Antioxidant nicotinoyl compounds

Abstract

Pyridinium antioxidant compounds that react with reactive oxygen species ("ROS") are disclosed, as are methods of their use. The pyridinium group has either a primary amide at the 3 position and a substitution at the 4 position; or non-primary amide at the 3 position, and optionally a substitution at the 4 position. The antioxidant compound does not produce 4-methyl pyridone, which is an undesirable side-product of some pyridinium compounds.

Description

ANTIOXIDANT NICOTINOYL COMPOUNDSRELATED APPLICATION DATA

[0001] The present application claims priority to United States Provisional Patent Application Number 63 / 449,159 filed May 17, 2024 which is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to biologically useful antioxidant compounds. Such antioxidant compounds as well as methods for use therewith are provided.BACKGROUND

[0003] Reactive oxygen species (ROS) have been implicated in numerous adverse health outcomes. Antioxidants and ROS-scavengers can reduce physiologic ROS (intracellular and extracellular ROS), which could reduce the severity of oxidation-related disease. However, to date there are few if any such agents that have shown clinical efficacy.

[0004] Nicotinamide adenine dinucleotide (NAD) is a recognized redox cofactor, and for this reason nicotinamide compounds have been investigated for possible clinical use as antioxidants. However, methyl nicotinamide is known to form methyl pyridone compounds, including 4-methyl pyridone (4-Me-PY). 4-Me-PY is suspected to be a potent pro- inflammatory agent that is correlated with risks of developing cardiovascular diseases, as well as being correlated with inflammation and associated diseases. Prevailing opinion is that methyl nicotinamides are poor candidates as clinical antioxidants. Extracellular ROS emerging from immune response and inflammation remain an unaddressed challenge.

[0005] There is a continuing need in the art for well-tolerated clinically effective antioxidants.SUMMARY

[0006] It has been discovered that methyl-ni cotinoyl compounds react with oxide species to form 2-Me-pyridone, 4-Me-pyridone and 6-Me-pyridone without enzymatic catalysis. Further, due to an interaction between a primary amide group at the 3 -carbon and the newly formed carbonyl at the 2 or 4 position, restricted rotation occurs. The present disclosure describes novel nicotinoyl compounds that react with intracellular and extracellular ROS but do not form 4-Me-PY. Such antioxidant compounds may comprise a pyridinium group having either: (a) an 1#124275915v4ester moiety at the 3 position; or (b) an amide moiety at the 3 position and a substitution at the 4 position. Preferred embodiments of the antioxidant compound do not have a primary amide moiety at the 3 position when the 4 carbon is unsubstituted.

[0007] In a preferred embodiment the antioxidant compound has one of the following five structures of Formula (I):

[0008] Examples of possible Al groups are: alkyl, aryl, alkenyl, alkynyl, or polyethylene glycol. Examples of possible A2 groups are: hydrogen , methyl, alkyl, aryl, heteroaryl, halogens, carbonyl, and nitrile. Examples of possible A3 groups are: substituted or unsubstituted alkyl, aryl, heteroaryl, hydroxylamine, alkoxide, basic amino acid, beta-amino acid, omega-amino acid, and polyethylene glycol. Examples of possible A4 groups are:2#124275915v4substituted or unsubstituted alkyl, aryl, and polyethylene glycol. Examples of possible A5 groups are: hydrogen, substituted or unsubstituted alkyl, aryl, heteroaryl, N-ethyl, alkoxide, alkyl amine, aryl amine, polyethylene glycol, ethylene glycol fatty acid, a B-vitamin, a modified B-vitamin, folate, alkoxyalkylfolate, thiamine, glycol-nicotinate, O-alkyoxy- nicotinate, PEG-nicotinate, PEG-folate, methyl-glycol nicotinate, PEG-N-methylnicotinate, and O-alkoxy N-methyl nicotinate. In some embodiments, A5 is a conjugate of vitamin Bl, vitamin B2, vitamin B3, vitamin B6, vitamin B7, or vitamin B9. Examples of possible A6 groups are methyl, alkyl, aryl, heteroaryl, halogens, carbonyl, and nitrile.

[0009] In a general embodiment, the antioxidant compound is a carboxyl-containing N-alkyl pyridinium compound. In some instances, the carboxyl-containing N-alkyl pyridinium compound is a substituted N-methyl-nicotinoyl or conjugate thereof. In some instances, the N- alkyl nicotinic acid is an N-alkyl trigonelline. In some instances, the antioxidant compound is glycine conjugate of the N-methyl-nicotinic acid, and wherein the conjugation is on the carboxyl group of N-methyl nicotinuric acid and an amino or alcohol containing amino acid. In some instances, the antioxidant compound is an amino acid conjugate of the N-methyl- ni cotinuric acid, and wherein the conjugation is on a carboxylic acid of the amino acid.

[0010] In a general embodiment, the antioxidant compound is a N-alkyl-nicotinic acid or conjugate thereof. In some embodiments, the antioxidant compound is an N-methyl-nicotinuric acid or conjugate thereof.

[0011] In a general embodiment, the antioxidant compound is a di-trigonelline conjugate. In some instances, the conjugate includes an alkyl or polyethylene glycol linker. In some instances, the linker is alkyl or polyethylene glycol. In some embodiments, the antioxidant compound is a di-trigonelline compound. In some embodiments, the antioxidant compound is a di-methyl-N-nicotinamide having an alkyl linker. In some embodiments, the antioxidant compound is a methyl-N-nicotinamide linked to methyl-nicotinic acid via an alkyl linker. In some embodiments, the antioxidant compound includes a methylated amide.

[0012] In another aspect of the present disclosure, there is presented an antioxidant compound that reacts with reactive oxygen species under physiological conditions, where the antioxidant compound comprises a pyridinium group having either: (a) a moiety at the 3 position that is not a primary amide; or (b) a primary amide moiety at the 3 position and a substitution at the 4 position.3#124275915v4

[0013] In a preferred embodiment the antioxidant compound has one of the following seven structures:

[0014] In a preferred embodiment, the antioxidant compound has one of the following three structures:4#124275915v4

[0015] Another aspect of the disclosure is a pharmaceutical composition comprising any of the antioxidant compounds described herein and a pharmaceutical agent, wherein the pharmaceutical agent promotes ROS when administered to a subject.

[0016] In another aspect of the present disclosure, there is presented a method of reducing inflammation in a biological subject in need thereof. The method includes administering any of the antioxidant compounds described above to the subject in an amount effective to reduce circulating or intracellular reactive oxygen species.

[0017] In some embodiments, the inflammation is associated with a disease or condition. In some instances, the disease is an inflammatory bowel disease or a neurodegenerative disease. In some embodiments, the antioxidant compound is administered topically.

[0018] In yet another aspect of the present disclosure, there is presented a method of reducing reactive oxygen species in a biological subject in need thereof. The method includes administering any the antioxidant compound described above to the subject in an amount effective to reduce circulating or intracellular reactive oxygen species.

[0019] In yet another aspect of the present disclosure, there is presented a method of treatment or prevention of a disease associated with reactive oxygen species. The method includes administering any of the antioxidant compounds described above to a subject in need thereof in a therapeutically effective amount.5#124275915v4

[0020] In some embodiments, at least a portion of the reactive oxygen species are promoted by a pharmaceutical agent, and wherein the pharmaceutical agent is administered to the subject for treatment or prevention of a disease or condition. In some instances, the pharmaceutical agent is acetaminophen.

[0021] In another aspect of the present disclosure, there is presented a method of treatment or prevention of a disease or condition in a biological subject in need thereof. The method includes administering a pharmaceutical agent to the subject in a therapeutically effective amount, where the pharmaceutical agent promotes reactive oxygen species in the subject. The method further includes administering any of the antioxidant compounds described above to the subject in an amount effective to reduce the reactive oxygen species.

[0022] In some embodiments, the pharmaceutical agent is acetaminophen. In some embodiments, the antioxidant compound is administered topically.

[0023] In other aspects of the present disclosure, the subject matter is described in the accompanying specification and drawings.

[0024] The above presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key or critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present disclosure can be better understood, by way of example only, with reference to the following drawings. The elements of the drawings are not necessarily to scale relative to each other, emphasis instead being placed upon clearly illustrating the principles of the disclosure.

[0026] FIG. 1 is a schematic describing B-vitamin conjugates that function as reactive oxygen species (ROS) trappers according to the present disclosure. Displayed are trigonelline-O- propyltriphenylphosphonium, methylnicotinamide-N-propyl-triphenylphosphonium, and trigonelline-Bl conjugate, ethyl-ditrigonelline, methyl-nicotinuric acid, O-ethyl-N-methyl- nicotinurate, N, N-alkyl-N, N-dimethyl-nicotinamide, N, N-alkylamino methylnicotinamide.6#124275915v4

[0027] FIG. 2 is a schematic describing exemplary syntheses of methyl nicotinamide propyltriphenylphosphinonium conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0028] FIG. 3 is a schematic describing an exemplary synthesis of methyl nicotinamide propyl triphenylphosphonium conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0029] FIG. 4A is a graphical analysis describing the starting materials and methyl nicotinamide propyl triphenylphosphine conjugate product that functions as ROS reacting antioxidant compounds according to the present disclosure.

[0030] FIG. 4B is a graphical analysis describing the starting materials and methyl nicotinamide propyl triphenylphosphine conjugate product that functions as ROS reacting antioxidant compounds according to the present disclosure.

[0031] FIG. 4C is a graphical analysis describing the starting materials and methyl nicotinamide propyl triphenylphosphine conjugate product that functions as ROS reacting antioxidant compounds according to the present disclosure.

[0032] FIG. 4D is a graphical analysis describing the starting materials and methyl nicotinamide propyl triphenylphosphine conjugate product that functions as ROS reacting antioxidant compounds according to the present disclosure.

[0033] FIG. 4E is a graphical analysis describing the starting materials and methyl nicotinamide propyl triphenylphosphine conjugate product that functions as ROS reacting antioxidant compounds according to the present disclosure.

[0034] FIG. 5A is a graphical analysis describing exemplary syntheses of nicotinic acid propyl triphenylphosphine and methyl nicotinic acid propyl triphenylphosphine conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0035] FIG. 5B is a schematic depiction of exemplary syntheses of nicotinic acid propyl triphenylphosphine and methyl nicotinic acid propyl triphenylphosphine conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0036] FIG. 6A is a graphical analysis of alternative reactions for forming methyl nicotinic acid triphenylphosphine conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.7#124275915v4

[0037] FIG. 6B is a graphical analysis of alternative reactions for forming methyl nicotinic acid triphenylphosphine conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0038] FIG. 6C is a graphical analysis of alternative reactions for forming methyl nicotinic acid triphenylphosphine conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0039] FIG. 7A is a schematic depiction of exemplary syntheses of a nicotinic acid conjugate.

[0040] FIG. 7B is a graphical representation of di-nicotinamide, di-nicotinic acid, di-A-methyl nicotinamide, and di-trigonelline conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0041] FIG. 7C is a schematic representation of di-nicotinamide, di-nicotinic acid, di-.V- methyl nicotinamide, and di-trigonelline conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0042] FIG. 8 is a schematic describing an exemplary synthesis of thiamine-trigonelline conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0043] FIG. 9 is a schematic describing syntheses of thiamine-methyl nicotinic acid conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0044] FIG. 10A is a schematic describing an exemplary synthesis of thiamine-nicotinuric acid and thiamine-methyl-nicotinuric acid conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0045] FIG. 10B is a graphical representation of an exemplary synthesis of thiamine- nicotinuric acid and thiamine-methyl-nicotinuric acid conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0046] FIG. 10C is a graphical representation of an exemplary synthesis of thiamine- nicotinuric acid and thiamine-methyl-nicotinuric acid conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0047] FIG. 11 A is a schematic describing use of a nicotinuric ethyl ester as a precursor to N- methyl-nicotinuric acid for the formation of conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.8#124275915v4

[0048] FIG. 1 IB is a graphical representation of use of a nicotinuric ethyl ester as a precursor to N-methyl-nicotinuric acid for the formation of conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0049] FIG. 11C is a graphical representation of use of a nicotinuric ethyl ester as a precursor to N-methyl-nicotinuric acid for the formation of conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0050] FIG. 1 ID is a graphical representation of use of a nicotinuric ethyl ester as a precursor to N-methyl-nicotinuric acid for the formation of conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0051] FIG. 1 IE is a graphical representation of use of a nicotinuric ethyl ester as a precursor to N-methyl-nicotinuric acid for the formation of conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0052] FIG. 12A is a schematic describing exemplary syntheses of glycolic acid conjugates of nicotinic acid and trigonelline that function as ROS reacting antioxidant compounds according to the present disclosure.

[0053] FIG. 12B is a graphical representation of exemplary syntheses of glycolic acid conjugates of nicotinic acid and trigonelline that function as ROS reacting antioxidant compounds according to the present disclosure.

[0054] FIG. 13 is a schematic describing exemplary syntheses of vitamin B2-trigonelline and vitamin B2-trigonelline-glycolic acid conjugates that function as ROS reacting antioxidant compounds according to the present disclosure.

[0055] FIG. 14 is a schematic describing exemplary conjugations of thiamine and biotin with trigonelline and trigonelline-glycolic acid that result in ROS reacting antioxidant compounds according to the present disclosure.

[0056] FIG. 15 is a schematic representation of the experimental setup for the CellTiter-Glo viability evaluation, where for co-treatments, the concentration of antioxidants of the present disclosure is 100 pM and the concentration of acetaminophen (APAP) is 2 mM.

[0057] FIG. 16 is a schematic representation of antioxidant compounds of the present disclosure for co-culture with APAP. The chloride salts are generated by anion exchange (Amberlite resin) from the iodide or bromide form of the pyridinium compounds. The top9#124275915v4antioxidant compound is denoted BE-01-06, the middle antioxidant compound is denoted BE- 01-09, and the bottom antioxidant compound is denoted. BE-01-13.

[0058] FIG. 17A is a graphical representation of the viability of HepG2 cells after culture with APAP, BE-01-06, or a combination thereof. Incubation with antioxidant compounds occurred over 72 hours. The vehicle represents phosphate buffered saline (PBS), and NRH represents dihydronicotinamide riboside. NRH is used as a control since it is a source of reductive power that works towards NADPH formation and glutathione maintenance.

[0059] FIG. 17B is a graphical representation of the viability of HepG2 cells after culture with APAP, BE-01-06, or a combination thereof. Incubation with antioxidant compounds occurred over 120 hours. The vehicle represents phosphate buffered saline (PBS), and NRH represents dihydronicotinamide riboside. NRH is used as a control since it is a source of reductive power that works towards NADPH formation and glutathione maintenance.

[0060] FIG. 18A is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HepG2 cells in a time dependent manner using BE-01-06. Incubation with antioxidant compounds occurred over 72 hours.

[0061] FIG. 18B is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HepG2 cells in a time dependent manner using BE-01-06. Incubation was over 120 hours.

[0062] FIG. 19A is a graphical representation of the viability of HepG2 cells after culture with APAP, BE-01-09, or a combination thereof. Incubation with antioxidant compounds occurred over 72 hours. The vehicle represents phosphate buffered saline (PBS), and NRH represents dihydronicotinamide riboside.

[0063] FIG. 19B is a graphical representation of the viability of HepG2 cells after culture with APAP, BE-01-09, or a combination thereof. Incubation was over 120 hours. The vehicle represents phosphate buffered saline (PBS), and NRH represents dihydronicotinamide riboside.

[0064] FIG. 20A is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HepG2 cells in a time dependent manner using BE-01-09. Incubation with antioxidant compounds occurred over 72 hours.

[0065] FIG. 20B is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HepG2 cells in a time dependent manner using BE-01-09. Incubation was over 120 hours.10#124275915v4

[0066] FIG. 21A is a graphical representation of the viability of HepG2 cells after culture with APAP, BE-01-13, or a combination thereof. Incubation with antioxidant compounds occurred over 72 hours. The vehicle represents phosphate buffered saline (PBS), and NRH represents dihydronicotinamide riboside.

[0067] FIG. 21B is a graphical representation of the viability of HepG2 cells after culture with APAP, BE-01-13, or a combination thereof. Incubation was over 120 hours. The vehicle represents phosphate buffered saline (PBS), and NRH represents dihydronicotinamide riboside.

[0068] FIG. 22A is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HepG2 cells in a time dependent manner using BE-01-13. Incubation with antioxidant compounds occurred over 72 hours.

[0069] FIG. 22B is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HepG2 cells in a time dependent manner using BE-01-13. Incubation was over 120 hours.

[0070] FIG. 23 is a schematic representation of the experimental setup for the CellTiter-Glo viability evaluation, where for co-treatments, the concentration of antioxidants of the present disclosure is 10 pM and the concentration of acetaminophen (APAP) is 2 mM.

[0071] FIG. 24A is a graphical representation of the viability of HEK293T cells after culture with APAP, BE-01-06, or a combination thereof. Incubation with antioxidant compounds occurred over 72 hours. The vehicle represents phosphate buffered saline (PBS).

[0072] FIG. 24B is a graphical representation of the viability of HEK293T cells after culture with APAP, BE-01-06, or a combination thereof. Incubation was over 120 hours. The vehicle represents phosphate buffered saline (PBS).

[0073] FIG. 25A is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HEK293T cells in a time dependent manner using BE-01-06. Incubation with antioxidant compounds occurred over 72 hours.

[0074] FIG. 25B is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HEK293T cells in a time dependent manner using BE-01-06. Incubation was over 120 hours.

[0075] FIG. 26A is a graphical representation of the viability of HEK293T cells after culture with APAP, BE-01-09, or a combination thereof. Incubation with antioxidant compounds occurred over 72 hours. The vehicle represents phosphate buffered saline (PBS).11#124275915v4

[0076] FIG. 26B is a graphical representation of the viability of HEK293T cells after culture with APAP, BE-01-09, or a combination thereof. Incubation was over 120 hours. The vehicle represents phosphate buffered saline (PBS).

[0077] FIG. 27A is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HEK293T cells in a time dependent manner using BE-01-09. Incubation with antioxidant compounds occurred over 72 hours.

[0078] FIG. 27B is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HEK293T cells in a time dependent manner using BE-01-09. Incubation was over 120 hours.

[0079] FIG. 28A is a graphical representation of the viability of HEK293T cells after culture with APAP, BE-01-13, or a combination thereof. Incubation with antioxidant compounds occurred over 72 hours. The vehicle represents phosphate buffered saline (PBS).

[0080] FIG. 28B is a graphical representation of the viability of HEK293T cells after culture with APAP, BE-01-13, or a combination thereof. Incubation was over 120 hours. The vehicle represents phosphate buffered saline (PBS).

[0081] FIG. 29A is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HEK293T cells in a time dependent manner using BE-01-13. Incubation with antioxidant compounds occurred over 72 hours.

[0082] FIG. 29B is a graphical representation of methyl nicotinamide protection against APA- induced cell death in HEK293T cells in a time dependent manner using BE-01-13. Incubation was over 120 hours.

[0083] FIG. 30 is a schematic representation of antioxidant compounds of the present disclosure, including a linker represented by the structure between parentheses. The linker may be as disclosed elsewhere in this disclosure, such as for example an alkyl group, a short polyethylene glycol, or spermine.DETAILED DESCRIPTIONDEFINITIONS

[0084] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art of this disclosure. It will be further understood that terms, such as those defined in commonly12#124275915v4used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well known functions or constructions may not be described in detail for brevity or clarity.

[0085] With reference to the use of the word(s) “comprise,” “comprises,” and “comprising” in the foregoing description and / or in the following claims, unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that each of those words is to be so interpreted in construing the foregoing description and / or the following claims.

[0086] The term “including” should be interpreted to mean “including but not limited to...” unless the context clearly indicate otherwise.

[0087] The term “consisting essentially of’ means that, in addition to the recited elements, what is claimed may also contain other elements (steps, structures, ingredients, components, etc.) that do not adversely affect the operability of what is claimed for its intended purpose. Such addition of other elements that do not adversely affect the operability of what is claimed for its intended purpose would not constitute a material change in the basic and novel characteristics of what is claimed.

[0088] The term “adapted to” means designed or configured to accomplish the specified objective, not simply able to be made to accomplish the specified objective.

[0089] The term “capable of’ means able to be made to accomplish the specified objective.

[0090] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well (i.e. “at least one”), unless the context clearly indicates otherwise.

[0091] The terms “prevention”, “prevent”, “preventing”, “suppression”, “suppress” and “suppressing” as used herein refer to a course of action (such as implanting a medical device) initiated prior to the onset of a clinical manifestation of a disease state or condition so as to prevent or reduce such clinical manifestation of the disease state or condition. Such preventing and suppressing need not be absolute to be useful.

[0092] The terms “treatment”, “treat” and “treating” as used herein refers a course of action (such as implanting a medical device) initiated after the onset of a clinical manifestation of a13#124275915v4disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition. Such treating need not be absolute to be useful.

[0093] In this disclosure terms such as “administering” or “administration” include acts such as prescribing, dispensing, giving, or taking a substance such that what is prescribed, dispensed, given, or taken is actually contacts the patient’s body externally or internally (or both). It is specifically contemplated that instructions or a prescription by a medical professional to another person to give, take, administer, or self-administer a substance is an act of administration.

[0094] The term “in need of treatment” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a condition that is treatable by a method or device of the present disclosure.

[0095] The term “in need of prevention” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a condition that is preventable by a method or device of the disclosure.

[0096] Terms such as “at least one of A and B” should be understood to mean “only A, only B, or both A and B.” The same construction should be applied to longer list (e.g., “at least one of A, B, and C”).

[0097] The term “individual”, “subject” or “patient” as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female.

[0098] None of the definitions above are intended to define what might be considered “equivalent” to anything that is claimed, under the “doctrine of equivalents” or analogous laws.ANTIOXIDANT COMPOUND

[0099] The present disclosure refers to antioxidant compounds that react with reactive oxygen species (ROS) under physiological conditions. Methyl nicotinamides may form methyl pyridone compounds, including 4-methyl pyridone (4-Me-PY), which is a suspected pro-14#124275915v4inflammatory agent associated with inflammation and related diseases. In some instances, the present disclosure describes novel nicotinoyl antioxidant compounds that react with ROS but do not form 4-Me-PY. Such antioxidant compounds are pyridinium compounds with or without N-substitution. When present, N- substitutions of the pyridinium compounds may include alkyl, aryl, alkenyl, alkynyl, and polyethylene glycol groups.

[0100] Further, the antioxidant compounds may include a pyridinium group with: (1) a moiety with a secondary amide or teriary amide at the 3 position, or (2) both a primary amide moiety at the 3 position and a substitution at the 4 position. Preferred embodiments of the antioxidant compound do not have a primary amide moiety at the 3 position when the 4 carbon is unsubstituted. In some instances, the antioxidant compound is a pyridinium group with an ester moiety or carboxylic acid moiety at the 3 position. Where an ester moiety or carboxylic acid moiety is present at the 3 position, the 4 position may be unsubstituted or substituted; preferred substitutions include a hydrogen, methyl, alkyl, aryl, heteroaryl, halogens, carbonyl, or nitrile group. Similarly, where a secondary or tertiary amide moiety is present at the 3 position, the 4 position may be unsubstituted or substituted; and preferred substitutions include a hydrogen, methyl, alkyl, aryl, heteroaryl, halogens, carbonyl, or nitrile group.

[0101] The antioxidant compounds may include carboxyl-containing pyridinium compounds with a carboxylic acid or ester moiety at the 3 position. Where an ester moiety is present at the 3 position, R groups may include: hydrogen, alkyl, aryl, N-ethyl, alkoxide, alkyl amine, aryl amine, polyethylene glycol, ethylene glycol fatty acid, a B-vitamin, a modified B-vitamin, folate, alkoxyalkylfolate, thiamine, glycol-nicotinate, O-alkyoxy-nicotinate, PEG-nicotinate, PEG-folate, methyl-glycol nicotinate, PEG-N-methylnicotinate, and O-alkoxy N-methyl nicotinate groups.

[0102] The antioxidant compounds may include amide-containing pyridinium compounds, where the amide moiety is a primary, secondary, or tertiary amide. Where the antioxidant compound is a secondary or tertiary amide, the R group may include a substituted or unsubstituted alkyl, aryl, heteroaryl, hydroxylamine, alkoxide, basic amino acid, beta-amino acid, omega-amino acid, and polyethylene glycol groups. Where the antioxidant compound is a tertiary amide, the R group may include a substituted or unsubstituted alkyl, aryl, and polyethylene glycol groups.15#124275915v4

[0103] Prophetic examples of antioxidant compounds capable of reacting with ROS under physiological conditions without forming 4-Me-PY are generally disclosed in FIG. 1 to FIG. 14.

[0104] In FIG. 1, exemplary trigonelline-O-triphenylphosphine, trigonelline-N- triphenylphosphine, and trigonelline-Bl conjugates are disclosed as ROS trapping antioxidant compounds. Further ROS trapping antioxidant compounds are disclosed in FIG. 2, which shows exemplary nicotinamide triphenylphosphine and methyl nicotinamide triphenylphosphine conjugates, along with their synthesis schemes. For example, methyl nicotinamide triphenylphosphine conjugates may be synthesized using (3 -aminopropyl) triphenylphosphonium bromide and nicotinic acid starting materials (also shown in FIG. 3).The NMR spectra of the starting materials and methyl nicotinamide triphenylphosphine product of FIG. 3 is shown in FIGS. 4A-8. In FIG. 5A and FIG. 5B, the syntheses of nicotinic acid- triphenylphosphine and methyl-nicotinic acid-triphenylphosphine conjugates are shown, along with NMR spectra for starting materials and the nicotinic acid-triphenylphosphine product. In this synthesis scheme, starting materials are (3-hydroxypropyl) triphenylphosphonium bromide and nicotinic acid, and alternative syntheses are compared in FIGS. 6A-13.

[0105] In FIG. 7B and FIG. 7C, exemplary di-nicotinamide, di-methyl-N-nicotinamide, dinicotinic acid, and di-trigonelline conjugates consistent with the present disclosure are shown. The di-nicotinamide, di-methyl-N-nicotinamide, di-nicotinic acid, and di-trigonelline conjugates may include linkers, including alkyl and polyethylene glycol linkers. In addition, FIG. 4D shows the synthesis of an ethylene glycol-nicotinic acid conjugate consistent with the present disclosure using ethylene glycol and nicotinic acid starting materials.

[0106] In FIG. 8 and FIG. 9, exemplary thiamine-trigonelline conjugates consistent with the present disclosure are shown, along with synthesis schemes. Similarly, in FIGS. 10A-20, exemplary thiamine-nicotinuric acid and thiamine-methyl-nicotinuric acid conjugates consistent with the present disclosure are shown, along with synthesis schemes and NMR spectra for the reaction and products.

[0107] In FIGS. 11 A-25, the conjugation of nicotinic acid and glycine ethyl ester are shown to produce a nicotinuric ethyl ester precursor to an N-methyl niconinuric ethyl ester compound of the present disclosure. Exemplary glycolic acid conjugates of nicotinic acid and trigonelline are shown in FIG. 12A and FIG. 12B, along with their synthesis schemes.16#124275915v4

[0108] Further, in FIG. 13 and FIG. 14, vitamin B-trigonelline-glycolic acid conjugates are disclosed, where conjugates include vitamin Bl, B2, and B7 conjugates.

[0109] Antioxidant compounds compatible with reducing ROS generated by a pharmaceutical agent, such as acetaminophen, are provided. These antioxidant compounds include methyl nicotinamide (BE-01-06), 2-carbon ester dimers (BE-01-09), and 2-carbon amide dimers (BE- 01-13).

[0110] The antioxidant compounds of the present disclosure may be administered according to the methods described below, either individually, in combination of two or more thereof, and / or in combination with another pharmaceutical agent. When used with a pharmaceutical agent, the antioxidant compounds of the present disclosure may be administered to reduce reactive oxygen species (ROS) produced by the pharmaceutical agent. In some instances, the antioxidant compounds of the present disclosure may be administered to reduce ROS produced by acetaminophen, chloroquine, amiodarone, dronedarone, azathioprine, buprenorphine, lovastatin, nefazodone, troglitazone, chlorpromazine, diclofenac, mefenamic acid, and clozapine. In some instances, the pharmaceutical agent may be acetaminophen, chloroquine, diclofenac, mefenamic acid, and clozapine.METHODS

[0111] A method of reducing reactive oxygen species (ROS) is provided, by reacting any of the antioxidant compounds disclosed above with the ROS. The ROS may be in a biological subject, in which case the antioxidant compound may be administered to the subject as part of a pharmaceutical composition. The subject may be in need of the administration - for example, the subject may have a condition or disease state associated with excessive ROS or be at risk of the same; or the subject may be undergoing treatment with a drug or biologic agent that promotes ROS formation. The antioxidant compound may be administered in an effective amount, which in this context means an amount sufficient to reduce circulating ROS, intracellular ROS, or both.

[0112] Diseases or conditions associated with ROS and compatible with ROS reduction by antioxidant compounds of the present disclosure include diseases and conditions with inflammatory effects. Such diseases and conditions include, but are not limited to, inflammatory arthritis, inflammatory bowel disease, and neurodegenerative disease. Examples of inflammatory arthritis include, but are not limited to, gout, rheumatoid arthritis, psoriatic17#124275915v4arthritis, and ankylosing spondylitis. Examples of inflammatory bowel diseases and conditions include, but are not limited to, Crohn’s disease and ulcerative colitis. Examples of neurodegenerative diseases and conditions include, but are not limited to, Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, amyotrophic lateral sclerosis, and Huntington’s disease.

[0113] The subject may have a condition or disease state associated with inflammation. The subject may have a condition or disease state that results in inflammation or negative effects to the gut biome. The subject may have a condition or disease state requiring the use of a pharmaceutical agent, where the pharmaceutical agent generates ROS. The subject may be in need of pain relief and may be administered acetaminophen and an antioxidant compound of the present disclosure to reduce or prevent a cytotoxic effect of ROS generation associated with acetaminophen.

[0114] A "pharmaceutical composition" refers to a mixture of any of the antioxidant compounds of the disclosure with other components, such as physiologically / pharmaceutically acceptable carriers and / or excipients. The purpose of a pharmaceutical composition is to facilitate administration of an antioxidant compound of formula I, including salts, tautomeric forms, hydrates and / or solvates to a subject (including pharmaceutically acceptable forms of the foregoing).

[0115] Some embodiments of the pharmaceutical composition comprise a pharmaceutical agent, such as a drug or biologic. The pharmaceutical agent may be of a type that promotes ROS in the subject. Examples of such pharmaceutical agents that promote ROS are acetaminophen, chloroquine, amiodarone, dronedarone, azathioprine, buprenorphine, lovastatin, nefazodone, troglitazone, chlorpromazine, diclofenac, mefenamic acid, and clozapine. In some embodiments of the pharmaceutical composition the pharmaceutical agent may be present in a therapeutically effective amount to treat or prevent a disease state or condition.

[0116] For example, if the pharmaceutical agent is acetaminophen, the acetaminophen may be present in an amount sufficient to exert an analgesic effect in the subject, an antipyretic effect in the subject, or both.

[0117] For example, if the pharmaceutical agent is chloroquine, it may be present in an amendment sufficient to treat or prevent malaria (such as certain strains of P. falciparum P.18#124275915v4ovale, P. vivax, and P. malariae , extraintestinal amebiasis; rheumatoid arthritis; and systemic lupus erythematosus.

[0118] For example, if the pharmaceutical agent is amiodarone, it may be present in an amendment sufficient to treat or prevent ventricular arrhythmias, supraventricular tachyarrhythmias, atrial fibrillation, or ventricular tachyarrhythmias.

[0119] For example, if the pharmaceutical agent is dronedarone, it may be present in an amendment sufficient to treat or prevent cardiac arrhythmias or atrial fibrillation.

[0120] For example, if the pharmaceutical agent is azathioprine, it may be present in an amendment sufficient to treat or prevent active rheumatoid arthritis, kidney transplant rejection, inflammatory bowel disease, Churg-Strauss syndrome, autoimmune hepatitis, chronic ITP, lupus nephritis, connective tissue disease-associated ILD, multiple sclerosis, severe myasthenia gravis, recurrent pericarditis, psoriasis, non-infectious uveitis, relapsing polychondritis, dermatomyositis / polymyositis, erythema multiforme, severe and refractory atopic dermatitis, chronic actinic dermatitis, pyoderma gangrenosum, Behcet disease, cutaneous vasculitis, pityriasis rubra pilaris, lichen planus, bullous pemphigoid, or pemphigus vulgaris.

[0121] For example, if the pharmaceutical agent is buprenorphine, it may be present in an amount sufficient to treat or prevent acute pain, chronic pain, opioid dependence, or opioid withdrawal.

[0122] For example, if the pharmaceutical agent is lovastatin, it may be present in an amount sufficient to treat or prevent coronary heart disease, hypercholesterolemia, heterozygous familial hypercholesterolemia, and non-cardioembolic stroke.

[0123] For example, if the pharmaceutical agent is nefazodone it may be present in an amount sufficient to treat or prevent depression.

[0124] For example, if the pharmaceutical agent is troglitazone, it may be present in an amount sufficient to treat or prevent diabetes, lower blood glucose, or improve cellular response to insulin.

[0125] For example, if the pharmaceutical agent is chlorpromazine, it may be present in an amount sufficient to treat or prevent psychotic disorders, schizophrenia, bipolar disorder, acute intermittent porphyria, tetanus, nausea, vomiting, restlessness, anxiety, persistent hiccups, and behavioral conditions.19#124275915v4

[0126] For example, if the pharmaceutical agent is diclofenac, it may be present in an amount sufficient to exert an analgesic effect in the subject. In some instances, if the pharmaceutical agent is diclofenac, it may be present in an amount sufficient to treat or prevent arthritis, migraines, and other painful conditions such as menstrual cramps.

[0127] For example, if the pharmaceutical agent is clozapine, it may be present in an amount sufficient to treat or prevent symptoms of schizophrenia or Parkinson’s disease.

[0128] For example, if the pharmaceutical agent is mefenamic acid, it may be present in an amount sufficient to exert an analgesic effect in the subject. In some instances, it may be present in an amount sufficient to treat or prevent arthritis, toothache, or menstrual cramps.

[0129] The term “pharmaceutically acceptable salts” as used herein includes salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science 1077, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Pharmaceutically acceptable salt forms may also include forms wherein the ratio of molecules comprising the salt is not 1: 1. For example, the salt may comprise more than one inorganic or organic acid20#124275915v4molecule per molecule of base, such as two hydrochloric acid molecules per inorganic or organic acid molecule. As another example, the salt may comprise less than one inorganic or organic acid molecule per molecule of base, such as two molecules of inorganic or organic acid molecule per molecule of tartaric acid. Salts may also exist as solvates or hydrates.

[0130] The term "acid" contemplates all pharmaceutically acceptable inorganic or organic acids. Inorganic acids include mineral acids such as hydrohalic acids, such as hydrobromic and hydrochloric acids, sulfuric acids, phosphoric acids and nitric acids. Organic acids include all pharmaceutically acceptable aliphatic, alicyclic and aromatic carboxylic acids, dicarboxylic acids, tricarboxylic acids, and fatty acids. Preferred acids are straight chain or branched, saturated or unsaturated C1-C20 aliphatic carboxylic acids, which are optionally substituted by halogen or by hydroxyl groups, or C6-C12 aromatic carboxylic acids. Examples of such acids are carbonic acid, formic acid, fumaric acid, acetic acid, propionic acid, isopropionic acid, valeric acid, alpha-hydroxy acids, such as glycolic acid and lactic acid, chloroacetic acid, benzoic acid, methane sulfonic acid, and salicylic acid. Examples of dicarboxylic acids include oxalic acid, malic acid, succinic acid, tataric acid and maleic acid. An example of a tricarboxylic acid is citric acid. Fatty acids include all pharmaceutically acceptable saturated or unsaturated aliphatic or aromatic carboxylic acids having 4 to 24 carbon atoms. Examples include butyric acid, isobutyric acid, sec-butyric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and phenylsteric acid. Other acids include gluconic acid, glycoheptonic acid and lactobionic acid.

[0131] Such compositions are administered to a subject in sufficient amounts to deliver a therapeutically effective amount of the antioxidant compound(s) so as to be effective in the treatment and prevention methods disclosed herein. The therapeutically effective amount may vary according to a variety of factors such as, but not limited to, the subject’s condition, weight, sex and age. Other factors include the mode and site of administration. The pharmaceutical compositions may be provided to the subject in any method known in the art. Exemplary routes of administration include, but are not limited to, subcutaneous, intravenous, topical, epicutaneous, oral, intraosseous, intramuscular, intranasal and pulmonary. The compositions of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the compositions are administered to the subject more than once, a variety of regimens may be used, such as, but not limited to, one per day, once per week, once per month or once per year. The compositions may also be21#124275915v4administered to the subject more than one time per day. The therapeutically effective amount of the nucleic acid molecules and appropriate dosing regimens may be identified by routine testing in order to obtain optimal activity, while minimizing any potential side effects. In addition, co-administration or sequential administration of other agents may be desirable.

[0132] The compositions of the present disclosure may be administered systemically, such as by intravenous administration, or locally such as by subcutaneous injection or by application of a paste or cream.

[0133] The compositions of the present disclosure may further comprise agents which improve the solubility, half-life, absorption, etc. of the antioxidant compound(s). Furthermore, the compositions of the present disclosure may further comprise agents that attenuate undesirable side effects and / or or decrease the toxicity of the antioxidant compounds(s). Examples of such agents are described in a variety of texts, such a, but not limited to, Remington: The Science and Practice of Pharmacy (20th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor).

[0134] The compositions of the present disclosure can be administered in a wide variety of dosage forms for administration. For example, the compositions can be administered in forms, such as, but not limited to, tablets, capsules, sachets, lozenges, troches, pills, powders, granules, elixirs, tinctures, solutions, suspensions, elixirs, syrups, ointments, creams, pastes, emulsions, or solutions for intravenous administration or injection. Other dosage forms include administration transdermally, via patch mechanism or ointment. Further dosage forms include formulations suitable for delivery by nebulizers or metered dose inhalers. Any of the foregoing may be modified to provide for timed release and / or sustained release formulations.

[0135] In the present disclosure, the pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffering agents, disintegrating agents and carriers, as well as accessory agents, such as, but not limited to, coloring agents and flavoring agents (collectively referred to herein as a carrier). Typically, the pharmaceutically acceptable carrier is chemically inert to the active antioxidant compounds and has no detrimental side effects or toxicity under the conditions of use. The pharmaceutically acceptable carriers can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may22#124275915v4differ depending on the particular dosage form employed and other characteristics of the composition.

[0136] For instance, for oral administration in solid form, such as but not limited to, tablets, capsules, sachets, lozenges, troches, pills, powders, or granules, the antioxidant compound(s) may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier, such as, but not limited to, inert fillers, suitable binders, lubricants, disintegrating agents and accessory agents. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthum gum and the like. Tablet forms can include the following alone or in combination: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid as well as the other carriers described herein. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.

[0137] For oral liquid forms, such as but not limited to, tinctures, solutions, suspensions, elixirs, syrups, the nucleic acid molecules of the present disclosure can be dissolved in diluents, such as water, saline, or alcohols. Furthermore, the oral liquid forms may comprise suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methylcellulose and the like. Moreover, when desired or necessary, suitable and coloring agents or other accessory agents can also be incorporated into the mixture. Other dispersing agents that may be employed include glycerin and the like.

[0138] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the patient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The antioxidant compound(s) may be administered in a23#124275915v4physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4- methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as, but not limited to, a soap, an oil or a detergent, suspending agent, such as, but not limited to, pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0139] Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkylbeta-aminopropionates, and 2- alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0140] Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain an nonionic surfactant having a hydrophile-lipophile balance (HLB) of from about 12 to about 17.

[0141] Topical dosage forms, such as, but not limited to, ointments, creams, pastes, emulsions, containing the nucleic acid molecule of the present disclosure, can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and24#124275915v4shampoos in cream or gel formulations. Inclusion of a skin exfoliant or dermal abrasive preparation may also be used. Such topical preparations may be applied to a patch, bandage or dressing for transdermal delivery or may be applied to a bandage or dressing for delivery directly to the site of a wound or cutaneous injury.

[0142] The antioxidant compound(s) of the present disclosure can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. Such liposomes may also contain monoclonal antibodies to direct delivery of the liposome to a particular cell type or group of cell types.

[0143] The antioxidant compound(s) of the present disclosure may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include, but are not limited to, polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the antioxidant compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.WORKING EXAMPLESExample 1 : HepG2 and HEK293T Cells co-cultured with acetaminophen (APAP)

[0144] Acetaminophen (APAP) is a ROS promoting drug. As such, its activity on cells is evaluated for the effects on cell viability.

[0145] Hep G2 cells were suspended in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% (v / v) heat-inactivated Fetal Bovine Serum (FBS). For HEK 293T cells, they were suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v / v) heat-inactivated FBS. Hep G2 cells were co-cultured with APAP and antioxidant compounds of the present disclosure, with cell viability measured using a CellTiter-Glo® cell viability assay (Promega). The viability assay experimental setup is depicted in FIG. 15. In FIG. 15, “Anti 1” is BE-01-06 of FIG. 8, “Anti 2” is BE-01-09 of FIG. 8, and “Anti 3” is BE- 01-13 of FIG. 16.25#124275915v4

[0146] The stock solutions for the series of antioxidant compounds depicted schematically in FIG. 16 is as follows hereafter: 50 mM APAP: a 40-mL stock was prepared by dissolving 303.6 mg APAP in 40 mL degassed PBS; 20 mM BE-01-06: a 1 -mL stock solution was prepared by dissolving 3.3 mg BE-01-06 in 1 mL degassed PBS; 20 mM BE-01-09: a 1-mL stock solution was prepared by dissolving 7.3 mg BE-01-09 in 1 mL degassed PBS; and 20 mM BE-01-13 : a 1-mL stock solution was prepared by dissolving 7.8 mg BE-01-13 in 1 mL degassed PBS.

[0147] The treatment solutions to accomplish the experimental treatment depicted in FIG. 15 were provided as follows: 22 mM (2 mM) APAP: a 0.5-mL stock was prepared by diluting 220 pL (50 mM) APAP in 280 pL degassed PBS; 1.1 mM (100 pM) Antioxidants: a 0.5-mL stock was prepared by diluting 27.5 pL (20 mM) antioxidant in 472.5 pL degassed PBS; and 22 mM APAP + 1.1 mM Antioxidants: a 0.5-mL stock was prepared by diluting 220 pL (50 mM) APAP and 27.5 pL (20 mM) antioxidant in 252.5 pL degassed PBS. For cell treatment, 10 pL of each of the treatment solutions above were added to 100 pL of cell solution to give the concentrations depicted in the layout of FIG. 15.

[0148] On Day 1 of the viability study, 100 pL of Hep G2 cells were seeded in a black 96- well plate at a density of 10,000 cells / well. After the cells attached to the plate while incubating at room temperature for approximately 30 minutes, the plates were placed in an incubator at 37°C and 5% CO2 for at least an hour. The plates were then removed from the incubator and 10 pL of antioxidant compound from a designated treatment solution were added to the wells designated on the plate layout. The plates were placed back in an incubator and remained in the same media over the course of 72 or 120 hours.

[0149] On Day 4 / 6, (after 72 or 120 hours), the plates were removed from the incubator. 60 pL of supernatant was aspirated from each well to leave a final volume of 50 pL per well. This volume was provided because the CellTiter-Glo assay requires a 1 : 1 volumetric ratio between supernatant and assay reagent according to the commercial kit instructions. 50 pL of the CellTiter-Glo (CTG) reagent, as provided by the commercial kit, were added to each well. The plates were then mixed in a motion similar to an orbital shaker for 2 minutes for cell lysing. The plates were then incubated at room temperature for 10 minutes. After 10 minutes, the one- second integrated luminescence of each well was measured using a Tecan Spark Plate Reader.

[0150] From the collected raw data, an average of the background signal (just media - no cells) was calculated. Further, an average of the vehicle (PBS) treatment was obtained. Viability as a percentage was determined by subtracting the average background value from the26#124275915v4raw measurement of a well. This difference was then divided by the value for the vehicle treatment and multiplied by 100 to obtain a percentage. Data was transferred to GraphPad Prism where data sets and statistical analysis using a 2Way Anova method were obtained.

[0151] FIG. 17A and FIG. 17B show the results of the co-culture of Me-NAM and APAP with HepG2 cells. These results indicate that BE-01-06 is not cytotoxic and does not affect HepG2 cell viability. FIG. 18A and FIG. 18B show that methyl nicotinamide (Me-NAM) protects against APAP-induced cell death in HepG2 cells in a time dependent manner.

[0152] FIG. 19A and FIG. 19B show the results of the co-culture of BE-01-09 (a 2-carbon ester dimer) and APAP with HepG2 cells. These results indicate that BE-01-09 is not cytotoxic and does not affect HepG2 cell viability. FIG. 20A and FIG. 20B show that the 2-carbon ester dimer (BE-01-09) protects against APAP-induced cell death in HepG2 cells in a time dependent manner.

[0153] FIG. 21 A and FIG. 2 IB show the results of the co-culture of BE-01-13 (a 2-carbon amide dimer) and APAP with HepG2 cells. These results indicate that BE-01-13 is not cytotoxic and does not affect HepG2 cell viability, and that BE-01-13 also rescues HepG2 cells from APAP cytotoxicity. FIG. 22A and FIG. 22B show that the 2-carbon amide dimer (BE-01- 13) rescues HepG2 cells from APAP cytotoxicity in a time dependent manner.

[0154] Next, HEK293T cells were co-cultured with APAP and the antioxidant compounds of the present disclosure, with cell viability measured with a CellTiter-Glo® cell viability assay (Promega). In the experimental setup depicted in FIG. 23, “Anti 1” is BE-01-06 of FIG. 16, “Anti 2” is BE-01-09 of FIG. 16, and “Anti 3” is BE-01-13 of FIG. 16.

[0155] The treatment solutions to accomplish the experimental treatment depicted in FIG. 23 were provided as follows: 22 mM (2 mM) APAP: a 0.5-mL stock was prepared by diluting 220 pL (50 mM) APAP in 280 pL degassed PBS; 22 mM APAP + 110 pM (10 pM) Antioxidants: a 0.5-mL stock was prepared by diluting 220 pL (50 mM) APAP and 50 pL (100 pM) antioxidants in 230 pL degassed PBS; and 22 mM APAP + 1.1 mM Antioxidants: a 0.5-mL stock was prepared by diluting 220 pL (50 mM) APAP and 27.5 pL (20 mM) antioxidant in 252.5 pL degassed PBS. For cell treatment, 10 L of each of the treatment solutions above were added to 100 pL of cell solution to give the concentrations depicted in the layout of FIG. 23.

[0156] On Day 1, 100 pL of HEK 293T cells were seeded in a black 96-well poly-D-lysine plate at a density of 5,000 cells / well. After the cells attached to the plate while incubating at27#124275915v4room temperature for approximately 30 minutes, the plates were placed in an incubator at 37°C and 5% CO2 for at least an hour. The plates were then removed from the incubator and 10 pL of antioxidant compound from a designated stock solution were added to the wells designated on the plate layout of FIG. 23. The plates were placed back in an incubator and remained in the same media over the course of 72 or 120 hours.

[0157] On Days 4 / 6 (72 or 120 hours), the plates were removed from the incubator. 60 pL of supernatant was aspirated from each well to leave a final volume of 50 pL per well. This volume was used because the CellTiter-Glo assay requires a 1 : 1 volumetric ratio between supernatant and assay reagent according to manufacturer’s instructions. 50 pL of the CellTiter- Glo (CTG) reagent were added to each well. The plates were then mixed in a motion similar to an orbital shaker for 2 minutes for cell lysing. The plates were then incubated at room temperature for 10 minutes. After 10 minutes, the one-second integrated luminescence of each well was measured using a Tecan Spark Plate Reader.

[0158] From the collected raw data, an average of the background signal (just media - no cells) was calculated. Further, an average of the vehicle (PBS) treatment was obtained.Viability as a percentage was determined by subtracting the average background value from the raw measurement of a well. This difference was then divided by the value for the vehicle treatment and multiplied by 100 to obtain a percentage. Data was transferred to GraphPad Prism where data sets and statistical analysis using a 2Way Anova method were obtained.

[0159] FIG. 24A and FIG. 24B show the results of the co-culture of Me-NAM and APAP with HEK293T cells. These results indicate that BE-01-06 is not cytotoxic and does not affect HEK293T cell viability. FIG. 25A and FIG. 25B show that methyl nicotinamide (Me-NAM) protects against APAP-induced cell death in HEK293T cells in a time dependent manner.

[0160] FIG. 26A and FIG. 26B show the results of the co-culture of BE-01-09 (a 2-carbon ester dimer) and APAP with HEK293T cells. This ester is a precursor to trigonelline (prodrug). These results indicate that BE-01-09 is not cytotoxic and does not affect HEK293T cell viability. FIG. 27A and FIG. 27B show that the 2-carbon ester dimer (BE-01-09) rescues HEK293T cells exposed to APAP even at low concentrations.

[0161] FIG. 28A and FIG. 28B show the results of the co-culture of BE-01-13 (a 4-carbon amide dimer) and APAP with HEK293T cells. These results indicate that BE-01-13 rescues HEK293T cells from APAP cytotoxicity. FIG. 29 A and FIG. 29B show that the BE-01-13 rescues HEK293T cells from APAP cytotoxicity.28#124275915v4CONCLUSIONS

[0162] It is to be understood that any given elements of the disclosed embodiments of the invention may be embodied in a single structure, a single step, a single substance, or the like. Similarly, a given element of the disclosed embodiment may be embodied in multiple structures, steps, substances, or the like. The foregoing description and accompanying drawings illustrate and describe certain processes, machines, manufactures, and compositions of matter, some of which embody the invention(s). Such descriptions or illustrations are not intended to limit the scope of what can be claimed, and are provided as aids in understanding the claims, enabling the making and use of what is claimed, and teaching the best mode of use of the invention(s). If this description and accompanying drawings are interpreted to disclose only a certain embodiment or embodiments, it shall not be construed to limit what can be claimed to that embodiment or embodiments. Any examples or embodiments of the invention described herein are not intended to indicate that what is claimed must be coextensive with such examples or embodiments. Where it is stated that the invention(s) or embodiments thereof achieve one or more objectives, it is not intended to limit what can be claimed to versions capable of achieving all such objectives. Any statements in this description criticizing the prior art are not intended to limit what is claimed to exclude any aspects of the prior art. Additionally, the disclosure shows and describes certain embodiments of the processes, machines, manufactures, compositions of matter, and other teachings disclosed, but it is to be understood that the teachings of the present disclosure are capable of use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the teachings as expressed herein. Any section headings herein are provided only for consistency with the suggestions of 37 C.F.R. § 1.77 or otherwise to provide organizational queues. These headings shall not limit or characterize the invention(s) set forth herein.29#124275915v4

Claims

CLAIMSThe following is claimed:

1. An antioxidant compound that reacts with reactive oxygen species under physiological conditions, having a structure selected from the group consisting of:wherein:Al is selected from the group consisting of: alkyl, aryl, alkenyl, alkynyl, or polyethylene glycol;A2is selected from the group consisting of: hydrogen, methyl, alkyl, aryl, heteroaryl, halogen, carbonyl, and nitrile;30#124275915v4A3is selected from the group consisting of: substituted or unsubstituted alkyl, aryl, heteroaryl, hydroxylamine, alkoxide, basic amino acid, beta-amino acid, omega-amino acid, and polyethylene glycol;A4is selected from the group consisting of: substituted or unsubstituted alkyl, aryl, and polyethylene glycol;A5 is selected from the group consisting of: hydrogen, substituted or unsubstituted alkyl, aryl, N-ethyl, alkoxide, alkyl amine, aryl amine, polyethylene glycol, ethylene glycol fatty acid, a B-vitamin, a modified B-vitamin, folate, alkoxy alkylfolate, thiamine, glycolnicotinate, O-alkyoxy-nicotinate, PEG-nicotinate, PEG-folate, methyl-glycol nicotinate, PEG- N-methylnicotinate, and O-alkoxy N-methyl nicotinate; andA6 is methyl, alkyl, aryl, heteroaryl, halogen, carbonyl, and nitrile.

2. The antioxidant compound of claim 1, wherein the antioxidant compound is a carboxyl- containing N-alkyl pyridinium antioxidant compound.

3. The antioxidant compound of claim 1, wherein the antioxidant compound is an N-alkyl- nicotinic acid or conjugate thereof.

4. The antioxidant compound of claim 1, wherein the antioxidant compound is an N-methyl- ni cotinuric acid or conjugate thereof.

5. The antioxidant compound of claim 2, wherein the antioxidant compound is a substituted N- methyl-nicotinoyl or conjugate thereof.

6. The antioxidant compound of claim 3, wherein the N-alkyl nicotinic acid is an N-alkyl trigonelline.

7. The antioxidant compound of claim 4, wherein the antioxidant compound is glycine conjugate of the N-methyl-nicotinic acid, and wherein the conjugation is on the carboxyl group of N-methyl nicotinuric acid and an amino or alcohol containing amino acid.

8. The antioxidant compound of claim 4, wherein the antioxidant compound is an amino acid conjugate of the N-methyl-nicotinuric acid, and wherein the conjugation is on a carboxylic acid of the amino acid.31#124275915v49. The antioxidant compound of claim 1, wherein the antioxidant compound is a di-trigonelline conjugate.

10. The antioxidant compound of claim 1, wherein the antioxidant compound is a ditrigonelline antioxidant compound.

11. The antioxidant compound of claim 9, wherein the conjugate includes an alkyl or polyethylene glycol linker.

12. The antioxidant compound of claim 1, wherein the antioxidant compound is a di-methyl-N- nicotinamide having an alkyl linker.

13. The antioxidant compound of claim 1, wherein the antioxidant compound is a methyl-N- ni cotinamide linked to methyl-nicotinic acid via an alkyl linker.

14. The antioxidant compound of claim 11, wherein the linker is alkyl or polyethylene glycol.

15. The antioxidant compound of claim 1, wherein the antioxidant compound includes a methylated amide.

16. The antioxidant compound of claim 1, wherein A5 is a conjugate of vitamin Bl, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, or a combination of two or more for the foregoing.

17. An antioxidant compound that reacts with reactive oxygen species under physiological conditions, wherein the antioxidant compound comprises a pyridinium group having either: (a) a moiety at the 3 position that is not a primary amide; or (b) a primary amide moiety at the 3 position and a substitution at the 4 position.

18. The antioxidant compound of claim 17, wherein the antioxidant compound is selected from one of the following:32#124275915v419. The antioxidant compound of claim 17, wherein the antioxidant compound is selected from one of the following:33#124275915v420. A pharmaceutical composition for the reduction of reactive oxygen species in a subject, the pharmaceutical composition comprising the antioxidant compound of any one of claims 1-19 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20 comprising a pharmaceutical agent that promotes ROS in the subject.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical agent is a drug.

23. The pharmaceutical composition of claim 21 wherein the pharmaceutical agent is selected from the group consisting of acetaminophen, chloroquine, amiodarone, dronedarone, azathioprine, buprenorphine, lovastatin, nefazodone, troglitazone, chlorpromazine, diclofenac, mefenamic acid, and clozapine.

24. A method of reducing inflammation in a subject in need thereof, comprising: administering antioxidant compound of any one of claims 1-19 to the subject in an amount effective to reduce circulating or intracellular reactive oxygen species.

25. The method of claim 24, wherein the inflammation is associated with a disease or a condition.34#124275915v426. The method of claim 25, wherein the disease is an inflammatory bowel disease or a neurodegenerative disease.

27. The method of claim 24, wherein the antioxidant compound is administered topically.

28. A method of reducing reactive oxygen species in a subject in need thereof, comprising: administering the antioxidant compound of any one of claims 1-19 to the subject in an amount effective to reduce circulating or intracellular reactive oxygen species.

29. The method of claim 28, wherein at least a portion of the reactive oxygen species are promoted by a pharmaceutical agent, and wherein the pharmaceutical agent is administered to the subject for treatment or prevention of a disease or condition.

30. The method of claim 29, wherein the pharmaceutical agent is acetaminophen.

31. A method of treatment or prevention of a disease or condition in a subject in need thereof, comprising: administering a pharmaceutical agent to the subject in a therapeutically effective amount, wherein the pharmaceutical agent promotes reactive oxygen species in the subject; and administering the antioxidant compound of any one of claims 1-19 to the subject in an amount effective to reduce the reactive oxygen species.

32. The method of claim 31, wherein the pharmaceutical agent is acetaminophen.

33. The method of claim 31, where the antioxidant compound is administered topically.

34. A method of treatment or prevention of a disease associated with reactive oxygen species, the method comprising: administering an antioxidant compound of any one of claims 1-19 to a subject in need thereof in a therapeutically effective amount.

35. The subject matter described in the accompanying specification and drawings.35#124275915v4

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