Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
  • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
  • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4415—Pyridoxine, i.e. Vitamin B6
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/06—Antiarrhythmics

Definitions

• Atrial fibrillation is the most common cardiac arrhythmia of clinical significance, and it often results in devastating outcomes. Because current treatment is frequently ineffective, there is a critical need for an improved understanding of the molecular mechanisms causing AF and novel strategies to treat it.
• antioxidants as "upstream therapy” including vitamins C and E, statins, and inhibitors of the renin-angiotensin-aldosterone system
• upstream therapy including vitamins C and E, statins, and inhibitors of the renin-angiotensin-aldosterone system
• ⁇ - KAs ⁇ -ketoaldehydes
• IsoLGs isolevuglandins
• ROS reactive oxygen species
• salicylamine is a natural product with an excellent safety profile in pre-clinical animal studies. Moreover, salicylamine prevents the formation of both ⁇ - KAs and toxic protein oligomers with remarkable therapeutic benefit in animal models of Alzheimer's disease and hypertension.
• the present inventors have identified protein oligomers and oxidative stress/formation of ⁇ -KAs in cellular and in vivo models associated with AF susceptibility, including rapidly-stimulated atrial cells, hypertension, obesity, and familial AF. Importantly, our preliminary data demonstrate a beneficial effect of scavenging ⁇ -KAs to reduce atrial protein oligomer formation and AF burden. Therefore, salicylamine, as well as its structural analogues that have been developed to date, represent a completely novel therapy to prevent and treat atrial arrhythmias, such as atrial
• Figure 1 is a flow chart that links oxidative stress with IsoLG/PAO formation, and ultimately atrial arrhythmogenesis.
• Figure 2 is shows the chemical mechanism for the scavenging of thel,4-dicarbonyl (red box) compounds known as isolevuglandins by pyridoxamine and its structural analog 2-HOBA (center box).
• Figure 4 shows IsoLGs in PAO formation for atrial HL-1 cells. After incubation with synthetic IsoLGs (isoketals) for 6hr, PAO formation was evident in unpaced cells (top panels).
• the anti-IsoLG adduct antibody (Dl l ScFv) is immunoreactive in paced but not control cells (bottom panels).
• Figure 6 shows ANPoligomers partially co-localize with atrial PAOs and are cytoxic to atrial cells.
• E Western blot of ANP after peptide incubation ( ⁇ ) for 24hr or 6d, compared to incubation with IsoLGs for 24hr.
• F ANP oligomers (2wk incubation) reduced ATP production by atrial HL-1 cells (24hr exposure).
• Figure 7 demonstrates that hypertension-mediated AF is suppressed by the dicarbonyl scavenger 2-HOBA.
• A Total inducible AF burden for normotensive (sham) and hypertensive (ang II) mice, as well as hypertensive mice treated with 2-HOBA, 4-HOBA or hydralazine/
• Figure 9 shows IsoLG adducts quantified in atrial HL-1 cells under cyclical 10% stretch (24hr) and unstretched conditions.
• FIG. 10 demonstrates that DIO-mediated AF is suppressed by 2-HOBA.
• B Weight data are illustrated for each experimental group over time.
• Figure 11 shows that obesity causes atrial PAOs.
• A, B. Images for merged and final PAO signal in LA of mice fed a low-fat (LFD) or high-fat diet (HFD; 8wk).
• LFD low-fat
• HFD high-fat diet
• Figure 14 shows Atrial HL-1 cell ATP production with WT and m-ANP incubated for variable time periods.
• Figure 15 is an example demonstrating a diet-induced obesity model.
• Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
• the terms "optional” or “optionally” means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
• the term "subject” refers to a target of administration.
• the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
• the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.
• the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
• a patient refers to a subject afflicted with a disease or disorder.
• patient includes human and veterinary subjects.
• treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
• This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
• this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
• prevent refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. As can be seen herein, there is overlap in the definition of treating and preventing.
• the term "diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.
• the phrase "identified to be in need of treatment for a disorder," or the like refers to selection of a subject based upon need for treatment of the disorder.
• a subject can be identified as having a need for treatment of a disorder (e.g., a disorder related to inflammation) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder.
• the identification can, in one aspect, be performed by a person different from the person making the diagnosis.
• the administration can be performed by one who subsequently performed the administration.
• administering refers to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.
• a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
• a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
• the term "effective amount” refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition.
• a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.
• the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts.
• the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose.
• the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be
• a preparation can be administered in a "prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition.
• pharmaceutically acceptable carrier refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for
• aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
• polyols such as glycerol, propylene glycol, polyethylene glycol and the like
• carboxymethylcellulose and suitable mixtures thereof include vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
• Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
• compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption.
• adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
• Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the inject
• Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide- polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
• the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
• Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
• the term “scavenger” or “scavenging” refers to a chemical substance that can be administered in order to remove or inactivate impurities or unwanted reaction products.
• the isoketals irreversibly adduct specifically to lysine residues on proteins.
• the isoketal scavengers of the present invention react with isoketals before they adduct to the lysine residues. Accordingly, the compounds of the present invention "scavenge” isoketals, thereby preventing them from adducting to proteins.
• the term "substituted" is contemplated to include all permissible substituents of organic compounds.
• the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds.
• Illustrative substituents include, for example, those described below.
• the permissible substituents can be one or more and the same or different for appropriate organic compounds.
• the heteroatoms, such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
• substitution or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. , a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
• alkyl as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, ⁇ -propyl, isopropyl, w-butyl, isobutyl, s-butyl, /-butyl, w-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.
• the alkyl group can be cyclic or acyclic.
• the alkyl group can be branched or unbranched.
• the alkyl group can also be substituted or unsubstituted.
• the alkyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.
• a "lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms.
• alkyl is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group.
• halogenated alkyl specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.
• alkoxyalkyl specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below.
• alkylamino specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like.
• alkyl is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.
• cycloalkyl refers to both unsubstituted and substituted cycloalkyl moieties
• the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g. , an "alkylcycloalkyl.”
• a substituted alkoxy can be specifically referred to as, e.g. , a "halogenated alkoxy”
• a particular substituted alkenyl can be, e.g. , an "alkenylalcohol,” and the like.
• cycloalkyl as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms.
• examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like.
• heterocycloalkyl is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.
• the cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted.
• the cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.
• polyalkylene group as used herein is a group having two or more CH 2 groups linked to one another.
• the polyalkylene group can be represented by a formula— (CH 2 ) a — , where "a" is an integer of from 2 to 500.
• Alkoxy also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as— OA 1 — OA 2 or— OA 1 — (OA 2 ) a — OA 3 , where "a” is an integer of from 1 to 200 and A 1 , A 2 , and A 3 are alkyl and/or cycloalkyl groups.
• amine or “amino” as used herein are represented by a formula NA X A 2 A 3 , where A 1 , A 2 , and A 3 can be, independently, hydrogen or optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
• hydroxyl as used herein is represented by a formula— OH.
• nitro as used herein is represented by a formula— NO2.
• pharmaceutically acceptable describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.
• Embodiments of the present invention include compounds of the following formula, and their use as agents in a method for treating, preventing, or ameliorating atrial fibrillation to a subject with or at risk of atrial fibrillation, thereby inhibiting or treating the atrial fibrillation:
• R is N or C
• R2 is independently H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C 3-1 o cycloalkyl, C 3- 8 membered ring containing C, O, S or N, optionally substituted with one or more R 2 , R3 and R4, and may cyclize with to one or more R 2i R3, or R 5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R3 is H, hydroxy, halogen, nitro, CF3, C1-0 alkyl, Ci-6 alkoxy, C3-10 cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R3 may cyclize with to one or more R2 or R5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R4 is H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C 3-1 o cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R3 may cyclize with to one or more R2 , R3, or R 5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R5 is a bond, H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C 3-1 o cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R3 may cyclize with to one or more R 2i R 3i or R 4 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• Another embodiment of the present invention includes compounds of the following formula, and their use in methods for treating, preventing, or ameliorating atrial arrhythmias to a subject with or at risk of an atrial arrhythmia:
• R is N or C
• R2 is independently H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C 3-1 o cycloalkyl, C 3- 8 membered ring containing C, O, S or N, optionally substituted with one or more R 2 , R3 and R ⁇ and may cyclize with to one or more R 2i R3, or R 5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R3 is H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C 3-1 o cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R3 may cyclize with to one or more R2 or R 5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R4 is H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C 3-1 o cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R3 may cyclize with to one or more R 2i R 3i or R 5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R5 is a bond, H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C 3-1 o cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R3 may cyclize with to one or more R2 , R 3, or R 4 to form an optionally substituted C3-8 membered ring containing C, O, S or N; and stereoisomers and analogs thereof.
• the compound may be selected from the compounds disclosed herein.
• the compound may be salicylamine.
• Another embodiment of the present invention is a method for treating, preventing, or ameliorating atrial fibrillation to a subject with or at risk of atrial fibrillation, thereby inhibiting or treating the atrial fibrillation, comprising the step of co-administering to the subject at least one compound in a dosage and amount effective to treat the dysfunction in the mammal, the compound having a structure represented by a compound of the following formula:
• R is N or C
• R2 is independently H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C3-10 cycloalkyl, C3- 8 membered ring containing C, O, S or N, optionally substituted with one or more R2, R 3 and R ⁇ and may cyclize with to one or more R2 , R 3 , or R 5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R 3 is H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C3-10 cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R 3 may cyclize with to one or more R2 or R5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R4 is H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C3-10 cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R 3 may cyclize with to one or more R2 , R3 , or R5 to form an optionally substituted C3-8 membered ring containing C, O, S or N;
• R5 is a bond, H, hydroxy, halogen, nitro, CF 3 , C 1-6 alkyl, C 1-6 alkoxy, C3-10 cycloalkyl, C3-8 membered ring containing C, O, S or N, optionally substituted with one or more R4, R2 and R 3 may cyclize with to one or more R2 , R 3, or R 4 to form an optionally substituted C3-8 membered ring containing C, O, S or N; and stereoisomers and analogs thereof; with a drug having a known side effect of treating, preventing, or ameliorating atrial fibrillation.
• Examples of compounds that may be used with the methods disclosed herein include, but are not limited to, compounds selected from the formula:
• R is N or C
• R2 is independently H, substituted or unsubstituted alkyl
• R3 is H, halogen, alkoxy, hydroxyl, nitro;
• R4 is H, substituted or unsubstituted alkyl, carboxyl; and pharmaceutically acceptable salts thereof.
• the compound is salicylamine (2-hydroxybenzylamine or 2-HOBA).
• the compound may be chosen from:
• the compound may also be chosen from:
• the compounds or analogs may also be chosen from:
• the compounds may also be chosen from:
• the compounds may also be chosen from
• AF atrial fibrillation
• AF a refractory arrhythmia that often results in devastating clinical outcomes.
• AF confers a 2-fold increased risk of dying, and substantial morbidity in the form of stroke, congestive heart failure, and treatment complications such as anticoagulant-related bleeding.
• the prevalence of AF continues to increase, highlighting its emergence as a growing epidemic and major public health challenge in the US and worldwide.
• the estimated annual cost of AF-related hospitalizations in 2010 was $3.46 billion dollars in the US alone.
• a major barrier of contemporary AF treatment has been the focus to reduce arrhythmia recurrence, using antiarrhythmic drugs and catheter ablation.
• pharmacologic therapy is associated with a -50% recurrence rate of AF at 6-12 months (and substantial side effects), while success of single-procedure ablation is 50-70%, with non-trivial procedural risks (1/1000 risk of death).
• therapy targeting the electrophysiologic/anatomic basis of AF has met with only limited success.
• Embodiments of the present invention include methods of treating atrial fibrillation and damage resulting therefrom.
• Another embodiment of the present invention is the concept that scavenging ⁇ -KAs can prevent/treat atrial arrhythmias such as atrial fibrillation/atrial flutter.
• Another embodiment of the present invention is the use of ⁇ - ⁇ scavengers such as salicylamine and compounds of the present invention to prevent/treat other types of atrial arrhythmias (eg, atrial tachycardia).
• Lipid peroxidation is a major component of ROS-mediated cellular damage, and the most reactive products generated, isolevuglandins (IsoLGs), react almost instantaneously with proteins to cause misfolding/ dysfunction.
• IsoLGs isolevuglandins
• Novel lipid dicarbonyl scavengers have been developed that preemptively bind IsoLGs before they can interact with biologic targets to damage cells, the best studied of which is 2-hydroxybenzylamine, or 2-HOBA.
• IsoLGs were identified as critical mediators in immune-mediated hypertension and Alzheimer's disease.
• preamyloid oligomers are now recognized to be the primary cytotoxic species that correlates with disease progression.
• IsoLGs markedly accelerate PAO formation for such proteins.
• the present inventors show that both IsoLGs and PAOs are biologically- relevant mediators that promote AF susceptibility ( Figure 1), making them potential therapeutic targets.
• the present inventors have identified the formation of IsoLGs and PAOs in rapidly-stimulated atrial cells, as well as models of oxidative stress-related AF susceptibility, specifically the common AF risk factors hypertension and obesity. Importantly, the data demonstrate a beneficial effect of scavenging IsoLGs to reduce AF burden, as well as oligomer formation.
• PAOs were frequently detected, and quantitative analysis revealed an association between atrial PAO burden and hypertension.
• a logical candidate for oligomer formation in the atrium is atrial natriuretic peptide (ANP), a known fibrillogenic protein encoded by the gene NPPA.
• ANP contributes to a common form of aging-related atrial amyloidosis linked to AF, and the present inventors have identified ANP in the oligomers present in human atrium.
• a mutation in NPPA that lengthens the ANP peptide is associated with familial AF.
• AF susceptibility occurs in the absence of gross or microscopic atrial structural remodeling.
• the proarrhythmic mechanisms whereby mutant ANP causes AF remain uncertain.
• mutant ANP is markedly more fibrillogenic than the wild- type peptide.
• These oligomers accumulating in the atria of mice expressing mutant ANP are cytotoxic and modulate atrial electrophysiology.
• the present inventors have also identified that in hypertension and obesity, oxidative stress- mediated isolevuglandins promotes atrial cell dysfunction/injury and AF susceptibility.
• the present inventors have also identified that oligomers derived from mutant ANP alter myocyte homeostasis to generate AF susceptibility, and they also promote oxidative stress/IsoLG formation that feedback in a positive manner to perpetuate the pathologic process.
• the present inventors have identified that AF risk factors linked to oxidative stress increase arrhythmia susceptibility through the generation of IsoLGs and cytotoxic PAOs.
• IsoLG and PAO formation may provide not only common mechanistic links between cardiac pathophysiology and AF, but also novel therapeutic targets in the prevention and/or treatment of this common and serious arrhythmia.
• Lipid peroxidation and isolevuglandins Lipid peroxidation and isolevuglandins.
• One of the most susceptible sites to ROS damage is polyunsaturated fatty acids in the cell membrane and circulation. Peroxidation of these lipids generates injurious reactive aldehydes, including malondialdehyde (MDA), 4-hydroxynonenal and related 4-oxo- 2-nonenal, acrolein, methylglyoxal, and isolevuglandins (IsoLGs; also called ⁇ -ketoaldehydes or isoketals).
• MDA malondialdehyde
• 4-hydroxynonenal and related 4-oxo- 2-nonenal 4-oxo- 2-nonenal
• crolein methylglyoxal
• IsoLGs isolevuglandins
• IsoLG adducts are covalent, irreversible modifications, and IsoLGs modifications include intramolecular crosslinks that cause dysfunction of proteins and structures relevant to cardiomyocyte homeostasis, including ion channels, mitochondria, and proteasomes.
• IsoLGs can also adduct to DNA. IsoLG adducts are increased in the diseased tissue in multiple conditions linked to oxidative injury/inflammation, and they were recently identified as critical mediators of oxidative injury in the brain in Alzheimer's disease and in the vasculature in HTN.
• Dicarbonyl scavengers to investigate mechanisms of oxidant stress-related diseases.
• An obvious first approach to protect against the assault of IsoLGs and other reactive dicarbonyls would be to reduce their production.
• contemporary antioxidants have been largely ineffective to reduce ROS, or oxidative stress-related diseases in general.
• Currently-available antioxidants are designed to prevent ROS-mediated injury by reacting with free radicals, inhibiting the activity of free radical- generating enzymes, or enhancing activity of intracellular antioxidant enzymes.
• an embodiment of the present invention is 2-hydroxybenzylamine (2- HOBA; also called salicylamine).
• 2-HOBA is not an antioxidant (i.e., it does not reduce ROS levels significantly). Rather, it reacts with IsoLGs at a much more rapid rate (by several orders of magnitude) than IsoLGs can bind to the ⁇ -amine of lysine.
• a structural requirement for this scavenging activity is the location of a hydroxyl group adjacent to a methylamine in the phenolic amine structure. For the related analog 4-HOBA, the structural proximity of these functional groups is lost - hence this compound cannot scavenge dicarbonyls and is inactive.
• 2-HOBA and its active analogs do not affect concentrations of 0 2 ⁇ or F 2 -Isoprostanes during in vitro oxidation, and the reduction in IsoLG adduct levels has been attributed directly to its dicarbonyl scavenging effect, and not to inhibition of ROS production and/or lipid peroxidation.
• An additional beneficial effect of compounds like 2-HOBA is that they can also scavenge other injurious lipid dicarbonyl compounds such as MDA, in addition to IsoLGs.
• MDA injurious lipid dicarbonyl compounds
• IsoLGs are preferentially targeted.
• 2-HOBA does not inhibit the COX1 or COX2 enzymes, and thus, the production of physiologic prostaglandins is preserved.
• 2-HOBA has been shown to prevent development of cognitive impairment in a mouse model of Alzheimer's disease, and it effectively lowered blood pressure (BP) in ang II-mediated HTN in mice.
• BP blood pressure
• 2-HOBA and its analogs represent a paradigm shift in pharmacologic strategy to prevent injurious oxidative protein modification.
• 2-HOBA is a natural product with an excellent safety profile based on in vitro studies, in vivo studies in thousands of mice, and recent pre-clinical toxicology studies (Metabolic Technologies, Inc., Ames, IA).
• the present inventors have discovered the role of novel mediators in the genesis of the AF substrate, thus challenging the current research paradigm for this common cardiac arrhythmia.
• the present inventors have accumulated multiple lines of evidence to support these hypotheses in cells, animal models, and humans.
• the present inventors have developed innovative imaging-based methods to quantitate PAO burden in small atrial samples in a reproducible manner, as a prerequisite for investigative studies in humans.
• the proposed experiments will establish the role of PAO-mediated proteotoxicity in promoting AF susceptibility using primarily a genetic model.
• AF begets AF An unexpected signal for protein misfolding.
• the progressive nature of AF is caused by electrical and structural remodeling due to rapid atrial activation that increases arrhythmia susceptibility - "AF begets AF".
• the present inventors found that atrial cells rapidly stimulated in culture undergo remodeling similar to that observed in human AF, with striking concordance of transcriptional changes.
• the present inventors found conserved transcriptional up-regulation for proteins implicated in protein misfolding/amyloidosis. This finding shows that proteotoxicity, a process linked to oxidative stress, can develop in human atrium as a novel mechanism to promote atrial injury.
• the present inventors have shown that in the setting of hypertension and obesity, oxidative stress- mediated isolevuglandins promote atrial cell dysfunction/injury and the substrate for AF. Histologic studies have demonstrated fibrosis and often atrial enlargement with established AF in the setting of HTN and obesity (with evidence of atrial contact/infiltration by pericardial fat in obesity). Nonetheless, little is known about molecular processes that lead to AF in its earliest stages.
• PAOs are not detected by Congo red. However, they share a common structural epitope irrespective of amino acid sequence, and conformation-specific antibodies, such as A-l 1, recognize oligomers generated by a wide variety of proteins.
• A-l 1 and a myocardial-specific antibody we developed a microscopic imaging-based method to enable robust and reproducible quantitative analysis of PAO burden in atrial samples harvested at the time of elective cardiac surgery. This method quantitated the relative area of myocardium containing PAOs (Figure 3), or Green/Red ratio (G/R), as a spatial representation of PAO burden.
• Protein oligomers andlsoLGs in a cellular model simulating AF The present inventors found that rapid pacing of cultured atrial HL-1 cells caused the accumulation of diffuse cytoplasmic PAOs (confirmed by Western blotting, with ANP a significant component), as well as enhanced superoxide (0 2 ⁇ ) production and abundant IsoLG adducts ( Figure 4, lower panels, detected by an anti-IsoLG lysyl adduct antibody D l l ScFv) that were absent in control, unpaced, spontaneously -beating cells.
• IsoLGs are mediators for AF susceptibility in HTN using a murine model (2wk mini-pump infusion of angiotensin II [ang II]), in which mice develop sustained HTN and inducible AF.
• IsoLGs are the most potent mediators of oxidative stress identified.
• IsoLG scavengers prevent AF susceptibility.
• IsoLGs markedly accelerate PAO formation for susceptible proteins, PAOs should develop in these ROS-mediated diseases, with prevention by IsoLG scavengers, consistent with our data to date.
• IsoLG formation and AF susceptibility should be reduced, compared to traditional DIO, providing further evidence for IsoLGs in the pathophysiologic process.
• Data associated with the present invention examines the role of cytotoxic mutant ANP oligomers in atrial pathophysiology and arrhythmia susceptibility for an NPPA mutation linked to familial AF.
• the present inventors show that mutant ANP oligomers alter atrial myocyte homeostasis to generate AF susceptibility, and they promote oxidative stress/IsoLG formation that feedback in a positive manner to perpetuate the pathologic process.
• Fibrillogenic proteins like ANP are diverse and unrelated in their primary amino acid structure. Factors that contribute to fibrillogenesis include variant protein structure, extensive ⁇ conformation of the precursor protein, and proteolytic processing of the precursor protein (as for amyloid ⁇ -42). In many cases, amyloidosis occurs as a consequence of a mutation or modification in the primary structure of a causative protein, or mutations/conditions causing its overproduction.
• NPPA encodes the precursor prepro-ANP, which undergoes proteolytic processing to generate N-terminal pro- ANP and ANP.
• Genetic studies have linked abnormal ANP production to familial AF. In a large family with Holt-Oram Syndrome, a missense mutation in T-box transcription factor 5 (TBx5) resulted in an atypical phenotype with early-onset AF and the overexpression of multiple genes, including NPPA. Familial AF has also been linked to mutations in NPPA itself. The best-studied example derives from a large family having multiple affected members with early-onset lone AF. A 2-base pair deletion was identified that abolished the normal stop codon, leading to a mature
• ANP protein containing the usual 28 amino acids plus an anomalous C-terminus of 12 additional residues.
• Plasma concentrations of mutant ANP in affected family members were 5-10 times higher than wild-type (WT) ANP due to abnormal proteolytic degradation.
• WT wild-type
• AF susceptibility occurred in the absence of gross atrial structural remodeling. Electrophysiologic mechanisms have been proposed to explain the pathogenesis of this mutation, but findings are controversial.
• the present inventors show that for mutant ANP, the altered amino acid sequence leads to accelerated protein misfolding and PAO development in the atria, as the proximal mechanism to increase arrhythmia susceptibility. Development of atrial PAOs in mice expressing mutant ANP.
• transgenic mice were generated that overexpress wild-type (WT) human ANP, or human ANP harboring the frame-shift mutation described above. These mice demonstrated phenotypic features reflective of the modeled human disease. Atrial volumes and left ventricular ejection fraction in the transgenic mice were similar to those of non-transgenic (NTG) animals (with a lower BP). For mice expressing mutant ANP, levels of circulating mutant ANP were elevated 5-6 fold (as in humans), and inducible AF was significantly increased, compared to WT-ANP or nontransgenic (NTG) mice.
• NTG non-transgenic mice
• Preliminary immunostaining revealed robust accumulation of PAOs in the atria of mutant ANP mice compared to NTG and WT-ANP mice ( Figure 12). These data support the concept of PAO formation as a driver of the AF substrate.
• Mutant ANP is highly fibrillogenic, generating cytotoxic PAOs.
• Western analysis was performed on peptides allowed to oligomerize at 23°C for variable time points.
• WT-ANP displayed time- and IsoLG-dependent fibrillogenesis; however, this process was markedly accelerated for the mutant peptide ( Figure 13).
• oligomer toxicity is a transient, time-dependent phenomenon, in the progression from monomers to PAOs to fibrils.
• peptides incubated in vitro to generate oligomers over different time periods were incubated with atrial HL-1 cells for 24hr.
• Mutant ANP PAOs cause potentially proarrhythmic electrophysiologic effects in atrial cells. Peptides were incubated in PBS for 24h: at this time point, mutant ANP develops oligomers but WT ANP does not ( Figure 13).
• Figure 15 is an example that shows a diet-induced obesity model.
• the following data are for 12 week-treated animals.
• Total AF (atrial fibrillation) burden is probably the best measure - the AF burden for animals treated with 2-HOBA was similar to that for the lean (low fat diet) mice, while 4-HOBA had no effect.
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• Circ Arrhythm a systematic review. Circ Arrhythm
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• Salomon RG Subbanagounder G, O'Neil J, Kaur K, Smith MA, Hoff HF, Perry G, Monnier VM. Levuglandin E2-protein adducts in human plasma and vasculature. Chem Res Toxicol 1997 May;10(5):536-45.
• Kirabo A Fontana V, de Faria AP, Loperena R, Galindo CL, Wu J, Bikineyeva AT, Dikalov S, Xiao L, Chen W, Saleh MA, Trott DW, Itani HA, Vinh A, Amamath V, Amamath K, Guzik TJ, Bernstein KE, Shen XZ, Shyr Y, Chen SC, Memaugh RL, Laffer CL, Elijovich F, Davies SS, Moreno H, Madhur MS, Roberts J, Harrison DG. DC isoketal-modified proteins activate T cells and promote hypertension. J Clin Invest 20 ⁇ 4 October l;124(10):4642-56.
• Zagol-Ikapite I Sosa IR, Oram D, Judd A, Amamath K, Amamath V, Stec D, Oates JA, Boutaud O. Modification of platelet proteins by malondialdehyde: prevention by dicarbonyl scavengers. J Lipid Res 2015 November;56(l l):2196-205. Amarnath V, Amarnath K. Scavenging 4-Oxo-2-nonenal. Chem Res Toxicol 2015 October 19;28(10): 1888-90.
• Glabe CG Kayed R. Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis. Neurology 2006 January 24;66(2 Suppl 1):S74-S78.
• Mielcarek M Inuabasi L, Bondulich MK, Muller T, Osborne GF, Franklin SA, Smith DL, Neueder A, Rosinski J, Rattray I, Protti A, Bates GP. Dysfunction of the CNS-heart axis in mouse models of Huntington's disease. PLoS Genet 2014 August;10(8):el004550.
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• Levuglandin forms adducts with histone H4 in a cyclooxygenase-2-dependent manner, altering its interaction with DNA.
• Female mice are protected against high-fat diet induced metabolic syndrome and increase the regulatory T cell population in adipose tissue.
• Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.
• N-proBNP N-terminal pro-brain natriuretic peptide
• Louros NN Iconomidou VA, Tsiolaki PL, Chrysina ED, Baltatzis GE, Patsouris ES,
• N-proANP N-terminal pro-atrial natriuretic peptide
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