WO2014063189A1

WO2014063189A1 - Methods and products for preventing and/or treating a stress induced cardiomyopathy

Info

Publication number: WO2014063189A1
Application number: PCT/AU2013/001224
Authority: WO
Inventors: John HOROWITZ; Yuliy CHIRKOV
Original assignee: Adelaide Research & Innovation Pty Ltd
Priority date: 2012-10-23
Filing date: 2013-10-23
Publication date: 2014-05-01

Abstract

The present disclosure relates to methods and products for preventing and/or treating a stress- induced cardiomyopathy. Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more of the following agents: a peroxynitrite inhibitor; an inhibitor of peroxynitrite synthesis; an inhibitor of peroxynitrite associated signalling; an inhibitor of peroxynitrite-induced DNA damage; a Tx NIP inhibitor; a beta 2 adrenoceptor antagonist; and a beta 3 adrenoceptor antagonist; thereby preventing and/or treating the stress-induced cardiomyopathy in the subject.

Description

METHODS AND PRODUCTS FOR PREVENTING AND/OR TREATING A STRESS INDUCED CARDIOMYOPATHY

PRIORITY CLAIM

[001] This application claims priority to Australian provisional patent application number 2012904658 filed on 23 October 2012, the contents of which is hereby incorporated by reference.

FIELD

[002] The present disclosure relates to methods and products for preventing and/or treating a stress-induced cardiomyopathy.

BACKGROUND

[003] Stress-induced cardiomyopathies are a type of non-ischaemic cardiomyopathy. They are due to a potentially reversible injury to part or all of the heart muscle, generally precipitated as a result of severe physical and/or emotional stress, and its concomitant neurohormonal responses, particularly the release of catecholamines. These conditions may also be induced by catecholamine administration in isolation, in susceptible persons. The most prevalent of the stress-induced cardiomyopathies is Tako- Tsubo cardiomyopathy (TTC). The condition is also frequently termed transient apical ballooning syndrome or broken heart syndrome, as it can be precipitated by acute emotional stress, such as the death of a loved one.

[004] TTC is characterised by the sudden development of regional left ventricular systolic dysfunction associated with a range of symptoms, including chest pain, dyspnoea, and collapse. The condition occurs predominantly in women aged greater than 50 years, and clinically may be associated with onset of a shock state, potentially lethal arrhythmias, and thrombo-embolism leading to the occurrence of embolic stroke.

[005] Stress-induced cardiomyopathy is difficult to differentiate from acute myocardial infarction (AMI), and accounts for at least 2% of cases initially diagnosed as evolving AMI. However the true incidence of the condition is unknown, due to diagnostic difficulties. Diagnosis of TTC is in large part by exclusion and as such it is believed that a significant proportion of patients remain undetected.

[006] Many of the "routine" treatments for AMI, and especially AMI complicated by shock, are inappropriate for stress-induced cardiomyopathy. For example, thrombolysis is inappropriate, because it risks severe adverse effects with no potential benefit, and administration of catecholamines for shock may potentially aggravate stress-induced cardiomyopathy.

[007] Whilst stress-induced cardiomyopathies only account for a small proportion of patients presenting with acute coronary syndrome, not only are they difficult to diagnose but there is no effective treatment. The correct clinical diagnosis of TTC is also particularly relevant, as inotropes which are commonly administered to patients with cardiomyopathy and other cardiac conditions can actually exacerbate TTC.

[008] Treatment of patients with TTC is currently largely empirical and supportive. In some cases, patients may be treated with medications for left ventricular systolic function, such as ACE inhibitors and beta blockers. Contrary to popular view, recovery from TTC is not rapid and takes at least 3 months, raising the possibility of permanent myocardial damage in affected patients.

[009] Given the lack of effective treatment for stress-induced cardiomyopathies, exacerbated by the difficulty in their diagnosis, there is a need to provide improved methods and/or products for preventing and/or treating such conditions, and/or to address one or more of the aforementioned and other problems relating to the prevention and/or treatment of these conditions, and/or to provide one or more advantages. SUMMARY

[0010] The present disclosure relates to the prevention and/or treatment of a stress- induced cardiomyopathy.

[0011] The present disclosure is based, in part, on recognition that patients with a stress-induced cardiomyopathy exhibit biochemical and physiological evidence indicating a propensity for both incremental release of nitric oxide (NO) and for increased tissue responsiveness to NO. Incremental release of NO interacts with a stress-induced catecholamine stimulus to predispose such patients to the occurrence of cardiac injury, and that the generation of peroxynitrite within the heart contributes to this injury.

[0012] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist;

thereby preventing and/or treating the stress-induced cardiomyopathy in the subject.

[0013] Certain embodiments of the present disclosure provide use of one or more of the following agents in the preparation of a medicament for prevention and/or treatment of a stress-induced cardiomyopathy in a subject:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage; (v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

[0014] Certain embodiments of the present disclosure provide a method of treating a subject having symptoms associated with an acute coronary syndrome and acute stress, the method comprising administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist

thereby treating the subject.

[0015] Certain embodiments of the present disclosure provide use of one or more of the following agents in the preparation of a medicament for treating a subject having symptoms associated with an acute coronary syndrome and acute stress:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor:

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

[0016] Certain embodiments of the present disclosure provide a method of treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy, the method comprising:

determining one or more of the following parameters in the subject: peroxynitrite concentration, the rate of peroxynitrite synthesis, the extent of peroxynitrite associated signalling, PARP level, myocardial 3-nitrotyrosine level, myocardial TxNIP level, and beta 2 and/or beta 3 adrenoceptor activation or signalling; and

treating the subject on the basis of the one or more parameters so determined.

[0017] Certain embodiments of the present disclosure provide a method of identifying a subject susceptible to, or suffering from, a stress-induced cardiomyopathy, the method comprising:

determining one or more of the following parameters in the subject: peroxynitrite concentration, the rate of peroxynitrite synthesis, the extent of peroxynitrite associated signalling, PARP level, myocardial 3-nitrotyrosine level, myocardial TxNIP level, beta 2 adrenoceptor activation or signalling and/or beta 3 adrenoceptor activation or signalling; and

identifying the subject as being susceptible to, or suffering from, a stress- induced cardiomyopathy on the basis of the one or more parameters so determined.

[0018] Certain embodiments of the present disclosure provide use of one or more of the following reagents in a kit for identifying a subject susceptible to, or suffering from, a stress-induced cardiomyopathy:

a reagent for detecting peroxynitrite;

a reagent for detecting peroxynitrite synthesis;

a reagent for detecting peroxynitrite associated signalling;

a reagent for detecting a PARP;

a reagent for detecting myocardial 3-nitrotyrosine;

a reagent for detecting myocardial TxNIP;

a reagent for detecting a beta 2 adrenoceptor activation and/or signalling; and a reagent for detecting a beta 3 adrenoceptor activation and/or signalling.

[0019] Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for preventing and/or treating a stress-induced cardiomyopathy, the method comprising:

identifying a candidate agent that inhibits peroxynitrite synthesis; and/or identifying a candidate agent that scavenges peroxynitrite; and/or identifying a candidate agent that promotes peroxynitrite decomposition; and/or

identifying a candidate agent that inhibits peroxynitrite associated signalling; and/or

identifying a candidate agent that inhibits peroxynitrite-induced DNA damage; and/or

identifying a candidate agent that inhibits TxNIP expression and/or activity; identifying a candidate agent that antagonises a beta 2 adrenoceptor; and identifying a candidate agent that antagonises a beta 3 adrenoceptor; and identifying one or more of the aforementioned candidate agents as a therapeutic agent for preventing and/or treating a stress-induced cardiomyopathy.

[0020] Other embodiments are disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

[0021] Certain embodiments are illustrated by the following figures. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the description.

[0022] Figure 1 shows platelet responses to nitric oxide (NO), asymmetric dimethylarginine (ADMA levels), and endothelial progenitor cell (EPC) counts at the acute phase and 3 months of TTC compared to controls. Figure 1A shows platelet responsiveness to :-- o-J ^ ·,:·-.- (SNP) [median 43% (interquartile range: 11 - 57) and 30% (12-64) for TTC vs. 13% (4-34) for controls, p = 0.0001]. Figure IB shows ADMA levels [mean 0.54 + 0.08 (SD) and 0.58 + 0.1 vs. 0.6 + 0.1 μΜ, p = 0.03]. Figure 1C shows EPC counts (p = NS) in the acute phase and 3 months of TTC compared to controls.

[0023] Figure 2 shows correlations between platelet responses to NO at the acute phase of TTC and normetanephrine concentrations (Fig. 1A) (r = 0.31, p = 0.03), and NT-proBNP concentrations (Fig. IB) (r = 0.31, p = 0.02). [0024] Figure 3 shows correlation between ADMA and N-terminal probrain natriuretic peptide (NT-proBNP) levels at 3 months (r = 0.5, p = 0.003).

[0025] Figure 4 shows baseline apical strain (A) and apical strain (B) 24 hours post- isoprenaline injection in a 5-month old female Sprague Dawley rat.

[0026] Figure 5 shows mean data for quantitation of a) 3-NT and b) TxNIP, in apical and basal myocardial sections from SD animals treated with saline vehicle (filled circles) or 5mg/kg of isoproteranole (filled squares). Data are expressed as a % of the microscopic field (control n=4, isoprenaline treated n=7).

[0027] Figure 6 shows examples of immuno staining for 3-NT (A,B and TxNip (C,D). Sections are from apical myocardium in all cases: A/C are from isoproterenol-treated animals, and B/D from saline vehicle treated controls.

[0028] Figure 7 shows plasma 3-NT concentrations in Takotsubo cardiomyopathy patients (TTC) and age-matched controls (C). There was no significant difference between the groups.

[0029] Figure 8 shows immunohistochemical stain for 3-NT (A, B) and TxNIP (C, D) in human left ventricular apical myocardium, evaluated at post-mortem. Sections A and C are from the heart of a patient dying of TTC; sections B and D are from a control heart.

[0030] Figure 9 shows blinded analysis of LV global longitudinal strain (GLS) in 12 rats treated with 3 aminobenzamide and isoprenaline or 12 rats treated with isoprenaline alone.

DETAILED DESCRIPTION

[0031] The present disclosure relates to the prevention and/or treatment of stress- induced cardiomyopathies. [0032] Certain embodiments of the present disclosure are directed to methods for preventing and/or treating a stress-induced cardiomyopathy, products for preventing and/or treating a stress-induced cardiomyopathy, methods for screening or identifying subjects susceptible to, or suffering from, a stress-induced cardiomyopathy, kits, and methods for identifying therapeutic agents for preventing and/or treating a stress- induced cardiomyopathy, that have one or more combinations of advantages. Other embodiments are disclosed herein.

[0033] For example, some of the advantages of the embodiments disclosed herein include one or more of the following: providing new methods of treatment of a stress- induced cardiomyopathy; providing improved efficacy of treatment of a stress-induced cardiomyopathy; providing new products for preventing and/or treating stress-induced cardiomyopathy; providing methods of screening for determining whether a subject may be susceptible to, or suffering from, a stress-induced cardiomyopathy; providing new methods of screening for candidate therapeutic agents for preventing and/or treating a stress-induced cardiomyopathy; to provide one or more advantages in the art; or to provide a useful commercial choice. Other advantages of certain embodiments are disclosed herein or may be appreciated in practicing one or more embodiments.

[0034] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject. Other embodiments are disclosed herein.

[0035] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, by administering to the subject a therapeutically effective amount of an agent as described herein, thereby preventing and/or treating the stress-induced cardiomyopathy in the subject.

[0036] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis; (iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage; and

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist;

[0037] In certain embodiments, the stress-induced cardiomyopathy comprises Takotsubo cardiomyopathy, a cardiomyopathy arising from exposure to a toxin (such as that arising from a jellyfish sting), a cardiomyopathy arising from administration of a drug or arising from surgical intervention, a cardiomyopathy triggered by severe burns, or a cardiomyopathy associated with a disease or disorder. Other forms of stress- induced cardiomyopathies are contemplated.

[0038] In certain embodiments, the stress-induced cardiomyopathy comprises Takotsubo cardiomyopathy.

[0039] The term "preventing", and related terms such as "prevention" and "prevent", refer to obtaining a desired pharmacologic and/or physiologic effect in terms of arresting or suppressing the appearance of one or more symptoms in the subject. The term "treatment", and related terms such as "treating" and "treat", refer to obtaining a desired pharmacologic and/or physiologic effect in terms of improving the condition of the subject, ameliorating, arresting, suppressing, relieving and/or slowing the progression of one or more symptoms in the subject, a partial or complete stabilization of the subject, a regression of the one or more symptoms, or a cure of a disease, condition or state in the subject.

[0040] In certain embodiments, the subject is human subject. In certain embodiments, the subject is a mammalian subject, a livestock animal (such as a horse, a cow, a sheep, a goat, a pig), a domestic animal (such as a dog or a cat) and other types of animals such as monkeys, rabbits, mice and laboratory animals. Veterinary applications of the present disclosure are contemplated. [0041] In certain embodiments, the subject is suffering from a stress-induced cardiomyopathy. Stress-induced cardiomyopathies are as described herein. In certain embodiments, the subject is suffering from Takotsubo cardiomyopathy.

[0042] In certain embodiments, the subject is susceptible to a stress-induced cardiomyopathy. In certain embodiments, the subject is susceptible to Takotsubo cardiomyopathy.

[0043] In certain embodiments, the subject has an increased risk or likelihood of suffering from a stress-induced cardiomyopathy. In certain embodiments, the subject has an increased risk or likelihood of suffering from Takotsubo cardiomyopathy.

[0044] In certain embodiments, the subject has symptoms associated with an acute coronary syndrome and acute stress.

[0045] In certain embodiments, the subject is a female human subject. In certain embodiments, the subject is a post-menopausal female human subject. Subjects with other characteristics are contemplated.

[0046] In certain embodiments, the methods of the present disclosure comprise administering to the subject an agent as described herein. Examples of agents include a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, a DNA, a RNA, an oligonucleotide, a ribozyme, a biologic, an aptamer, a peptide, a cofactor, a ligand, a receptor, an enzyme, a kinase, a phosphatase, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, a siRNA, an antibody, an amino acid, an antagonist, an agonist, an inhibitor, a suppressor, a modulator and an inducer. Other types of agents are contemplated.

[0047] In certain embodiments, the agent comprises one or more of the following agents: a peroxynitrite inhibitor; an inhibitor of peroxynitrite synthesis; an inhibitor of peroxynitrite associated signalling; an inhibitor of peroxynitrite-induced DNA damage; a TxNIP inhibitor; a beta 2 adrenoceptor antagonist; and a beta 3 adrenoceptor antagonist.

[0048] In certain embodiments, the agent comprises a peroxynitrite inhibitor. [0049] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of a peroxynitrite inhibitor.

[0050] A peroxynitrite inhibitor may be synthesized, produced or obtained commercially.

[0051] The term "inhibitor" as used herein refers to an agent, treatment, or intervention that results directly or indirectly in a reduction of peroxynitrite expression, concentration, level, activity or function, including for example a decrease in expression, a decrease in expression, a decrease in activity, an inhibitory alteration in the timing and/or location of activity, or otherwise provide inhibitory control over activity. Other forms of inhibition are contemplated.

[0052] In certain embodiments, the inhibitor comprises an antagonist. In certain embodiments, the antagonist comprises a non-selective antagonist. In certain embodiments, the antagonist comprises a selective antagonist.

[0053] The term "activity" as used herein refers to the function of a species and includes, for example, the level, the specificity, the ability to interact (directly and/or indirectly) with and/or modify other species, the ability to signal, and the ability to cause changes (directly and/or indirectly) in other cellular and/or non-cellular events. Examples of modulating the activity of a species include, for example, changes in the level of the species, changes in the localisation of the species, changes in the synthesis and/or degradation rates of the species, changes in the timing of activity, changes in the ability to interact with other species (such as a change in the ability of a ligand and a receptor to interact), changes in the chemical composition of the species, changes in signalling, and changes in cellular and/or non-cellular events affected by the species.

[0054] In certain embodiments, the methods as described herein comprise administering a peroxynitrite inhibitor to a subject. In certain embodiments, pharmaceutical compositions or medicaments as described herein comprise a peroxynitrite inhibitor. [0055] In certain embodiments, the peroxynitrite inhibitor comprises a peroxynitrite scavenger.

[0056] The term "peroxynitrite scavenger" as used herein includes an agent that decreases the level peroxynitrite, directly and/or indirectly. Examples of peroxynitrite scavengers include a peroxiredoxin, a scavenger of peroxynitrite radicals, a thiol-based antioxidant (such as mercaptoalkylguanidines, N-acetylcysteine, and dihyrdolipoic acid), ebselen, a selenium containing protein (such as glutathione peroxidise), selenocysteine, selenomethionine, Tempol, cabergoline, acetaminophin, Nebivolol, hydralazine, pindolol, zileuton, pencillamine, simvastin, edaravone, propofol, deprenyl, rasagiline, desferoxamine, a tyrosine containing peptide, and a peroxynitrite decomposition catalyst (such as a metalloporphyrin). In certain embodiments, the methods as described herein comprise administering a peroxynitrite scavenger to the subject.

[0057] A peroxynitrite scavenger may be synthesized, produced or obtained commercially.

[0058] In certain embodiments, the peroxynitrite inhibitor comprises an agent that promotes peroxynitrite decomposition.

[0059] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that promotes peroxynitrite decomposition.

[0060] The term "agent that promotes peroxynitrite decomposition" as used herein includes an agent that causes the breakdown of peroxynitrite directly and/or indirectly. Examples of agents that promote peroxynitrite decomposition include agents such as a metalloporphyrin. An agent that promotes peroxynitrite decomposition may be synthesized, produced or obtained commercially.

[0061] In certain embodiments, the peroxynitrite inhibitor comprises an agent that promotes peroxynitrite decomposition. [0062] In certain embodiments, the agent comprises an inhibitor of peroxynitrite synthesis. An inhibitor of peroxynitrite synthesis may be synthesized, produced or obtained commercially.

[0063] In certain embodiments, the methods as described herein comprise administering an inhibitor of peroxynitrite synthesis to the subject. In certain embodiments, pharmaceutical compositions or medicaments as described herein comprises an inhibitor of peroxynitrite synthesis.

[0064] In certain embodiments, the inhibitor of peroxynitrite synthesis comprises an inhibitor of nitric oxide synthesis. In certain embodiments, the inhibitor of nitric oxide synthesis comprises an inhibitor of a nitric oxide synthase. In certain embodiments, the inhibitor of nitric oxide synthase comprises an inhibitor of one or more NOS-1, NOS-2 and NOS-3. Other types of specific or non-specific nitric oxide synthase subtypes are contemplated. Inhibitors of nitric oxide synthesis are as described in, for example, V tecek J. (2012) Mediators of Inflammation 2012 (2012), Article ID 318087,

[0065] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of peroxynitrite synthesis.

[0066] In certain embodiments, the inhibitor of peroxynitrite synthesis comprises an inhibitor of superoxide synthesis. An inhibitor of superoxide synthesis may be synthesized, produced or obtained commercially.

[0067] In certain embodiments, the agent comprises an inhibitor of peroxynitrite associated signalling. In certain embodiments, methods as described herein comprise administering an inhibitor of peroxynitrite associated signalling. In certain embodiments, pharmaceutical compositions or medicaments as described herein comprise an inhibitor of peroxynitrite associated signalling. [0068] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of peroxynitrite associated signalling.

[0069] An inhibitor of peroxynitrite associated signalling may be synthesized, produced or obtained commercially.

[0070] In certain embodiments, the inhibitor of peroxynitrite associated signalling comprises an inhibitor of a signalling pathway comprising a signalling pathway involving one or more of the following: nuclear factor kappa B, protein kinase C, phosphatidylinositol 3-kinase; extracellular-regulated kinase, Janus kinase, epidermal growth factor receptor, platelet-derived growth factor receptor, truncated tyrosine protein kinase, and p38. In certain embodiments, the methods as described herein comprise administering an inhibitor of peroxynitrite associated signalling to a subject.

[0071] In certain embodiments, the agent comprises an inhibitor of peroxynitrite- induced oxidative damage. In certain embodiments, methods as described herein comprise administering an inhibitor of peroxynitrite-induced oxidative damage to a subject. In certain embodiments, pharmaceutical compositions or medicaments as described herein comprises an inhibitor of peroxynitrite-induced oxidative damage.

[0072] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of peroxynitrite-induced oxidative damage.

[0073] An inhibitor of peroxynitrite-induced oxidative damage may be synthesized, produced or obtained commercially.

[0074] In certain embodiments, the inhibitor of peroxynitrite-induced oxidative damage comprises an inhibitor of peroxynitrite-induced DNA damage. In certain embodiments, the agent comprises an inhibitor of peroxynitrite-induced DNA damage. [0075] In certain embodiments, the agent comprises an inhibitor of peroxynitrite- induced DNA damage.

[0076] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of a peroxynitrite induced DNA damage.

[0077] An inhibitor of peroxynitrite-induced DNA damage may be synthesized, produced or obtained commercially.

[0078] In certain embodiments, methods as described herein comprise administering an inhibitor of peroxynitrite-induced DNA damage to a subject. In certain embodiments, pharmaceutical compositions or medicaments as described herein comprises an inhibitor of peroxynitrite-induced DNA damage.

[0079] In certain embodiments, the inhibitor of peroxynitrite-induced DNA damage comprises a PARP inhibitor. In certain embodiments, the inhibitor of peroxynitrite- induced DNA damage comprises an inhibitor of one or more of PARP concentration, activity, expression or level. A PARP inhibitor may be synthesized, produced or obtained commercially.

[0080] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of a PARP inhibitor.

[0081] In certain embodiments, the inhibitor of peroxynitrite-induced DNA damage comprises a PARP- 1 and/or a PARP-2 inhibitor.

[0082] The term "PARP inhibitor" as used herein refers to an inhibitor (or antagonist) of Poly(ADP-ribose) polymerases (PARP 1 and/or PARP-2). Examples of PARP inhibitors are described in Penning, Current Opinion In Drug Discovery & Development 2010 13 (5): 577-586 and WO 2012/151701. PARP inhibition may be determined using conventional methods, including for example dot blots (Affar EB et al., Anal Biochem. 1998; 259(2):280-3), and BER assays that measure the direct activity of PARP to form poly ADP-ribose chains for example by using radioactive assays with tritiated substrate NAD or specific antibodies to the polymer chains formed by PARP activity (Dillon K.J. et al, Journal of Biomolecular Screening, 8(3): 347-352 (2003)).

[0083] Examples of compounds which are PARP inhibitors include compounds and derivatives thereof from the class of Nicotinamides, Benzamides, Isoquinolinones, Dihydroisoquinolinones, Benzimidazoles, indoles, Phthalazin-1 (2H)-ones, quinazolinones, Isoindolinones, Phenanthridines, phenanthhdinones, Benzopyrones, Unsaturated hydroximic acid derivatives and Pyridazines.

[0084] Examples of compounds which are PARP inhibitors include: (i) nicotinamides, such as 5-methyl nicotinamide and 0-(2-hydroxy-3-piperidino- propyl)-3-carboxylic acid amidoxime, and analogues and derivatives thereof; (ii) benzamides, including 3- substituted benzamides such as 3-aminobenzamide, 3-hydroxybenzamide, 3- nitrosobenzamide, 3-methoxybenzamide and 3- chloroprocainamide, and 4- aminobenzamide, 1 , 5-di[(3- carbamoylphenyl)aminocarbonyloxy] pentane, and analogues and derivatives thereof; (iii) Isoquinolinones and Dihydroisoquinolinones, including 2H-isoquinolin- 1 -ones, 3H-quinazolin-4-ones, 5-substituted dihydroisoquinolinones such as 5-hydroxy dihydroisoquinolinone, 5-methyl dihydroisoquinolinone, and 5-hydroxy isoquinolinone, 5-amino isoquinolin-1 -one, 5- dihydroxyisoquinolinone, 3, 4 dihydroisoquinolin-1 (2H)- ones such as 3, 4 dihydro-5- methoxy-isoquinolin-1 (2H)-one and 3, 4 dihydro-5-methyl- 1 (2H)isoquinolinone, isoquinolin-1 (2H)-ones, 4,5-dihydro-imidazo[4,5,l -ij]quinolin-6- ones, 1 , 6,- naphthyridine-5(6H)-ones, 1 ,8-naphthalimides such as 4-amino-l ,8- naphthalimide, isoquinolinone, 3, 4-dihydro-5-[4-l (1 -piperidinyl) butoxy]-l (2H)- isoquinolinone, 2, 3 -dihydrobenzo[de] isoquinolin-1 -one, 1 -1 1 b-dihydro- [2H]benzopyrano[4, 3, 2- de]isoquinolin-3-one, and tetracyclic lactams, including benzpyranoisoquinolinones such as benzopyrano[4,3,2-de] isoquinolinone, and analogues and derivatives thereof; (iv) benzimidazoles and indoles, including benzoxazole-4-carboxamides, benzimidazole-4-carboxamides, such as 2-substituted benzoxazole 4-carboxamides and 2-substituted benzimidazole 4-carboxamides such as 2-aryl benzimidazole 4- carboxamides and 2-cycloalkylbenzimidazole-4-carboxamides including 2-(4- hydroxphenyl) benzimidazole 4-carboxamide, quinoxalinecarboxamides, imidazopyridinecarboxamides, 2-phenylindoles, 2-substituted benzoxazoles, such as 2- phenyl benzoxazole and 2-(3-methoxyphenyl) benzoxazole, 2-substituted benzimidazoles, such as 2-phenyl benzimidazole and 2-(3-methoxyphenyl) benzimidazole, 1 , 3, 4, 5 tetrahydro-azepino[5, 4, 3-cd]indol-6-one, azepinoindoles and azepinoindolones such as 1 , 5 dihydro-azepino[4, 5, 6-cd]indolin-6-one and dihydrodiazapinoindolinone, 3-substituted dihydrodiazapinoindolinones, such as 3-(4- thfluoromethyl phenyl )-dihydrodiazapinoindolinone, tetrahydrodiazapinoindolinone and 5,6,-dihydroimidazo[4, 5, 1 -j, k][l , 4]benzodiazopin-7(4H)-one, 2-phenyl-5,6- dihydro- imidazo[4,5,l - j k] [ 1 ,4]benzodiazepin-7(4H)-one and 2, 3, dihydro-isoindol- 1 -one, and analogues and derivatives thereof; (v) phthalazin-1 (2H)-ones and quinazolinones, such as 4-hydroxyquinazoline, phthalazinone, 5-methoxy-4-methyl-l (2) phthalazinones, 4-substituted phthalazinones, 4-(l -piperazinyl)-l (2H)- phthalazinone, tetracyclic benzopyrano[4, 3, 2-de] phthalazinones and tetracyclic indeno [1 , 2, 3-de] phthalazinones and 2-substituted quinazolines, such as 8-hydroxy-2- methylquinazolin-4-(3H) one, tricyclic phthalazinones and 2-aminophthalhydrazide, and analogues and derivatives thereof; (vi) Isoindolinones and analogues and derivatives thereof; (vii) phenanthridines and phenanthridinones, such as 5[H]phenanthridin-6-one, substituted 5[H] phenanthridin-6-ones, especially 2-, 3- substituted 5[H] phenantridin-6- ones and sulfonamide/carbannide derivatives of 6(5H)phenanthridinones, thieno[2, 3- c]isoquinolones such as 9-annino thieno[2, 3-c]isoquinolone and 9-hydroxythieno[2, 3- c]isoquinolone, 9-methoxythieno[2, 3-c]isoquinolone, and N-(6-oxo-5, 6- dihydrophenanthridin-2-yl]-2-(N,N-dimethylannino}acetannide, substituted 4,9- dihydrocyclopenta[lmn]phenanthridine-5-ones, and analogues and derivatives thereof;

(viii) benzopyrones such as 1 , 2-benzopyrone, 6-nitrosobenzopyrone, 6-nitroso 1 , 2- benzopyrone, and 5-iodo-6-aminobenzopyrone, and analogues and derivatives thereof;

(ix) unsaturated hydroximic acid derivatives such as 0-(3-piperidino-2-hydroxy-l - propyl)nicotinic amidoxime, and analogues and derivatives thereof; (x) pyridazines, including fused pyridazines and analogues and derivatives thereof; and (xi) other compounds such as caffeine, theophylline, and thymidine, and analogues and derivatives thereof.

[0085] In certain embodiments, the PARP inhibitor comprises 3-aminobenzamide, NU1025, ABT-888 (Veliparib), Olaparib (was AZD-2281), CEP 9722, MK4827, AG014699, Iniparib (previously BSI 201 ), LT-673, and E7016. [0086] In certain embodiments, the agent comprises a TxNIP inhibitor. A TxNIP inhibitor may be synthesized, produced or obtained commercially.

[0087] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of a TxNIP inhibitor.

[0088] In certain embodiments, the TxNIP inhibitor comprises an inhibitor of one or more of TxNIP activity, expression or level. In certain embodiments, the TxNIP inhibitor comprises an inhibitor of TxNIP expression.

[0089] In certain embodiments, the TxNIP inhibitor comprises an antibody directed against a TxNIP, or an siRNA which inhibits TXNIP expression or activity. For example, a TxNIP antibody is TxNIP Antibody (JY2) (NBP 1-54578) from Novus Biologicals.

[0090] Methods for preparing, formulating and administering pharmaceutical compositions comprising an antibody are known, and include for example "Handbook of Therapeutic Antibodies" ed. S. Dubel (2007) Wiley- VCH. Methods for producing and delivering antisense nucleic acids, microRNAs and siRNAs for therapeutic purposes are known, for example, as described in 'Nucleic Acid Therapeutics in Cancer" 2004 ed. Alan M. Gewirtz, Humana Press Inc.

[0091] In certain embodiments, the agent comprises a beta 2 adrenoceptor inhibitor. A beta-2 adrenoceptor inhibitor may be synthesized, produced or obtained commercially. In certain embodiments, the beta 2 adrenoceptor inhibitor comprises a beta 2 adrenoceptor antagonist.

[0092] In certain embodiments, the agent comprises a beta 2 adrenoceptor antagonist.

[0093] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of a beta 2 adrenoceptor antagonist. [0094] In certain embodiments, the antagonist is a selective antagonist. In certain embodiments, the antagonist is non-selective antagonist. In certain embodiments, the beta 2 adrenoceptor antagonist comprises an antagonist of a beta 2 adrenoceptor ligand. In certain embodiments, the beta 2 adrenoceptor antagonist comprises an antagonist of beta 2 adrenoceptor signalling. In certain embodiments, the beta 2 adrenoceptor antagonist comprises one or more of Butaxamine and 3(-isopropylamino)-l-[(7-methyl- 4-indanyl)oxy]butan-2-ol. A beta 2 adrenoceptor antagonist may be synthesized, produced or obtained commercially.

[0095] In certain embodiments, the methods as described herein comprise administering a beta 2 adrenoceptor antagonist to the subject. In certain embodiments, pharmaceutical compositions or medicaments as described herein comprise a beta 2 adrenoceptor antagonist.

[0096] In certain embodiments, the agent comprises a beta 3 adrenoceptor inhibitor. A beta 3 adrenoceptor inhibitor may be synthesized, produced or obtained commercially. In certain embodiments, the beta 3 adrenoceptor inhibitor comprises a beta 3 adrenoceptor antagonist.

[0097] In certain embodiments, the agent comprises a beta 3 adrenoceptor antagonist.

[0098] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of a beta 3 adrenoceptor antagonist.

[0099] In certain embodiments, the antagonist is a selective antagonist. In certain embodiments, the antagonist is non-selective antagonist. In certain embodiments, the beta 3 adrenoceptor antagonist comprises an antagonist of a beta 3 adrenoceptor ligand. In certain embodiments, the beta 3 adrenoceptor antagonist comprises an antagonist of a beta 3 adrenoceptor signalling. In certain embodiments, the antagonist of beta 3 adrenoceptor comprises one or more of SR 59230A ((2S)-l-(2-ethylphenoxy)-3-{ [(lS)- 1,2,3, 4-tetrahydronaphthalen-l-yl] amino }propan-2-ol), L-748,337 (N-[[3-[(2S)-2- Hydroxy-3-[[2-[4-[(phenylsulfonyl) amino]phenyl] ethyl] amino] propoxy] phenyl] methyl] -acetamide), L-748,328 (((S)-N-[4-[2-[[3-[3-(ami-nosulphonyl)phenoxy]-2- hydroxypropyl]amino]ethyl]benze-nesulfonamide), and CL 31624 (5-[(2R)-2-[[(2R)-2- (3-Chlorophenyl)-2-hydroxyethyl] amino] propyl]- l,3-benzodioxole-2,2-dicarboxylic acid disodium salt). A beta 3 adrenoceptor antagonist may be synthesized, produced or obtained commercially.

[00100] In certain embodiments, the methods as described herein comprise administering a beta 3 adrenoceptor antagonist to the subject. In certain embodiments, pharmaceutical compositions or medicaments as described herein comprise a beta 3 adrenoceptor antagonist.

[00101] In certain embodiments, the beta 2 and/or beta 3 adrenoceptor antagonist comprises an arrestin. In certain embodiments, the arrestin comprises an a-arrestin. In certain embodiments, the arrestin comprises a β-arrestin.

[00102] In certain embodiments, the beta 3 adrenoceptor antagonist comprises a- arrestin Arrdc3. In certain embodiments, the methods as described herein comprise administering an a-arrestin Arrdc3 to the subject. The accession number for human a- arrestin Arrdc3 protein is UniProtKB/Swiss-Prot: ARRD3_HUMAN, Q96B67.

[00103] In certain embodiments, the methods of prevention and/or treatment described herein comprise determining one or more clinical characteristics and/or determining one or more parameters in the subject.

[00104] In certain embodiments, the methods of preventing and/or treatment comprise determining one or more parameters from a processed sample obtained from the subject. Examples of samples include one or more biological fluids, such as blood, plasma, urine, amniotic fluid, tears, saliva, hair, skin, and one or more tissue samples, such as a cervical sample, a buccal sample or a chorionic villous sample. Other types of biological samples are contemplated. In certain embodiments, the sample comprises a blood sample, a plasma sample and/or a serum sample.

[00105] In certain embodiments, the clinical characteristics comprise one or more of chest pain and/or dyspnoea > 30min; ST/T wave changes; periapical or midventricular akinesis/hypokinesis; and no evidence for a diagnosis of myocardial infarction on coronary angiography or cardiovascular magnetic resonance. Other clinical characteristics are contemplated.

[00106] In certain embodiments, the one or more parameters comprise one or more of peroxynitrite concentration, the rate of peroxynitrite synthesis, the extent of peroxynitrite associated signalling, PARP level, plasma N-terminal pro-brain natriuretic concentration, myocardial 3-nitrotyrosine levels, myocardial TxNIP level, and beta 2 and/or beta 3 adrenoceptor activation or signalling. Methods for determining such parameters are known.

[00107] In certain embodiments, a rate of peroxynitrite synthesis and/or an extent of peroxynitrite associated signalling which is maintained or increased is indicative that the subject is suitable for treatment.

[00108] In certain embodiments, one or more of increased PARP level, increased myocardial 3-nitrotyrosine expression, and increased myocardial TxNIP expression is indicative that the subject is suitable for treatment.

[00109] In certain embodiments, one or more of increased beta 2 and/or beta 3 adrenoceptor activation or signalling is indicative that the subject is suitable for treatment.

[00110] In certain embodiments, methods of preventing and/or treating as described herein further comprise determining one or more of the following parameters in the subject: peroxynitrite concentration, the rate of peroxynitrite synthesis, the extent of peroxynitrite associated signalling, PARP level, plasma N-terminal pro-brain natriuretic concentration, myocardial 3-nitrotyrosine levels, myocardial TxNIP level, and beta 2 and/or beta 3 adrenoceptor activation or signalling.

[00111] In certain embodiments, a method of preventing and/or treating a stress- induced cardiomyopathy comprises determining one or more of the following parameters in the subject: peroxynitrite concentration, the rate of peroxynitrite synthesis, the extent of peroxynitrite associated signalling, PARP level, plasma N- terminal pro-brain natriuretic concentration, myocardial 3-nitrotyrosine levels, myocardial TxNIP level, and beta 2 and/or beta 3 adrenoceptor activation or signalling.

[00112] In certain embodiments, methods of prevention and/or treatment as described herein comprise administering one or more agents as described herein on the basis of the one or more parameters and/or clinical characteristics so determined.

[00113] In certain embodiments, a method of preventing and/or treating a stress- induced cardiomyopathy comprises determining one or more of the following parameters in the subject: peroxynitrite concentration, the rate of peroxynitrite synthesis, the extent of peroxynitrite associated signalling, PARP level, plasma N- terminal pro-brain natriuretic concentration, myocardial 3-nitrotyrosine levels, myocardial TxNIP level, and beta 2 and/or beta 3 adrenoceptor activation or signalling; and administering one or more agents on the basis of the one or more parameters so determined.

[00114] The term "therapeutically effective amount" as used herein refers to that amount of an agent that is sufficient to effect prevention and/or treatment, when administered to a subject. The therapeutically effective amount will vary depending upon a number of factors, including for example the specific activity of the agent being used, the severity of the disease, condition or state in the subject, the age, physical condition, existence of other disease states, and nutritional status of the subject.

[00115] In certain embodiments, an agent as described herein is administered to the subject in an amount ranging from one of the following selected ranges: 1 g/kg to 100 mg/kg; 1 μg/kg to 10 mg/kg; 1 μg/kg to 1 mg/kg; 1 μg/kg to 100 μg/kg; 1 μg/kg to ^g/kg; 10 μg/kg to 100 mg/kg; 10 μg/kg to 10 mg/kg; 10 μg/kg to 1 mg/kg; 10 μg/kg to 100 μg/kg; 100 μg/kg to 100 mg/kg; 100 μg/kg to 10 mg/kg; 100 μg/kg to 1 mg/kg; 1 mg/kg to 10 mg/kg; and 10 mg/kg to 100 mg/kg body weight. Other ranges are contemplated.

[00116] In certain embodiments, the agent is administered to the subject at a concentration of 1 μg/kg to 100 mg/kg. [00117] In certain embodiments, the agent is administered to the subject for a period of time of 12 months or less, 9 months or less, 6 months or less, 3 months or less, or 1 month or less following occurrence of the stress-induced cardiomyopathy. In certain embodiments, the agent is administered to the subject for a period of time of 12 months or more, 9 months or more, 6 months or more, 3 months or more, or 1 month or more following occurrence of the stress-induced cardiomyopathy. In certain embodiments, the agent is administered to the subject for a period of time of 3 months or less following occurrence of the stress-induced cardiomyopathy.

[00118] The agent may be administered to the subject in a suitable form. In this regard, the terms "administering" or "providing" include administering the agent, or administering a prodrug of the agent, or a derivative of the agent that will form an effective amount of the agent within the body of the subject. The terms include routes of administration that are systemic (e.g., via injection such as intravenous injection, orally in a tablet, pill, capsule, or other dosage form useful for systemic administration of pharmaceuticals), and topical (e.g., creams, solutions, and the like, including solutions such as mouthwashes, for topical oral administration).

[00119] In certain embodiments, the agent is administered orally. In certain embodiments, the agent is administered intravenously. In certain embodiments, the agent is administered via injection (such as intravenous injection). In certain embodiments, the agent is administered by nebulized administration, by aerosolized administration or by being instilled into the lung.

[00120] The agent may be administered alone or may be delivered in a mixture with other therapeutic agents and/or agents that enhance, stabilise or maintain the activity of the agent for administration. In certain embodiments, an administration vehicle (e.g., pill, tablet, implant, injectable solution, etc.) would contain both the agent and additional drug(s)/compound(s) etc.

[00121] The methods as described herein may also include combination therapy. In this regard, the subject is treated or given another drug or treatment modality in conjunction with an agent as described herein. This combination therapy can be sequential therapy where the subject is treated first with one and then the other, or where the two or more treatment modalities are given simultaneously, or substantially simultaneously.

[00122] "Co-administering" or "co-administration" refers to the administration of two or more therapeutic agents together at one time or substantially the same time. Two or more therapeutic agents can be co-formulated into a single dosage form or "combined dosage unit", or formulated separately and subsequently combined into a combined dosage unit, typically for intravenous administration or oral administration.

[00123] When administered to a subject the therapeutically effective dosage may vary depending upon the particular agent utilized, the mode of administration, the condition, and severity thereof, as well as the various physical factors related to the subject being treated. As discussed herein, suitable daily doses range from 1 μg/kg to 100 mg/kg. The daily dosages are expected to vary with route of administration, and the nature of the modulator administered. In certain embodiments the methods as described herein comprise administering to the subject escalating doses of agent and/or repeated doses. In certain embodiments, the agent is administered orally. In certain embodiments, the agent is administered via injection (such as intravenous injection). In certain embodiments, the agent is administered parenterally. In certain embodiments, the agent is administered by direct introduction to the lungs, such as by aerosol administration, by nebulized administration, and by being instilled into the lung. In certain embodiments, the agent is administered by implant. In certain embodiments, the agent is administered by subcutaneous injection, intraarticularly, rectally, intranasally, intraocularly, vaginally, or transdermally.

[00124] "Intravenous administration" is the administration of substances directly into a vein.

[00125] "Oral administration" is a route of administration where a substance is taken through the mouth, and includes buccal, sublabial and sublingual administration, as well as enteral administration. A typical form for the oral administration of therapeutic agents includes the use of tablets or capsules. [00126] In certain embodiments, the agent is administered as an immediate release formulation. The term "immediate release formulation" is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time. In certain embodiments, the agent is administered as a sustained release formulation. The term "sustained release formulation" is a formulation which is designed to slowly release a therapeutic agent in the body over an extended period of time.

[00127] In certain embodiments, one or more of the agents as described herein may be used in a pharmaceutical composition or in a medicament.

[00128] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

[00129] In certain embodiments, the agent as described herein may be used in a pharmaceutical composition or a medicament for use in the methods of the present disclosure.

[00130] In certain embodiments, the present disclosure provides a pharmaceutical composition when used to prevent and/or treat a stress-induced cardiomyopathy, as described herein.

[00131] In certain embodiments, the present disclosure provides a pharmaceutical composition when used to prevent and/or treat a stress-induced cardiomyopathy, the pharmaceutical composition or medicament comprising a therapeutically effective amount of an agent as described herein. [00132] In certain embodiments, the pharmaceutical composition or medicament further comprises a pharmaceutically acceptable carrier, as described herein

[00133] Certain embodiments of the present disclosure provide a method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a pharmaceutical composition or medicament as described herein.

[00134] Certain embodiments of the present disclosure provide a method of treating a subject having symptoms associated with an acute coronary syndrome and acute stress, the method comprising administering to the subject one or more agents as described herein.

[00135] Certain embodiments of the present disclosure provide a method of treating a subject having symptoms associated with an acute coronary syndrome and acute stress, the method comprising administering to the subject a pharmaceutical composition or medicament as described herein.

[00136] Certain embodiments of the present disclosure provide a method of treating a subject having symptoms associated with an acute coronary syndrome and acute stress, the method comprising administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage; and

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist;

thereby treating the subject.

[00137] Certain embodiments of the present disclosure provide a method of treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy, the method comprising administering to the subject one or more agents as described herein. [00138] Certain embodiments of the present disclosure provide a method of treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy, the method comprising administering to the subject a pharmaceutical composition or a medicament as described herein.

[00139] Certain embodiments of the present disclosure provide a method of treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy, the method comprising administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage; and

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist;

thereby treating the subject.

[00140] Certain embodiments of the present disclosure provide use of one or more of the agents as described herein for use in preventing and/or treating a stress-induced cardiomyopathy.

[00141] Certain embodiments of the present disclosure provide use of one or more agents as described herein in the preparation of a medicament for preventing and/or treating a stress-induced cardiomyopathy.

[00142] Certain embodiments of the present disclosure provide use of one or more of the following agents in the preparation of a medicament for prevention and/or treatment of a stress-induced cardiomyopathy in a subject:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor; (vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist

[00143] Certain embodiments of the present disclosure provide use of one or more of the agents as described herein for use in treating a subject having symptoms associated with an acute coronary syndrome and acute stress.

[00144] Certain embodiments of the present disclosure provide use of one or more agents as described herein in the preparation of a medicament for treating a subject having symptoms associated with an acute coronary syndrome.

[00145] Certain embodiments of the present disclosure provide use of one or more of the following agents in the preparation of a medicament for in treating a subject having symptoms associated with an acute coronary syndrome and acute stress:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

[00146] Certain embodiments of the present disclosure provide use of one or more of the agents as described herein for treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy.

[00147] Certain embodiments of the present disclosure provide use of one or more agents as described herein in the preparation of a medicament for treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy.

[00148] Certain embodiments of the present disclosure provide use of one or more of the following agents in the preparation of a medicament for treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy:

(i) a peroxynitrite inhibitor; (ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

[00149] In certain embodiments, the agent as described herein is present in a pharmaceutical composition or medicament for administration to the subject so as to provide the agent in an amount ranging from one of the following selected ranges: 1 μg/kg to 100 mg/kg; 1 μg/kg to 10 mg/kg; 1 μg/kg to 1 mg/kg; 1 μg/kg to 100 μg/kg; 1 μg/kg to 10μg/kg; 10 μg/kg to 100 mg/kg; 10 μg/kg to 10 mg/kg; 10 μg/kg to 1 mg/kg; 10 μg/kg to 100 μg/kg; 100 μg/kg to 100 mg/kg; 100 μg/kg to 10 mg/kg; 100 μg/kg to 1 mg/kg; 1 mg/kg to 10 mg/kg; and 10 mg/kg to 100 mg/kg body weight.

[00150] In certain embodiments, the agent as described herein is present in a pharmaceutical composition or medicament in an amount ranging from one of the following amounts: 10 μg - 1000 mg, 10 μg - 100 mg, 10 μg - 10 mg, 10 μg - 1 mg, 10 μg - 100 μg, 100 μg - 1000 mg, 100 μg - 100 mg, 100 μg - 10 mg, 100 μg - 1 mg, 1 mg - 1000 mg, 1 mg - 100 mg, 1 mg - lOmg, and 10 mg - 1000 mg.

[00151] In certain embodiments, the medicament is suitable for delivery to the subject by one or more of intravenous administration, intratracheal administration, by nebulized administration, by aerosolized administration, by instillation into the lung, by oral administration, by parenteral administration, by implant, by subcutaneous injection, intraarticularly, rectally, intranasally, intraocularly, vaginally, or transdermally. Methods of administration are as described herein.

[00152] In certain embodiments, the agent is provided in a pharmaceutically acceptable carrier suitable for administering the pharmaceutical composition or medicament to a subject. The carriers may be chosen based on the route of administration as described herein, the location of the target issue, the agent being delivered, the time course of delivery of the drug, etc. The term "pharmaceutically acceptable carrier" refers to a substantially inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. An example of a pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known in the art. Some examples of materials which can serve as pharmaceutically acceptable carriers include, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN 80; buffering agents such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colouring agents, releasing agents, coating agents, sweetening, flavouring and perfuming agents, preservatives and antioxidants can also be present.

[00153] In certain embodiments, the agent as described herein may be administered or present in a pharmaceutical composition as a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to acid addition salts or metal complexes which are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like.

[00154] In certain embodiments, the pharmaceutical compositions or medicament comprises other therapeutic agents and/or agents that enhance, stabilise or maintain the activity of the active. In certain embodiments, a pharmaceutical composition or medicament comprises one or more agents as described herein and one or more of a pharmaceutically acceptable carrier, vehicle and excipient. [00155] Oral formulations containing the agent as described herein may comprise any conventionally used oral forms, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions. Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharmaceutically acceptable starches (e.g. corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc. Useful tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar. Surface modifying agents include nonionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminium silicate, and triethanolamine. Oral formulations may utilize standard delay or time-release formulations to alter the absorption of the peptides. The oral formulation may also consist of administering the active ingredient in water or a fruit juice, containing appropriate solubilizers or emulsifiers as needed.

[00156] In certain embodiments, it may be desirable to administer the agents as described herein directly to the airways in the form of an aerosol. Formulations for the administration of aerosol forms are known.

[00157] In certain embodiments, the agents as described herein may also be administered parenterally (such as directly into the joint space) or intraperitoneally. For example, solutions or suspensions of these compounds in a non-ionised form or as a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations typically contain a preservative to prevent the growth of microorganisms.

[00158] In certain embodiments, the agents as described herein may also be administered by injection. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[00159] In certain embodiments, the agents as described herein may also be administered intravenously. Compositions containing the agent described herein suitable for intravenous administration may be formulated by a skilled person.

[00160] In certain embodiments, the agent may also be administered transdermally. Transdermal administrations are understood to include all administrations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administrations may be carried out using the agent as described herein, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (eg rectal and vaginal).

[00161] Transdermal administration may also be accomplished through the use of a transdermal patch containing the active compound and a carrier that is inert to the active compound, is non toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable. A variety of occlusive devices may be used to release the active ingredient into the blood stream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient. [00162] In certain embodiments, the agent as described herein may also be administered by way of a suppository. Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.

[00163] Additional numerous various excipients, dosage forms, dispersing agents and the like that are suitable for use in connection with the administration of the agent as described herein and/or the formulation into medicaments or pharmaceutical compositions. Formulations are known and described in, for example, Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference in its entirety.

[00164] Certain embodiments of the present disclosure provide a method of treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy, by treating the subject on the basis of one or more parameters and/or clinical characteristics determined. Examples of parameters and clinical characteristics are as described herein.

[00165] Certain embodiments of the present disclosure provide a method of treating a subject susceptible to, or suffering from, a stress-induced cardiomyopathy, the method comprising:

determining one or more of the following parameters in the subject: peroxynitrite concentration, the rate of peroxynitrite synthesis, the extent of peroxynitrite associated signalling, PARP level, myocardial 3-nitrotyrosine level, myocardial TxNIP level, beta 2 adrenoceptor activation or signalling and beta 3 adrenoceptor activation or signalling; and

treating the subject on the basis of the one or more parameters so determined.

[00166] Methods for determining peroxynitrite concentration, rate of peroxynitrite synthesis, extent of peroxynitrite associated signalling, PARP level, myocardial 3- nitrotyrosine level, myocardial TxNIP level, and beta 2 and/or beta 3 adrenoceptor activation or signalling are known. [00167] In certain embodiments, the methods as described herein comprise obating a sample from the subject. In certain embodiments, the methods as described herein comprise processing the sample obtained from the subject to allow the determination of the one or more parameters. In certain embodiments, the methods as described herein comprises processing a sample obtained from the subject to allow the determination of the one or more parameters.

[00168] In certain embodiments, the measurement of one or more parameters in the methods as described herein comprises processing a sample obtained from the subject to allow the determination of the one or more parameters. Examples of samples are as described herein. Methods for processing samples to detect and/or determine a parameter are known.

[00169] In certain embodiments, a rate of peroxynitrite synthesis and/or an extent of peroxynitrite associated signalling which is maintained or increased is indicative that the subject is to be treated for a stress-induced cardiomyopathy.

[00170] In certain embodiments, one or more of increased PARP level, increased myocardial 3-nitrotyrosine expression, increased myocardial TxNIP expression is indicative that the subject is to be treated for a stress-induced cardiomyopathy.

[00171] In certain embodiments, an increased beta 2 and/or beta 3 adrenoceptor activation or signalling is indicative that the subject is to be treated for a stress-induced cardiomyopathy.

[00172] In certain embodiments, treating a subject comprises administering to the subject a therapeutically effective amount of one or more agents, as described herein.

[00173] In certain embodiments, treating a subject comprises administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage; (v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

[00174] Certain embodiments of the present disclosure provide a method of identifying, and/or screening for, a subject susceptible to, or suffering from, a stress-induced cardiomyopathy by determining one or more parameters as described herein in the subject.

[00175] Certain embodiments of the present disclosure provide a method of identifying a subject susceptible to, or suffering from, a stress-induced cardiomyopathy, the method comprising:

[00176] Detection and/or determination of the one or more parameters is as described herein.

[00177] In certain embodiments, the method comprises determining the one or more parameters from a processed sample obtained from the subject.

[00178] In certain embodiments, one or more of a rate of peroxynitrite synthesis which is maintained or increased, an extent of peroxynitrite associated signalling which is maintained or increased, increased PARP level, increased myocardial 3-nitrotyrosine expression, increased myocardial TxNIP expression, and increased beta 2 and/or beta 3 adrenoceptor activation or signalling is indicative that the subject is susceptible to, or suffering from, a stress-induced cardiomyopathy. [00179] Certain embodiments of the present disclosure provide a method of screening a subject to determine whether the subject is susceptible to, or suffering from, a stress- induced cardiomyopathy.

[00180] Certain embodiments of the present disclosure provide a method of screening a subject to determine whether the subject is susceptible to, or suffering from, a stress- induced cardiomyopathy by determining one or more parameters as described herein in the subject.

[00181] Certain embodiments of the present disclosure provide a method of screening to determine whether a subject is susceptible to, or suffering from, a stress-induced cardiomyopathy, the method comprising:

determining whether the subject is susceptible to, or suffering from, a stress- induced cardiomyopathy on the basis of the one or more parameters so determined.

[00182] In certain embodiments, one or more of a rate of peroxynitrite synthesis which is maintained or increased, an extent of peroxynitrite associated signalling which is maintained or increased, increased PARP level, increased myocardial 3-nitrotyrosine expression, increased myocardial TxNIP expression, and increased beta 2 and/or beta 3 adrenoceptor activation or signalling is indicative that the subject is susceptible to, or suffering from, a stress-induced cardiomyopathy.

[00183] Certain embodiments of the present disclosure comprise a kit for screening and/or identifying a subject susceptible to, or suffering from, a stress-induced cardiomyopathy.

[00184] A kit may comprise one or more components, reagents, markers and/or instructions as described herein. For example, reagents for processing samples obtained from a subject and reagents for detecting and/or determining one or more parameters in the processed samples are as described in the Examples.

[00185] Examples of reagents include a reagent for detecting peroxynitrite; a reagent for determining peroxynitrite synthesis; a reagent for detecting peroxynitrite associated signalling; a reagent for detecting a PARP; a reagent for detecting myocardial 3- nitro tyro sine; a reagent for detecting myocardial TxNIP; and a reagent for detecting beta 2 adrenoceptor activation or signalling and/or beta 3 adrenoceptor activation or signalling. Examples of such reagents are as described herein.

[00186] Certain embodiments of the present disclosure provide use of one or more of the following reagents in a kit for screening and/or identifying a subject susceptible to, or suffering from, a stress-induced cardiomyopathy:

[00187] Certain embodiments of the present disclosure provide use of one or more of the following reagents in a kit for screening and/or identifying a subject susceptible to, or suffering from, a stress-induced cardiomyopathy:

(i) a reagent for detecting peroxynitrite;

(ii) a reagent for detecting peroxynitrite synthesis;

(iii) a reagent for detecting peroxynitrite associated signalling;

(iv) a reagent for detecting a PARP;

(v) a reagent for detecting myocardial 3-nitrotyrosine;

(vi) a reagent for detecting myocardial TxNIP;

(vii) a reagent for detecting beta 2 adrenoceptor activation or signalling;

(viii) a reagent for detecting beta 3 adrenoceptor activation or signalling.

[00188] Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for preventing and/or treating a stress-induced cardiomyopathy.

[00189] Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for preventing and/or treating a stress-induced cardiomyopathy, the method comprising:

identifying a candidate agent that inhibits peroxynitrite synthesis; and/or identifying a candidate agent that promotes peroxynitrite decomposition; and/or

identifying a candidate agent that scavenges peroxynitrite; and/or

identifying a candidate agent that inhibits TxNIP expression and/or activity; and/or

identifying a candidate agent that antagonises a beta 2 adrenoceptor; and/or identifying a candidate agent that antagonises a beta 3 adrenoceptor; and identifying one or more of the aforementioned candidate agents as a therapeutic agent for preventing and/or treating a stress-induced cardiomyopathy.

[00190] Examples of candidate agents include a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, an aptamer, a peptide, a cofactor, a ligand, a receptor, an enzyme, a kinase, a phosphatase, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, a siRNA, an antibody, an amino acid, an antagonist, an agonist, an inhibitor, a suppressor, and an inducer. Other types of candidate agents are contemplated.

[00191] In vitro and/or in vivo tests for identifying a candidate agent that inhibits peroxynitrite synthesis are known. In vitro and/or in vivo tests for identifying a candidate agent that scavenges peroxynitrite are known. In vitro and/or in vivo tests for identifying a candidate agent that promotes peroxynitrite decomposition are known. In vitro and/or in vivo tests for identifying a candidate agent that inhibits peroxynitrite associated signalling are also known. In vitro and/or in vivo tests for identifying a candidate agent that inhibits peroxynitrite-induced DNA damage are known. In vitro and/or in vivo tests for identifying a candidate agent that inhibits TXNIP expression and/or activity are known. In vitro and/or in vivo tests identifying a candidate agent that antagonises a beta 2 adrenoceptor are known. In vitro and/or in vivo tests for identifying a candidate agent that antagonises a beta 3 adrenoceptor are known. [00192] Identifying a candidate agent as a therapeutic agent for preventing and/or treating a stress-induced cardiomyopathy may be accomplished by a suitable method. For example, the ability of the candidate agent to prevent and/or treat a stress-induced cardiomyopathy may be conducted in a suitable animal model and/or in a human clinical trial. Examples of animal models are as described herein.

[00193] Certain embodiments of the present disclosure provide a kit for performing the methods as described herein. As described herein, the kit may comprise one or agents, reagents, components, compositions, formulations, products and instructions as described herein. Kits may optionally comprise one or more instructions for administering the agent to a subject to prevent and/or treat a stress-induced cardiomyopathy.

[00194] Certain exemplary embodiments are illustrated by some of the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

EXAMPLE 1 - Paradoxical preservation of NO signalling in Takotsubo Cardiomyopathy

[00195] 1.1 Methods

[00196] The study was designed as a comparison between TTC cases and healthy postmenopausal female controls.

[00197] 1.1.1 Patient population. The patient characteristics are shown in Table 1. Table 1: Patient characteristics

[00198] (a) TTC group. 56 consecutive TTC patients were prospectively identified on the basis of the following criteria: (1) chest pain and/or dyspnoea > 30min, (2) ST/T wave changes and/or biomarker elevation, (3) periapical or midventricular akinesis/hypokinesis, and (4) no evidence for a diagnosis of myocardial infarction on coronary angiography or cardiovascular magnetic resonance (CMR).

[00199] (b) Control group. A control group of 110 consecutive females were randomly selected from a population-based study as previously described (Grant, J.F., et al., "The North West Adelaide Health Study: Detailed methods and baseline segmentation of a cohort for selected chronic diseases" Epidemiol Perspect Innov, 2006. 3: p. 4.). Subjects with current or previous symptomatic myocardial ischaemia were excluded. [00200] The study was approved by the institutional Ethics of Human Research Committee and written informed consent was obtained before study entry.

[00201 ] 1.1.2 Parameters of NO signalling

[00202] Assessment of integrity of the NO/ cyclic guanosine monophosphate (cGMP) system was undertaken in 3 ways:- (a) Platelet NO signalling was evaluated utilising anti-aggregatory effects of the NO donor sodium nitroprusside (SNP) (Chirkov, Y.Y., et al., "Stable angina and acute coronary syndromes are associated with nitric oxide resistance in platelets" Am Coll Cardiol, 2001. 37(7): p. 1851-7); (b) Plasma concentrations of the NOS inhibitor ADMA were determined as a marker/mediator of endothelial dysfunction (Boger, R.H., "Asymmetric dimethylarginine (ADMA) modulates endothelial function— therapeutic implications" Vase Med, 2003. 8(3): p. 149- 5); (c) Circulating endothelial progenitor cell (EPC) counts were determined via flow cytometry: EPC counts are substantially, although not entirely, NO-modulated (Fleissner, F. and T. Thum, "Critical role of the nitric oxide/reactive oxygen species balance in endothelial progenitor dysfunction" Antioxid Redox Signal, 2011. 15(4): p. 933-48).

[00203] Venous blood specimens were collected into corresponding tubes for measurements of inhibition of platelet aggregation by SNP, ADMA levels, and EPC counts. In the TTC patients, these evaluations occurred at the time of diagnosis and 3 months thereafter.

[00204] For platelet aggregation studies, blood samples were drawn by venesection from an antecubital vein and collected in plastic tubes containing 1: 10 volume of acid citrate anticoagulant (2 parts of 0.1 mol/L citric acid to 3 parts of 0.1 mol/L trisodium citrate); acidified citrate was utilized in order to minimize deterioration of platelet function during experiments. Aggregation in whole blood was examined utilizing a dual-channel impedance aggregometer (Model 560, Chrono-Log, Havertown, PA, USA) as described in Chirkov, Y.Y., et ah, "Nitrate resistance in platelets from patients with stable angina pectoris" Circulation, 1999. 100(2): p. 129-34. In brief, tests were performed at 37°C and stirring speed of 151 RCF. Samples of blood were diluted twofold with normal saline (final volume 1 ml) and warmed for 5 min at 37°C. Aggregation was induced with ADP (final concentrations of 2.5μηιο1/¾ and monitored continuously for 7 min, and responses were recorded for electrical impedance, in Ohms. In order to determine platelet responsiveness to NO, inhibition of aggregation by the NO donor sodium nitroprusside (SNP) (expressed as a percentage of ADP aggregation response) was utilized. A final concentration of ΙΟμιηοΙ/L of SNP was added to samples 1 minute before ADP. Duration of the incubation was estimated as the optimal in preliminary experiments. In control tests, physiological saline was added in appropriate volumes. Inhibition of aggregation was evaluated as a percentage comparing the extent of maximal aggregation in the presence and absence of SNP.

[00205] Plasma concentrations of ADMA were determined as described in Heresztyn, T., M.I. Worthley, and J.D. Horowitz, "Determination of 1-arginine and NG, NG - and NG, NG' -dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives" Chromatogr B Analyt Technol Biomed Life Sci, 2004. 805(2): p. 325-9. Blood was collected into heparinised tubes, centrifuged at 2 °C at 1680 RCF, and plasma was stored at -80 °C until assay. Concentrations of plasma ADMA were measured by high-performance liquid chromatography (HPLC) using the derivatisation reagent AccQ-Fluor after solid phase extraction as described in Heresztyn, T., M.I. Worthley, and J.D. Horowitz, "Determination of 1-arginine and NG, NG - and NG, NG' -dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives" Chromatogr B Analyt Technol Biomed Life Sci, 2004. 805(2): p. 325-9.

[00206] For determination of EPC counts, blood samples were collected in tubes containing potassium ethylene diamine tetra acetic acid (EDTA). Flow cytometric analysis (FACScan, Becton Dickinson) of mononuclear cells positive for both cell surface antigens, CD34 fluorescein isothiocyanate and CD133 phycoerythrin (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) was assessed as described in Rajendran, S., et al., "Polycystic ovary syndrome is associated with severe platelet and endothelial dysfunction in both obese and lean subjects" Atherosclerosis, 2009. 204(2): p. 509-14. Corresponding negative controls with IgG2a and IgGl were obtained. [00207] 1.1.3 Relationships between NO signalling and severity of TTC

[00208] (a) Biochemical correlates. Plasma concentrations of normetanephrine, a non- acidic derivative of norepinephrine, were measured as an index of catecholamine exposure in TTC. Venous blood at diagnosis was drawn into tubes containing EDTA and centrifuged at 2 °C at 1680 RCF for plasma storage at -80°C for subsequent assay of normetanephrine by liquid chromatography/tandem mass spectrometry (LC-MS/MS) as described in Lagerstedt, S.A., D.J. O'Kane, and R.J. Singh, "Measurement of plasma free metanephrine and normetanephrine by liquid chromatography-tandem mass spectrometry for diagnosis of pheochromocytoma" Clin Chem, 2004. 50(3): p. 603-11.

[00209] Serial routine biochemical investigations including assays of plasma NT- proBNP using immunoassay (Elecsys E 170, Roche Diagnostics, Mannheim, Germany), high sensitive plasma C-reactive protein (hs CRP) concentrations utilizing a latex- enhanced immunoturbidometric assay (Olympus au5400, Dallas, Texas, USA), CK, and troponin T were performed.

[00210] (b) Pathophysiological correlates. Appropriate global indices of LV deficit and tissue inflammation were derived from echocardiographic and cardiovascular magnetic resonance imaging studies, respectively, in acute and follow-up phases. Wall motion score index, as a measure of the overall extent of wall motion impairment, was calculated according to a sixteen-segment model, as described in Nguyen, T.H., et al., N-terminal pro-brain natriuretic protein levels in takotsubo cardiomyopathy. Am J Cardiol, 2011. 108(9): p. 1316-21, and Schiller, N.B., et al., "Recommendations for quantitation of the left ventricle by two-dimensional echocardiography" American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms. J Am Soc Echocardiogr, 1989. 2(5): p. 358-67. T2-weighted CMR was performed as described in Neil, C.J., et al., "Slowly resolving global myocardial inflammation/oedema in Tako-Tsubo cardiomyopathy: evidence from T2-weighted cardiac magnetic resonance imaging" Heart, 2012. utilizing a Philips Achieva 1.5 Tesla machine (Best, Netherlands). T2- weighted signal intensity (T2w-SI) was utilized as an index of myocardial edema/inflammation. T2w-SI was therefore the ratio of myocardial signal intensity to that of the upper pole of the spleen (myocardium : spleen), corrected for normals. [00211] 1.2 Statistics

[00212] Data were analyzed using the SPSS software (version 15, Chicago, Illinois, USA) and presented as mean and SD or median and interquartile range depending on data distributions.

[00213] In order to test the hypothesis that the severity of TTC attacks and/or the speed of recovery from TTC are correlated with markers of NO signalling, both SNP responses and ADMA concentrations were correlated with (1) markers of TTC severity: wall motion score index, T2-SI, NT-proBNP release, normetanephrine concentrations, peak CRP and troponin T levels (2) markers of recovery at 3 months: plasma NT- proBNP concentrations via linear regression or Spearman's correlation as appropriate.

[00214] Comparisons between TTC and control data were performed utilizing Bonferroni and Kruskal-Wallis multiple comparison tests as appropriate. Post-hoc analyses based categorically on SNP responses and ADMA concentrations were partitioned relative to median/mean values. A value of p < 0.05 was considered significant.

[00215] 1.3 Results

[00216] 1.3.1 Patients' characteristics. Clinical characteristics of the study population (both TTC and normal groups) are described in the Table. Fifty six patients (54 females, 2 males), diagnosed with TTC over a three-year period were studied. Potential emotional or physical stressors were identified in 46 patients (82%). Hypertension (56%) and dyslipidemia (48%) were the most common coronary risk factors in this study group. While there was no difference in Framingham 10 year coronary artery disease risk score between the 2 groups, hypertension and diabetes occurred more frequently in TTC than control subjects.

[00217] 1.3.2 NO signalling in TTC.

[00218] (a) Comparisons with controls and changes post TTC; (i) Figure 1 summarises SNP responses at admission and 3 months compared with control values. NO effect on platelet aggregation was increased in acute TTC relative to controls and this difference persisted at 3 months [median 43% (interquartile range: 11 - 57) and 30% (12-64) for TTC vs. 13% (4-34) for controls, p = 0.0001]; (ii) Plasma ADMA levels were lower in the acute phase and after 3 months post TTC compared to controls [mean 0.54 + 0.08 (SD) and 0.58 + 0.1 vs. 0.6 + 0.1 μΜ, p = 0.03]; Although ADMA concentrations increased significantly with patient age, TTC was associated with incremental lower ADMA levels at any particular age; (iii) EPC counts did not differ significantly between TTC and control subjects, nor fluctuate significantly between admission and 3 months in TTC patients (Figure 1).

[00219] (b) Correlations with acute markers of severity of TTC. There were direct correlations between platelet responsiveness to SNP at the acute phase of TTC and both peak normetanephrine and peak NT-proBNP (Figure 2) (r = 0.31, p = 0.03; r = 0.31, p = 0.02, respectively).

[00220] At no stage was there any significant correlation between either SNP responses or ADMA concentrations and either: (1) wall motion score index on echocardiography and (2) extent of edema via CMR T2 signal intensity.

[00221] (c) Correlations with extent of recovery at 3 months. At 3 months, ADMA levels were directly correlated with simultaneous NT-pro BNP concentrations (r = 0.5, p = 0.003; Figure 3).

[00222] Discussion

[00223] This study establishes that TTC is associated, not with the impairment in NO signalling expected on the basis of putative catecholamine-induced redox stress, but with a "paradoxical" finding: increased platelet responsiveness to SNP relative to population norms, together with significantly lowered ADMA levels. Furthermore, circulating EPC counts, which partially reflect NO stimulation, were not reduced relative to control values, either acutely or during recovery from TTC. The observed changes were not restricted to the acute phase of TTC, but persisted for at least 3 months thereafter. [00224] A secondary observation was that the extent of SNP response was directly correlated with some, but not all, markers of severity of individual TTC attacks: - while there was no significant correlation with extent of wall motion impairment or edema, there were significant correlations with normetanephrine and NT-proBNP concentrations. As regards ADMA concentrations, these were directly and strongly correlated with levels of NT-proBNP at 3 months (Figure 3). Thus, intact NO signalling tended to be associated with more severe episodes of TTC, while ADMA, a marker of impaired NO generation, was correlated with biochemical evidence of impaired recovery. It is therefore appropriate to consider the possible mechanisms and implications of the surprising findings.

[00225] In retrospect, no association has previously been demonstrated between TTC and endothelial dysfunction or other disorders of NO signalling. The tendency to diagnose TTC primarily in patients with normal epicardial coronary arteries might have introduced some selection bias in this regard. In the current study, Framingham risk scores were similar for TTC and control populations.

[00226] In theory, exposure to high concentrations of catecholamines might have increased NOS activation in platelets, but this would not have increased response to SNP. Furthermore, both the activity and even presence of NOS in platelets are open to question, while catecholamines may increase generation of reactive oxygen species, thus inducing "scavenging" of NO and/or inactivation of sGC, the main biochemical components underlying impaired response to NO. Similarly, catecholamines may increase ADMA concentrations. The direct correlation between normetanephrine concentrations and SNP response is therefore surprising.

[00227] However, we have previously demonstrated that both extent of NT-proBNP elevation and of increase in normetanephrine concentrations are markers of severity of TTC attacks. We have postulated that NT-proBNP release may be predominantly inflammatory in origin. Furthermore, elevation of catecholamine levels after onset of TTC may represent both cause and consequence, in uncertain proportions. Recent findings that the generation of cyclic GMP, whether from particulate guanylate cyclase by BNP or from sGC by NO, may increase catecholamine release, adds a further dimension to this complex interrelationship. Considering the 3 month data, the main conclusion to be drawn is that patients with TTC have paradoxically preserved NO signalling, which may well have antedated as well as extended beyond the acute illness. Whether this supra-normal NOS signalling in some way predisposes towards TTC is an issue raised by the current data.

[00228] As regards the correlation between 3-month ADMA concentrations and incomplete recovery from TTC, this is partially consistent with existing data, which indicates that there is early activation of the Reperfusion Injury Salvage Kinase (RISK) pathway, which limits extent of cellular necrosis under oxidant stress. NO is a major physiological activator of this pathway, and integrity of eNOS would therefore tend to limit long-term impairment post TTC.

[00229] Previous studies have utilized the cold pressor test and mental stress to precipitate "paradoxical" vasoconstrictor responses in patients with previous TTC, a finding which suggests the presence of endothelial dysfunction in such patients. Given that acute assessment of endothelial function is problematic in TTC patients due to the frequent presence of systemic hypotension, it is difficult to resolve the disparate implications of our current findings with those of previous studies.

[00230] The major limitation of this study is that the pathogenic significance of the current findings is uncertain (and cannot easily be determined in humans). However, the differences observed between TTC patients and controls, especially as regards SNP responses, are both large and highly statistically significant, this difference was present despite the fact that the TTC patients were somewhat older than the controls, and a higher proportion of TTC patients had diabetes and/or hypertension (Table 1), all conditions associated with elevation of ADMA concentrations and impairment of NO signalling.

[00231] One possible consequence of supranormal NO signalling in TTC is the precipitation of early hypotension, a common finding in TTC irrespective of the presence or absence of pulmonary congestion. While this may be primarily due to BNP release, NO may also contribute. [00232] In summary, NO signalling is paradoxically greater than normal in TTC both acutely and for at least 3 months thereafter, and is correlated with some markers of TTC severity. The possible role of NO as a modulator of the pathogenesis of TTC is worthy of further exploration.

EXAMPLE 2 - Further observations

[00233] NO, by inducing the formation of cGMP, may potentially magnify catecholamine release. Hence, if patients with stress-induced cardiomyopathy have exaggerated NO formation (via low ADMA) and/or increased cGMP formation from NO (as suggested by increased SNP responsiveness), this could potentially magnify the consequences of an initial catecholamine release stimulus.

[00234] Catecholamines may induce secondary activation of NO synthesis (via eNOS), via both beta 2 and beta 3 adrenoceptor stimulation in the heart. Hence if patients with stress-induced cardiomyopathy have more active eNOS, the release of NO in response to catecholamine effect in the heart would be increased.

[00235] Finally, peroxynitrite formation occurs via an interaction between superoxide anion (released via catecholamine effect in the heart) and NO. Hence increased NO release in the presence of catecholamine stimulus would be expected to generate more peroxynitrite.

EXAMPLE 3 - Association of Tako-Tsubo (stress) Cardiomyopathy with evidence of increased nitrosative stress

[00236] 1. Methods

[00237] 1.1 Rat model

[00238] In view of the predominant occurrence of TTC in ageing women, we sought to develop an animal model in ageing female rats, using single intraperitoneal injections of isoproterenol. The study was approved by the institutional Ethics Committee, and was performed in female Sprague-Dawley rats. Preliminary experiments were conducted to evaluate the impact of age and isoproterenol dose on tolerability:- it was found that in rats aged > 12 months mortality rates following intraperitoneal injection of 5mg/kg isoproterenol were unacceptably high. Therefore all experiments were performed in animals aged 4 - 5 months, weighing 230 to 280 grams.

[00239] Effects of isoprenaline on left ventricular regional and global function were evaluated by echocardiography, performed during anaesthesia with 2% isofluorane before and 24hours after isoproterenol injection. Animals were secured on a warming pad and limb-lead electrocardiograms recorded throughout echocardiography. A GE 10s 2298589 sector array probe with a frequency range of 4.0 - 10.5 MHz was utilized for acquisition of echocardiographic views. Imaging was performed in the shallow left lateral decubitus position. For analysis of images, parasternal long axis views as well as short axis views of both apex and base of the left ventricle were utilized.

[00240] Measurement of fractional area shortening (FAS) was performed in the parasternal long axis view at three pre-defined levels in the left ventricle: (I) basal measurements were performed 3 mm below the mitral annulus (ii) mid-ventricular FAS was recorded at the level of the papillary muscle and (iii) apical measurements 3 mm from the left ventricular apex. Radial strain was measured from short axis apical and basal views utilizing speckle-tracking software, with means of triplicate estimates utilized. Experienced investigators blinded to treatment status and phase of treatment performed all echocardiographic analyses. Control rats treated with saline vehicle alone were also included in this blinded process.

[00241] All rats were euthanized 24 hours post isoproterenol injection. Heart were excised and divided into apical and basal left ventricular sections, which were then fixed in 9% formaldehyde and paraffin embedded.

[00242] 1. 2. Determination of plasma 3-nitrotyrosine (3-NT) concentrations

[00243] In order to determine whether there might be systemic circulatory evidence of nitrosative stress in patients with TTC, blood samples were taken from patients by venesection within 48 hours of onset of symptoms of TTC (n = 23) and from 16 age- matched healthy women. The Institutional Ethics of Human Research Committee approved the protocol and informed consent was obtained in all cases. Venous blood was collected into tubes containing potassium ethylene diamine tetra acetic acid (EDTA) and centrifuged at 1680 relative centrifugal force (RCF) for 10 minutes at 2°C and frozen plasma was stored at -80°C until analysis. Determination of plasma 3-NT was performed by mass spectrometry (Finnigan MAT, San Jose, CA) coupled with gas chromatography .

[00244] 1.3. Evaluation of nitrosative stress: post mortem studies in TTC patients

[00245] Over a period of 3 years, there were 6 in-hospital deaths of patients after diagnosis of an acute episode of TTC. In 4 of these cases, permission was obtained from patients' families for performance of limited autopsy to evaluate changes within the myocardium. Control myocardial samples were also obtained from the left ventricles of 4 female patients who had died of non-cardiac causes.

[00246] In all cases, left ventricular biopsies were taken at the levels of apex, mid- ventricle and base, and were embedded in paraffin as for the rat ventricular biopsies for subsequent immunohistological study.

[00247] 1.4. Immunohistochemical studies

[00248] Immunohistochemical studies for evaluation of regional left ventricular content of 3-NT and of TxNIP were performed in a similar manner for both rat and human left ventricular biopsies.

[00249] Paraffin sections of 4 micrometers were cut and positioned on Superfrost Plus slides for drying. They were then heated to 45°C for 15 minutes to removed air bubbles, cooled to room temperature, dewaxed in xylene and rehydrated. Sections were then boiled in 0.01 mol/L citrate butter (pH 6.0) for 20 minutes in a microwave oven and allowed to cool for 30 minutes in a water bath at 23°C, then washed twice in phosphate- buttered saline (PBS). Sections were then incubated in a humidity chamber with blocking buffer (2% serum, 5% bovine serum albumin) for 40 minutes, and then patted dry.

[00250] Primary antibody was applied at a dilution of 1:300 for 3-NT (Abeam) or 1:500 for TxNIP (MBL International Co., Woburn, MA) and incubated overnight, followed by repeated PBS washes. Sections were incubated for 30 minutes with 3% hydrogen peroxide to block endogenous peroxidases and then washed again with PBS. Secondary antibody (Santa Cruz Biotechnology) was applied at a dilution of 1:200 and incubated for 60 minutes, followed by repeated PBS washing.

[00251] Nuclei were counter- stained with Gill's haematoxylin and slides were mounted in DePex. Sections were visualised utilizing the NOVA Red kit (Vector Laboratories, Burlingame, CA).

[00252] Quantification of both 3-NT and TxNIP immunostaining was performed utilizing images of sections viewed at 4x magnification, utilizing Image J software. In all cases, evaluation was performed by investigators blinded as to treatment status (in the case of rat myocardium) or diagnosis (in the case of human myocardium).

[00253] In the case of 3-NT estimates, because of regional heterogeneity of immunostaining, content estimates represented the mean of 8 regional data points per slide examined, while for TxNIP content determinations the mean of 4 data points was utilized.

[00254] 1. 5. Statistical methodology

[00255] For studies performed in rats, comparisons of left ventricular structure and function before and after isoproterenol were made utilizing Student's paired t-test. Comparisons of myocardial 3-NT and TxNIP content between treated and control rats and between TTC and control patients were made utilizing non-paired t-tests, while evaluation of apex versus base heterogeneity of 3-NT and TxNIP content was performed by 2-way ANOVA. All data are expressed as mean +SEM unless otherwise stated.

[00256] 2. Results

[00257] 2.1. Rat experiments

[00258] Data are compared for the 7 rats who survived 24 hours post isoproterenol (3 died, all within the first 2 hours) and 4 control rats. [00259] 2.1a. Effects on LV structure and function

[00260] Echocardiographic data for isoprenaline treated rats are summarized in Table 1. In the majority of animals, predominantly apical hypokinesis was evident 24 hours post isoproterenol. There was no significant change in heart rate or in apical IVSD, while the reduction in apical FAS was inconsistent. There was a mean reduction of approximately 35% (p = 0.010) in apical strain (exemplified in Figure 4).

Table 1 : Echocardiographic parameters of isoprenaline-treated rats at baseline and at 24 hours post isoprenaline injection.

va ue

[00261] 2.1b. Immunostaining

[00262] Inter-individual coefficients of variability for estimates of 3 -NT and TxNIP were 13% and 9% respectively, in isoproterenol-treated hearts. Mean data for treated and control rats are summarized in Figure 5, while individual examples are provided in Figure 6.

[00263] 3-NT immunostaining was increased approximately 10-fold (P<0.01) in apical myocardium of isoproterenol treated rats (Figure 5A). While 3-NT content tended to be greater in apical than basal myocardium, this difference did not reach statistical significance. Tissue staining for 3-NT was heterogeneous, with predominantly perivascular localisation (Figure 6A).

[00264] TxNIP immunostaining was increased approximately 30-fold (P<0.01) in myocardium of isoproterenol-treated rats (Figure 5B), with no evidence of any differential expression between apex and base, and with homogeneous staining throughout the myocardium (Figure 6B).

[00265] 2.2 Human plasma 3-NT concentrations

[00266] TTC patients (n = 23) and controls (n = 16) were well matched for age. Of the TTC patients, 33% presented with S-T elevation, and mean left ventricular ejection fraction at presentation was 49+16%. Consistent with previous observations, plasma NT-proBNP concentration was markedly elevated beyond population norms in the TTC patients (mean 8650+2800).

[00267] Data regarding plasma 3-NT concentration determinations are shown in Figure 7. There was no significant difference between patients (mean 2.26+0.22 nmol/L) and control subjects (mean 2.20+0.25 nmol/L).

[00268] 2.3. Human autopsy samples

[00269] Data for the 4 TTC patients whose hearts were available for study are summarized in Table 2. In all cases, autopsy had been performed at least 24 hours post patients' death. Control autopsy data were obtained from 4 women of similar ages (mean 59.5+0.5 years).

Table 2: Basic characteristics and biochemical parameters of 4 patients who underwent autopsy after dying during an attack of TTC

[00270] Left ventricular apical myocardium from TTC patients exhibited approximately 6-fold elevation of 3-NT content relative to controls (mean 2.4 1+0.9% vs 0.4+0.3%; Figure 5).

[00271] Furthermore apical TxNIP staining in hearts of TTC patients was greater (1.1+0.1%) than at in control hearts (0.3+0.03%; Figure 8).

[00272] 3. Discussion

[00273] In this investigation, performed both in a rat model of TTC and in humans in the acute stages of the disease, we sought to test whether TTC is associated with the development of nitrosative stress, signifying increased generation of ONOO- within the myocardium. We utilized the generation of 3-NT as a marker of ONOO- effect. The results of the investigation indicate that 3-NT formation is increased, particularly in periapical left ventricular myocardium, both in the rat model of TTC and in the hearts of patients dying early in the course of the disease, although plasma 3-NT concentrations did not differ significantly between TTC patients and age-matched female controls.

[00274] The observations which led to our initiating the current investigation included the findings that TTC is associated with intense and prolonged inflammatory activation, with predominantly peri-apical myocardial edema persisting for at least 12 weeks after onset of symptoms, together with extensive release of BNP, NT-proBNP and CRP as systemic manifestations of inflammatory activation. Furthermore, patients with TTC exhibit evidence of increased platelet responsiveness to NO and also have lower ADMA concentrations than age-matched female controls, raising the possibility of enhanced NO signalling.

[00275] We postulated that TTC might be associated with catecholamine-induced stimulation of myocardial β2 (and/or β3) adrenoceptors, as demonstrated in recent studies in rats and mice, and that the coupling of these receptors to NO synthase might lead to increased generation of ONOO-, particularly within apical myocardium. Furthermore, given that TxNIP activation is partially dependent on ONOO- mediated activation of the peroxynitrite/poly (ADP-Ribose) Polymerase [PARP] pathway, we chose to evaluate myocardial content not only of 3-NT, a marker of nitrosative stress, but also of TxNIP, a pivotal activator of the inflammasome.

[00276] In our experiments, rats were particularly sensitive to 5 mg/kg isoproterenol, with mortality rates of >50% in rats aged >1 year, while mortality rates in 5 month old female rats were of the order of 30%. Consistent with the results of previous investigations, isoproterenol induced substantial impairment of left ventricular apical strain.

[00277] The associated increase in myocardial 3-NT content was predominantly, but not entirely, perivascular in distribution. This finding raises the possibility that the NO and/or 0₂ ^~ which combine to generate ONOO- are predominantly formed in vascular endothelium or smooth muscle. Interestingly, it has previously been postulated that material(s) released from endocardial endothelium may account for negative inotropic changes in TTC. Peroxynitrate also may stimulate soluble guanylate cyclase, releasing cyclic GMP, which may contribute to further release of catecholamines.

[00278] Increases in myocardial TxNIP content were substantial in the rat model. TxNIP is an a-arrestin which exerts a pivotal role as an inflammatory activator. While it has previously been shown that TxNIP expression may be increased by redox stress, hyperglycaemia or non-laminar vascular flow, it has recently emerged that ONOO- (via PARP) is also a modulator of TxNIP activation. The current data therefore demonstrate that TxNIP expression is increased in this model.

[00279] The results of human studies revealed that there was no increase in plasma 3- NT concentrations in patients with TTC, suggesting that there are no substantial systemic manifestations of nitrosative stress. Protein nitration appears to be a relatively transient effect of ONOO- release in many models and the current data do not exclude transient early systemic 3-NT generation. This is potentially relevant because ONOO- generation and resultant PARP activation tends to be present in many inflammatory disease models. On the other hand, the autopsy data revealed analogous changes to those seen in rats, with both 3-NT and TxNIP accumulation in apical myocardium. These data therefore confirm that nitrosative stress occurs in humans with TTC, as well as in the rat model.

[00280] To date, no effective treatment options for TTC have emerged. The current findings point to the potential for interventions to reduce myocardial inflammation in TTC, with possible consequences of limiting hemodynamic disturbances in the early stage of the disease and accelerating recovery.

EXAMPLE 5 -Treatment of rats with a PARP inhibitor in a TTC model improves LV mechanics

[00281] Left ventricular (LV) global longitudinal strain (GLS) is a measure of the active shortening of the LV in the longitudinal direction and represents a sensitive measure of LV mechanics. [00282] We tested the ability of a 3-aminobenazamide (3AB), a known inhibitor of PARP-1, to improve left ventricular mechanics in the isoprenaline rat model of TTC in a blinded analysis.

[00283] Methods: Experiments were performed in female Sprague-Dawley rats (12 rats for isoprenaline treatment alone and 12 rats for both isoprenaline and 3AB treatment), aged 4 - 5 months, weighing 230 to 280 grams. A single dose of 50mg/kg a PARP inhibitor (3AB) was intraperitoneally administered to 12 rats 30 minutes prior to isoproterenol (5mg/kg) injection.

[00284] Left ventricular regional and global functions were evaluated by echocardiography, performed during anaesthesia with 2% isofluorane before and 24hours after isoproterenol injection.

[00285] The results are shown in Figure 9.

[00286] As shown in Figure 9, consistent with earlier studies described herein, treatment of rats with isoprenaline alone after 24 hours resulted in a reduction in LV global longitudinal strain (GLS). Treatment with 3-aminobenzamide increased LV global longitudinal strain (GLS) in rats treated with isoprenaline, demonstrating the ability of inhibition of PARP to improve LV mechanics in the rat model of TTC.

EXAMPLE 6 -Treatment of a stress-induced cardiomyopathy in human subjects

[00287] Patients having Takotsubo cardiomyopathy may be identified on the basis of the following criteria: (1) chest pain and/or dyspnoea > 30min, (2) ST/T wave changes and/or biomarker elevation, (3) periapical or midventricular akinesis/hypokinesis, and (4) no evidence for a diagnosis of myocardial infarction on coronary angiography or cardiovascular magnetic resonance (CMR). Additional criteria as described herein may be used to assist with the identification of patients with TTC.

[00288] Patients with identified Takotsubo cardiomyopathy may be treated with an agent as described herein, including a therapeutic agent from one of the following classes:

(i) an inhibitor of peroxynitrite synthesis (eg a nitric oxide synthase inhibitor); (ii) a peroxynitrite inhibitor, including a peroxynitrite scavenger and/or an agent that promoted peroxynitrite decomposition;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage, such as a PARP-1 and/or PARP-2 inhibitor;

(v) a TxNIP inhibitor, such as an inhibitor of TxNIP expression;

(vi) a beta 2 adrenoceptor antagonist (eg Butaxamine or 3(-isopropylamino)-l- [(7-methyl-4-indanyl)oxy]butan-2-ol); and

(vi) a beta 3 adrenoceptor antagonist (eg SR 59230A ((2S)-l-(2-ethylphenoxy)- 3-{ [(1S)-1,2,3, 4-tetrahydronaphthalen-l-yl] amino }propan-2-ol), L-748,337 (N- [ [3 - [(2S )-2-Hydroxy- 3 - [ [2- [4- [(phenylsulfonyl) amino]phenyl] ethyl] amino] propoxy] phenyl] methyl] -acetamide), L-748,328 (((S)-N-[4-[2-[[3-[3- (ami-nosulphonyl)phenoxy]-2-hydroxypropyl]amino]ethyl]benze- nesulfonamide) or CL 31624 (5-[(2R)-2-[[(2R)-2-(3-Chlorophenyl)-2- hydroxyethyl] amino] propyl]-l,3-benzodioxole-2,2-dicarboxylic acid disodium salt).

[00289] The agents may be administered to patients, for example being administered twice daily, either orally or intravenously, to the subjects. Treatment may be undertaken for a period of up to 3 months. The dosage of the agent may be selected by a skilled physician. Suitable formulations may be prepared for administration, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985. In some circumstances, agents may be administered prophylatically to patients considered to be susceptible to TTC, for example on the basis of one or more parameters and/or clinical characteristics as described herein.

[00290] The effectiveness of treatment may be assessed on the basis of a combination of the following criteria: (1) chest pain and/or dyspnoea > 30min, (2) ST/T wave changes and/or biomarker elevation, and (3) periapical or midventricular akinesis/hypokinesis. As used herein, the singular forms "a," "an," and "the" may refer to plural articles unless specifically stated otherwise.

[00291] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[00292] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[00293] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[00294] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[00295] Although the present disclosure has been described with reference to particular examples, it will be appreciated by those skilled in the art that the disclosure may be embodied in many other forms.

[00296] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.

Claims

1. A method of preventing and/or treating a stress-induced cardiomyopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage; and

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist;

2. The method according to claim 1, wherein the stress-induced cardiomyopathy comprises Takotsubo cardiomyopathy.

3. The method according to claims 1 or 2, wherein the peroxynitrite inhibitor comprises a peroxynitrite scavenger and/or an agent that promotes peroxynitrite decomposition.

4. The method according to any one of claims 1 to 3, wherein the inhibitor of peroxynitrite synthesis comprises an inhibitor of nitric oxide synthesis.

5. The method according to claim 4, wherein the inhibitor of nitric oxide synthesis comprises an inhibitor of a nitric oxide synthase.

6. The method according to any one of claims 1 to 5, wherein the inhibitor of peroxynitrite synthesis comprises an inhibitor of superoxide synthesis.

7. The method according to any one of claims 1 to 6, wherein the inhibitor of peroxynitrite associated signalling comprises an inhibitor of a signalling pathway comprising a signalling pathway involving one or more of the following: nuclear factor kappa B, protein kinase C, phosphatidylinositol 3-kinase; extracellular-regulated kinase, Janus kinase, epidermal growth factor receptor, platelet-derived growth factor receptor, truncated tyrosine protein kinase, and p38.

8. The method according to any one of claims 1 to 7, wherein the inhibitor of peroxynitrite-induced DNA damage comprises a PARP inhibitor.

9. The method according to any one of claims 1 to 8, wherein the TxNIP inhibitor comprises an inhibitor of TxNIP expression.

10. The method according to any one of claims 1 to 9, wherein the beta 2 adrenoceptor antagonist comprises one or more of Butaxamine and 3(-isopropylamino)- l-[(7-methyl-4-indanyl)oxy]butan-2-ol.

11. The method according to any one of claims 1 to 10, wherein the beta 3 adrenoceptor antagonist comprises one or more of SR 59230A ((2S)-l-(2- ethylphenoxy)-3-{ [(1S)-1,2,3, 4-tetrahydronaphthalen-l-yl] amino }propan-2-ol), L- 748,337 (N-[[3-[(2S)-2-Hydroxy-3-[[2-[4-[(phenylsulfonyl) amino]phenyl] ethyl] amino] propoxy] phenyl] methyl] -acetamide), L-748,328 (((S)-N-[4-[2-[[3-[3-(ami- nosulphonyl)phenoxy]-2-hydroxypropyl]amino]ethyl]benze-nesulfonamide), and CL 31624 (5-[(2R)-2-[[(2R)-2-(3-Chlorophenyl)-2-hydroxyethyl] amino] propyl]-l,3- benzodioxole-2,2-dicarboxylic acid disodium salt).

12. The method according to any one of claims 1 to 11, wherein the agent is administered to the subject for a period of time of 3 months or less following occurrence of the stress-induced cardiomyopathy.

13. The method according to any one of claims 1 to 12, wherein the subject is a post-menopausal female subject.

14. The method according to any one of claims 1 to 13, wherein the method further comprises:

determining one or more of the following parameters in the subject: peroxynitrite concentration, the rate of peroxynitrite synthesis, the extent of peroxynitrite associated signalling, PARP level, plasma N-terminal pro-brain natriuretic concentration, myocardial 3-nitrotyrosine levels, myocardial TxNIP level, and beta 2 and/or beta 3 adrenoceptor activation or signalling; and administering the one or more agents on the basis of the one or more parameters so determined.

15. Use of one or more of the following agents in the preparation of a medicament for prevention and/or treatment of a stress-induced cardiomyopathy in a subject:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

16. A method of treating a subject having symptoms associated with an acute coronary syndrome and acute stress, the method comprising administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist;

thereby treating the subject.

17. Use of one or more of the following agents in the preparation of a medicament for treating a subject having symptoms associated with an acute coronary syndrome and acute stress:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling; (iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor;

(vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

18. A method of treating a subject susceptible to, or suffering from, a stress- induced cardiomyopathy, the method comprising:

treating the subject on the basis of the one or more parameters so determined.

19. The method according to claim 18, wherein the method comprises processing a sample obtained from the subject to allow the determination of the one or more parameters.

20. The method according to claims 18 or 19, wherein a rate of peroxynitrite synthesis and/or an extent of peroxynitrite associated signalling which is maintained or increased is indicative that the subject is to be treated.

21. The method according to any one of claims 18 to 20, wherein one or more of increased PARP level, increased myocardial 3-nitrotyrosine expression, increased myocardial TxNIP expression is indicative that the subject is to be treated.

22. The method according to any one of claims 18 to 21, wherein the treating comprises administering to the subject a therapeutically effective amount of one or more of the following agents:

(i) a peroxynitrite inhibitor;

(ii) an inhibitor of peroxynitrite synthesis;

(iii) an inhibitor of peroxynitrite associated signalling;

(iv) an inhibitor of peroxynitrite-induced DNA damage;

(v) a TxNIP inhibitor; (vi) a beta 2 adrenoceptor antagonist; and

(vii) a beta 3 adrenoceptor antagonist.

23. A method of identifying a subject susceptible to, or suffering from, a stress- induced cardiomyopathy, the method comprising:

24. The method according to claim 23, wherein the method comprises determining the one or more parameters from a processed sample obtained from the subject.

25. The method according to claims 23 or 24, wherein one or more of a rate of peroxynitrite synthesis which is maintained or increased, an extent of peroxynitrite associated signalling which is maintained or increased, increased PARP level, increased myocardial 3-nitrotyrosine expression, and increased myocardial TxNIP expression is indicative that the subject is susceptible to, or suffering from, a stress-induced cardiomyopathy.

26. A kit for performing the method of any one of claims 23 to 25.

27. Use of one or more of the following reagents in a kit for identifying a subject susceptible to, or suffering from, a stress-induced cardiomyopathy:

a reagent for detecting peroxynitrite;

a reagent for detecting peroxynitrite synthesis;

a reagent for detecting peroxynitrite associated signalling;

a reagent for detecting a PARP;

a reagent for detecting myocardial 3-nitrotyrosine;

a reagent for detecting myocardial TxNIP; and a reagent for detecting beta 2 adrenoceptor activation or signalling; and a reagent for detecting beta 3 adrenoceptor activation or signalling.

28. A method of identifying a therapeutic agent for preventing and/or treating a stress-induced cardiomyopathy, the method comprising:

identifying a candidate agent that inhibits peroxynitrite synthesis; and/or identifying a candidate agent that scavenges peroxynitrite; and/or

identifying a candidate agent that promotes peroxynitrite decomposition; and/or

identifying a candidate agent that inhibits TxNIP expression and/or activity; and

identifying a candidate agent that antagonises a beta 2 adrenoceptor; and/or identifying a candidate agent that antagonises a beta 3 adrenoceptor, and identifying one or more of the aforementioned candidate agents as a therapeutic agent for preventing and/or treating a stress-induced cardiomyopathy.