WO2017185142A1

WO2017185142A1 - Method for preventing and/or treating atrial fibrillation

Info

Publication number: WO2017185142A1
Application number: PCT/AU2017/050393
Authority: WO
Inventors: Prashanthan SANDERS; Dennis Lau; Rajiv Mahajan; Darragh TWOMEY
Original assignee: Adelaide Research & Innovation Pty Ltd
Priority date: 2016-04-29
Filing date: 2017-04-28
Publication date: 2017-11-02

Abstract

The present disclosure relates to methods and products for preventing and/or treating atrial fibrillation. Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial fibrillation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

Description

METHOD FOR PREVENTING AND/OR TREATING ATRIAL FIBRILLATION PRIORITY CLAEVI

[001] This application claims priority to Australian provisional patent application number 2016901586 filed on 29 April 2016, the content of which is hereby incorporated by reference.

FIELD

[002] The present disclosure relates to methods and products for preventing and/or treating atrial fibrillation.

BACKGROUND

[003] Atrial fibrillation is the most prevalent arrhythmia, the incidence of which increases with age and tends to occur more in males than females. Approximately 4% of people over the age of 60 have experienced an episode of atrial fibrillation and this disorder accounts for one-third of hospital admissions for cardiac rhythm disturbances. For example, over 2.2 million people are believed to have AF in the Unites States alone.

[004] Although atrial fibrillation is often asymptomatic, it may cause palpitations or chest pain. Prolonged atrial fibrillation often results in the development of congestive heart failure and/or stroke. Heart failure develops as the heart attempts to compensate for the reduced cardiac efficiency, while stroke may occur when thrombi form in the atria, pass into the blood stream and lodge in the brain. Pulmonary emboli may also develop in this manner.

[005] Clinically atrial fibrillation is diagnosed by irregular rhythm and an absence of P waves on an ECG. In addition, the ECG of a patient with atrial fibrillation will usually show a narrow QRS complex, although it may be wide if abnormal conduction or partial or full interruption of electrical conduction in the bundle blocks is present.

[006] Current methods for treating atrial fibrillation include electric and/or chemical cardioversion and laser ablation. Anticoagulants, such as warfarin, dabigatran, and heparin, are also typically prescribed in order to avoid stroke.

[007] Chemotherapeutic treatment of atrial fibrillation includes heart rate control drugs, cardiac glycosides, beta-blockers, and calcium channel blockers which seek to reduce the heart rate to one that is closer to normal to reduce symptoms, and rhythm control drugs which seek to restore and maintain the regular heart rhythm. However, many of the common agents used to treat atrial fibrillation are relatively toxic and/or have a range of undesirable side effects. In addition, clinical studies have indicated that management of atrial fibrillation with rhythm control offers no survival advantage over rate control, and that rate control potentially offers advantages, such as a lower risk of adverse events.

[008] Accordingly, there is a need to provide improved or alternative methods and/or products for preventing and/or treating atrial fibrillation, and/or to provide one or more advantages.

SUMMARY

[009] The present disclosure relates to the prevention and/or treatment of atrial fibrillation.

[0010] Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial fibrillation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0011] Certain embodiments of the present disclosure provide an endothelin receptor antagonist for use in the prevention and/or treatment of atrial fibrillation.

[0012] Certain embodiments of the present disclosure provide use of an endothelin receptor antagonist in the preparation of a medicament for preventing and/or treating atrial fibrillation. [0013] Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial remodelling due to increased weight gain, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0014] Certain embodiments of the present disclosure provide a method for preventing and/or treating reduced and/or slowed atrial conduction in subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0015] Certain embodiments of the present disclosure provide a method for preventing and/or treating reduced atrial conduction velocity in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0016] Certain embodiments of the present disclosure provide a method for preventing and/or treating increased atrial conduction heterogeneity in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0017] Certain embodiments of the present disclosure provide a method for preventing and/or treating increased atrial fibrosis in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0018] Certain embodiments of the present disclosure provide a method for preventing and/or treating increased atrial inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist. [0019] Certain embodiments of the present disclosure provide a method of treating a subject, the method comprising:

identifying a subject suffering from or susceptible to atrial fibrillation; and administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0020] Certain embodiments of the present disclosure provide a method for reducing the likelihood of stroke in a subject suffering from or susceptible to atrial fibrillation, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0021] Certain embodiments of the present disclosure a method for reducing the dose of an agent for preventing and/or treating atrial fibrillation administered to a subject, the method comprising administering to the subject an endothelin receptor antagonist and thereby reduce the dose of the agent for preventing and/or treating atrial fibrillation.

[0022] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from or susceptible to atrial fibrillation and suitable for treatment with an endothelin receptor antagonist, the method comprising identifying a subject with one or more of the following characteristics: an increased body mass index; a body mass index of 25 kgm-2 or greater; a body mass index of 25 to 29.9 kgm-2; a body mass index of 30 kgm^"2 or greater; overweight; obesity; an increased level of intra-myocardial lipid; an increased level of inter-myocardial lipid; an increased pericardial fat/lipid volume; an increased level of intra-atrial myocardial lipid; an increased level of interatrial myocardial lipid; reduced and/or slowed atrial conduction; reduced atrial conduction velocity; and increased atrial conduction heterogeneity.

[0023] Certain embodiments of the present disclosure a pharmaceutical composition when used to prevent and/or treat atrial fibrillation, the pharmaceutical composition comprising a therapeutically effective amount of an endothelin receptor antagonist.

[0024] Certain embodiments of the present disclosure provide a pharmaceutical composition when used to prevent and/or treat atrial fibrillation, the pharmaceutical composition comprising a therapeutically effective amount of bosentan and/or macitentan, and/or a pharmaceutically acceptable salt or hydrate thereof.

[0025] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of an endothelin receptor antagonist, and an inhibitor of the renin-angiotensin-aldosterone axis and/or a TGF-β antagonist.

[0026] Certain embodiments of the present disclosure provide a combination product comprising:

one or more endothelin receptor antagonists; and

instructions for administering the antagonist to a subject to prevent and/or treat atrial fibrillation.

[0027] Certain embodiments of the present disclosure provide a method for identifying an agent for preventing and/or treating atrial fibrillation, the method comprising identifying an endothelin receptor antagonist as an agent for preventing and/or treating atrial fibrillation.

[0028] Certain embodiments of the present disclosure provide a method for identifying an agent for preventing and/or treating atrial fibrillation, the method comprising:

determining the ability of a test agent to antagonise activity of an endothelin receptor activity; and

identifying the test agent as an agent for preventing and/or treating atrial fibrillation.

[0029] Certain embodiments of the present disclosure provide method of preventing and/or treating atrial fibrillation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist and/or tranilast.

[0030] Other embodiments are disclosed herein. BRIEF DESCRIPTION OF THE FIGURES

[0031] 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.

[0032] Figure 1 shows a diagrammatic representation of the study outline. EPS: electrophysiology study. CMR: cardiac magnetic resonance imaging.

[0033] Figure 2 shows representative CMR cines of atrial volumes (top panel) and pericardial fat volume (bottom panel) from baseline, overweight and obese cohorts (across). Maximum atrial volumes shown at ventricular end-systole.

[0034] Figure 3 shows activation isochronal maps of LAA pacing at 400 msec. Demonstrated are representative examples of SI (top row) and S2 (bottom row) for an animal at baseline (left column), overweight (middle column) and obese (right column) cohorts.

[0035] Figure 4 shows the effect of progressive obesity on regional slowing in conduction velocity at the 4 pre-defined pacing sites. This relationship persisted following adjustment for hemodynamic variables. RAA: right atrial appendage, RAFW: right atrial free wall, LAA: left atrial appendage, LAFW: left atrial free wall. CLs of 500ms and 200ms are presented.

[0036] Figure 5 shows pacing train (SI) and premature extra stimulus (S2) impact on CV decrement with increasing weight.

[0037] Figure 6 shows changes in biatrial conduction heterogeneity index with increasing adiposity. CLs of 500ms and 200ms are presented.

[0038] Figure 7 shows histology from left atrial tissue. From top panel; Inflammatory infiltrates on H&E, myocardial lipidosis on oil-red-0 and myocardial fibrosis on Picrosirius red staining. [0039] Figure 8 shows Endothelin Receptor A (ETA) receptor (63kDa) OD, Endothelin Receptor B (ETB) receptor (30kDa) OD and a- Tubulin (55kDa). Two bands are shown per group.

[0040] Figure 9 shows the study design for an interventional study of rhe role of endothelin-receptor blockade in the prevention of the substrate for atrial fibrillation.

[0041] Figure 10 shows the conduction velocity (CV) at the study baseline, midpoint and endpoint (left) and by region at endpoint study (right)

[0042] Figure 11 shows the epicardial conduction velocity (CV) at each corner of the LA plaque (top) and conduction heterogeneity (bottom), both with representative examples.

[0043] Figure 12 shows the effective refractory periods (ERP). The upper panel shows endocardial results at study baseline, midpoint and end point, the lower panel shows epicardial results at study endpoint. Left panels are with a drivetrain of 400ms, right at 200ms.

[0044] Figure 13 shows proportion of fractionated signal at baseline, midpoint and endpoint (top). Representative examples of LA voltage and fractionation maps in both groups at baseline and endpoint (bottom). Markers represent points with fractionated signal.

[0045] Figure 14 shows AF inducibility at end point study. Total AF duration (log transformed for statistical analysis) is shown on the left, number of episodes >2s is shown on the right.

[0046] Figure 15 shows Masson's tri chrome staining of LAA tissue at 40x magnification (upper panels). Immunohistochemistry of gap junction protein connexin43 at 40x magnification (lower panels).

[0047] Figure 16 shows LAA immunohistochemistry with representative examples on the left and comparison of treatment and control groups on the left. TGF-β = Transforming growth factor; Angll = Angiotensin II; CTGF = Connective tissue growth factor; PDGF = Platelet derived growth factor.

[0048] Figure 17 shows AF inducibility at end point study. The proportion of episodes >2s is shown on the left, the total AF duration (log transformed for statistical analysis) on the left.

[0049] Figure 18 shows study design for treatment studies with tranilast to prevent atrial remodelling and AF in an obese ovine model.

[0050] Figure 19 shows conduction velocity (CV) at the study baseline, 32 weeks and 64 weeks (left) and by region at endpoint study (right).

[0051] Figure 20 shows epicardial conduction velocity (CV) at each site (left) and conduction heterogeneity (right).

[0052] Figure 21 shows effective refractory periods (ERP). Endocardial results at each timepoint (upper) and epicardial results at study endpoint (lower). Left panels are at a 400ms cycle length, right at 200ms.

[0053] Figure 22 shows proportion of fractionated signal at baseline, midpoint and endpoint.

[0054] Figure 23 shows AF inducibility at end point study. DETAILED DESCRIPTION

[0055] The present disclosure relates to methods and products for the prevention and/or treatment of atrial fibrillation.

[0056] Certain embodiments of the present disclosure are directed to methods for preventing and/or treating atrial fibrillation, pharmaceutical compositions for preventing and/or treating atrial fibrillation, kits and products for preventing and/or treating atrial fibrillation, and methods for identifying agents for preventing and/or treating atrial fibrillation.

[0057] Certain disclosed embodiments provide methods, compositions, products and kits for preventing and/or treating atrial fibrillation that have one or more combinations of advantages. For example, some of the advantages of some of the embodiments disclosed herein include one or more of the following: providing another or alternative form of treatment for subjects suffering from, or susceptible to, atrial fibrillation; providing improved efficacy of treatment; providing a treatment which has improved efficacy for specific types of subjects; providing a treatment that allows reduced dosages of existing drugs to treat atrial fibrillation; providing a treatment that reduces the likelihood of stroke occurring; providing a treatment for atrial fibrillation in overweight or obese subjects; to address one or more problems in the art; to provide one or more advantages in the art; and/or to provide a useful commercial choice. Other advantages of certain embodiments of the present disclosure are also disclosed herein.

[0058] The present disclosure is based, at least in part, on the determination that in an animal model, intervention with an endothelin receptor antagonist prevents atrial electrical remodelling due to obesity.

[0059] Certain embodiments provide a method for preventing and/or treating atrial fibrillation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[0060] Atrial fibrillation occurs when the heart's two upper chambers (the right and left atria) quiver instead of beating and contracting rhythmically. Electrocardiographically, atrial fibrillation is characterized by a highly disorganized atrial electrical activity that often results in fast and irregular beating of the heart's two lower chambers (the right and left ventricles). Methods for assessing atrial fibrillation are known in the art.

[0061] In certain embodiments, the atrial fibrillation is atrial fibrillation associated with obesity. In certain embodiments, the atrial fibrillation is atrial fibrillation associated with weight gain. [0062] In certain embodiments, the atrial fibrillation is acute atrial fibrillation, spontaneous fibrillation or chronic atrial fibrillation.

[0063] In certain embodiments, the atrial fibrillation comprises spontaneous atrial fibrillation, paroxysmal atrial fibrillation, recurrent atrial fibrillation, persistent atrial fibrillation, or permanent atrial fibrillation.

[0064] 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.

[0065] In certain embodiments, the subject is human subject.

[0066] 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. Other types of animals are contemplated. Veterinary applications of the present disclosure are contemplated.

[0067] In certain embodiments, the subject is suffering from atrial fibrillation. In certain embodiments, the subject is suffering from acute atrial fibrillation, chronic atrial fibrillation, spontaneous atrial fibrillation, paroxysmal atrial fibrillation, recurrent atrial fibrillation, persistent atrial fibrillation, or permanent atrial fibrillation.

[0068] In certain embodiments, the subject is susceptible to atrial fibrillation.

[0069] In certain embodiments, the subject is susceptible to acute atrial fibrillation, chronic atrial fibrillation, spontaneous atrial fibrillation, paroxysmal atrial fibrillation, recurrent atrial fibrillation, persistent atrial fibrillation, or permanent atrial fibrillation.

[0070] In certain embodiments, the subject has an increased risk or likelihood of suffering from atrial fibrillation. In certain embodiments, the subject has an increased risk or likelihood of suffering from acute atrial fibrillation, chronic atrial fibrillation, spontaneous atrial fibrillation, paroxysmal atrial fibrillation, recurrent atrial fibrillation, persistent atrial fibrillation, or permanent atrial fibrillation.

[0071] In certain embodiments, the subject is suffering from or susceptible to one or more of reduced and/or slowed atrial conduction, reduced atrial conduction velocity, increased atrial conduction heterogeneity, atrial remodelling due to increased weight, atrial fibrosis and atrial inflammation.

[0072] In certain embodiments, the subject has an increased risk or likelihood of suffering from one or more of reduced and/or slowed atrial conduction, reduced atrial conduction velocity, increased atrial conduction heterogeneity, atrial remodelling due to increased weight, atrial fibrosis and atrial inflammation.

[0073] In certain embodiments, the subject has an increased body mass index.

[0074] In certain embodiments, the subject has a body mass index of 25 kgm^"2 or greater. In certain embodiments, the subject has a body mass index of 25 to 29.9 kgm^"2. In certain embodiments, the subject is overweight.

[0075] In certain embodiments, the subject has a body mass index of 30 kgm^"2 or greater.

[0076] In certain embodiments, the subject is obese. In certain embodiments, the subject is overweight. In certain embodiments, the subject is overweight or obese.

[0077] In certain embodiments, the subject has one or more of an increased level of intra-myocardial lipid, an increased level of inter-myocardial lipid and/or an increased pericardial fat or lipid volume. In certain embodiments, the subject has an increased level of intra-atrial myocardial lipid and/or an increased level of inter-atrial myocardial lipid.

[0078] In certain embodiments, the subject has one or more of the following characteristics: elevated ethnicity-specific waist circumference, type II diabetes, glucose intolerance, fasting hyperinsulinemia, increased cardiac MRI-determined atrial fibrosis by delayed enhancement, elevated high sensitivity CRP, low adiponectin levels and diabetic dyslipidemia.

[0079] In certain embodiments, the subject has one or more of the following characteristics: an increased body mass index; a body mass index of 25 kgm^"2or greater; a body mass index of 25 to 29.9 kgm^"2; a body mass index of 30 kgm^"2or greater; overweight; obesity; an increased level of intra-myocardial lipid; an increased level of inter-myocardial lipid; an increased pericardial fat volume; an increased level of intra- atrial myocardial lipid; an increased level of inter-atrial myocardial lipid; reduced and/or slowed atrial conduction; reduced atrial conduction velocity; and increased atrial conduction heterogeneity; elevated ethnicity-specific waist circumference, type II diabetes, glucose intolerance, fasting hyperinsulinemia, increased cardiac MRI- determined atrial fibrosis by delayed enhancement, elevated high sensitivity CRP, low adiponectin levels and diabetic dyslipidemia picture.

[0080] In certain embodiments, the present disclosure provides a method of treating a subject, the method comprising:

identifying a subject suffering from or susceptible to atrial fibrillation with one or more of the following characteristics: an increased body mass index; a body mass index of 25 kgm^"2 or greater; a body mass index of 25 to 29.9 kgm^"2; a body mass index of 30 kgm^"2 or greater; overweight; obesity; an increased level of intra-myocardial lipid; an increased level of inter- myocardial lipid; an increased pericardial fat volume; an increased level of intra-atrial myocardial lipid; an increased level of inter-atrial myocardial lipid; reduced and/or slowed atrial conduction; reduced atrial conduction velocity; and increased atrial conduction heterogeneity; and

administering to the subject a therapeutically effective amount of an endothelin receptor antagonist. [0081] The term "antagonist" as used herein refers to an agent, treatment, or intervention that results directly or indirectly in a change in endothelin receptor activity so as to cause a decrease, an inhibition, a reduction, and/or an inability to be stimulated, in endothelin receptor activity, including for example a decrease in activity, an alteration in the timing and/or location of activity, or otherwise provide some form of negative control over activity.

[0082] For example, an antagonist may (i) act directly to decrease the activity of an endothelin receptor, such as by altering the level of expression of the receptor, altering localisation of the receptor, altering signalling by the receptor, altering internationalisation of the receptor, and/or altering timing of receptor function; (ii) act to decrease the activity of a signalling pathway associated with an endothelin receptor, such as by altering the activity of a Gn protein; (iii) act to alter the level and/or the activity of a ligand that binds to an endothelin receptor, such as by competing with the binding of a ligand, or by altering the synthesis, breakdown, and/or localisation of the ligand. Other forms of action are contemplated.

[0083] Examples of antagonists 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, a ligand mimetic, a ligand antagonist, a dominant negative, a competitor, an inhibitor, and/or a suppressor.

[0084] 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, a change in the level of the species, a change in the localisation of the species, a change in the synthesis and/or degradation rates of the species, a change in the timing of activity, a change in the ability to interact with other species (such as a change in the ability of a ligand and a receptor to interact), a change in the chemical composition of the species, a change in signalling, and a change in cellular and/or non-cellular events affected by the species. Other forms of action are contemplated.

[0085] In certain embodiments, the endothelin receptor antagonist comprises one or more of A-186086, Ro-61-6612 (tezosentan), SB-209670, SB-217242 (enrasentan), SB- 217242, PD 142,893, PD 145,065, Ro 47-0203 (bosentan), R0 48-5033, macitentan, ACT-132577, A-127722, A-147627 (atrasentan), A-216546, BQ-123, BQ-610, FR 139317, Lu-135252 (darusentan), PD 151,242, PD 156,707, TBC-11251 (saitaxsentan), A-192621, BQ-788, RES-701-1, Ro 46-8443, and/or a pharmaceutically acceptable salt, hydrate, tautomer, isomer, pre-drug and/or derivative of any one or more of the aforementioned. The aforementioned antagonists are either commercially available or may be synthesized by a method known in the art.

[0086] In certain embodiments, the antagonist is a selective antagonist. In certain embodiments, the antagonist is a non-selective antagonist.

[0087] In certain embodiments, the antagonist is a non-selective of endothelin receptor antagonist.

[0088] In certain embodiments, the antagonist is a selective endothelin receptor antagonist.

[0089] In certain embodiments, the antagonist is selected from one or more of an endothelin A receptor antagonist, an endothelin receptor B antagonist, an antagonist of synthesis of a ligand for an endothelin receptor, an antagonist of signalling via an endothelin receptor (such as a modulator of Gn protein coupled signalling), and an antagonist of endothelin receptor level or expression.

[0090] In certain embodiments, the antagonist is an endothelin receptor A antagonist and/or an endothelin receptor B antagonist. In certain embodiments, the antagonist is an endothelin receptor A antagonist and an endothelin receptor B antagonist.

[0091] In certain embodiments, the antagonist is a selective antagonist of endothelin receptor A activity. In certain embodiments, the antagonist is a non-selective antagonist of endothelin receptor A activity. In certain embodiments, the antagonist is a selective antagonist of endothelin receptor B activity. In certain embodiments, the antagonist is a non-selective antagonist of endothelin receptor B activity.

[0092] In certain embodiments, the antagonist is a non-selective endothelin receptor antagonist. In certain embodiments, the non-selective antagonist comprises one or more of A- 186086, Ro-61-6612 (tezosentan), SB-209670, SB-217242 (enrasentan), SB- 217242, PD 142,893, PD 145,065, Ro 47-0203 (bosentan), R0 48-5033, macitentan, ACT- 132577 or a pharmaceutically acceptable salt, hydrate, tautomer, isomer, pre-drug and/or derivative of any one or more of the aforementioned. The aforementioned antagonists are either commercially available or may be synthesized by a method known in the art.

[0093] In certain embodiments, the antagonist is a selective antagonist of endothelin receptor A activity. In certain embodiments, the selective antagonist comprises one or more of A-127722, A-147627 (Atrasentan), A-216546, BQ-123, BQ-610, FR 139317, Lu-135252 (darusentan), PD 151,242, PD 156,707, TBC-11251 (saitaxsentan), and a pharmaceutically acceptable salt, hydrate, tautomer, isomer, pre-drug and/or derivative of any one or more of the aforementioned. The aforementioned antagonists are either commercially available or may be synthesized by a method known in the art.

[0094] In certain embodiments, the antagonist is a selective antagonist of endothelin receptor B activity. In certain embodiments, the selective antagonist comprises one or more of A-192621, BQ-788, RES-701-1, Ro 46-8443 and a pharmaceutically acceptable salt, hydrate, tautomer, isomer, pre-drug and/or derivative of any one or more of the aforementioned. The aforementioned antagonists are either commercially available or may be synthesized by a method known in the art.

[0095] In certain embodiments, the endothelin receptor antagonist comprises bosentan and/or a pharmaceutically acceptable salt or hydrate thereof. Bosentan is available commercially. Methods for synthesizing bosentan are known in the art, for example Kompella et al. (2014) Science Journal of Chemistry 2(6-1): 9-15.

[0096] Bosentan has the formula C₂₇H₂₉N₅0₆S^»H₂0 and is designated chemically as 4-tert-butyl-N- [6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-[2,2']-bipyrimidin-4- yl]- benzenesulfonamide monohydrate, and has the following structural formula:

[0097] In certain embodiments, the endothelin receptor antagonist comprises macitentan and/or a pharmaceutically acceptable salt or hydrate thereof. Macitentan is available commercially. Methods for synthesizing macitentan are known in the art, for example Bolli et al. (2012) J. Med Chem. 55(17): 7849-7861.

[0098] Macitentan has the formula Ci H₂oBr₂N60₄S and is designated chemically as N-[5-(4-Bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-4-pyrimidinyl]-N'- propylsulfamide, and has the following structural formula:

[0099] In certain embodiments, the antagonist comprises an antagonist of a ligand of an endothelin receptor.

[00100] In certain embodiments, the antagonist comprises an antagonist of one or more of endothelin-1, endothelin-2, and endothelin-3. In certain embodiments, the antagonist comprises an antagonist of endothelin-1.

[00101] In certain embodiments, the antagonist inhibits the binding of a ligand of an endothelin receptor.

[00102] In certain embodiments, the antagonist inhibits the synthesis of a ligand for an endothelin receptor.

[00103] In certain embodiments, the antagonist inhibits the processing of a species that forms a ligand for an endothelin receptor.

[00104] In certain embodiments, the antagonist inhibits processing of a precursor of one or more of endothelin-1, endothelin-2, and endothelin-3. In certain embodiments, the antagonist is an inhibitor of an endothelin converting enzyme.

[00105] In certain embodiments, the method further comprises administering a further active agent to the subject.

[00106] In certain embodiments, the method further comprises administering to the subject an effective amount of an inhibitor of the renin-angiotensin-aldosterone axis. In certain embodiments, the inhibitor of the renin-angiotensin-aldosterone axis is selected from one or more of a Renin inhibitor, an Angiotensin converting enzyme inhibitor, an Angiotensin II receptor antagonist, an Aldosterone receptor antagonist and a selective aldosterone synthase inhibitor. Inhibitors of the renin-angiotensin-aldosterone axis are known in the art and commercially available.

[00107] In certain embodiments, the method further comprises administering to the subject an effective amount of a TGF-β antagonist. TGF-β antagonists are known in the art and commercially available

[00108] In certain embodiments, the method further comprises administering to the subject an effective amount of another agent, such as an anti-fibrotic agent or anti- fibrogenic agent, such as tranilast. [00109] In certain embodiments, the method of preventing and/or treating atrial fibrillation further comprises a weight reduction intervention. Examples of weight loss interventions are known in the art. In certain embodiments, the weight reduction intervention comprises administering to the subject an effective amount of a weight loss agent.

[00110] In certain embodiments, the method comprises identifying a subject suffering from or susceptible to atrial fibrillation and treating the subject with an endothelin receptor antagonist. Methods for identifying a subject suffering from or susceptible to atrial fibrillation are known in the art.

[00111] In certain embodiments, the method comprises identifying a subject suffering from or susceptible to atrial fibrillation and having one or more characteristics.

[00112] In certain embodiments, the one or more characteristics comprise an increased body mass index; a body mass index of 25 kgm^"2 or greater; a body mass index of 25 to 29.9 kgm^"2; a body mass index of 30 kgm^"2 or greater; overweight; obesity; an increased level of intra-myocardial lipid; an increased level of inter-myocardial lipid; an increased pericardial fat volume; an increased level of intra-atrial myocardial lipid; an increased level of inter-atrial myocardial lipid; reduced and/or slowed atrial conduction; reduced atrial conduction velocity; and increased atrial conduction heterogeneity; atrial fibrosis; atrial inflammation; elevated ethnicity-specific waist circumference, type II diabetes, glucose intolerance, fasting hyperinsulinemia, increased cardiac MRI-determined atrial fibrosis by delayed enhancement, elevated high sensitivity CRP, low adiponectin levels and diabetic dyslipidemia picture.

[00113] Certain embodiments of the present disclosure provide a method of treating a subject, the method comprising:

[00114] In certain embodiments, the method comprises identifying a subject suffering from or susceptible to atrial fibrillation and having one or more characteristics, and treating the subject with an endothelin receptor antagonist on the basis of the one or more characteristics.

[00115] In certain embodiments, the one or more characteristics comprise one or more of an increased body mass index; a body mass index of 25 kgm-2or greater; a body mass index of 25 to 29.9 kgm-2; a body mass index of 30 kgm^"2or greater; overweight; obesity; an increased level of intra-myocardial lipid; an increased level of inter- myocardial lipid; an increased pericardial fat volume; an increased level of intra-atrial myocardial lipid; an increased level of inter-atrial myocardial lipid; reduced and/or slowed atrial conduction; reduced atrial conduction velocity; and increased atrial conduction heterogeneity; atrial fibrosis, atrial inflammation, elevated ethnicity-specific waist circumference, type II diabetes, glucose intolerance, fasting hyperinsulinemia, increased cardiac MRI-determined atrial fibrosis by delayed enhancement, elevated high sensitivity CRP, low adiponectin levels and diabetic dyslipidemia picture.

[00116] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from or susceptible to atrial fibrillation and suitable for treatment with an endothelin receptor antagonist.

[00117] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from or susceptible to atrial fibrillation and suitable for treatment with an endothelin receptor antagonist, the method comprising identifying a subject with one or more of the following characteristics: an increased body mass index; a body mass index of 25 kgm-² or greater; a body mass index of 25 to 29.9 kgm^"2; a body mass index of 30 kgm^"2 or greater; overweight; obesity; an increased level of intra-myocardial lipid; an increased level of inter-myocardial lipid; an increased pericardial fat/lipid volume; an increased level of intra-atrial myocardial lipid; an increased level of interatrial myocardial lipid; reduced and/or slowed atrial conduction; reduced atrial conduction velocity; increased atrial conduction heterogeneity, atrial fibrosis and atrial inflammation.

[00118] Endothelin receptor antagonists are as described herein. [00119] 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. A suitable amount may be readily selected by a medical practitioner.

[00120] In certain embodiments, the antagonist is administered to the subject in an amount ranging from one of the following selected ranges: 1 μg/kg to 1000 mg/kg, 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 ^g/kg; 10 μg/kg to 1000 mg/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; 10 μg/kg to 1000 mg/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 1000 mg/kg, 1 mg/kg to 100 mg/kg; 1 mg/kg to 10 mg/kg; 10 mg/kg to 1000 mg/kg; 10 mg/kg to 100 mg/kg; and 100 mg/kg to 1000 mg/kg body weight. Other ranges are contemplated.

[00121] In certain embodiments, the antagonist is administered to the subject in an amount ranging from one of the following selected ranges: 0.1 mg/kg to 10 mg/kg, 0.5 mg to 10 mg/kg, 1 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg. 0.1 mg/kg to 5 mg/kg, 0.5 mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, 0.1 mg/kg to 1 mg/kg or 0.5 mg/kg to 1 mg/kg.

[00122] In certain embodiments, the antagonist is administered to the subject in an amount ranging from one of the following selected ranges: 1 μg/kg/day to 1000 mg/kg/day, 1 μg/kg/day to 100 mg/kg/day; 1 μg/kg/day to 10 mg/kg/day; 1 μg/kg/day to 1 mg/kg/day; 1 μg/kg/day to 100 μg/kg/day; 1 μg/kg/day to 10μg/kg/day; 10 μg/kg/day to 1000 mg/kg/day, 10 μg/kg/day to 100 mg/kg/day; 10 μg/kg/day to 10 mg/kg/day; 10 μg/kg/day to 1 mg/kg/day; 10 μg/kg/day to 100 μg/kg/day; 10 μg/kg/day to 1000 mg/kg/day, 100 μg/kg/day to 100 mg/kg/day; 100 μg/kg/day to 10 mg/kg/day; 100 μg/kg/day to 1 mg/kg/day; 1 mg/kg/day to 1000 mg/kg/day, 1 mg/kg/day to 100 mg/kg/day; 1 mg/kg/day to 10 mg/kg/day; 10 mg/kg/day to 1000 mg/kg/day; 10 mg/kg/day to 100 mg/kg/day; and 100 mg/kg/day to 1000 mg/kg/day body weight. Other ranges are contemplated.

[00123] In certain embodiments, the antagonist is administered to the subject in an amount ranging from one of the following selected ranges: 0.1 mg/kg/day to 10 mg/kg/day, 0.5 mg/kg/day to 10 mg/kg/day, 1 mg/kg/day to 10 mg/kg/day, 5 mg/kg/day to 10 mg/kg/day, 0.1 mg/kg/day to 5 mg/kg/day, 0.5 mg/kg/day to 5 mg/kg/day, 1 mg/kg/day to 5 mg/kg/day, 0.1 mg/kg/day to 1 mg/kg/day, or 0.5 mg/kg/day to 1 mg/kg/day.

[00124] The antagonist may be administered to the subject in a suitable form. In this regard, the terms "administering" or "providing" includes administering the antagonist, or administering a prodrug of the antagonist, or a derivative of the antagonist that will form an effective amount of the antagonist 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, pastes, ointment, including solutions such as mouthwashes, for topical oral administration). Other routes of administration are contemplated.

[00125] In certain embodiments, the antagonist is administered orally. In certain embodiments, the antagonist is administered intravenously. In certain embodiments, the antagonist is administered via injection such as intravenous injection. In certain embodiments, the antagonist is administered by nebulized administration, by aerosolized administration or by being instilled into the lung. Other forms of administration are contemplated.

[00126] The antagonist may be administered alone or may be delivered in a mixture with other therapeutic agents and/or agents that, for example, enhance, stabilise or maintain the activity of the antagonist. In certain embodiments, an administration vehicle (e.g., pill, tablet, implant, injectable solution, etc.) would contain both the antagonist and additional agent(s). [00127] The methods as described herein may also include combination therapy. In this regard, the subject may be treated or given another drug or treatment modality in conjunction with the antagonist as described herein. This combination therapy can be sequential therapy where the subject is treated first with one and then the other, or the two or more treatment modalities are given simultaneously.

[00128] "Co-administering" or "co-administration" refers to the administration of two or more therapeutic agents together at one time. The 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.

[00129] When administered to a subject the therapeutically effective dosage of an agent 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. The daily dosages are expected to vary with route of administration, and the nature of the antagonist administered and any other agents administered. In certain embodiments, the methods comprise administering to the subject escalating doses of antagonist and/or repeated doses. In certain embodiments, the antagonist is administered orally. In certain embodiments, the antagonist is administered via injection, such as intravenous injection. In certain embodiments, the antagonist is administered parenterally. In certain embodiments, the antagonist 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 antagonist is administered by implant. In certain embodiments, the antagonist is administered by subcutaneous injection, intra- articularly, rectally, intranasally, intraocularly, vaginally, or transdermally. Methods of administration are known in the art.

[00130] "Intravenous administration" is the administration of substances directly into a vein.

[00131] "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. Typical forms for the oral administration of therapeutic agents includes the use of tablets or capsules.

[00132] In certain embodiments, the antagonist 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. Immediate release formulations are known in the art.

[00133] In certain embodiments, the antagonist 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. Sustained release formulations are known in the art.

[00134] In certain embodiments, the antagonist may be used in a pharmaceutical composition. In certain embodiments, the antagonist may be used in a pharmaceutical composition for use in the methods of the present disclosure as described herein.

[00135] Certain embodiments of the present disclosure provide a pharmaceutical composition

[00136] In certain embodiments, the pharmaceutical composition comprises a pharmaceutical composition for use to prevent and/or treat a condition as described herein. In certain embodiments, the pharmaceutical composition comprises a pharmaceutical composition for use to prevent and/or treat atrial fibrillation.

[00137] Certain embodiments of the present disclosure provide a pharmaceutical composition when used to prevent and/or treat atrial fibrillation, the pharmaceutical composition comprising a therapeutically effective amount of an endothelin receptor antagonist.

[00138] Endothelin receptor antagonists are as described herein.

[00139] In certain embodiments, the pharmaceutical composition comprises a further agent as described herein. [00140] In certain embodiments, a pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or suitable excipients.

[00141] In certain embodiments, the present disclosure provides use of an endothelin receptor antagonist in the preparation of a medicament for preventing and/or treating a condition as described herein.

[00142] In certain embodiments, the present disclosure provides use of an endothelin receptor antagonist in the preparation of a medicament for preventing and/or treating atrial fibrillation.

[00143] Certain embodiments of the present disclosure provide a pharmaceutical composition when used to prevent and/or treat atrial fibrillation, the pharmaceutical composition comprising a therapeutically effective amount of bosentan and/or a pharmaceutically acceptable salt and/or hydrate thereof.

[00144] Certain embodiments of the present disclosure provide a pharmaceutical composition when used to prevent and/or treat atrial fibrillation, the pharmaceutical composition comprising a therapeutically effective amount of macitentan and/or a pharmaceutically acceptable salt and/or hydrate thereof.

[00145] Certain embodiments of the present disclosure provide a pharmaceutical composition when used to prevent and/or treat atrial fibrillation, the pharmaceutical composition comprising a therapeutically effective amount of bosentan and/or macitentan, and/or a pharmaceutically acceptable salt and/or hydrate thereof.

[00146] In certain embodiments, the antagonist is present in a composition or medicament so as to provide an amount of antagonist for administration to the subject in an amount ranging from one of the following selected ranges: 1 μg/kg to 1000 mg/kg, 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 ^g/kg; 10 μg/kg to 1000 mg/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; 10 μg/kg to 1000 mg/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 1000 mg/kg, 1 mg/kg to 100 mg/kg; 1 mg/kg to 10 mg/kg; 10 mg/kg to 1000 mg/kg; 10 mg/kg to 100 mg/kg; and 100 mg/kg to 1000 mg/kg body weight. Other ranges are contemplated.

[00147] In certain embodiments, the antagonist is present in a composition or a medicament to provide an amount of antagonist for administration to the subject in an amount ranging from one of following selected ranges: 0.1 mg/kg to 10 mg/kg, 0.5 mg to 10 mg/kg, 1 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg. 0.1 mg/kg to 5 mg/kg, 0.5 mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, 0.1 mg/kg to 1 mg/kg or 0.5 mg/kg to 1 mg/kg.

[00148] In certain embodiments, the antagonist is present in a composition or medicament so as to provide an amount of antagonist for administration to the subject in an amount ranging from one of the following selected ranges: 1 μg/kg/day to 1000 mg/kg/day, 1 μg/kg/day to 100 mg/kg/day; 1 μg/kg/day to 10 mg/kg/day; 1 μg/kg/day to 1 mg/kg/day; 1 μg/kg/day to 100 μg/kg/day; 1 μg/kg/day to 10μg/kg/day; 10 μg/kg/day to 1000 mg/kg/day, 10 μg/kg/day to 100 mg/kg/day; 10 μg/kg/day to 10 mg/kg/day; 10 μg/kg/day to 1 mg/kg/day; 10 μg/kg/day to 100 μg/kg/day; 10 μg/kg/day to 1000 mg/kg/day, 100 μg/kg/day to 100 mg/kg/day; 100 μg/kg/day to 10 mg/kg/day; 100 μg/kg/day to 1 mg/kg/day; 1 mg/kg/day to 1000 mg/kg/day, 1 mg/kg/day to 100 mg/kg/day; 1 mg/kg/day to 10 mg/kg/day; 10 mg/kg/day to 1000 mg/kg/day; 10 mg/kg/day to 100 mg/kg/day; and 100 mg/kg/day to 1000 mg/kg/day body weight. Other ranges are contemplated.

In certain embodiments, the antagonist is present in a composition or medicament so as to provide an amount of antagonist for administration to the subject in an amount ranging from one of the following selected ranges: 0.1 mg/kg/day to 10 mg/kg/day, 0.5 mg/kg/day to 10 mg/kg/day, 1 mg/kg/day to 10 mg/kg/day, 5 mg/kg/day to 10 mg/kg/day, 0.1 mg/kg/day to 5 mg/kg/day, 0.5 mg/kg/day to 5 mg/kg/day, 1 mg/kg/day to 5 mg/kg/day, 0.1 mg/kg/day to 1 mg/kg/day, or 0.5 mg/kg/day to 1 mg/kg/day.

[00149] In certain embodiments, the composition or 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. Other routes of administration are contemplated.

[00150] In certain embodiments, the antagonist is provided with a pharmaceutically acceptable carrier suitable for administering the pharmaceutical composition to a subject. The carriers may be chosen based on various considerations including the route of administration, the agent(s) being delivered and the time course of delivery of the agents. The term "pharmaceutically acceptable carrier" refers to a substantially inert solid, semi-solid or liquid filler, diluent, excipient, 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.

[00151] In certain embodiments, the antagonist is provided with a pharmaceutically acceptable excipient.

[00152] For example, suitable excipients for use with an antagonist in the form of a tablet include a tablet with a tablet core and a film coat as follows: tablet core - maize starch, pregelatinised starch, sodium starch glycollate, povidone, glycerol dibehenate, magnesium stearate; film coat - hypromellose, glycerol triacetate, talc, titanium dioxide (E171), iron oxide yellow (E172), iron oxide red (E172), ethylcellulose. Other excipients are contemplated. [00153] For example, a tablet with the endothelin receptor antagonist bosentan may include an amount of the agent as a monohydrate and a tablet core with maize starch, pregelatinised starch, sodium starch glycollate, povidone, glycerol dibehenate, and magnesium stearate, and a film coat with hypromellose, glycerol triacetate, talc, titanium dioxide (E171), iron oxide yellow (E172), iron oxide red (E172), and ethylcellulose.

[00154] In certain embodiments, the antagonist 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, palmoic, 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. Other pharmaceutically acceptable salts are contemplated.

[00155] In certain embodiments, the antagonist may be administered, or present in a pharmaceutical composition, as a pharmaceutically acceptable hydrate. A hydrate is a solid adduct containing both the parent compound (e.g., the anhydrate of a drug or excipient) and water.

[00156] In certain embodiments, the pharmaceutical compositions or medicaments as described herein comprise other therapeutic agents and/or agents that enhance, stabilise or maintain the activity of the active.

[00157] Oral formulations 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 dodecyl sulfate, 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. Oral formulation are known in the art and may be formulated by a skilled person. .

[00158] In certain embodiments, it may be desirable to administer the antagonist directly to the airways in the form of an aerosol. Formulations for the administration of aerosol forms are known in the art and may be formulated by a skilled person. .

[00159] In certain embodiments, the antagonist may also be administered parenterally (such as directly into the joint space) or intraperitoneally. For example, solutions or suspensions of agents 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. Parenteral formulations are known in the art and may be formulated by a skilled person.

[00160] In certain embodiments, the antagonist 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, isotonic saline, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Injectable formulations are known in the art and may be formulated by a skilled person.

[00161] In certain embodiments, the antagonist may also be administered intravenously. Compositions suitable for intravenous administration are known in the art and may be formulated by a skilled person. For example, isotonic saline may be used in an intravenous composition containing an antagonist.

[00162] In certain embodiments, the antagonist 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 an antagonist as described herein, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).

[00163] 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. Transdermal formulations are known in art and may be formulated by a skilled person.

[00164] In certain embodiments, the antagonist 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. Suppository formulations are known in the art and may be formulated by a skilled person.

[00165] Additional numerous various excipients, dosage forms, dispersing agents and the like that are suitable for use in connection with administration 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.

[00166] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of bosentan and/or a pharmaceutically acceptable salt and/or hydrate thereof for use for preventing and/or treating atrial fibrillation.

[00167] Certain embodiments of the present disclosure provide bosentan and/or a pharmaceutically acceptable salt and/or hydrate thereof for preventing and/or treating atrial fibrillation.

[00168] Certain embodiments of the present disclosure provide use of bosentan and/or a pharmaceutically acceptable salt and/or hydrate thereof in the preparation of a medicament for preventing and/or treating atrial fibrillation.

[00169] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of macitentan and/or a pharmaceutically acceptable salt and/or hydrate thereof for use for preventing and/or treating atrial fibrillation.

[00170] Certain embodiments of the present disclosure provide macitentan and/or a pharmaceutically acceptable salt and/or hydrate thereof for preventing and/or treating atrial fibrillation.

[00171] Certain embodiments of the present disclosure provide use of macitentan and/or a pharmaceutically acceptable salt and/or hydrate thereof in the preparation of a medicament for preventing and/or treating atrial fibrillation.

[00172] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of an endothelin receptor antagonist and an inhibitor of the renin-angiotensin-aldosterone axis.

[00173] In certain embodiments, the inhibitor of the renin-angiotensin-aldosterone axis is selected from one or more of a renin inhibitor, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, an aldosterone receptor antagonist and a selective aldosterone synthase inhibitor.

[00174] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutically effective amount of an endothelin receptor antagonist and an anti-fibrotic agent and/or an anti-fibrogenic agent, such as tranilast.

[00175] Certain embodiments of the present disclosure provide a method for preventing and/or treating a condition as described herein, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or medicament as described herein.

[00176] Certain embodiments of the present disclosure provide a method of preventing and/or treating atrial fibrillation, the method comprising administering to the subject of a therapeutically effective amount of a pharmaceutical composition or medicament as described herein.

[00177] Certain embodiments of the present disclosure provide other uses as described herein of an endothelin receptor antagonist. Endothelin receptor antagonists are as described herein. In certain embodiments, an endothelin receptor antagonist is used to reduce the frequency and/or duration of episodes of atrial fibrillation and/or reducing the severity and/or effects of atrial fibrillation, to prevent and/or treat atrial remodelling due to weight gain, to prevent and/or treat atrial remodelling due to obesity, to prevent and/or treat one or more of reduced and/or slowed atrial conduction, to prevent and/or treat reduced atrial conduction velocity, to prevent and/or treat increased atrial conduction heterogeneity, to modulate atrial rhythm, to prevent and/or treat atrial fibrosis, to prevent and/or treat atrial inflammation, to reduce the likelihood of stroke, to reduce the probability of a subject developing atrial fibrillation, making an arrhythmia in a subject more responsive to pharmacological intervention, and to reduce the dose of another agent for preventing and/or treating atrial fibrillation. Endothelin receptor antagonists and methods for administering endothelin receptor antagonists are as described herein. Pharmaceutical compositions comprising an endothelin receptor antagonist may be used for the aforementioned uses.

[00178] Certain embodiments of the present disclosure provide a method of reducing the frequency and/or duration of episodes of atrial fibrillation and/or reducing the severity and/or effects of atrial fibrillation.

[00179] Certain embodiments of the present disclosure provide a method of reducing the frequency and/or duration of episodes of atrial fibrillation and/or reducing the severity and/or effects of atrial fibrillation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[00180] Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial remodelling due to weight gain.

[00181] Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial remodelling due to weight gain in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[00182] Certain embodiments of the present disclosure provide a method for preventing and/or treating reduced and/or slowed atrial conduction.

[00183] Certain embodiments of the present disclosure provide a method for preventing and/or treating reduced and/or slowed atrial conduction in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist. [00184] Certain embodiments of the present disclosure provide a method for preventing and/or treating reduced atrial conduction velocity.

[00185] Certain embodiments of the present disclosure provide a method for preventing and/or treating reduced atrial conduction velocity in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[00186] Certain embodiments of the present disclosure provide a method for preventing and/or treating reduced atrial conduction heterogeneity.

[00187] Certain embodiments of the present disclosure provide a method for preventing and/or treating reduced atrial conduction heterogeneity in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[00188] Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial fibrosis.

[00189] Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial fibrosis in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[00190] Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial inflammation.

[00191] Certain embodiments of the present disclosure provide a method for preventing and/or treating atrial inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[00192] Certain embodiments of the present disclosure provide a method for reducing the likelihood of stroke in a subject suffering from or susceptible to atrial fibrillation.

[00193] Certain embodiments of the present disclosure provide a method for reducing the likelihood of stroke in a subject suffering from or susceptible to atrial fibrillation, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[00194] Certain embodiments of the present disclosure provide a method of reducing the probability of a subject developing atrial fibrillation.

[00195] Certain embodiments of the present disclosure provide a method of reducing the probability of a subject developing atrial fibrillation, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

[00196] Certain embodiments of the present disclosure provide a method of reducing the dose of an agent administered for preventing and/or treating atrial fibrillation.

[00197] Certain embodiments of the present disclosure provide a method of reducing the dose of an agent administered to a subject for preventing and/or treating atrial fibrillation, the method comprising administering to the subject an effective amount of an endothelin receptor antagonist, and thereby reduce the dose of the agent for preventing and/or treating atrial fibrillation..

[00198] Examples of such agents include beta blockers (such as metoprolol, atenolol, bisoprolol, nebivolol), non-dihydropyridine calcium channel blockers (such as diltiazem or verapamil), cardiac glycosides (such as digoxin), amiodarone, diltiazem, amiodarone, dronedarone, procainamide, dofetilide, ibutilide, propafenone, flecainide, and vernakalant.

[00199] Selection of the appropriate reduction in the dose may be determined by a suitably skilled medical practitioner.

[00200] In certain embodiments, the administration of an endothelin receptor antagonist may be used to reduce the dose of another agent for preventing and/or treating stroke associated with atrial fibrillation. Examples of such agents include warfarin, vitamin-K epoxide reductase inhibitors (such as tecarfarin), direct thrombin inhibitors (such as AZD-0837; dabigatran etexilate, dabigatran, ximelagatran; melagatran, and argatroban), Factor Xa inhibitors (such as apixaban, rivaroxaban, YM466, betrixaban, and edoxaban).

[00201] Certain embodiments of the present disclosure provide a product comprising an endothelin receptor antagonist.

[00202] In certain embodiments, a product comprising an endothelin receptor antagonist is provided for use in the methods as described herein.

[00203] Certain embodiments of the present disclosure provide a combination product comprising:

an endothelin receptor antagonist; and

instructions for administering the antagonist to a subject to prevent and/or treat a condition as described herein.

[00204] Certain embodiments of the present disclosure provide a combination product comprising:

an endothelin receptor antagonist; and

[00205] In certain embodiments, the combination product may optionally further comprise an inhibitor of the renin-angiotensin-aldosterone axis and/or instructions for weight loss intervention.

[00206] Certain embodiments of the present disclosure provide a kit for performing a method as described herein.

[00207] The kit may comprise one or more antagonists, agents, reagents, components, compositions, formulations, products and instructions, as described herein.

[00208] Certain embodiments of the present disclosure provide a kit for preventing and/or treating atrial fibrillation, the kit comprising an endothelin receptor antagonist and optionally comprising one or more of instructions for administering the antagonist to a subject.

[00209] Typically, a kit for administration to a subject contains the active agent (ie an endothlin receptor antagonist) in a form suitable for administration to the subject and one or more instructions, data sheets, and information on dosages, side effects, contraindications, and drug monitoring.

[00210] Certain embodiments of the present disclosure provide a method of identifying active agents.

[00211] For example, in certain embodiments methods may be used to identify an agent for preventing and/or treating atrial fibrillation, to identify an agent suitable to reduce the frequency and/or duration of episodes of atrial fibrillation and/or reducing the severity and/or effects of atrial fibrillation, to prevent and/or treat atrial remodelling due to weight gain, to prevent and/or treat atrial remodelling due to obesity, to prevent and/or treat one or more of reduced and/or slowed atrial conduction, to prevent and/or treat reduced atrial conduction velocity, to prevent and/or treat increased atrial conduction heterogeneity, to modulate atrial rhythm, to prevent and/or treat atrial fibrosis, to prevent and/or treat atrial inflammation, to reduce the likelihood of stroke, to reduce the probability of a subject developing atrial fibrillation, to identify an agent for making an arrhythmia in a subject more responsive to pharmacological intervention, and to reduce the dose of another agent needed for preventing and/or treating a condition as described herein, such as atrial fibrillation.

[00212] Certain embodiments of the present disclosure provide a method of identifying an agent for preventing and/or treating atrial fibrillation.

[00213] Certain embodiments of the present disclosure provide a method for identifying an agent for preventing and/or treating atrial fibrillation, the method comprising:

determining the ability of a test agent to antagonise endothelin receptor activity; and

identifying the test agent as an agent for preventing and/or treating atrial fibrillation. [00214] Examples of test 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, aa ligand antagonist, a ligand mimetic, a dominant negative, a competitor, an inhibitor, and/or a suppressor. Other types of agents are contemplated.

[00215] The test agent may be a candidate drug agent.

[00216] Determination of the ability of a test agent to antagonise endothelin receptor activity may be accomplished in an appropriate system. For example, the ability of the test agent to modulate endothelin receptor activity may be determined in one or more of an in vitro cell free system, an in vitro cell system, an animal model and a human subject. Methods for assessing endothelin receptor activity in vitro and/or in vivo are known in the art, for example as described in Ihara et al. (1991) Biochem Biophys Res Commun. 178(1): 132-7, Miyata et al. (1992) J. Antibiotics 45:74-82, and Urade et al. (1992) FEBS Lett. 311(1): 12-6.

[00217] For example, the ability of a test agent to antagonise endothelin receptor activity may be tested in a suitable animal model. Examples of animal models include a horse, a cow, a sheep, a goat, a pig, a dog, a cat, a primate, a monkey, a rabbit, a mouse and laboratory animals.

[00218] Determination of the ability of a test agent to modulate endothelin receptor activity may also be accomplished (independently or after testing in an animal model) in a human subject in an appropriate clinical trial. Examples of human subjects are as described herein.

[00219] Methods for assessing the ability of an agent to prevent and/or treat atrial fibrillation are known in the art. Identification that the test agent is as an agent for preventing and/or treating atrial fibrillation may be accomplished using a method known in the art, for example using an animal model as described herein and/or in a human clinical trial.

[00220] In certain embodiments, the test agent comprises a derivative of a molecule that is an endothelin receptor antagonist as described herein.

[00221] In certain embodiments, the test agent comprises a derivative of A-186086, Ro- 61-6612 (tezosentan), SB-209670, SB-217242 (enrasentan), SB-217242, PD 142,893, PD 145,065, Ro 47-0203 (bosentan), R0 48-5033, macitentan, ACT-132577, A-127722, A- 147627 (atrasentan), A-216546, BQ-123, BQ-610, FR 139317, Lu- 135252 (darusentan), PD 151,242, PD 156,707, TBC-11251 (saitaxsentan), A-192621, BQ-788, RES-701-1, or Ro 46-8443.

[00222] In certain embodiments, the test agent comprises a derivative of bosentan.

[00223] In certain embodiments, the test agent comprises a derivative of macitentan.

[00224] Certain embodiments of the present disclosure provide a method of screening endothelin receptor antagonists for their ability to provide a therapeutic effect as described herein.

[00225] Certain embodiments of the present disclosure provide a method of screening endothelin receptor antagonists for their ability to provide a therapeutic effect for one or more of the following: to reduce the frequency and/or duration of episodes of atrial fibrillation and/or reducing the severity and/or effects of atrial fibrillation, to prevent and/or treat atrial remodelling due to weight gain, to prevent and/or treat atrial remodelling due to obesity, to prevent and/or treat one or more of reduced and/or slowed atrial conduction, to prevent and/or treat reduced atrial conduction velocity, to prevent and/or treat increased atrial conduction heterogeneity, to modulate atrial rhythm, to prevent and/or treat atrial fibrosis, to prevent and/or treat atrial inflammation, to reduce the likelihood of stroke, to reduce the probability of a subject developing atrial fibrillation, making an arrhythmia in a subject more responsive to pharmacological intervention, and to reduce the dose of another agent for preventing and/or treating atrial fibrillation. [00226] Certain embodiments of the present disclosure provide a method of identifying an agent for preventing and/or treating of atrial fibrillation, the method comprising identifying an endothelin receptor antagonist, and/or a derivative thereof, as an agent for preventing and/or treating atrial fibrillation.

[00227] Certain embodiments of the present disclosure provide a method for identifying an agent for preventing and/or treating atrial fibrillation, the method comprising:

providing an endothelin receptor antagonist and/or a derivative thereof; and identifying the endothelin receptor antagonist and/or a derivative thereof as an agent for preventing and/or treating atrial fibrillation.

[00228] Endothelin receptor antagonists are as described herein.

[00229] In certain embodiments, the endothelin receptor antagonist to be screened/tested is a derivative of an endothelin receptor antagonist as described herein. In certain embodiments, the endothelin receptor antagonist to be screened/tested is a derivative of bosentan. In certain embodiments, the endothelin receptor antagonist to be screened/tested is a derivative of macitentan.

[00230] Certain embodiments of the present disclosure provide a method for identifying an agent for preventing and/or treating atrial fibrillation, the method comprising:

determining the ability of a derivative of bosentan to antagonise endothelin receptor activity; and

identifying the derivative as an agent for preventing and/or treating atrial fibrillation.

[00231] Certain embodiments of the present disclosure provide a method for identifying an agent for preventing and/or treating atrial fibrillation, the method comprising:

determining the ability of a derivative of macitentan to antagonise endothelin receptor activity; and

[00232] Certain embodiments of the present disclosure provide an agent identified by a method as described herein.

[00233] Certain embodiments of the present disclosure provide a method of treating a condition or disorder as described herein, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist and/or tranilast. Methods for administering agents to a subject are as described herein.

[00234] Subjects suitable for treatment are as described herein. In certain embodiments, the subject is obese.

[00235] In certain embodiments, there is provided a method for preventing and/or treating atrial fibrillation in a subject, the method comprising administering to the subject a therapeutically effective amount of tranilast, for example for preventing and/or treating atrial fibrillation in obese patients.

[00236] The present disclosure is further described by 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 - Progressive weight gain results in abnormal Endothelin Receptor expression, atrial fibrosis and the substrate for atrial fibrillation

[00237] Methods

[00238] (i) Animals

[00239] Thirty-six Merino-cross wethers were studied. All procedures were conducted in accordance with guidelines outlined in the "Position of the American Heart Association on Research Animal Use", adopted November 11 1984. This study was approved by the Animal Ethics Committees of the University of Adelaide and the Institute of Medical and Veterinary Services, Adelaide, Australia.

[00240] (ii) Study Protocol [00241] Animals were acclimatized to University housing pens for 7-days prior to study. Thirty animals underwent ad-libitum feeding to induce obesity as previously described (see below for details). At baseline, 4 months and 8 months, 10 of the cohort were randomly selected for CMR followed by open chest electrophysiology study. An additional 6 sheep were studied (3 at each of the 2 time-points; 4 months and 8 months) as controls. Intravenous sodium thiopentone (15 - 20 mg/kg) was used for induction before endotracheal intubation. Isoflurane in oxygen (2 - 4%) was used for maintenance. Invasive arterial blood pressure, heart rate, pulse oximetry, end-tidal C0₂ and temperature were monitored continuously.

[00242] (iii) Ad-Libitum Feeding Obese Ovine Model

[00243] A previously characterized ovine model of progressive weight gain, utilizing an ad-libitum regimen of hay and high energy pellets, was used to induce progressive weight gain. This model has shown an approximate increase of 10kg monthly up to 36 weeks during which weight gain plateaued (terminal obesity). At baseline, 30 healthy animals were commenced on a high caloric diet of unlimited supply of energy-dense soybean oil (2.2%) and molasses fortified grain and maintenance hey with weekly weight measurement. Excess voluntary intake was predominantly of grass alfalfa silage and hay. For the obese sheep, pellets were gradually introduced at 8% excess basal energy requirements, and rationed to >70% of total dry matter intake. Blood samples were periodically collected to ensure electrolyte and acid-base homeostasis. To maintain the 6 controls at their baseline weight, hay was distributed for maintenance, while energy dense pellets were rationed at 0.75% of live weight daily to maintain weight tightly between 50-60%). Nutritional content of food and housing conditions were identical between both groups, but merely the amount was varied. Shorn weight was recorded immediately prior to surgery. The study outline is illustrated in Figurel .

[00244] (ii) Cardiac Magnetic Resonance Imaging

[00245] Cardiac MRI was performed to assess bi-atrial and bi -ventricular volumes and ejection fractions (Siemens Sonata 1.5 Telsa, MR Imaging Systems, Siemens Medical Solutions, Erlangen Germany) with 6 mm slices through the atria and 10 mm through the ventricles without inter-slice gaps. Animals were placed and secured in the dorsal recumbent position for scanning. Mechanical ventilation was maintained to facilitate ECG-gated image acquisition with periodic breath holding. All analyses were performed offline by blinded operators using proprietary software, QMass MR (Medis medical imaging systems, Leiden, Netherlands). Chamber size, ventricular mass and pericardial fat volumes were measured using previously described methods (Wong CX, Abed HS, Molaee P, Nelson AJ, Brooks AG, Sharma G, et al. "Pericardial fat is associated with atrial fibrillation severity and ablation outcome" J Am Coll Cardiol. 2011 Apr 26;57(17): 1745-51)

[00246] (iii) Hemodynamic Recordings

[00247] Continuous invasive mean arterial pressure (MAP) monitoring was undertaken at the time of electrophysiology study. In addition, to quantify the diastolic functional disturbance of obesity, direct LA catheterization was performed to measure LA pressure (LAP).

[00248] (iv) Electrophysiology Study

[00249] Electrophysiology study was performed under general anesthesia. Midline sternotomy was used to facilitate cardiac exposure, pericardial cradle formation and epicardial application of the multi-electrode plaques. Custom designed 128-electrode plaques with 5mm spacing were applied over the right and left atria spanning the appendage and free wall on each chamber as previously described (Lau DH, Mackenzie L, Kelly DJ, Psaltis PJ, Worthington M, Rajendram A, et al. "Short-term hypertension is associated with the development of atrial fibrillation substrate: a study in an ovine hypertensive model." Heart Rhythm. 2010 Mar;7(3):396-404; Lau DH, Mackenzie L, Kelly DJ, Psaltis PJ, Brooks AG, Worthington M, et al. "Hypertension and atrial fibrillation: evidence of progressive atrial remodeling with electrostructural correlate in a conscious chronically instrumented ovine model." Heart Rhythm. 2010 Sep;7(9): 1282-90.). Plaque were sutured to adjacent myocardium ensuring consistent contact and attached to a computerized signal digital analyzer (Lab System Pro, Bard Electrophysiology, Lowell MA). Surface ECG and epicardial electrograms were continuously recorded and stored for offline analysis. All electrograms were filtered between 30-500Hz and measured with computer assisted calipers at a sweep speed of 200mm/sec.

[00250] (v) Atrial Effective Refractory Period (ERP)

[00251] Atrial ERP was measured at twice the diastolic threshold (tissue capture threshold) at CL (SI) of 500, 400, 300 and 200ms from four sites (right atrial appendage [RAA], right atrial free wall [RAFW], left atrial appendage [LAA] and left atrial free wall [LAFW]). Eight basic (SI) stimuli were followed by a premature (S2) Stimulus in 10 ms decrements. Atrial ERP was defined as the longest S1-S2 interval not resulting in a propagated response. Each measurement was repeated three times. If there was greater than 10ms variability, two further measurements were taken and the total averaged.

[00252] (vi) Atrial Conduction Velocity (CV)

[00253] Conduction velocity was calculated from each site in both atria, during stable capture of SI pacing train and the shortest coupled S2 that captures the atria at each CL. Activation maps were created using semi-automated custom designed software (Nucleus Medical, Adelaide, Australia; Lau DH, Mackenzie L, Kelly DJ, Psaltis PJ, Worthington M, Rajendram A, et al. "Short-term hypertension is associated with the development of atrial fibrillation substrate: a study in an ovine hypertensive model." Heart Rhythm. 2010 Mar; 7(3):396-404. Each annotation was manually verified with the local activation time annotated to the peak of the largest amplitude deflection on bipolar electrograms. Local conduction velocity was calculated from the local vectors within each triangle of electrodes. Mean CV was then derived for each map.

[00254] (vii) Atrial Conduction Heterogeneity

[00255] Conduction heterogeneity was assessed using established phase mapping techniques during SI and S2 pacing (Lammers WJ, Schalij MJ, Kirchhof CJ, Allessie MA. "Quantification of spatial inhomogeneity in conduction and initiation of reentrant atrial arrhythmias." Am J Physiol. 1990 Oct;259 (4 Pt 2):H1254-63). In brief, the largest activation time difference between every four adjacent electrodes was first determined and divided by inter-electrode distances. The largest value at each site was then used to create a phase map, with values also displayed as histograms. Absolute conduction phase delay was calculated as the difference between the 5th and 95th percentile of the phase difference distribution. Conduction heterogeneity index is then determined by dividing the absolute phase delay by the median (P50).

[00256] (viii) AF Induction

[00257] Spontaneous and induced AF was recorded. Spontaneous AF was defined as episodes of irregular atrial rhythm lasting >5sec occurring during the electrophysiology study in the absence of pacing after stable plaque application. Inducibility of AF was determined by ramp burst pacing commencing at CL 250 msec with progressive 5 ms decrements until AF was induced or there was no longer 1 : 1 conduction. This manoeuvre was repeated 5 times from each of the 4 per-determined pacing sites and undertaken after the completion of electrophysiologic evaluation. When AF was induced and persisted for >5sec, the episode and its duration was recorded. Electrical cardioversion was performed only when hemodynamic compromise occurred or if AF became sustained (>5mins). If cardioversion was required or arrhythmia sustained, no further electrophysiologic recordings were made.

[00258] (ix) Structural Analysis

[00259] At the completion of all electrophysiologic and hemodynamic recordings, the heart was removed. From each of the 4 chambers and interventricular septum, 1cm interval, 6 micron sections were harvested and fixed in 10% buffered formalin or frozen at -70°C.

[00260] Quantitative analysis of wax embedded specimens for percentage interstitial extracellular matrix was performed using Picrosirius red staining. Six stained sections from each individual myocardial chamber of every animal were digitally captured (20 sections/animal) with an area of picrosirius red selected for its colour range and the proportional area of tissue with this range of colour quantified, as previously described (DH, Mackenzie L, Kelly DJ, Psaltis PJ, Brooks AG, Worthington M, et al. "Hypertension and atrial fibrillation: evidence of progressive atrial remodeling with electrostructural correlate in a conscious chronically instrumented ovine model" Heart Rhythm. 2010 Sep;7(9): 1282-90). [00261] For lipid content analysis, 1cm cubes of frozen endocardial and intramural myocardium from each chamber were air dried and formalin fixed, after careful epicardial fat stripping. Sections 5-7μπι thick were prepared with Oil-red-0 0.3% w/v isopropanol and distilled water for 15 minutes, then washed with 60%isopropanol. Five random non-overlapping fields were captured and digitized using a Carl Zeiss microscope attached to AxioCamMRc5 digital camera (Carl Zeiss, North Ryde, NSW, Australia) at X200 magnification. For digital images, an area of red was selected for its colour range and the proportional area of tissue with this range of colour then quantified. Calculation of the proportional area stained red (adipose tissue) was then determined using image analysis (AxioVision Release 4.8.1; Carl Zeiss, North Ryde, NSW, Australia) and expressed as proportional per area, as previously described(Lehr HA, van der Loos CM, Teeling P, Gown AM. "Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis" J Histochem Cytochem. 1999 Jan;47(l): 119-26). Cellular infiltrates were visualized using hematoxylin and eosin staining and semi-quantitatively graded using a previously described scale of 0-2 (Geboes K, Riddell R, Ost A, Jensfelt B, Persson T, Lofberg R. "A reproducible grading scale for histological assessment of inflammation in ulcerative colitis" Gut. 2000 Sep;47(3):404-9).

[00262] (xi) Endothelin Receptor

[00263] Western blot methods were used to assess changes in ET receptor expression in atrial myocardium between weight groups. Total protein was extracted from the atria as described before (Woessner JF, Jr. "Quantification of matrix metalloproteinases in tissue samples." Methods Enzymol. 1995;248:510-28) and quantified by Bradford protein assay. To determine changes in endothelin (ET) receptor expression between groups, protein extracts (containing an equal amount of 10-15 μg of total protein/lane) were then electrophoresed under reducing conditions on 10.5% acrylamide gels. Western blot analyses were performed with polyclonal antibodies to the ETA receptor (ab30536; 1 :500 dilution; Abeam) and ETB receptor (AF4496; 1 :300 dilution; R&D Systems), in addition to a goat anti-sheep secondary antibody. A monoclonal antibody to the house keeping protein, a-tubulin (1 :8000 dilutiuon; Millipore Corp.) was used to demonstrate equivalent loading of protein samples. Densitometry of ETA (63kDa) and ETB (~30kDa) receptor bands was performed using Bio-Rad GS710 Calibrated Imaging Densitometer and Quantity-OneTM software (Bio-Rad Laboratories). The density of each receptor was then corrected for corresponding a-tubulin and expressed as an absolute optical density (arbitrary units).

[00264] (xii) Statistical Analysis

[00265] Continuous variables are expressed as mean ± standard deviation. Hemodynamic variables, histologic indices and AF episode number/duration are expressed as median and IQR. One-way ANOVA was used to determine differences in CMR measures across weight cohorts. Kruskal-Wallis test was used to determine differences in AF inducibility/duration, quantitative histology and hemodynamic variables between weight cohorts. To investigate the effect of progressive obesity on electrophysiological parameters, a linear mixed-effects model was used with each electrophysiological parameter (ERP, CV and CHI) entered as the dependent variable to determine the effects of weight cohort, for both SI and S2-derived recordings. Fixed factors of weight cohort, CL and site (appendage and free wall of each corresponding atrium) were entered for main effects. Animal ID was entered as a random factor to account for nested data within each experiment. Bonferroni method was use for pairwise comparisons. Hemodynamic variables (MAP and LAP) were entered as covariates. To determine any possible time-related changes in electrophysiology, baseline, 4 month and 8 month time-point controls were similarly entered into a linear mixed-effects model. Variables were log transformed to satisfy model assumptions. All analyses were performed using SPSS/PASW versionl8 (SPSS Inc. Chicago, IL).

[00266] Results

[00267] There was progressive weight gain with increasing feeding duration from 58±7 kg at baseline to 77±5 kg (overweight) at 4 months and 105±13 kg (obese) at 8 months (P<0.001). In contrast, there was no weight change in the control group; 58±6 kg at baseline, 50±4 kg at 4 months and 54±5 kg at 8 months (P=0.2). Table 1 shows the changes in atrial size, atrial structure and hemodynamic parameters with progressive weight gain. There was a graded increase in MAP and LAP (P=0.02 and P<0.001 respectively), with increasing weight gain. Table 1

BASELINE OVERWEIGHT OBESE

(4 months) (8 months)

Weight 58±7 77±5 105±13 P<0.001

LA Volume (mL) 31.8±6.0 37.6±7.3 41.6±.3 P=0.01

RA Volume (mL) 34.0±4.8 36.7±6.8 40.8±5.1 P=0.04

LVEDV (mL) 86.4±15.4 96.4±14.3 97.4±16.6 P=0.2

LVEF (%) 42.0±5.0 44.6±4.7 42.3±3.7 P=0.4

RVEDV (mL) 64.9±11.1 70.9±14.2 71.3±16.0 P=0.5

RVEF (%) 40.7±9.0 45.9±5.4 40.7±6.6 P=0.2

Ventricular

108.3±13.6 138.5±12.7 146.9±10.7 P<0.001 myocardial mass (g)

Pericardial adipose

141.4±20.4 278.0±46.4 281.4±44.3 P<0.001 tissue (cm³)

5.0 4.5 12.7

LA fibrosis (%)† P=0.007

(3.3-6.3) (3.6-8.7) (7.1-15.1)

6.2 5.2 12.0

RA fibrosis (%)† P=0.01

(3.8-8.1) (4.3-8.2) (7.7-13.7)

Atrial infiltrates grade 1.0 1.5 2.0

P=0.04 (0-2)† (0.8-1.5) (1.0-1.6) (1.3-2.0)

Atrial myocardial 1.4 4.8 5.8

P=0.049 lipid (%)† (0.08-6.4) (3.8-8.5) (2.9-9.0)

591 2469 1145

ETA OD*† P=0.001

(492-659) (2207-2664) (952-1484)

513 2609 1990

ETB OD*† P=0.001

(276-602) (2248-2825) (1601-2066)

73.8 74.1 91.1

MAP (mmHg)† P=0.02

(66.4-76.3) (69.6-77.0) (79.4-104.4)

4.5 6.1 8.8

LAP (mmHg)† P<0.001

(3.7-4.9) (5.3-6.7) (6.7-9.6) Table 1 : Data expressed as mean±l SD.†Presented as median and IQR. *0D Optical Densitometry.

[00268] (i) Functional and Structural Changes

[00269] Twenty-two CMR scans were available for analysis. The remaining 8 were excluded due to either rumen contents creating artifact rendering them unanalyzable, or physical inability to fit the recumbent animal within the scanner. Table 1 shows a significant progressive increase in LA (P=0.01) and RA (P=0.04) volumes with increasing weight gain [see Figure 2] and likewise, a progressive increase in ventricular myocardial mass (P<0.001) and pericardial fat volume (P<0.001); however, there were no statistical differences in ventricular volumes or function. Interclass correlation coefficient between 2 independent observers for LA and RA volumes were 0.87 and 0.92, respectively. For other CMR measures, intra-ob server and inter-observer coefficients of variation were 3.5% and 4.9%, respectively.

[00270] (ii) Atrial Electrophysiology

[00271] Effective Refractory Period - With increasing adiposity there were no significant differences in ERP between groups at any site; LAA P=0.2, LAFW P=0.8, RAA P=0.08, RAFW P=0.2. Likewise, there was a non-significant increase in ERP between baseline, 4 month and 8 month controls (LAA: baseline: 195±10 msec, overweight: 216±18 msec, obese: 217±18, P=0.4).

[00272] (iii) Atrial Conduction

[00273] Figure 3 shows representative examples of activation maps during pacing from the LAA at baseline, overweight and obese animals. Note the progressive conduction slowing demonstrated by the progressive isochronal crowding and delayed activation. In addition, evolution of circuitous wavefront propagation is observed with increasing weight.

[00274] Figure 4 explores the changes in CV with increasing weight. There was progressive slowing in conduction with increasing weight which was most profound in the obese group; LAA PO.001, LAFW P=0.001, RAA P=0.001, RAFW P=0.001. Following adjustment for hemodynamic changes, conduction slowing remained significant; LAA P=0.01, LAFW P=0.01, RAA P=0.01, RAFW P=0.03. While baseline and overweight animals demonstrated no significant differences in CVs between CLs, obese animals demonstrated marked CL-dependency of CV with significantly greater slowing at shorter CLs; conduction slowing was significantly greater at CL 200ms compared to CL 500ms only in the obese cohort; RAFW P=0.03, RAA P=0.004, LAFW P=0.04, LAA ns. Extending these observations, Figure 5 demonstrates the differences in CV at baseline, overweight and obese animals in terms of SI and S2. As expected, the S2 coupling interval demonstrated slower conduction. However, with increasing obesity, the extent of the conduction slowing with S2 compared to SI was greater (P<0.001). These features suggest a functional component to conduction slowing observed as a result of obesity and potentially a greater interaction of triggers with the underlying substrate. Associated with atrial conduction slowing was a progressive increase in regional conduction heterogeneity, with increasing adiposity as shown in Figure 6.

[00275] There was an increase in CHI with progressive weight gain. At slower pacing (CL 500ms), while CHI was progressive with increasing weight; LAA P=0.008, LAFW P=0.001, RAA P=0.001, RAFW PO.001, after adjustment for hemodynamic variables, at some sites this relationship between obesity and CHI was ameliorated; LAA P=0.3, LAFW P=0.2, RAA P=0.1, RAFW P=0.003. However, with faster pacing (CL 200ms) there was a global increase in heterogeneous conduction (P<0.05 for all sites) and this effect largely persisted despite statistical adjustment for hemodynamic variables; LAA P=0.2, LAFW P=0.03, RAA P=0.02, RAFW P=0.001. This illustrates the contribution of co-morbid hemodynamic stress and rate-stress on deleterious atrial myocardial conduction.

[00276] In the control cohorts, there were no significant changes in CV and CHI with increasing adiposity at all sites and pacing CLs.

[00277] (v) AF Burden

[00278] With increasing weight, there was a progressive increase in spontaneous AF episodes during electrophysiology testing; baseline 2 (0-4), overweight 122 (33-376), obese 688 (90-834), P<0.001, see Figure 3 which shows an example of spontaneous left atrial ectopic activity accelerating/degenerating into atrial fibrillation. There was a significant dose-response association between increasing weight and inducible AF as quantified by the number of episodes; baseline 6 (0-32), overweight 100 (12-396), obese 830 (370-1454), P=0.001 and cumulative episode duration of AF (minutes); baseline 11 (0-69), 288 (109-465), obese 696 (238-1170), PO.001.

[00279] (vi) Fibrosis, Cellular Infiltrates and Lipidosis

[00280] Right and left atrial tissue demonstrated distorted myocyte arrangement and widening of the interstitium with obesity. Quantitative histology showed increased perivascular collagen deposition with increasing adiposity. This was most profound in the obese group, but not the baseline and overweight groups, (P=0.01), suggesting evolution with longer exposure to obesity. Alternatively, myocardial lipidosis occurred early in the overweight group with a progressive dose-dependent increase (P=0.04). Figure 7 demonstrates representative examples of atrial histology using Picrosirius staining and Oil-red-0 for animals at baseline, overweight and obese. On H&E staining, with increasing weight there were greater inflammatory cellular infiltrates in atrial tissue (P=0.04) and ventricular tissue (P=0.01).

[00281] (vii) Endothelin Receptor Expression

[00282] Figure 8 shows the changes in ET receptor subtype expression with increasing weight. The bars represent the optical density of the western blot analysis of the ET receptor proteins and comparison internal control a-tubulin. With increasing weight, there was a profound increase in ETA (panel A) and ETB receptor expression (panel B) on atrial myocardium (P=0.001 for both receptor subtypes). For ETA, there was a 4-fold and 2-fold increase in receptor density, relative to baseline in the overweight and obese groups, respectively. For ETB, there was a 5-fold and 4-fold increase in receptor density, relative to baseline in the overweight and obese groups, respectively.

[00283] Conclusion [00284] Progressive obesity predisposes to a greater burden of AF by forming a dose- dependent vulnerable electro-anatomical substrate. This occurs temporally disproportionate to the progressive hemodynamic impact of obesity, providing evidence for a direct pathogenic role of obesity on the AF substrate. These changes may be mediated through ET-dependent mechanisms.

EXAMPLE 2 - The role of endothelin-receptor blockade in the prevention of the substrate for atrial fibrillation - an interventional study in an obese ovine model

[00285] We hypothesised that direct inhibition with an endothelin receptor antagonist (ERA) would prevent the substrate for AF in obesity.

[00286] Methods: Obesity was induced in 20 sheep over 60 weeks using a high-calorie diet. Animals were randomized in equal groups to be: 1. treated with an ERA (bosentan 125mg bd) during this period; or 2. Controls. Endocardial EP studies, DEXA and CMR were performed at 0, 30 and 60 weeks to determine conduction (CV), refractoriness (ERP), AF inducibility, body fat and cardiac structure. Following terminal epicardial EP studies, tissue was harvested to determine fibrosis and protein expression.

[00287] Results: At 60 weeks, ERA treatment attenuated obesity related conduction slowing (1.15 vs 0.94m/s, p<0.001). ERP and atrial voltage did not differ but there was reduced fractionation (p=0.05) with ERA treatment. The ERA treated group had less AF induced (52.5 vs 77.5%, p=0.04) and shorter AF episodes (20.5 vs 47.0s, p=0.04) as compared to obese controls. There was reduction in atrial fibrosis (3.8 vs 6.0%, p=0.02), increased connexin-43 expression (p=0.01) and reduced expression of Angll (p=0.05), CTGF (p=0.003), PDGF (p=0.04) and IL-6 (p=0.02) with ERA treatment.

[00288] Conclusion: Intervention with an endothelin receptor antagonist prevented atrial electrical remodelling due to obesity. In particular, there was attenuation of conduction slowing, reduction in AF burden and inhibition of atrial fibrosis and inflammation. This may represent a unique therapeutic target in AF associated with obesity.

[00289] Introduction [00290] Atrial fibrillation (AF) is the most common heart rhythm disorder in adults with its prevalence reaching epidemic proportions. Patients are often highly symptomatic and notable complications include stroke, cardiomyopathy and dementia. This has resulted in significant increase in hospitalization and cost to the community. Several factors have been implicated for the burgeoning frequency of AF. In addition to the classical risk factors such as aging and hypertension, obesity has more recently emerged as an independent association with both the development and progression of the disease. Obesity is also at epidemic levels and confers a 49% increased risk of developing AF, the risk increasing in parallel with increasing BMI.4

[00291] Previous studies have provided direct evidence for the role of obesity in establishing the substrate predisposing to AF. In an ovine model, progressive weight gain was associated with conduction slowing and more inducible AF. Importantly this occurred without the usual confounding factors seen in humans, namely obstructive sleep apnoea and glucose intolerance.

[00292] Bosentan is an orally active non-peptide endothelin receptor antagonist (ERA) of both receptor subtypes, with an affinity for ET-A. It is used clinically in the management of patients with primary pulmonary hypertension. With the progressive upregulation of ET receptors associated with weight gain as demonstrated in Example 1, we hypothesized that intervention to block this pathway would prevent the development of the substrate for AF. Therefore, the present study investigated the effect of ERA on the electrophysiological and structural atrial abnormalities associated with obesity in an ovine model.

[00293] Methods

[00294] Animals were studied in accordance with the National Health and Medical Research Council of Australia guidelines on animal research. Approval for the study was granted by animal research committees of the University of Adelaide and South Australian Health and Medical Research Institute, Adelaide, Australia.

[00295] Study Protocol [00296] The study design is shown in Figure 9. The obese model and intervention are described below. Animals underwent sequential percutaneous endocardial electrophysiological and electroanatomical evaluation at baseline, 30 and 60 weeks. In addition at each time point, cardiac MRI and DEXA scans were performed. To allow more detailed electrophysiological characterization, at the terminal study a high density plaque was utilized to perform epicardial mapping. Following the terminal study, tissue was harvested to characterize the degree of atrial fibrosis, inflammation and pro-fibrotic protein expression.

[00297] Obese Ovine model

[00298] Twenty sheep (Merino Cross) had obesity induced using an ad libitum diet of high fat pellets, predominantly consisting of wheat, barley and canola seed. Excess voluntary intake was of grass alfalfa silage and hay. The pellets were gradually introduced at 8% excess basal energy requirements and rationed to 70% of the total dry matter intake. Plasma samples were collected at intervals to ensure stability of hematological and biochemical indices. This was continued for the duration of the study, with gradual increase in weights in all animals over 60 weeks. Weights were recorded weekly and reported weights were those taken in a shorn and fasted state immediately prior to electrophysiological procedures.

[00299] Animals were randomly allocated to being either in the control group or to undergo treatment with bosentan. In both groups, feeding was titrated identically to maintain a constant and gradual weight gain.

[00300] Treatment (ET Receptor Antagonist) group

[00301] Ten animals were randomized to receive bosentan treatment during the 60- week period of weight gain. Pharmacokinetic studies were performed to ensure twice daily dosing was appropriate and that significant hypotension did not occur. Therefore, treatment was given orally at the standard human dose of 125 mg twice daily. Bosentan powder (Synthesis Med Chem Pty Ltd, Melbourne, Australia) was suspended with tragacanth mucilage (125 mg/40ml). Animal age, housing conditions and feeding regimen were identical in treatment and control groups. [00302] Animal anesthesia

[00303] All procedures were performed under general anesthetic. A pre-med of diazepam 0.4 mg/kg was given prior to induction with ketamine 5 mg/kg. Isoflurane 2.5% with 4 liters/minute of oxygen was used for maintenance. Non-invasive blood pressure, heart rate, pulse oximetry and end-tidal C0₂ were continuously monitored.

[00304] Sequential endocardial electrophysiological study

[00305] Internal jugular and femoral venous access were obtained using a conventional percutaneous approach. A 10-pole coronary sinus (CS) catheter (2-5-2 mm inter- electrode spacing) was positioned with the proximal bipole at the CS ostium in best septal LAO projection. Transseptal puncture was performed using an SL0 sheath and BRK needle using conventional techniques, as described in Mahajan R, Lau DH, Brooks AG, et al. Electrophysiological, Electroanatomical, and Structural Remodeling of the Atria as Consequences of Sustained Obesity. J Am Coll Cardiol Jul 7 2015; 66: 1- 11.

[00306] Surface ECG and bipolar endocardial electrograms were monitored continuously and stored on a computer-based digital amplifier/recorder system (Lab System Pro, Bard Electrophysiology, Lowell, MA) with storage for offline analysis. Intracardiac electrograms were filtered from 30 to 500 Hz and measured with computer-assisted calipers at a sweep speed of 200mm/s.

[00307] Electroanatomical mapping

[00308] Electroanatomical maps were created in sinus rhythm using the Carto XP system (Biosense Webster, Diamond Bar, CA, USA) as described in Sanders P, Morton JB, Davidson NC, Spence SJ, Vohra JK, Sparks PB, Kalman JM. Electrical remodeling of the atria in congestive heart failure: electrophysiological and electroanatomic mapping in humans. Circulation Sep 23 2003; 108: 1461-1468. Both right and left atria were mapped using a 3.5-mm tip catheter ablation catheter (Navistar, Biosense Webster, Diamond Bar, CA, USA), with a minimum of 100 equally distributed points collected in each chamber. Endocardial contact was confirmed with a combination of electrogram stability, fluoroscopy and the CARTO point stability criteria (<6mm stability in space, <5ms in local activation time). Location, voltage and activation timing were recorded at each point. For analysis the atria was divided as follows: Septal right atrium (RA); High lateral RA; Low lateral RA; Posterior left atrium (LA); Lateral LA; and Inferior LA. The following were determined:

1. Conduction velocity in each region, determined by the mean velocity between 3- 5 pairs of points in the direction of conduction determined by isochronal maps;

2. Signal fractionation, defined as a signal greater than 50ms duration with at least four deflections;

3. Atrial voltage. Low voltage was defined as an area with three contiguous points with a bipolar voltage of <0.5mV. Scar was an area with three contiguous points <0.05mV.

[00309] Effective refractory periods

[00310] Effective refractory period (ERP) was measured at three times the tissue capture threshold at a pulse width of 2ms. This was performed from the following 8 sites: Septal RA; High lateral RA; Low lateral RA; Posterior LA; Lateral LA; Inferior LA; Proximal coronary sinus (CS); and Distal CS. Eight SI stimuli were delivered with SI cycle lengths of 400 and 200ms, with incremental S2 (5ms increments) commencing at a coupling interval of 100ms using a Micropace EPS 320 stimulator (Micropace, Canterbury, Australia). The ERP was the longest S1-S2 interval without atrial capture. The mean of two attempts was recorded. An additional attempt was made if the difference between attempts was greater than 10ms.

[00311] AF induction

[00312] Inducibility of AF was determined using a burst pacing protocol from the left atrial appendage, performed at the terminal study. Twenty impulses were delivered at the lowest cycle length with 1 : 1 atrial capture. An AF episode was defined as irregular atrial activity lasting >2 seconds. This protocol was repeated five times and the number of episodes and total duration were recorded. Sustained AF was defined as an episode lasting >20 minutes. In the event of sustained AF, no further testing was performed.

[00313] Hemodynamic recordings

[00314] Non-invasive systolic blood pressure and invasive mean right atrial and systolic right ventricular pressures were recorded at the start of each procedure. Mean left atrial pressure was recorded immediately following transseptal puncture.

[00315] Terminal epicardial electrophysiological study

[00316] Left lateral thoracotomy was performed to access the left atrium. A custom made 90-pole plaque was placed on the left atrial appendage. Pacing was performed from each of the four corners of the plaque at 400ms. Epicardial electrograms were recorded and the following determined:

1. Effective refractory periods were measured using the same protocol as the endocardial study.

2. Conduction velocity was determined using activation maps using custom software, as described in Lau DH, Mackenzie L, Kelly DJ, et al. Hypertension and atrial fibrillation: evidence of progressive atrial remodeling with electrostructural correlate in a conscious chronically instrumented ovine model. Heart Rhythm Sep 2010; 7: 1282-1290. The peak of the largest amplitude deflection on each bipolar electrogram was automatically annotated and manually verified. Local conduction velocity was calculated for each point from triangulated local vectors, allowing subsequent calculation of mean velocity for each map.

3. Conduction heterogeneity was calculated using established phase mapping techniques integrated into the software, as described in Lau DH, Mackenzie L, Kelly DJ, et al. Hypertension and atrial fibrillation: evidence of progressive atrial remodeling with electrostructural correlate in a conscious chronically instrumented ovine model. Heart Rhythm Sep 2010; 7: 1282-1290. Absolute conduction phase delay was calculated by subtracting the 5th from the 95th percentile of the phase difference distribution (P5.95). Conduction heterogeneity index was derived from dividing P5.95 by the median (P₅₀).

[00317] Cardiac MRI

[00318] Chamber volumes were measured using MRI (Siemens Sonata 1.5 Tesla, MR Imaging Systems, Siemens Medical Solutions, Erlangen, Germany). 6-mm slices were taken through the left atrium and ventricle. Images were taken using electrocardiogram- gating and periodic breath holding. Analyses were performed offline using CVI42 (Circle Cardiovascular Imaging Inc., Calgary, Canada).

[00319] Dual -Energy X-ray Absorptiometry (DEXA)

[00320] Scans were acquired using a GE-Lunar Prodigy Vision DXA bone densitometer using Encore 13.60.033 software (GE-Lunar, Madison, WI, USA). Scans were performed using Total Body scan protocol in standard or thick mode to allow for variation in tissue depth.

[00321] Tissue analysis

[00322] For the immunohistochemical study, polyclonal antibodies to TGF beta (Sigma-Aldrich, St Louis, MO, USA, Cat #SAB4502954), Angiotensin II (Abbiotec, San Diego, CA, USA, Cat#251229), CTGF (Abbiotec, San Diego, CA, USA, Cat#251261), IL-6 (Abbiotec, San Diego, CA, USA, Cat#250717) and PDGF (Abeam, Cambridge, UK, Cat#ab61219) were used at dilutions of 1 :500, 1 :250, 1 :400, 1 :500 and 1 :6400, respectively, in a standard streptavidin-biotinylated immunoperoxidase technique. All sections underwent antigen retrieval using citrate buffer pH6.0 prior to overnight antibody incubation. This was followed by a biotinylated anti-rabbit secondary (Vector Laboratories, Burlingame, CA, USA. Cat # BA-1000), then washing in phosphate buffered saline. Slides were then incubated with a streptavidin-conjugated peroxidase tertiary (Pierce, Pasadena, CA, USA. Cat # 21127). Sections were visualised using diaminobenzidine tetrahydrochloride, washed, counterstained with haematoxylin, dehydrated, cleared, and mounted on glass slides. A minus primary control, as well as a control showing the normal pattern of expression of the antigen in question, was run with each batch of slides.

[00323] Images of the tissue were digitally captured from 5 random fields per section at 40x magnification using NanoZoomer Digital Pathology System software (Hamamatsu Phonetics, Japan). Quantitative assessments of collagen content and immuno-staining were made with the appropriate color range selection using Image-Pro Premium v9.1 (Media Cybernetics Inc., Rockville, MD). Results were calculated in percentage relative to the whole tissue area.

[00324] Statistical Analysis

[00325] Normally distributed data was expressed as mean ± standard deviation and compared with unpaired t-test. Differences between groups over time were tested using analysis of variance. Conduction velocity and ERP across regions were analyzed using a mixed effects model ANOVA with sheep ID used as a random effect to account for the dependence in observations from the same animal. Fixed effects included combinations of group, time-point and region with a maximum of two fixed effects entered into the statistical model at one time. Main effects and their interaction were tested. If a significant interaction was present, post-hoc testing was performed. AF duration was non-parametric, therefore log-transformation was performed prior to t-test comparison, with values expressed as median and interquartile range. P-values of less than 0.05 were considered statistically significant. Analyses were performed using SPSS version 23 (SPSS Inc. Chicago, IL).

[00326] Results

[00327] Weight, structural and hemodynamic parameters

[00328] Table 2 presents that changes in anthropometry, hemodynamic and cardiac structure in the two groups. Both groups increased in weight and body fat equally over 60 weeks. Left and right atrial pressures significantly increased with increasing weight, accompanied by a non-significant increase in systolic blood pressure and RV systolic pressure. There was no increase in chamber volumes with increasing weight and a trend to increased LV mass with weight gain. There was no significant difference in hemodynamics, chamber volumes, LV mass or function between treatment and control groups at any timepoint.

Table 2. Structural and hemodynamic characteristics of ERA treated and control groups.

[00329] Electrophysiological parameters

[00330] Conduction velocity and conduction heterogeneity

[00331] Sequential endocardial electrophysiological studies demonstrated that there was progressive endocardial conduction slowing seen with increasing weight in both groups (p<0.001). The ERA group demonstrated significant attenuation of conduction slowing when compared with controls (p=0.001) at both 30 weeks (1.24±0.05 vs 1.00±0.05m/s) and 60 weeks (1.15±0.05 vs 0.94±0.05m/s) as shown in Figure 10 panel A. This improvement in conduction velocity was homogeneous across all atrial sites (panel B) (Site*Group interactiona l 7).

[00332] At the end of the study period, high density mapping of the LA confirmed the marked differences in the conduction properties of the atria. Conduction velocity at 60 weeks was higher in the ERA group than controls (0.98±0.03 vs 0.92±0.03m/s, p=0.001) as shown in Figure 11. Epicardial conduction was also less heterogeneous with ERA treatment (1.28±0.05 vs 1.47±0.05, p=0.02). Figure 11 shows representative examples of activation maps and phase histograms in each of the groups.

[00333] Effective refractory periods

[00334] There was no significant change in ERP with increasing weight, overall or when analysed by chamber. There was no difference in ERP between groups at mid- or endpoint at either cycle length (Figure 12). Terminal epicardial ERP determination demonstrated that there was no significant difference between the groups.

[00335] Signal fractionation

[00336] Signal fractionation increased with increasing weight in both groups (p<0.001) as shown in Figure 13. ERA treated animals had significantly less signal fractionation than control animals at 60 weeks (15.2±3.6 vs 28.6±8.0%, p=0.0001).

[00337] Signal voltage

[00338] There was no significant reduction in endocardial voltage with increasing weight or between groups (5.81±0.99 vs 5.77±1.07mV at endpoint). No areas of low voltage (<0.5mV) were seen in any animal.

[00339] AF inducibility

[00340] Figure 14 shows the AF burden at endpoint study. The ERA treated group had significantly fewer AF episodes (52.5±23.8 vs 77.5±19.8%, p=0.04). The total AF duration was also reduced by ERA treatment (20.5 [IQR 38.2] vs 47.0s [IQR 93.0], p=0.04). Three (1 ERA treated, 2 control) animals had sustained AF (>20 mins) and were excluded from inducibility and duration analysis.

[00341] Histological parameters

[00342] Tissue fibrosis

[00343] There was a reduction in interstitial fibrosis in animals treated with ERA when compared with controls. Masson's tri chrome staining of the LA demonstrated a fibrotic area of 3.81±1.73% vs 6.02±1.62% in the ERA treated and control groups respectively (p=0.02). Figure 15 (upper panel) shows representative sections of Masson's trichrome stained tissue from both groups.

[00344] Immunohistochemistry

[00345] Results of Immunohistochemistry of left atrial tissue for pro-fibrotic protein expression are shown in Figure 16. ERA treated animals had reduced expression of angiotensin II (0.65±0.13 vs 0.84±0.18%, p=0.05), connective tissue growth factor (0.89±0.30 vs 1.60±0.36%, p=0.003), and platelet derived growth factor (1.01±0.44 vs 1.47±0.22%, p=0.04) compared to obese controls.

[00346] There was no difference in TGF-β expression between groups (2.17±0.64 vs 2.21±0.94%, p=NS). There was also reduced inflammatory protein expression with ERA treatment (figure 17) with ERA treatment, with reduction in CD68 (0.35±0.09 vs 0.53±0.18, p=0.03) and IL6 expression (1.89±1.12 vs 3.42±1.28, p=0.02).. Additionally, there was increased expression of the gap junction protein connexin 43 (2.79±0.67 vs 1.94±0.54%, p=0.01) with ERA treatment, as demonstrated by immunohistochemistry (Figure 15, lower panel).

[00347] Discussion

[00348] Obesity is increasingly recognized as an important determinant of the "rising tide" in epidemic of AF. Previous studies have demonstrated the evolution of the substrate recognized to predispose to AF with progressive weight gain; however, the mechanisms for these changes have been elusive. The current study undertakes an interventional study to evaluate the role of endothelin receptor pathways in the development of the substrate for AF in obesity to provide new information on the pathophysiological links between these conditions.

[00349] Endothelin receptor blockade prevented the development of atrial substrate in an obese ovine model. Treatment with bosentan during weight gain was associated with:

1. Prevention of the marked conduction abnormalities that result from weight gain characterised by prevention of conduction slowing, conduction heterogeneity and signal fractionation;

2. Prevention of the structural changes associated with obesity. In particular there was marked attenuation of the interstitial fibrosis and preservation of gap- junctional content;

3. As a result of these changes there was a reduced vulnerability to AF.

[00350] Importantly, these effects observed with ERA, seem to be mediated through a reduction in inflammatory (IL6) and profibrotic markers (Connective tissue growth factor, Platelet derived growth factor, and Angiotensin II) but not TGF-β. Finally, these beneficial effects were observed despite the evolution of progressive obesity.

[00351] Substrate for AF

[00352] Several studies have evaluated the substrate predisposing to the development of AF. These have consistently identified the presence of diffuse atrial fibrosis with the associated conduction abnormalities as being central to the remodelling associated with conditions predisposing to AF. In particular, this has been demonstrated in the preclinical setting in models of heart failure, hypertension, sleep apnea, and coronary ischaemia. Importantly similar observations have also been confirmed in the clinic with areas of low voltage and electrical silence, conduction slowing, and fractionated electrograms. Interestingly similar changes were observed in patients with "lone AF".

[00353] Previous animal studies investigating the effect of obesity have demonstrated progressive atrial conduction slowing, increased heterogeneity, signal fractionation and increased propensity to AF. The findings were associated with diffuse, interstitial atrial fibrosis and upregulation of pro-fibrotic proteins. In the clinical setting, atrial electrophysiology has been shown to differ in obese patients with AF, with shorter ERPs and slower conduction into pulmonary veins than in lean patients with AF.

[00354] The present study confirms the findings of these prior studies demonstrating the presence of interstitial atrial fibrosis, heterogeneous and slowed conduction, with up regulation of fibrotic and inflammatory markers associated with obesity.

[00355] Intervention using an endothelin receptor blockade, partially prevented the development of these abnormalities with reduced collagen staining on Masson's trichrome, reduced inflammation and increased connexin 43 expression associated with resolution of the conduction abnormalities associated with obesity. Importantly, it reduced the atrial vulnerability to AF. Notably, there was no significant difference in haemodynamic parameters between the groups, suggesting that endothelin receptor blockade in obesity may prevent atrial fibrosis directly, without an effect on either systemic or intracardiac pressures.

[00356] Mechanisms for the formation of the atrial substrate for AF

[00357] In obese ovine subjects, there was upregulation of other profibrotic molecules, namely transforming growth factor beta (TGF- β), connective tissue growth factor (CTGF), platelet derived growth factor (PDGF), all of which have been directly implicated in atrial fibrosis. Our study investigated these protein and grants some insight into the mechanisms by which obesity causes fibrosis.

[00358] TGF-β is directly involved cardiac fibrosis. Fibroblasts exposed to TGF-β had a dose and time dependent increase in collagen production. In a canine heart failure model, atrial TGF-β was significantly upregulated in association with atrial fibrosis and blockade of TGF-β receptors resulted in reduced fibrosis and propensity to AF.

[00359] In our study, endothelin receptor blockade reduced fibrosis without an effect on TGF-β expression. This suggests that TGF-β acts independently upstream to endothelin. It is likely that TGF-β continues to have a profibrotic effect despite endothelin receptor blockade and could explain why fibrosis is not completely prevented. TGF-β antagonism may represent an alternative therapeutic option for obesity related atrial fibrosis, potentially in combination with ERA treatment.

[00360] Mice with overexpression of Angll have increased atrial fibrosis with more inducible AF. Atrial Angll expression is increased in dogs with atrial fibrosis associated with heart failure. In our study, animals treated with ERA showed downregulation of Angll receptors.

[00361] CTGF has previously been found to be a regulator of the effects of endothelin- 1 in cardiac tissue. Our results showed a significant downregulation of CTGF with ERA treatment. This is strongly suggestive that CTGF acts downstream to endothelin in the fibrotic cascade.

[00362] PDGF is associated with atrial fibrosis and AF, as previously demonstrated by a pressure overloaded mouse model. Previous studies have also shown that administration of endothelin has no significant effect on PDGF in rat aortic tissue, but blockade of endothelin receptors reduced PDGF expression in the coronary arteries of rats undergoing cardiac transplantation. We have demonstrated reduced expression in the atrial tissue of animals treated with ERA than in controls. This offers new insight into the mechanisms of fibrosis in obesity, and is suggestive that PDGF acts downstream to endothelin.

[00363] IL-6 exposure is associated with cardiac fibrosis and hypertrophy in a mouse model and serum IL-6 has been shown to correlate with human AF more than several other inflammatory markers, including c-reactive protein. ET-1 induces IL-6 release from human vascular smooth muscle cells. Our study is in keeping with this; ERA treatment was associated with reduction in IL-6 expression, suggestive of an antiinflammatory action.

[00364] Clinical Implications

[00365] Using an interventional design this study demonstrates that ERA can attenuate the formation of the AF substrate due to weight gain and obesity. It demonstrates the central role of the endothelin pathways in the formation of the substrate for AF.

[00366] Recent studies have highlighted the importance of weight and risk factor management in the management of AF. However, in the clinic, fluctuation in weight is often observed and has a proven negative impact on AF burden. Treatment with an endothelin receptor antagonist may play an important role in the clinical management of patients with AF and in particular in patents with obesity related AF.

[00367] Conclusion

[00368] Endothelin receptor pathways are important determinants of the substrate for AF in obesity. Blockade of endothelin receptors partially prevents the development of atrial substrate due to weight gain. This may represent a unique therapeutic target in AF associated with obesity.

EXAMPLE 3 - Treatment of a patient with atrial fibrillation

[00369] A patent suffering from atrial fibrillation and suitable for treatment with an endothelin receptor antagonist may be identified by a suitably qualified medical practitioner.

[00370] For example, bosentan or macitentan may be selected as a suitable endothelin receptor antagonist.

[00371] For administration of bosentan to the patient, film coated tablets or dispersible tables containing a suitable amount of bosentan may be used. Typical dosages of bosentan (as a monohydrate) in tablet form include 32 mg in a dispersible tablet, or 62.5 mg or 125 mg in film coated tablets. For example a suitable dose of bosentan for a human may be in the range from 30 to 125 mg per day.

[00372] Methods for formulation of drugs are known in the art, for as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985.

[00373] Tablets may be taken orally at a suitable frequency, for example morning and evening, with or without food. The frequency of administration may be selected by a qualified practitioner. For example, a dose of 62.5 mg to 125 mg may be administered twice daily. Suitable doses and frequency of administration for paediatric patients may be selected.

[00374] Excipients - Tablet core: maize starch, pregelatinised starch, sodium starch glycollate, povidone, glycerol dibehenate, magnesium stearate. Excipients - Film coat: hypromellose, glycerol triacetate, talc, titanium dioxide (El 71), iron oxide yellow (E172), iron oxide red (E172), ethylcellulose.

[00375] For a dispersible tablet: cellulose microcrystalline, calcium hydrogen phosphate anhydrous, croscarmellose sodium, silica colloidal anhydrous, tartaric acid, tutti frutti flavour, aspartame (E951), acesulfame potassium, magnesium stearate.

[00376] Another endothelin receptor antagonist is niacitentan. For example a suitable dose of niacitentan for a human may be in the range from 1 to 30 mg per day, and administered by way of a film coated, immediate release tablet.

[00377] Effectiveness of the endothelin receptor antagonist as a treatment for atrial fibrillation may be assessed by a medical practitioner, and any modification to dose, frequency, efficacy, side effects, and co-morbidities assessed by the practitioner.

EXAMPLE 4 - The role of endothelin-receptor blockade in the prevention of the substrate for atrial fibrillation - an interventional study in an obese ovine model

[00378] Introduction:

[00379] Endothelin-1 has been implicated in the pathogenesis of atrial fibrillation (AF), with upregulation of endothelin receptors in atrial tissue following weight gain. We hypothesised that direct inhibition with an endothelin receptor antagonist (ERA) would prevent the substrate for AF.

[00380] Methods:

[00381] Obesity was induced in 20 sheep over 60 weeks using a high-calorie diet. Animals were randomized in equal groups to be: 1. treated with an ERA (bosentan 125mg bd) during this period; or 2. as controls. Endocardial EP studies and CMR were performed at 0, 30 and 60 weeks to determine conduction (CV), refractoriness (ERP), AF inducibility and cardiac structure. Following terminal epicardial EP studies, tissue was harvested to determine fibrosis and protein expression.

[00382] Results. The results are provided in Table 3.

Table 3. Comparison of ERA treated and control animals at 60 weeks.

[00383] At 60 weeks, the following were observed with ERA treatment compared to obese controls: improved conduction velocity(1.15 vs 0.94m/s p<0.001); no change in ERP and atrial voltage; reduced fractionation (p=0.05); less AF induced (52.5 vs 77.5% p=0.04) and shorter AF episodes (20.5 vs 47.0s p=0.04); reduction in atrial fibrosis (3.8 vs 6.0% p=0.02); increased connexin-43 expression (p=0.01); and reduced expression of Angll (p=0.05), CTGF (p=0.003) and IL-6 (p=0.02).

[00384] Conclusion:

[00385] Intervention with an endothelin receptor antagonist prevented atrial electrical remodelling due to obesity. In particular, there was attenuation of conduction slowing, reduction in AF burden and inhibition of atrial fibrosis and inflammation. This may represent a therapeutic target in AF associated with obesity.

EXAMPLE 5 - Treatment with tranilast prevents atrial remodelling and AF in an obese ovine model

[00386] We sought to examine the effect of tranilast treatment on atrial electrophysiology in an obese animal model. Tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) is an anti-inflammatory/anti-fibrotic compound, predominantly affecting TGF-β.

[00387] 1. Methods

[00388] Animals were studied according to National Health and Medical Research Council of Australia guidelines on animal research.

[00389] 1.1 Study Protocol

[00390] Animals underwent sequential investigations at baseline, 32 and 64 weeks, as shown in Figure 18. Percutaneous endocardial electrophysiological and electroanatomical evaluation was performed on all animals, along with cardiac MRI and DEXA assessment of fat content. Additionally at terminal study, high-density mapping of the epicardial surface was performed using a 90-pole plaque, to allow more detailed electrophysiological study. Following this, cardiac tissue samples were taken to assess atrial fibrosis, inflammation and pro-fibrotic protein expression. [00391] 1.2 Obesity induction

[00392] Obesity was induced in sixteen Merino Cross sheep using a diet of high fat pellets consisting of wheat, barley and canola seed. Excess voluntary intake was of grass alfalfa silage and hay. The pellets were gradually introduced at 8% excess basal energy requirements and rationed to 70% of the total dry matter intake. This was continued for the duration of the study, to allow gradual increase in weights in all animals over 64 weeks. Weights were recorded weekly and reported weights were those taken in a shorn and fasted state immediately prior to electrophysiological procedures. Plasma samples were collected at intervals to ensure stability of haematological and biochemical indices.

[00393] At study commencement, animals were randomly allocated to being either in the control group or to undergo treatment with tranilast. Feeding was titrated identically in all animals to maintain a constant and gradual weight gain.

[00394] 1.3 Treatment (tranilast) group

[00395] Eight animals were randomised to receive tranilast treatment during the 60- week period of weight gain. Pharmacokinetic studies have previously been performed to ensure twice daily dosing was appropriate (unpublished). Treatment was given orally at a dose of lg twice daily. Tranilast powder (Synthesis med chem Pty Ltd, Melbourne, Australia) was suspended with tragacanth mucilage (lg/40ml).

[00396] 1.4 Animal anaesthesia

[00397] All procedures were performed under general anaesthetic. A pre-med of diazepam 0.4mg/kg was given prior to induction with ketamine 5mg/kg. Isoflurane 2.5%) with 4 litres/minute of oxygen was used for maintenance. Non-invasive blood pressure, heart rate, pulse oximetry and end-tidal C0₂ were continuously monitored.

[00398] 1.5 Endocardial electrophysiological study

[00399] Internal jugular and femoral venous access were obtained using a conventional percutaneous approach. A 10-pole coronary sinus (CS) catheter (2-5-2 mm inter- electrode spacing) was positioned via the internal jugular vein, with the proximal bipole at the CS ostium. Left atrial mapping was performed after transseptal puncture using an SLO sheath and BRK needle, as previously described.2

[00400] Surface ECG and bipolar endocardial electrograms were monitored continuously and stored on a computer-based digital amplifier/recorder system (Lab System Pro, Bard Electrophysiology, Lowell, MA) with storage for offline analysis. Intracardiac electrograms were filtered from 30 to 500 Hz and measured with computer-assisted calipers at a sweep speed of 200mm/s.

[00401] Electroanatomical mapping

[00402] Electroanatomical maps were created in sinus rhythm using the Carto XP system (Biosense Webster, Diamond Bar, CA). Right and left atrial maps were recorded using a 3.5-mm tip catheter ablation catheter (Navistar, Biosense Webster, Diamond Bar, CA) with a minimum of 80 equally distributed points collected in each chamber. Endocardial contact was confirmed with a combination of electrogram stability, fluoroscopy and the Carto point stability criteria (< 6mm stability in space, < 5ms in local activation time). Location, voltage and activation timing were recorded at each point. For analysis the atria was divided as follows: Septal right atrium (RA); High lateral RA; Low lateral RA; Posterior left atrium (LA); Lateral LA; and Inferior LA. The following parameters were determined:

[00403] Regional Conduction velocity

[00404] The direction of conduction was determined from examination of isochronal maps. 3-5 pairs of points were taken in the direction of conduction, with velocity being calculated using the distance and timing as reported by the mapping system.

[00405] Signal fractionation

[00406] This was defined as a signal greater than 50ms duration with at least four deflections.

[00407] Bipolar voltage.

[00408] Low voltage was defined as an area with three contiguous points with a bipolar voltage of <0.5mV. [00409] Scar was an area with three contiguous points <0.05mV. [00410] Effective refractory periods

[00411] Effective refractory period (ERP) was measured at three times the tissue capture threshold at a pulse width of 2ms, using a Micropace EPS 320 stimulator (Micropace, Canterbury, Australia). This was performed from the following 8 sites: Septal RA; High lateral RA; Low lateral RA; Posterior LA; Lateral LA; Inferior LA; Proximal coronary sinus (CS); and Distal CS. Eight SI stimuli were delivered with SI cycle lengths of 400 and 200ms, with incremental S2 impulses (10ms increments), commencing at a coupling interval of 100ms. The ERP was the longest S1-S2 interval without atrial capture. The mean of two attempts was recorded. An additional attempt was made if the difference between attempts was greater than 10ms.

[00412] AF induction

[00413] Inducibility of AF was determined using a burst pacing protocol from the left atrial appendage, performed at the terminal study. Twenty impulses were delivered at the lowest cycle length with 1 : 1 atrial capture. An episode was defined as irregular atrial activity lasting greater than or equal to 3 seconds. This protocol was repeated five times and the total duration was recorded. Sustained AF was defined as an episode lasting >10 minutes. In the event of sustained AF, no further testing was performed.

[00414] Hemodynamic recordings

[00415] Non-invasive systolic blood pressure and invasive mean right atrial and systolic right ventricular pressures via the SR0 sheath were recorded at the start of each procedure. Mean left atrial pressure was recorded immediately following transseptal puncture.

[00416] 1.6 Terminal epicardial electrophysiological study

[00417] Left lateral thoracotomy was performed to access the left atrium. A custom made 90-pole plaque was placed on the left atrial appendage and connected to the computer-based digital amplifier/recorder system (Lab System Pro, Bard Electrophysiology, Lowell, MA). Pacing was performed from each of the four corners of the plaque at 400ms. Epicardial electrograms were recorded and the following determined:

[00418] Effective refractory periods were measured using the same protocol as the endocardial study.

[00419] Conduction velocity was determined using activation maps analysed by custom software. The peak of the largest amplitude deflection on each bipolar electrogram was automatically annotated and manually verified. Local conduction velocity was calculated for each point from triangulated local vectors, allowing subsequent calculation of mean velocity for each map.

[00420] Conduction heterogeneity was calculated using established phase mapping techniques integrated into the software. Absolute conduction phase delay was calculated by subtracting the 5th from the 95th percentile of the phase difference distribution (P5- 95). Conduction heterogeneity index was derived from dividing P5-95 by the median (P50).

[00421] 1.7 Cardiac MRI

[00422] Chamber volumes were measured using MRI (Siemens Sonata 1.5 Tesla, MR Imaging Systems, Siemens Medical Solutions, Erlangen, Germany). 6-mm slices were taken through the left atrium and ventricle. Images were taken using electrocardiogram- gating and periodic breath holding. Analyses were performed offline using CVI42 (Circle Cardiovascular Imaging Inc., Calgary, Canada).

[00423] 1.8 Dual-Energy X-ray Absorptiometry (DEXA)

[00424] Scans were acquired using a GE-Lunar Prodigy Vision DXA bone densitometer using Encore 13.60.033 software (GE-Lunar, Madison, WI, USA). Scans were performed using Total Body scan protocol in standard or thick mode to allow for variation in tissue depth.

[00425] 1.8 Statistical Analysis

[00426] Normally distributed data was expressed as mean ± standard deviation and compared using unpaired t-test. Analysis of variance was used to detect differences between groups over time. Due to the dependence in observations from the same animal, conduction velocity and ERP across regions were analysed using a mixed effects model ANOVA. Sheep ID was used as a random effect and combinations of group, timepoint and region were used as fixed effects. A maximum of two fixed effects were entered into the statistical model at one time, with testing of both main effects and their interaction. If a significant interaction was present, post-hoc testing was performed. AF duration was non-parametric; therefore log-transformation was performed prior to t-test comparison, with values expressed as median and interquartile range. P-values of less than 0.05 were considered statistically significant. Analyses were performed using SPSS version 23 (SPSS Inc. Chicago, IL).

[00427] 2. Results

[00428] 2.1 Weight, structural and hemodynamic parameters

[00429] Table 4 presents the changes in anthropometry, hemodynamic and cardiac structure in the two groups. Both groups increased in weight equally over 64 weeks. Left and right atrial pressures significantly increased with increasing weight, accompanied by a non-significant increase in systolic blood pressure and RV systolic pressure. There was a non-significant increase in left atrial volume and left ventricular mass with increasing weight but no change in LV volume or function.

[00430] There was no significant difference in hemodynamics, chamber volumes, LV mass or function between treatment and control groups at any timepoint.

Table 4. Weight, structural and hemodynamic parameters

[00431] 2.2 Electrophysiological parameters [00432] Atrial conduction

[00433] With increasing weight, progressive slowing of atrial conduction occurred in both groups (p<0.001). Animals treated with tranilast had significantly less endocardial conduction slowing than controls (p<0.001) at both midpoint and endpoint, as shown in Figure 19. On analysis by region at end point study, this change was found to be heterogeneous, with significant differences in conduction across atrial regions. In the left atrium, tranilast treatment was associated with relative preservation of conduction on the posterior (1.18±0.10 vs 0.96±0.06m/s, p<0.001) and lateral (1.19±0.06 vs 0.95±0.05m/s, p<0.001) walls. In the right atrium, the upper lateral (1.15±0.05 vs 0.89±0.12m/s, p<0.001) wall showed significantly higher conduction velocities in tranilast treated animals.

[00434] Similarly, epicardial conduction at the 64-week end point study was higher in the tranilast treated group (1.19±0.06 vs 0.95±0.05m/s, p<0.001), as shown in Figure 20. This improvement in conduction velocity was homogeneous across all four plaque sites (site*group interaction p=0.21). Notably, there was no difference in conduction heterogeneity between treatment and control groups at any epicardial site.

[00435] Effective refractory periods

[00436] There was no significant change in ERP with increasing weight. At midpoint study, there was a significant reduction in atrial ERP following 200ms drivetrain in the tranilast treated animals, when compared with controls (136± 10 vs 146±10ms). This was not seen with a 400ms drive train and was also not apparent at end point study (Figure 21)

[00437] Signal fractionation

[00438] Signal fractionation increased with increasing weight in both groups (p<0.001) as shown in Figure 22. Tranilast treated animals had significantly less signal fractionation than control animals at 60 weeks (16.7±6.2 vs 29.3±8.8%, p=0.01).

[00439] Signal voltage

[00440] There was no significant reduction in endocardial voltage with increasing weight or between groups. No areas of low voltage (<0.5mV) were seen in any animal.

[00441] AF inducibility

[00442] The tranilast treated group had significantly less AF during the induction protocol (12s [IQR 36] vs 43s [IQR 110], p=0.05), as shown in Figure 23. Two animals (1 tranilast treated, 1 control) had sustained AF (>10mins) during testing and were excluded from inducibility analysis. [00443] 3. Discussion

[00444] This study gives us new information on the mechanism of the abnormalities in atrial electrophysiology seen in obesity. The following effects were seen with tranilast treatment:

[00445] 1) Attenuation of the marked reduction in endocardial conduction velocity associated with increasing obesity, with associated reduction in signal fractionation. Interestingly, the reduction in conduction velocities with increased weight was seen across all atrial regions, whereas the animals treated with tranilast demonstrated a more heterogeneous pattern, with sparing of certain regions in both atria.

[00446] 2) Higher epicardial conduction velocity at end point study. This was consistent across all 4 sites on the plaque. There was, however, no difference in local conduction heterogeneity at any site.

[00447] 3) As a result of this, there was a significant reduction in AF inducibility in the treatment group.

[00448] Importantly, these results occurred without effect on haemodynamics or chamber volumes. There was also a minor, but significant, reduction in atrial ERP at midpoint study. However, this was only at one cycle length and similar differences were not seen at endpoint study. Contrary to this, when used in a canine heart failure model, this effect was not seen; rather tranilast appeared to protect against the reduction in ERP associated with the development of heart failure.

[00449] Atrial substrate

[00450] Changes in atrial electrophysiology have been demonstrated with a wide variety of conditions. These were first noted secondary to prolonged rapid atrial pacing, resulting in reductions in atrial refractory periods and increased susceptibility to AF. Subsequent studies used a heart failure model to induce structural remodelling, with increases in localised conduction slowing and fibrosis of atrial tissue, neither of which were seen in animals subjected to rapid atrial pacing alone We have previously demonstrated the development of conduction slowing, without ERP change in an obese ovine model. Our study again demonstrated this progressive conduction slowing with increasing weight, without changes in ERP. [00451] TGF-β and cardiac fibrosis

[00452] TGF-β has long been recognised to be a central mediator of the fibrotic pathway. Its effect on infarcted, hypertrophic and cardiomyopathic ventricular tissue has been well characterised. More recent work has investigated its relationship with atrial fibrosis. Overexpression of TGF-β in a transgenic mouse model induced heterogeneous conduction in the left atrium, slowed conduction in the right atrium and increased susceptibility to AF. The cardiac profibrotic effects of TGF- β may be enhanced in atrial tissue. Previous studies have demonstrated TGF- β inhibition to have a disease- modifying role. Pressure overloaded rats treated with anti TGF- β antibodies demonstrated dramatically reduced myocardial fibrosis.1 Tranilast was found, in conjunction with TGF- β mRNA downregulation, to reduce ventricular fibrosis in one study of hypertensive rats, 12 along with reduced mortality. It also prevented cardiac fibrosis in rats with diabetic cardiomyopathy, despite persistent hypertension and hyperglycaemia. Although initial human studies of its effect on coronary restenosis were encouraging, a subsequent multicentre, randomised trial reported no significant effect. Recent investigation has found that it reduces both atrial fibrosis and AF inducibility when used in a canine heart failure model. Our study is in keeping with these findings. Atrial remodelling was prevented by tranilast, as demonstrated by significantly improved endocardial and epicardial atrial conduction velocities in the treatment group. This corresponded with a reduced susceptibility to AF with tranilast treatment.

[00453] TGF- β and obesity

[00454] We have previously demonstrated the progressive up-regulation of TGF- β receptors in atrial tissue during weight gain, in both the intermediate and long term. This was associated with both increased atrial fibrosis and an increased susceptibility to AF. TGF- β is expressed in adipose tissue and the degree of expression correlates with BMI in human subjects. A recent study used an original atrial organo-culture model to demonstrated that TGF- β was secreted by human epicardial fat in small amounts and this was less than by subcutaneous fat. However, Activin A, a member of the TGF superfamily, was secreted by epicardial fat significantly more than in non-cardiac fat, and its presence induced TGF- β expression.

[00455] The distribution of epicardial fat is not uniform. One study showed the SVC, left atrial appendage and left AV groove are adjacent to the largest fat depots. Another found most left atrial epicardial fat was located in the inferior and posterior region, with minimal appendage and lateral fat. Animal studies have also demonstrated infiltration of fat into cardiac tissue, in particularly in the posterior wall of the left atrium, which showed marked differences in our study, when compared with the inferior LA. This may account for the heterogeneous nature of the conduction velocities in tranilast treated animals.

[00456] Tranilast effects

[00457] Although the effect of Tranilast is predominantly on the TGF- β pathway, it has several other potentially anti-fibrotic and anti-inflammatory effects that may account for some of the observed effects. Macrophages exposed to tranilast demonstrated upregulation of the anti-inflammatory molecule heme oxygenase- 1, downregulation of the pro-inflammatory substance cyclooxygenase-2. This resulted in inhibition of prostaglandins, tumour necrosis factor and interleukin-lb and demonstrates the potential confounding effects of tranilast treatment. It also acts antagonistically on angiotensin II, inhibiting the its effects in vascular smooth muscle. In a hypertensive rat model, it inhibited PDGF as well as TGF- β induced collagen synthesis. It may also attenuate the effect of connective tissue growth factor, as shown in renal tissue. It is unclear whether these effects are directly due to tranilast, or whether they are secondary to TGF- β blockade, but further study may be warranted to isolated the exact mechanisms of pure TGF- β blockade.

[00458] 4. Conclusion

[00459] Treatment with tranilast attenuates the conduction slowing associated with obesity with minor effects on refractory periods. These conduction changes are heterogeneous across both atria. This reduction in atrial substrate results in reduced inducibility of AF. Tranilast treatment may be of benefit in obese patients with AF. [00460] 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.

[00461] As used herein, the singular forms "a," "an," and "the" may refer to plural articles unless specifically stated otherwise.

[00462] 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.

[00463] 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.

[00464] 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.

[00465] 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.

[00466] 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 for preventing and/or treating atrial fibrillation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

2. The method according to claim 1, wherein the subject is overweight or obese.

3. The method according to claims 1 or 2, wherein the atrial fibrillation comprises spontaneous atrial fibrillation, paroxysmal atrial fibrillation, persistent atrial fibrillation, or permanent atrial fibrillation.

4. The method according to any one of claims 1 to 3, wherein the method comprises reducing the frequency and/or duration of episodes of atrial fibrillation and/or reducing the severity and/or effects of atrial fibrillation.

5. The method according to any one of claims 1 to 4, wherein the subject is suffering from or susceptible to one or more of the following: reduced and/or slowed atrial conduction; reduced atrial conduction velocity; increased atrial conduction heterogeneity; atrial remodelling due to increased weight gain; atrial fibrosis; and inflammation.

6. The method according to any one of claims 1 to 5, wherein the antagonist comprises an antagonist of an endothelin- A receptor and/or an antagonist of an endothelin-B receptor.

7. The method according to any one of claims 1 to 6, wherein the antagonist comprises an antagonist of a ligand of an endothelin receptor.

8. The method according to any one of claims 1 to 7, wherein the antagonist comprises an antagonist of endothelin- 1.

9. The method according to any one of claims 1 to 8, wherein the antagonist is a non-selective endothelin receptor antagonist.

10. The method according to any one of claims 1 to 9, wherein the antagonist comprises one or more of A-186086, Ro-61-6612 (tezosentan), SB-209670, SB-217242 (enrasentan), SB-217242, PD 142,893, PD 145,065, Ro 47-0203 (bosentan), Ro 48- 5033, macitentan, ACT-132577, and/or a pharmaceutically acceptable salt, hydrate, tautomer, isomer, pre-drug and/or derivative of any one or more of the aforementioned.

11. The method according to any one of claims 1 to 10, wherein the antagonist comprises bosentan and/or a pharmaceutically acceptable salt or hydrate thereof.

12. The method according to any one of claims 1 to 10, wherein the antagonist comprises macitentan and/or a pharmaceutically acceptable salt or hydrate thereof.

13. The method according to any one of claims 1 to 8, wherein the antagonist is a selective antagonist.

14. The method according to claim 13, wherein the antagonist is a selective antagonist of an endothelin receptor A.

15. The method according to claims 13 or 14, wherein the antagonist comprises one or more of A- 127722, A- 147627 (atrasentan), A-216546, BQ-123, BQ-610, FR 139317, Lu-135252 (darusentan), PD 151,242, PD 156,707, TBC-11251 (saitaxsentan), and a pharmaceutically acceptable salt, hydrate, tautomer, isomer, pre-drug and/or derivative of any one or more of the aforementioned.

16. The method according to claim 13, wherein the antagonist is a selective antagonist of an endothelin receptor B.

17. The method according to claim 16, wherein the antagonist comprises one or more of A-192621, BQ-788, RES-701-1, Ro 46-8443 and a pharmaceutically acceptable salt, hydrate, tautomer, isomer, pre-drug and/or derivative of any one or more of the aforementioned.

18. The method according to any one of claims 1 to 17, wherein the method further comprises administering to the subject an inhibitor of the renin-angiotensin-aldosterone axis and/or a TGF-β antagonist.

19. The method according to any one of claims 1 to 18, wherein the method is used to prevent and/or treat reduced and/or slowed atrial conduction; to prevent and/or treat reduced atrial conduction velocity; to prevent and/or treat increased atrial conduction heterogeneity; to modulate atrial rhythm; to prevent and/or treat atrial remodelling due to increased weight gain; to prevent and increased atrial fibrosis; to prevent and/or treat increased atrial inflammation; to prevent and/or treat spontaneous atrial fibrillation; prevent and/or treat paroxysmal atrial fibrillation; to prevent and/or treat persistent atrial fibrillation; and/or to prevent and/or treat permanent atrial fibrillation.

20. An endothelin receptor antagonist for use in the prevention and/or treatment of atrial fibrillation.

21. Use of an endothelin receptor antagonist in the preparation of a medicament for preventing and/or treating atrial fibrillation.

22. A method for preventing and/or treating atrial remodelling due to increased weight gain, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

23. A method for preventing and/or treating reduced and/or slowed atrial conduction in subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

24. A method for preventing and/or treating reduced atrial conduction velocity in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

25. A method for preventing and/or treating increased atrial conduction heterogeneity in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

26. A method for preventing and/or treating increased atrial fibrosis in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

27. A method for preventing and/or treating increased atrial inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

28. A method of treating a subject, the method comprising:

29. The method according to claim 28, wherein the subject comprises one or more of the following characteristics: an increased body mass index; a body mass index of 25 kgm^"2 or greater; a body mass index of 30 kgm^"2 or greater; a body mass index of 25 to 29.9 kgm^"2; overweight; obesity; an increased level of intra-myocardial lipid; an increased level of inter-myocardial lipid; an increased pericardial fat volume; an increased level of intra-atrial myocardial lipid; an increased level of inter-atrial myocardial lipid; reduced and/or slowed atrial conduction; reduced atrial conduction velocity; and increased atrial conduction heterogeneity.

30. A method for reducing the likelihood of stroke in a subject suffering from or susceptible to atrial fibrillation, the method comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist.

31. A method for reducing the dose of an agent for preventing and/or treating atrial fibrillation administered to a subject, the method comprising administering to the subject an endothelin receptor antagonist and thereby reduce the dose of the agent for preventing and/or treating atrial fibrilllation.

32. A method of identifying a subject suffering from or susceptible to atrial fibrillation and suitable for treatment with an endothelin receptor antagonist, the method comprising identifying a subject with one or more of the following characteristics: an increased body mass index; a body mass index of 25 kgm^"2 or greater; a body mass index of 25 to 29.9 kgm^"2; a body mass index of 30 kgm^"2 or greater; overweight; obesity; an increased level of intra-myocardial lipid; an increased level of inter- myocardial lipid; an increased pericardial fat/lipid volume; an increased level of intra- atrial myocardial lipid; an increased level of inter-atrial myocardial lipid; reduced and/or slowed atrial conduction; reduced atrial conduction velocity; and increased atrial conduction heterogeneity.

33. A pharmaceutical composition when used to prevent and/or treat atrial fibrillation, the pharmaceutical composition comprising a therapeutically effective amount of an endothelin receptor antagonist.

34. A pharmaceutical composition when used to prevent and/or treat atrial fibrillation, the pharmaceutical composition comprising a therapeutically effective amount of bosentan and/or macitentan, and/or a pharmaceutically acceptable salt or hydrate thereof.

35. A pharmaceutical composition comprising a therapeutically effective amount of an endothelin receptor antagonist, and an inhibitor of the renin-angiotensin- aldosterone axis and/or a TGF-β antagonist.

36. A method for preventing and/or treating atrial fibrillation in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 34 to 36.

37. A kit for performing the method according to any one of claims 1 to 32.

38. A kit for preventing and/or treating atrial fibrillation, the kit comprising an endothelin receptor antagonist.

39. A combination product comprising:

one or more endothelin receptor antagonists; and

40. A method for identifying an agent for preventing and/or treating atrial fibrillation, the method comprising identifying an endothelin receptor antagonist as an agent for preventing and/or treating atrial fibrillation.

41. A method for identifying an agent for preventing and/or treating atrial fibrillation, the method comprising:

42. The method according to claims 40 or 41, wherein the test agent comprises a derivative of bosentan or macitentan.

43. An agent identified by the method according to any one of claims 40 to 42.