control male mice (8 to 10 per group) were studied at 24-25 months of age. A few comparisons also include young male mice (3 to 4 months of age, n=3-6). The mice were maintained on a 12 h light/dark cycle and were fed ad libitum normal chow diet. Their water consumption and body weight were monitored weekly to allow for dose adjustments, if necessary, and to determine how much of the drug was actually consumed (on average 150.58 mg/kg/day). One OKN-007-treated mouse clustered with untreated controls and was dropped from further analysis as a non responder, since it would not provide information on OKN-007-induced transcriptional changes. [0084] Immunofluorescence. Spinal cord immunofluorescent staining was done as previously described [11] The following antibodies were used: anti-NeuN - Cell Signaling #D3S3I, rabbit mAB, 1:500; anti-Ibal - Wako #011-27991, goat, 1:250; anti- GFAP - Abeam, ab7260, rabbit, 1:1000; Cy3-conjugated AffmiPure goat anti-rabbit IgG (H+L) - Jackson ImmunoResearch, #111-165-003, 1:200; Alexa Fluor 488-conjugated AffmiPure donkey anti-rabbit IgG (H+L) - Jackson ImmunoResearch, #711-545-152, 1:200; Cy™3 AffmiPure donkey anti-goat IgG (H+L) - Jackson ImmunoResearch, #705-165-147, 1:200. The slides were imaged using Nikon C2 confocal microscope. Alpha-motor neurons were counted as described previously [11] (total number of cells counted - 3,293). Microglia proliferation was assessed by counting all Ibal -positive cells per ventral hom (both gray and white matter; 6,825 cells in total) and microglia activation was determined by counting the number of non-activated, ramified microglia and expressed as the percentage of all Ibal -positive cells per ventral hom. Astrocyte proliferation was measured by counting all GFAP-positive cells per ventral hom (protoplasmic astrocytes in gray matter; 7,346 cells in total). Neuromuscular junction (NMJ) staining was performed as previously described [11] with a modification consisting in using frozen gastrocnemius muscle, instead of fresh one. Briefly, mouse gastrocnemius muscle was dissected, cleaned from connective tissue and cut in small flat pieces at the time of the sacrifice. Muscle pieces were then fixed (lh RT in 10% StuMOL in ddH20), washed twice in PBS (5 min each time), cryoprotected by incubating overnight in 30% sucrose in PBS, frozen on dry ice and stored at -80°C. On the day of staining, tissues were thawed, and staining was continued as previously described, starting from the permeabilization step. The person that analyzed all IF images was blinded to the identity of the samples. Antibodies used: anti-SV2, DSHB, #2315387, mouse monoclonal, 1:50; anti-2H3, DSHB, #2314897, mouse monoclonal, 1:50; a-BTX-Alexa 488, Invitrogen, #B13422, 1:1000; goat anti-mouse Cy3, Jackson ImmunoResearch, #115-165-146, 1:250. The scorer was blinded to the identity of the samples.

[0085] Immunoblotting. Western blotting was done as previously described [11] with the use of the following antibodies: anti-cleaved caspase-3, (Aspl75, 5A1E) rabbit mAb CST #9664, 1:1000; goat anti-rabbit IgG-HRP, Santa Cruz, sc-2004, 1:10,000.

[0086] Blood-spinal cord barrier permeability assay. HRP (type II, #P8250, Sigma, St. Louis, MO, 75 mg/ kg in 0.9% saline) was injected IP into anaesthetized mice as previously described [11] HRP extravasation regions per spinal cord hemi-section were then counted.

[0087] RNA-seq. Total RNA was isolated from 30 mg of the spinal cord of young, old control mice, and old OKN-007-treated mice using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The samples were prepared then and processed by the Clinical Genomics Center at OMRF (omrf.org/research-faculty/core- facilities/next-generation-sequencing/). TruSeq Stranded mRNA Library Kit (Illumina) was used for library preparation, and the samples were sequenced on Illumina NextSeq 500. The results were then processed and analyzed by OMRF Genomics and Data Science group. Raw sequencing reads (in a FASTQ format) were trimmed of residual adaptor sequences using Scythe software. Low-quality bases at the beginning or the end of sequencing reads were removed using sickle; then the quality of remaining reads was confirmed with FastQC. Trimmed sequencing reads were aligned to the Mus musculus genome reference (GRCm38/mml0) using STAR v2.4.0h. Gene-level read counts were determined using HTSeq v0.5.3p9 with the GENCODE Release M10 (GRCm38) annotation. Read-count normalization and differentially expressed analyses were performed using the edgeR package from Bioconductor. Expression values normalized with the voom function were analyzed for differential expression using the standard functions of the limma package. Moderate t-test p-values were adjusted for multiple testing using the false discovery rate (FDR) method and FDR<0.05 was used to filter significand differentially expressed transcripts between conditions considered. Ingenuity Pathway Analysis (IP A, QIAGEN, Redwood City CA) was used to identify and explore significant pathways, functional sets and gene networks interactively.

[0088] Statistical analysis. Statistical analyses were done in Graphpad Prism 9. To compare two groups, after confirming normality, unpaired two-tailed t-test was used, while to assess the difference between treatments and timepoints, ordinary two-way ANOVA with post hoc multiple comparisons test was used.

[0089] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. [0090] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0091] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0092] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0093] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only. As used herein, the phrase “consisting essentially of’ requires the specified features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps as well as those that do not materially affect the basic and novel characteristic(s) and/or function of the claimed invention.

[0094] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0095] As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0096] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. [0097] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (1), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0098] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A method for increasing longevity of a cell or organism comprising administering to the cell or organism a composition comprising a therapeutically effective amount of 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof to increase a longevity of a cell or organism.

2. The method of claim 1, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or intraperitoneally.

3. The method of claim 1, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof are from about 40 to 1,200 mg/kg body weight/day, 100 to 450 mg/kg body weight/day, 200 to 400 mg/kg body weight/day, 300 to 800 mg/kg body weight/day, 350 to 1,000 mg/kg body weight/day, or 400 to 1,100 mg/kg body weight/day.

4. The method of claim 1, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone, or a pharmaceutically acceptable salt thereof, is provided in a sustained-release formulation.

5. The method of claim 1, wherein the composition at least one of: reduces free radicals in the brain, protects neurons, prevents microglial cell activation, or prevents a decrease in muscle mass.

6. The method of claim 1 , wherein the composition inhibits the transforming growth factor bΐ (TGF-bI) pathway.

7. The method of claim 1, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is at least one of: (1) administered at least one of continuously, intermittently, systemically, or locally, (2) administered one or more times a day; (3) sequentially or concomitantly, with another pharmaceutical agent; or (4) as a single agent or in combination with another pharmaceutical agent.

8. A method for increasing cellular resistance to stress, aging or DNA damage in a cell comprising contacting the cell with a composition that inhibits the transforming growth factor bΐ (TGF-bI) pathway.

9. The method of claim 8, wherein the composition comprises a therapeutically effective amount of 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof to increase a longevity of a cell or organism.

10. The method of claim 8, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or intraperitoneally.

11. The method of claim 8, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof are from about 40 to 1,200 mg/kg body weight/day, 100 to 450 mg/kg body weight/day, 200 to 400 mg/kg body weight/day, 300 to 800 mg/kg body weight/day, 350 to 1,000 mg/kg body weight/day, or 400 to 1,100 mg/kg body weight/day.

12. The method of claim 8, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone, or a pharmaceutically acceptable salt thereof, is provided in a sustained-release formulation.

13. The method of claim 8, wherein the composition at least one of: reduces free radicals in the brain, protects neurons, prevents microglial cell activation, or prevents a decrease in muscle mass.

14. The method of claim 8, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is at least one of: (1) administered at least one of continuously, intermittently, systemically, or locally, (2) administered one or more times a day; (3) sequentially or concomitantly, with another pharmaceutical agent; or (4) as a single agent or in combination with another pharmaceutical agent.

15. A method for increasing cellular resistance to stress, aging or DNA damage in a cell comprising contacting the cell with a composition comprising a therapeutically effective amount of 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4-disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof to increase a longevity of a cell or organism.

16. The method of claim 15, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or intraperitoneally.

17. The method of claim 15, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof are from about 40 to 1,200 mg/kg body weight/day, 100 to 450 mg/kg body weight/day, 200 to 400 mg/kg body weight/day, 300 to 800 mg/kg body weight/day, 350 to 1,000 mg/kg body weight/day, or 400 to 1,100 mg/kg body weight/day.

18. The method of claim 15, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone, or a pharmaceutically acceptable salt thereof, is provided in a sustained-release formulation.

19. The method of claim 15, wherein the composition at least one of: reduces free radicals in the brain, protects neurons, prevents microglial cell activation, or prevents a decrease in muscle mass.

20. The method of claim 15, wherein the 2,4-Disulfonyl-N-Tert-Butylnitrone, 2,4- disulfonyl a-phenyl tertiary butyl nitrone or a pharmaceutically acceptable salt thereof is at least one of: (1) administered at least one of continuously, intermittently, systemically, or locally, (2) administered one or more times a day; (3) sequentially or concomitantly, with another pharmaceutical agent; or (4) as a single agent or in combination with another pharmaceutical agent.