Cross-reference to related applications

The present application claims priority from Australian Provisional Patent Application No. 2021900882 filed on 25 March 2021, the contents of which are incorporated herein by reference in their entirety.

Technical field

The present disclosure relates to the field of therapeutic methods for the prevention or treatment of pulmonary hypertension (PH), and more particularly pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension.

Background

Pulmonary hypertension (PH) defines a group of clinical conditions in which patients present with an abnormal elevation in their pulmonary circulation pressure. The recognition of subgroups of patients sharing specific features has led to the most recent classification of PH. The current World Health Organisation (WHO) classification system for PH groups patients into classes (Classes I to V) with similar pathological findings, haemodynamic profiles, and management strategies (see Yaghi et al., J Investig Med 2020 for review). A number of therapies have been utilised to treat different classes of PH, nevertheless the morbidity and mortality of PH remains high. Critically, therapies that are effective in certain classes of PH may not be effective in other classes owing to fundamental differences in aetiology and pathobiology. For instance, treatments for class 1 PH (or PAH) are either ineffective or harmful in class 2 PH (Rosenkranz et al., Eur Heart J, 2016; Cao et al., Int J Cardiol, 2018). Accordingly, there remains an unmet clinical need for effective drug therapies for PH, and in particular certain classes thereof.

Summary

The present disclosure is based on the surprising finding that β3-adrenergic receptor agonist therapy is effective in treating pre-capillary pulmonary hypertension, and more particularly pulmonary arterial hypertension, in a mouse model thereof.

In a first aspect, the present disclosure provides a method of preventing or treating precapillary pulmonary hypertension in a subject, said method including the step of administering a therapeutically effective amount of a β3-adrenergic receptor agonist to the subject to thereby prevent or treat pre-capillary pulmonary hypertension in the subject.

In a second aspect, the present disclosure provides the use of a β3-adrenergic receptor agonist in the manufacture of a medicament for the prevention or treatment of pre-capillary pulmonary hypertension in a subject.

Suitably, the pulmonary hypertension of the above aspects is or comprises pulmonary arterial hypertension (PAH) or chronic thromboembolic pulmonary hypertension. In an example, the pulmonary hypertension is pulmonary arterial hypertension, such as idiopathic PAH, heritable PAH, drug-induced PAH, toxin-induced PAH, PAH associated with connective tissue diseases, PAH associated with human immunodeficiency virus, PAH associated with portal hypertension, PAH associated with congenital heart disease, PAH associated with schistosomiasis, persistent PH of the newborn and any combination thereof. In particular examples, the chronic thromboembolic pulmonary hypertension is due to one or more inoperable small distal pulmonary emboli.

Suitably, the β3-adrenergic receptor agonist is selected from the group consisting of mirabegron (YM178), CL 316,243, AZ002, BRL-37344, BRL-35135, amibegron (SR- 58611A), SR59104A, SR59119A, solabegron (GW427353), vibegron, MK-0634, ritobegron, BMS-187257, CGP 12177, L-755,507, L-742,791, L-750,355, L-749,372, L-796,568, LY- 368,842, SB-226552, SB-251023, ICI-D 7114, FR 149175, Ro40-2148, rafabegron, BMS- 196085, trecadrine, SB-418790, Nebivolol and pharmaceutically acceptable salts or solvates thereof. In one particular example, the β3-adrenergic receptor agonist is or comprises CL316,243. In another example, the β3-adrenergic receptor agonist is or comprises mirabegron.

With respect to the first aspect, said method can further include the step of administering to the subject a therapeutically effective amount of an endothelin receptor antagonist. With respect to the second aspect, said medicament can be formulated to be administered with an endothelin receptor antagonist.

Suitably, the endothelin receptor antagonist is selected from the group consisting of macitentan, bosentan, darusentan, sitaxsentan, tezosentan, ambrisentan, atrasentan, avosentan, clazosentan, zibotentan, edonentan, enrasentan, danusentan, A- 182086, A- 192621, ABT-627, BMS193884, BQ- 123, BQ-788, CI 1020, FR-139317, S-0139, CPU0213, J- 104132, SB- 209670, TA-0201, TAK-044, TBC3711, YM-598, ZD-1611, ZD-4054 and pharmaceutically acceptable salts or solvates thereof. In some examples, the endothelin receptor antagonist is or comprises bosentan. In a third aspect, the present disclosure provides a kit for use in the method of the first aspect, said kit comprising a β3-adrenergic receptor agonist, optionally an endothelin receptor antagonist and optionally instructions for use.

Brief description of the drawings

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Figure 1. A. Pressure-Volume Loops and Right Ventricular-Pulmonary Artery Coupling Efficiency in the Hypoxia Model. Representative right ventricular (RV) pressure- volume loops in a normoxic mouse treated with vehicle, a hypoxic mouse treated with vehicle and a hypoxic mouse treated with CL316243 are shown. Calculation of end-systolic elastance (Ees) and effective arterial elastance (Ea) is performed by decreasing preload via temporary occlusion of inferior vena cava. Ees is determined from a linear approximation to the end- systolic pressure-volume relationship while Ea is the slope of the line that connects the ventricular end-systolic point to the ventricular end-diastolic volume projected on the volume axis. In hypoxic mice, Ees increased in response to increased Ea, but as shown in the panel, this adaptation was inadequate, and Ees/Ea ratio significantly dropped compared with the normoxic mice. Treatment with CL3106243, sildenafil or CL3106243 plus bosentan resulted in significant increase in the Ees/Ea ratio compared with the hypoxic control mice. N=5-8 mice per group; * p<0.0001 compared with normoxia controls, t p <0.05 compared with hypoxia controls. B. Effects of Treatments on Right Ventricular Hypertrophy in the Hypoxia Model. Hypoxia increased RV hypertrophy as assessed by the Fulton’s index. All treatments led to a significant reduction in the RV hypertrophy, reflecting the improved RV-PA coupling efficiency and homeometric reverse remodelling of RV. N = 5-10 mice per group; * p<0.0001 compared with normoxic controls, t p<0.0001 compared with hypoxic controls.

Figure 2. A. Collagen Content in the Right Ventricle in the Hypoxia Model. Representative right ventricular sections stained with Picrosirius red (PSR) are shown. Collagen content (red colour, expressed as percentage of the area per field of view) was increased in the hypoxic mice treated with vehicle compared with normoxic mice treated with vehicle. Treatment with CL316243 (CL) alone or in combination with bosentan reduced the collagen content compared with the hypoxic control mice. N=5-6 mice/group, * p=0.0008 compared with normoxic controls, t p <0.05 compared with hypoxic controls. B. Effects of Treatments on Pulmonary Vascular Remodelling in Hypoxic Mice. Representative images of pulmonary vasculature are shown. Arrows point to a-smooth muscle actin (SMA)-positive alveolar vessels. The number of a-SMA positive intraacinar vessels were higher in hypoxic mice treated with vehicle compared with the normoxic control mice. All treatments resulted in a significant reduction in the number of a-SMA positive vessels compared with the hypoxic control mice. N=5-6 mice/group. * p<0.0001 compared with normoxic controls, t p<0.05 compared with hypoxic controls.

Figure 3. Effects of Treatments on Cell Proliferation in the Lungs of Hypoxic Mice. Representative figures for proliferating cell nuclear antigen (PCNA) and a-smooth muscle actin (SMA) immunostaining, and summary of the data for different treatment groups are shown. Hypoxia increased the proliferation index in the a-SMA positive cells in the lungs of mice treated with vehicle, while the proliferation index was reduced by all treatments. N=5 mice/group, * p=0.0007 compared with normoxic controls, t p <0.001 compared with the hypoxic controls.

Figure 4. A. Effects of Treatments on Expression of Endothelial Nitric Oxide Synthase in the Lungs of Hypoxic Mice. Immunoblots of endothelial nitric oxide synthase (eNOS) in the lungs are shown. 0 actin was used as the loading control. Mean densitometries for eNOS normalised to 0 actin are shown in the panel. Expression of eNOS was higher in the lungs of hypoxic mice compared with the normoxic control mice. N=5-6 mice. * p=0.008 compared with the normoxic controls on Kruskal-Wallis test. B. Effects of Treatments on Ghitathionylation of eNOS. Immunoblots (IB) of eNOS and GSH performed on eNOS immunoprecipitate (IP) fiom the lungs of mice are shown. Mean densitometries for GSH IB normalised to eNOS IB are shown in the panel. Glutathionylation of eNOS (eNOS-GSH) was increased in the lungs of hypoxic mice treated with vehicle compared with the normoxic controls. Treatment with CL316243 alone or in combination with bosentan reduced eNOS-

GSH compared with the hypoxic control mice. N=5-6 mice. * p=0.009 compared with the normoxic controls and t p <0.02 compared with the hypoxic controls. C. Effects of Treatments on Expression of Soluble Guanylyl Cyclase Subunits. Immunoblots of soluble guanylyl cyclase (sGC) al and 01 subunits are shown. 0 actin was used as the loading control. Mean densitometries for sGC α 1 and β 1 subunits normalised to β actin are shown in the panels. Expression of sGC α1, not 01 subunit, was reduced in the lungs of hypoxic mice compared with the normoxic controls. All treatment increased the expression of sGC α 1 compared with the hypoxic controls. N=5-7 mice. * p=0.03 compared with the normoxic controls, + p <0.03 compared with the hypoxic controls.

Figure 5. A. HPLC Analysis of Dihydroethidium Oxidation Products in the Lungs of Hypoxic Mice. Representative chromatograms from a normoxic control and a hypoxic control mouse are shown. On HPLC, the peaks for the specific (2-OH-E ⁴ ) and nonspecific products (E ⁺ ) resulting from oxidation of dihydroethidium (DHE) are detected. B. O ₂ - Levels in the Lungs. Mean values of 2-OH-E ⁺ normalised over protein concentration are shown in the panel. Hypoxia increased the O ₂ - levels in the lungs compared with the normoxic controls, and treatments led to a significant reduction in O ₂ - levels compared with the hypoxic control mice. N=5-6 control, * p=0.002 compared with the normoxic control, t p <0.03 compared with the hypoxic control. CL=CL316243

Figure 6. Effects of β3 Adrenergic Receptor Agonism in Experimental Pulmonary Arterial Hypertension. A. In pulmonary arterial hypertension, endothelial nitric oxide synthase is “uncoupled” via glutathionylation (GSS) of reactive cysteine residues, which leads to decreased generation of nitric oxide (NO) and increased generation of Oz". These changes lead to impaired downstream NO-dependent signalling in smooth muscle cells, resulting in increased vascular proliferation and vasoconstriction, and increased pulmonary arterial (PA) pressure. Consequently, the right ventricular (RV)-PA coupling efficiency is impaired, and RV becomes hypertrophied and fibrosed. B. Stimulation of β3-adrenergic receptors (beta3 ARs) by CL316243 leads to a decrease in the glutathionylation-mediated uncoupling of eNOS, decreased Oz" and increased NO-dependent signalling in smooth muscle cells, which lead to decreased proliferation and increased vasodilation, hence decrease in PA pressure. Sildenafil and riociguat target downstream cellular effectors in NO signalling. The reduction in RV afterioad results in improved RV-PA coupling efficiency and reverse remodelling of RV (reduced hypertrophy, and with C316243, reduced fibrosis). cGMP=cyclic guanosine monophosphate, GMP=guanosine monophosphate, GTP=guanosine triphosphate, PDE5=phosphodiesterase type 5, sGC-soluble guanylyl cyclase

Figure 7. Study Protocol. FiOz= fraction of inspired Oz

Figure 8. A. Fulton Index in the Sugen Hypoxia Model. Right ventricular hypertrophy was significantly reduced by all treatments compared with the Sugen hypoxia control mice treated with vehicle. N=5-8 mice/group; p<0.05 vs. normoxic control; f p<0.05 vs. hypoxic control. B. Pulmonary Vascular Remodelling in the Sugen Hypoxia Model. The number of α smooth muscle actin positive vessels in the pulmonary microvasculature was significantly increased in the Sugen hypoxia control mice, and was significantly reduced by treatments compared with vehicle. SHx=Sugen hypoxia, a SMA= a smooth muscle actin. N=5 mice/group; p<0.05 vs. normoxic control; f p<0.05 vs. hypoxic control.

Figure 9. β3AR Expression in Human PAH. On RNA microarray analysis, expression levels of β3ARs were significantly lower in the explanted lungs of patients with PAH compared with healthy controls (PAH: 6.36±0.15, n=15 vs. control: 6.5610.1, n=ll; p=0.002 on impaired t-test). RMA=robust multiarray average.

Figure 10. Design of the β3-Adrenergic Agonists in Pulmonary Hypertension Trial. (BEAT-PH Trial) The BEAT-PH trial is a proof-of-concept, investigator-initiated, single-arm, acute haemodynamic study of Mirabegron, a selective β3 AR agonist. The study consists of two parts: Part A where a single dose (25 mg) of Mirabegron is tested, and Part B where a higher dose (50 mg) of Mirabegron is tested in patients with PAH.

Detailed description

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e g., in genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the pinpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

For the present disclosure, the database accession number or unique identifier provided herein for a gene or protein, as well as the gene and/or protein sequence or sequences associated therewith, are incorporated by reference herein. Methods for preventing and treating pulmonary hypertension

The inventors have surprisingly shown for the first time that β3-adrenergic receptor agonist therapy can positively modulate cardiovascular functioning and remodelling in a mouse model of PAH, and therefore offers promise as a treatment for classes of human PH that are due to pre-capillary pathologic changes (i.e., Class 1 or Class 4 PH or PAH).

Accordingly, there is provided herein a method of preventing or treating pre-capillary pulmonary hypertension in a subject, said method including the step of administering a therapeutically effective amount of a β3-adrenergic receptor agonist to the subject to thereby prevent or treat pre-capillary pulmonary hypertension in the subject.

In a related form there is provided use of a β3-adrenergic receptor agonist in the manufacture of a medicament for the prevention or treatment of pre-capillary pulmonary hypertension in a subject.

In yet another form, there is provided a β3-adrenergic receptor agonist or a composition comprising same for use in the prevention or treatment of pre-capillary pulmonary hypertension in a subject.

With respect to the aspects described herein, the term “subject' includes but is not limited to mammals inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Preferably, the subject is a human.

Administration

Methods of treating pre-capillary PH may be prophylactic, preventative or therapeutic and suitable for treatment of pre-capillary PH in mammals, particularly humans. As used herein, “treating”, “treat' or “treatment' refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of pre-capillary PH after the pre-capillary PH and/or its symptoms have at least started to develop. As used herein, “preventing”, “prevent” or “prevention” refers to a therapeutic intervention, course of action or protocol initiated prior to the onset of pre-capillary PH and/or a symptom thereof so as to prevent, inhibit or delay the development or progression of the pre-capillary PH or a symptom thereof.

The term “therapeutically effective amount” describes a quantity of a specified agent, such as a β3-adrenergic receptor agonist or the composition described herein, sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a β3-adrenergic receptor agonist and optionally one or more further therapeutic agents, such as an endothelin antagonist, necessary to reduce, alleviate and/or prevent pre- capillary PH. Suitably, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of pre-capillary PH. More particularly, a “therapeutically effective amount” may be an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression, such as cardiovascular remodelling.

Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent usefill for reducing, alleviating and/or preventing pre-capillary PH will be dependent on the subject being treated, the type and severity of any associated disease, disorder and/or condition (e.g., disease progression), and the manner of administration of the therapeutic composition.

The amount of a β3-adrenergic receptor agonist and/or any fiuther therapeutic agents (e.g., an endothelin antagonist) contained within an individual dose or kit may be expressed in terms of milligrams per kilogram of patient body weight (i.e., mg/kg). For example, a β3- adrenergic receptor agonist, an endothelin antagonist and/or a fiuther agent, may be administered to a patient at a dose of about 0.0001 to about 50 mg/kg of patient body weight, or about 0.1 to about 100 mg/kg of patient body weight (e.g., 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5,

I.75, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,

II.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100 mg/kg, and any range therein).

In particular examples, administration of the β3-adrenergic receptor agonist described herein can inhibit cardiovascular remodelling associated with pre-capillary PH in the subject. By way of example, β3-adrenergic receptor agonist therapy may inhibit hypertrophy of the right ventricle, such as right ventricular wall thickening, in the subject. Such therapy can also inhibit or prevent muscularisation and/or thickening of the pulmonary artery and/or other pulmonary vessels (e.g., pulmonary microvasculature or microvessels) in the subject, such as inhibit fiuther muscularisation and/or thickening of the pulmonary artery and/or other pulmonary vessels in the subject from baseline (e.g., at diagnosis or the commencement of therapy). The thickening of the pulmonary artery and/or right ventricular hypertrophy may be determined by, for example, chest CT (such as, unenhanced axial 10 mm CT sections) and/or ultrasound/echocardiography, and used to calculate main pulmonary artery diameter (mPA) and/or right ventricular wall thickness. Moreover, such therapy may prevent or inhibit collagen deposition (i.e., fibrosis) in the right ventricle of subjects with PH. Accordingly, in another form, the present disclosure provides a method for preventing or treating right ventricular fibrosis in a subject having pulmonary hypertension, including the step of administering a therapeutically effective amount of a β3-adrenergic receptor agonist to the subject to thereby prevent or treat right ventricular fibrosis in the subject. In one example, the subject has pre-capillary pulmonary hypertension, such as PAH. In another example, the method further includes the step of administering a therapeutically effective amount of an endothelin receptor antagonist, such as those described herein, to the subject. To this end, the endothelin receptor antagonist can finther reduce the pulmonary pressure and/or prevent or treat right ventricular fibrosis in a subject having pulmonary hypertension.

Additionally, administration of the selective β3-adrenergic receptor agonist described herein may increase stroke volume and/or stroke volume to end systolic volume ratio (“SV7ESV”) in the subject. “Stroke volume” (“SV”) is the volume of blood pumped fiom the right or left ventricle per single contraction. Stroke volume may be calculated using measurements of ventricle volumes fiom an echocardiogram and calculated by subtracting the volume of the blood in the ventricle at the end of a beat (called “end-systolic volume,” “ESV”) fiom the volume of blood just prior to the beat (called “end-diastolic volume,” “EDV”). The term stroke volume can apply to each of the two ventricles, but more particularly right ventricular stroke volume, of the heart.

In some examples, administration of the selective β3-adrenergic receptor agonist described herein increases right ventricle cardiac output and/or ejection fraction in the subject. “Cardiac output” (“CO”) is defined as the amount of blood pumped by a ventricle, such as the right ventricle, per unit time. “Ejection fraction” is the percentage of blood that is pumped out of a filled ventricle with each heartbeat. Echocardiographic techniques and radionuclide imaging techniques can be used to estimate real-time changes in ventricular dimensions, thus computing stroke volume, which when multiplied by heart rate, gives cardiac output. Ejection fraction may be calculated using any one of the formulas known to one of ordinary skill in the art.

The therapeutic methods provided herein may improve other hemodynamic measurements in a subject having pre-capillary PH, including PAH and chronic thromboembolic PH, such as, for example, pulmonary acceleration time, the ratio of pulmonary acceleration time to ejection time, pulmonary velocity-time integral, right ventricular systolic pressure, right ventricular diastolic pressure, arterial elastance, end systolic elastance, preload recruitable stroke work, ratio of end systolic elastance to arterial elastance, right atrial pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, systemic arterial pressure, heart rate, pulmonary vascular resistance, and/or systemic vascular resistance. Methods and devices for measuring such readouts of cardiovascular and haemodynamic function are known to one of ordinary skill in the art.

As such, in still another form, the present disclosure provides a method for enhancing or improving cardiovascular function in a subject having pulmonary hypertension, including the step of administering a therapeutically effective amount of a β3-adrenergic receptor agonist to the subject to thereby enhance or improve cardiovascular function in the subject. In one example, the subject has pre-capillary pulmonary hypertension, such as PAH. In another example, the method further includes the step of administering a therapeutically effective amount of an endothelin receptor antagonist (i.e., in addition to the β3-adrenergic receptor agonist), such as those described herein, to the subject.

The methods described herein may also improve other clinical parameters, such as pulmonary function, in the subject being treated. For example, dining or following a treatment period a subject may have an increased exercise capacity or activity, as measured by, for example, a test of 6-minute walking distance (6 MWD) or measure of activity, or lowering Borg dyspnea index (BDI).

Administration of the β3-adrenergic receptor agonist provided herein may improve or increase endothelial nitric oxide synthase (eNOS) coupling, expression and/or activity in subjects having pre-capillary PH (e.g., reduce glutathionylation of eNOS). To this end, such treatment may further improve nitric oxide (NO) production, bioavailability and signalling, such as by increased activity and/or expression of effector molecules of eNOS signalling (e.g., soluble guanylyl cyclase al subunit).

The therapeutic methods described herein can also prevent or inhibit oxidative stress, and hence the production of reactive oxygen species (ROS), which can lead to the formation of a number of oxidized derivatives within one or more organs, tissues and/or cells of the subject. The presence of oxidative stress may be tested in one of three ways: (1) direct measurement of the ROS; (2) measurement of the resulting damage to biomolecules; and (3) detection of antioxidant levels. Exemplary methods of measuring oxidative stress in a cell, tissue or biological sample from a subject are well known in the art.

It is envisaged that the current methods can also improve the prognosis of the subject being treated. For example, administration of the β3-adrenergic receptor agonist to the subject with pre-capillary PH may reduce the probability of a clinical worsening event (e.g., death, lung transplantation, hospitalization for the pre-capillary PH, atrial septostomy, initiation of additional pulmonary hypertension therapy or a combination thereof) during the treatment period, and/or a modulation (i.e., an increase or decrease) from baseline in one or more biomarkers of disease progression (e.g., a reduction from baseline in serum brain natriuretic peptide (BNP) orNT-pro-BNP).

In some examples, the methods described herein provide a reduction of at least about 25%, at least about 50%, at least about 75% or at least about 80%, in probability of a clinical worsening event during the treatment period. In other examples, the current methods prevent a change of at least about 25%, at least about 50%, at least about 75% or at least about 80% of the concentration of one or more biomarkers of pre-capillary PH disease progression.

Based on the foregoing, therapy with the selective β3-adrenergic receptor agonist described herein may increase survival time of the subject. For example, the methods described herein may prolong the life of a subject having pre-capillary PH, such as PAH or chronic thromboembolic PH, from a time of initiation of treatment by, for example, at least about 15 days, at least about 30 days, at least about 60 days, at least about 90 days, at least about 120 days, at least about 150 days, at least about 180 days, at least about 210 days, at least about 240 days, at least about 270 days, about least about 300 days, at least about 330 days, at least about 360 days, at least about 1.5 years, at least about 2 years, at least about 2.5 years, at least about 3 years, at least about 3.5 years, at least about 4 years, at least about 4.5 years, or at least about 5 years inclusive of any range therein.

Pre-capillary pulmonary hypertension

As used herein, the term “pulmonary hypertension” refers to increased blood pressure with the arteries of the lungs from any cause. PH is typically defined hemodynamically as a systolic pulmonary arterial pressure greater than 30 mmHg or mean pulmonary arterial pressure (mPAP) of >20 mmHg at rest and consists of a heterogeneous group of conditions classified based on the underlying diagnostic features into 5 classes.

Owing to the increased pressure, PH damages both the large pulmonary artery and the small pulmonary artery. The wall of the pulmonary capillaries typically becomes thicker and can no longer transfer oxygen and carbon dioxide between the blood and lungs normally. Pulmonary hypertension also causes the pulmonary arteries to thicken and the channels through which blood flows narrow. Once pulmonary hypertension occurs, the right side of the heart works harder to compensate; however, the increased effort makes it enlarged and thickened. The proliferation of smooth muscle and endothelial cells, which normally exist in a resting state, leads to vascular remodelling and occlusion of the pulmonary vascular lumen. This can elicit a progressive increase in lung pressure as blood is pumped through the reduced lumen area of the pulmonary vasculature.

The term “pre-capillary pulmonary hypertension” as used herein refers to pulmonary hypertension that is not caused by elevated left-sided filling pressures in the heart. Accordingly, and as the skilled artisan will appreciate, pre-capillary pulmonary hypertension includes the clinical groups or classes 1 (pulmonary arterial hypertension), 3 (pulmonary hypertension due to lung diseases and/or hypoxia), 4 (chronic thrombo-embolic pulmonary hypertension) and 5 (pulmonary hypertension with unclear and/or multifactorial mechanisms). Accordingly, the subject’s pre-capillary pulmonary hypertension does not encompass or include pulmonary hypertension caused by or associated with left-sided heart disease (i.e., Class 2 PH), such as left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular disease, congenital/acquired left heart inflow/outflow tract obstruction, congenital cardiomyopathies).

PCT/ES2013/070611 describes the administration of a single dose of the β3-adrenergic receptor agonist BRL 37344 to a pig model of Type 4 PH (/. e., pulmonary arterial embolization with synthetic microspheres). This preliminary data demonstrates a trend for a decrease in mean and systemic pulmonary arterial pressure in the treated animals, but this decrease was not statistically significant, and the size of the error bars suggest significant variability of the data and/or a small number of animals being assessed. Furthermore, PCT/ES2013/070611 failed to disclose any improvements in pulmonary vascular resistance and right atrial pressure, which are important indicators of PH in addition to pulmonary arterial pressure. Based on the foregoing, the skilled person would appreciate that this document fails to provide any teaching that administration, and more particularly chronic or regular administration, of a β3-adrenergic receptor agonist can be useful in the treatment of Class 4 PH and more broadly, pre-capillary PH. Additionally, this data fiiils to teach or suggest to the skilled artisan that β3-adrenergic receptor agonist therapy can be successfully applied to other classes of PH, including Classes 1, 3 and 5, which differ significantly from Class 4 PH in their pathogenesis.

The inventors for PCT/ES2013/070611 further investigated the effect of β3-adrenergic receptor agonist therapy in a pig model of class 2 PH (i.e., PH caused by left-sided heart disease). It is noted that PH due to left-sided heart disease is of post-capillary origin, characterised by an increase in pulmonary capillary pressure, which is unlike that for PH of pre-capillary origin. Furthermore, it is well established that treatments for post-capillary PH are considerably different owing to these differences in aetiology and pathogenesis of this class of PH versus that for pre-capillary PH and in particular class 2 PH. To this end, treatments for pre-capillary PH can have no effect or even harm patients with post-capillary PH owing to left- sided heart disease (see, e.g., Cao etal., hit J Cardiol, 2018, volume 273, P213-220). Therefore, even though this document posits that β3-adrenergic receptor agonists may be usefol in pre- capillary PH, it fails to provide any conclusive data that might indicate or suggest that there is a reasonable expectation of such therapeutic agents being beneficial in this regard. Indeed, the art of record demonstrating that treatments for post-capillary PH do not also translate to being beneficial, but may actually be detrimental, in pre-capillary PH would suggest otherwise.

Patients in class 3 PH (PH due to lung disease and/or hypoxia) and class 4 PH (chronic thromboembolic PH or CTEPH) develop a phenotype that is distinct from PH due to left heart failure (i.e., increase in pressure at post-capillary venules and “back-pressure” through to pulmonary arteries) and may thus also benefit from the therapeutic methods described herein.

In particular examples, the subject’s pre-capillary pulmonary hypertension belongs to Class 1 PH (i.e., pulmonary arterial hypertension) or Class 4 PH (i.e., chronic thromboembolic pulmonary hypertension). More particularly, the subject’s pre-capillary pulmonary hypertension belongs to Class 1 PH (i.e., the subject has PAH). In some examples, the subject’s pre-capillary pulmonary hypertension does not belong to Class 4 PH (i.e., the subject does not have chronic thromboembolic pulmonary hypertension).

The term “pulmonary arterial hypertension” (“PAH”) is used herein to indicate a progressive lung disorder which is characterized by sustained elevation of pulmonary artery pressure. Those patients with PAH typically have pulmonary artery pressure that is equal to or greater than 25 mmHg with a pulmonary capillary or left atrial pressure equal to or less than 15 mmHg. Exemplary forms of PAH include idiopathic PAH, heritable PAH, drug-induced PAH, toxin-induced PAH, PAH associated with connective tissue diseases, PAH associated with human immunodeficiency virus, PAH associated with portal hypertension, PAH associated with congenital heart disease PAH associated with schistosomiasis, and persistent PH of the newborn.

For the present disclosure, the subject with pre-capillary PH, such as PAH, may be undergoing a treatment regimen (preventative and/or therapeutic). In this regard, the subject may have been determined to either (i) have existing pre-capillary PH; or (ii) be predisposed to pre-capillary PH. Accordingly, the methods of the present disclosure may further include the step of diagnosing or selecting subjects having or being pre-disposed to pre-capillary hypertension for treatment with the β3-adrenergic receptor agonist described herein.

P3-adrenergic receptor agonists The term “β3-adrenergic receptor agonist” refers to a compound that binds to the β3- adrenergic receptor and activates it. The activity of a compound as an adrenergic β3-receptor agonist can be assessed, for example, by the adenosine monophosphate accumulation assay (see, e.g., Takasu et al., J Pharmacol Exp Ther., 2007, 321: 642-647) or by the ability to stimulate the production of adenyl cyclase activity in adipocytes (see, e.g., Cooper et al., J. Biol. Chem., 1979, 254: 8927-8931).

Non-limiting examples of β3-adrenergic receptor agonists that may be used in the methods described herein include mirabegron (YM178), CL 316,243, AZ002, BRL-37344, BRL-35135, amibegron (SR-58611A), SR59104A, SR59119A, solabegron (GW427353), vibegron, MK-0634, ritobegron, BMS-187257, CGP 12177, L-755,507, L-742,791, L- 750,355, L-749,372, L-796,568, LY-368,842, SB-226552, SB-251023, ICI-D 7114, FR 149175, Ro40-2148, rafabegron, BMS-196085, trecadrine, SB-418790, Nebivolol and pharmaceutically acceptable salts or solvates thereof. In some examples, the β3-adrenergic receptor agonist is not BRL-37344.

In some examples, the β3-adrenergic receptor agonist can be considered to be a selective β3-adrenergic receptor agonist. The term “agonist” as used herein refers to a molecule having the ability to promote or activate a biological function of a target protein, such as a β3- adrenergic receptor. The terms “selectively agonise” or “selectively activate” refers to the agent's ability to preferentially increase or promote the target protein or receptor’s signalling activity, such as activation of an associated G protein, as compared to off-target signalling activity, via direct or indirect interaction with the target (e.g., the β3-adrenergic receptor).

Suitably, the β3-adrenergic receptor agonist can be administered at a daily dosage ranging from about 1 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 20 mg, about 20 mg to about 30 mg, about 30 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg, about 350 mg to about 400 mg, about 400 mg to about 450 mg, or about 450 mg to about 500 mg. More particularly, the β3- adrenergic receptor agonist may be administered to a subject, such as a human subject, at a daily dosage ranging from about 5 mg to about 300 mg (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 mg inclusive of any range therein) and more particularly from about 25 mg to about 50 mg. In various examples, the β3-adrenergic receptor agonist can be administered at a daily dosage of about 25 mg. In other examples, the β3-adrenergic receptor agonist can be administered at a daily dosage of about 50 mg. The β3-adrenergic receptor agonist can be derived from phenylethanolamine, with the following general formula: wherein R1 and R2 can represent various meanings, as detailed below.

In a particular example, R1 is selected from hydrogen and halogen (F, Cl, Br or I); the halogen is preferably chlorine. R1 can be in any position (ortho, meta or para); in a preferred example, R1 is in the meta position.

In another example, R2 is an aralkyl, being able to be substituted in the aryl part and/or in the alkyl part, or a radical selected from: and

Particular R2 radicals are indicated below:

In a preferred example, R1 represents chlorine in meta position and R2 is an optionally phenyl-substituted l-methyl-2-phenylethyl radical.

In some examples, the β3-adrenergic receptor agonist is or comprises CL316,243. CL- 316,243 (CAS No. 138908-40-4) has the chemical name of disodium 5-[(2R)-2-[[(2R)-2-(3- Chlorophenyl)-2-hydroxyethyl]amino]propyl]-l,3-benzodioxole- 2,2-dicarboxylate and whose formula is:

In other examples, the β3-adrenergic receptor agonist is or comprises mirabegron. Mirabegron has the chemical name 2-(2-aminothiazol-4-yl)-4'-(2-((2-hydroxy-2- phenylethyl)amino)ethyl)acetanilide and is commercially available under the trade name Myrbetriq™. A pharmaceutically acceptable salt or solvate of mirabegron (e.g., mirabegron hydrochloride) may be used. Mirabegron has the below chemical formula:

Mirabegron may be administered via routes known in the ait, such as by oral administration. The amount of mirabegron or a pharmaceutically acceptable salt or solvate thereof administered to the patient can be in the range of, for example, about 1 mg to about 500 mg per day. In certain examples, mirabegron is administered (e.g., orally) at a daily dosage ranging from about 1 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 20 mg, about 20 mg to about 30 mg, about 30 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg, about 350 mg to about 400 mg, about 400 mg to about 450 mg, or about 450 mg to about 500 mg.

In one particular example, mirabegron is administered to a subject, such as a human subject, at a daily dosage ranging from about 5 mg to about 300 mg (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 mg inclusive of any range therein) and more particularly from about 25 mg to about 50 mg. In one specific example, mirabegron is administered to the subject at an initial daily dosage of about 25 mg, such as for at least about 1, 2, 3, 4, 5, 6, 7 or 8 days, followed by a subsequent daily dosage of about 50 mg to about 100 mg. As such, the expected initial dose can be about 25 mg/24 hours, being titrated to about 50 mg/24 hours. This dosage can then be titrated higher, such as to about 100 mg/24 hours, or lower as is appropriate for the subject. The dose of 50 mg/24 hours is an example of an average dose that can be easily extrapolated to patients with whom an efficacy study could be conducted knowing that the side effect profile is favourable. In another example, mirabegron may be administered to the subject at a daily dosage of any of 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, or 200 mg, for the duration of treatment. The total treatment duration may be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days. The total treatment duration may be, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more. Suitably, mirabegron is administered to the subject at a daily dose of at least about 25 mg or at least about 50 mg. In this regard, mirabegron may be administered to the subject at a daily dose of about 25 mg or about 50 mg. In various examples, mirabegron is administered at a daily dosage of about 25 mg. In other examples, mirabegron is administered at a daily dosage of about 50 mg. In some examples, mirabegron is administered at a daily dosage of about 75 mg. In other examples, mirabegron is administered at a daily dosage of about 100 mg. In certain examples, mirabegron is administered at a daily dosage of about 125 mg. In particular examples, mirabegron is administered at a daily dosage of about 150 mg. In various examples, mirabegron is administered at a daily dosage of about 175 mg. In some examples, mirabegron is administered at a daily dosage of about 200 mg. In other examples, mirabegron is administered at a daily dosage of about 225 mg. In certain examples, mirabegron is administered at a daily dosage of about 250 mg. In particular examples, mirabegron is administered at a daily dosage of about 275 mg. In specific examples, mirabegron is administered at a daily dosage of about 300 mg.

In one example, mirabegron may be administered to the subject at an initial daily dosage of 25 mg, followed by a daily dose of 50 mg for 8 or 9 days. In any of the methods disclosed herein, the daily dosage of mirabegron may not exceed 300 mg per day.

In some examples of the methods described herein, the β3-adrenergic receptor agonist may be administered to the subject as a monotherapy (i.e., as the only therapeutic agent). It is envisaged, however, that the β3-adrenergic receptor agonist may alternatively be administered in combination with one or more further or additional therapeutic agents.

Accordingly, the methods described herein can finther comprise administering a therapeutically effective amount of one or more further therapeutic agents. In particular examples, the further therapeutic agent is selected from the group consisting of an anticoagulant (e.g., warfarin), a diuretic (e.g., chlorthalidon, indapamid, bendrofhimethiazid, metolazon, cyclopenthiazid, polythiazid, mefrusid, ximapid, chlorothiazid and hydrochlorothiazide), a cardiac glycoside, a calcium channel blocker (e.g., diltiazem, felodipine, amlodipine and nifedipine), a vasodilator (e.g., prostacyclin, epoprostenol, treprostinil and nitric oxide (NO)), a prostacyclin analogue (e.g., ilomedin, treprostinil and epoprostenol), an endothelin antagonist (e.g., an endothelin receptor antagonist), a phosphodiesterase (PDE) inhibitor (e.g., a phosphodiesterase V inhibitor, such as tadalafil, sildenafil and vardenafil), a soluble guanylyl cyclase activator (e.g., riociguat), an endopeptidase inhibitor, a lipid lowering agent (e.g., HMG CoA reductase inhibitors such as simvastatin, pravastatin, atorvastatin, lovastatin, itavastatin, fluvastatin, pitavastatin, rosuvastatin, ZD-4522 and cerivastatin), an angiotensin converting enzyme (ACE) inhibitor (e.g., enalapril, ramipril, captopril, cilazapril, trandolapril, fosinopril, quinapril, moexipril, lisinopril and perindopril), an angiotensin receptor inhibitor or blocker (ARB) (e.g., losartan, candesartan, irbesartan, embusartan, valsartan and telmisartan) and a thromboxane inhibitor. In some examples, the current methods comprise administering a therapeutically effective amount of at least one or more therapeutic antibodies, or fragments thereof, such as an anti- Grem 1 antibody, an anti-PDGFRβ antibody, an anti-TLR4 antibody, an anti-TLR2 antibody, an anti-endothelin (EDN1) antibody, an anti-endothelin receptor (ETA and/or ETB) antibody, an anti-osteoprotegerin (OPG) antibody and an anti-ASICl antibody.

In particular examples, however, the β3-adrenergic receptor agonist is not to be administered with a therapeutic agent that modulates the NO pathway. For example, the β3- adrenergic receptor agonist is not to be administered with a PDE inhibitor (e.g., a PDE type 5 inhibitor), such as sildenafil, tadalafil and vardenafil, a soluble guanylyl cyclase (sGC) activator or stimulator, such as riociguat, NO, an NO donor (e.g., a nitrate), such as nitroglycerin, or any combination thereof.

Example 1 herein demonstrates that administration of a β3-adrenergic receptor agonist recoupled eNOS function by reducing glutathionylation - an oxidative modification that is a well-established mechanism of eNOS uncoupling. This effect, along with other biological effects of β3-adrenergic receptor agonist therapy described herein may confer advantages compared with current PH therapies that target the downstream cellular effectors of NO, such as PDE-5 inhibitors (e.g., sildenafil, tadalafil) and soluble guanylate cyclase (sGC) activators (e.g., riociguat).

The skilled person will appreciate that PDE5 inhibitors (e.g., sildenafil and tadalafil) competitively bind to the catalytic domain of PDE5 and prevent cGMP binding and hydrolysis, while sGC activators, such as riociguat, bind to the reduced (heme-bound) form of sGC to activate sGC via two different mechanisms: stabilizing the NO-sGC complex in the presence of NO, or activating sGC directly in the absence of NO by binding to a separate regulatory site. The efficacy of PDE5 inhibitors is typically dependent on the presence of cGMP, which in turn is dependent on the bioavailability of NO. Since NO can be progressively depleted as PH worsens, the efficacy of PDE5 inhibitors can be diminished. Similarly, so can the NO- dependent activity of sGC stimulators. The effect of β3-adrenergic receptor stimulation to recouple eNOS means that this pathway exerts its effect upstream of the aforementioned therapies, and, contrary to PDE5 inhibitors and sGC stimulators, by increasing NO levels (see Figure 6). Additionally, β3-adrenergic receptor agonists can reduce the levels of oxidative stress and, as a result, the levels of peroxynitrite (ONOO"), whereas sGC stimulators and PDE5 inhibitors tend not to impact the levels of ONOO ". Thus, when these other agents become ineffective in the treatment of PH, β3-adrenergic receptor agonists could be considered as an effective alternative treatment.

In addition to the above, PDE5 inhibitors and sGC stimulators can cause systemic hypotension, a side effect that can limit the dose up-titration in clinical practice. Conversely, β3-adrenergic receptor agonists do not typically decrease the systemic arterial pressure.

In view of the above, β3-adrenergic receptor agonists may prove to be suitable alternative therapies in subjects with PH, and more particularly PAH, in whom PDE5 inhibitors and sGC stimulators are ineffective (e.g., due to the progressive decrease in NO bioavailability as PAH progresses) or poorly tolerated (e.g. , due to side effects, such as systemic hypotension).

Accordingly, in some examples, the subject may have been previously or is currently being treated with a PDE5 inhibitor and/or a sGC stimulator. In this regard, the current methods may, but do not necessarily require, the initial step of determining the efficacy and/or tolerability (e.g., monitoring for any side effects) of one or more sGC inhibitors and/or one or more PDE5 inhibitors in the subject with pre-capillary PH before administering the β3- adrenergic agonists as disclosed herein. Thus, the methods disclosed herein may be performed on a subject in which one or more sGC inhibitors and/or one or more PDE5 inhibitors have been determined to be ineffective or have reduced efficacy and/or have minimal or low tolerability (e.g., demonstrate adverse or side effects) in the subject.

In other examples, the β3-adrenergic receptor agonist is not to be administered to patients with one or more of the following diseases, disorders or conditions:

• Significant hepatic (e.g., transaminases or bilirubin x 3 above upper reference level) or renal impairment (e.g., GFR< 30 ml/min/1,73 m2)

• Significant hypotension (defined as symptomatic systolic blood pressure < 80 mmHg) or hypertension (defined as systolic >180 mmHg and/or diastolic blood pressure >110 mmHg)

• Right heart failure defined with PH associated with systemic venous congestion and fluid retention (e.g., peripheral oedema, ascites)

• Left heart failure (e.g., LVEF <50% and clinical signs of left heart failure)

• Severe interstitial lung disease with forced vital capacity (FVC) < 70%

• Congenital or drug-induced QT prolongation

• Patients with known bladder outlet obstruction and patients taking antimuscarinic medications for overactive bladder syndrome • Anaemic patients with Hb <100 mg/dL and iron deficiency or gastrointestinal bleed within 6 months

• Treatment with a tricyclic antidepressant or CYP2D6 substrates other than beta- blockers or treatment with digoxin

• Pregnant and/or breast-feeding females

In particular examples which relate to combination therapies, the β3-adrenergic receptor agonist and the finther therapeutic agent, such as the endothelin antagonist, may be administered simultaneously, concurrently, sequentially, successively, alternately or separately in any particular combination and/or order. By way of example, the β3-adrenergic receptor agonist can be administered (i) prior to; (ii) after; or (iii) simultaneously with, the administration of the finther therapeutic agent. In one particular example, the β3-adrenergic receptor agonist can be administered (i) prior to; (ii) after; or (iii) simultaneously with, the administration of the endothelin antagonist. In some examples, administration of the β3- adrenergic receptor agonist and the finther therapeutic agent, such as the endothelin antagonist (either sequentially, concurrently etc.) results in treatment or prevention of pre-capillary PH that is greater than such treatment or prevention fiom administration of said agents in isolation.

Simultaneous administration typically includes administration at substantially the same time. This form of administration may also be referred to as “concomitant” administration. Concurrent administration includes administering the active agents within the same general time period, such as on the same day(s), but not necessarily at the same time. Alternate administration includes administration of one agent during a time period (e.g., over the course of a few days or a week), followed by administration of another agent dining a subsequent period of time (e.g., over the course of a few days or a week), and then repeating the pattern for one or more cycles. Sequential or successive administration includes administration of one agent during a first time period (e.g., over the course of a few days or a week) using one or more doses, followed by administration of another agent during a second and/or additional time period (e.g., over the course of a few days or a week) using one or more doses. An overlapping schedule may also be employed, which includes administration of the active agents on different days over the treatment period, but not necessarily according to a regular sequence. Variations on these general guidelines may also be employed, such as is appropriate for the agents used and the condition of the subject.

In some examples, a subject has a diagnosis of pre-capillary pulmonary hypertension in class 1 PH (i.e., PAH) or class 4 PH (i.e., chronic thromboembolic PH), such as chronic pulmonary hypertension due to inoperable small distal pulmonary emboli. In particular examples, the subject has a diagnosis of class 1 PH. In other examples, the subject has a diagnosis of class 4 PH. In this regard, the subject may have been diagnosed with chronic pulmonary hypertension due to one or more inoperable small distal pulmonary emboli.

Suitably, right heart catheterisation is clinically indicated for the subject. In some examples, the subject has class 1 PH and right heart catheterisation is clinically indicated for the subject. In other examples, the subject has class 4 PH and right heart catheterisation is clinically indicated for the subject.

Suitably, the subject has not received prior treatment with an endothelin receptor antagonist (ERA) (e.g. , Bosentan, Macitentan or Ambrisentan), a PDE5 inhibitor (e.g., tadalafil or sildenafil) or a soluble guanylyl cyclase activator (e.g., Riociguat). In some examples, the subject has class 1 PH and has not been previously treated with an ERA, a PDE5 inhibitor or a soluble guanylyl cyclase activator. In other examples, the subject has class 4 PH and has not been previously treated with an ERA, a PDE5 inhibitor or a soluble guanylyl cyclase activator.

Suitably, the subject is currently being treated with an ERA as a monotherapy. In some examples, the subject has class 1 PH and is currently being treated with an ERA as a monotherapy. In other examples, the subject has class 4 PH and is currently being treated with an ERA as a monotherapy.

Suitably, the subject is currently being treated with an ERA and either a PDE5 inhibitor or a soluble guanylyl cyclase activator as a combination therapy, wherein treatment with the PDE5 inhibitor or the soluble guanylyl cyclase activator is withheld for a period of time (e.g., 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 etc days) prior to administration of the therapeutically effective amount of the β3-adrenergic receptor agonist to the subject. In particular examples, the PDE5 inhibitor or the soluble guanylyl cyclase activator is withheld for a period of time of about 2.0 days prior to administration of the therapeutically effective amount of the β3- adrenergic receptor agonist to the subject. In certain examples, the subject is being treated with ambrisentan and sildenafil, wherein treatment with the sildenafil is withheld for about 2.0 days prior to administration of the therapeutically effective amount of the β3-adrenergic receptor agonist (e.g., mirabegron) to the subject. In some examples, the subject has class 1 PH and is currently being treated with an ERA and either a PDE5 inhibitor or a soluble guanylyl cyclase activator as a combination therapy, wherein treatment with the PDE5 inhibitor or the soluble guanylyl cyclase activator is withheld for a period of time prior to administration of the therapeutically effective amount of the β3-adrenergic receptor agonist to the subject. In some examples, the subject has class 4 PH and is currently being treated with an ERA and either a PDE5 inhibitor or a soluble guanylyl cyclase activator as a combination therapy, wherein treatment with the PDE5 inhibitor or the soluble guanylyl cyclase activator is withheld for a period of time prior to administration of the therapeutically effective amount of the β3- adrenergic receptor agonist to the subject.

Suitably, the β3-adrenergic receptor agonist is mirabegron. In various examples, the subject has class 1 PH and mirabegron is administered to the subject at a daily dose of about 25 mg or about 50 mg. In some examples, the subject has class 1 PH and mirabegron is administered to the subject at a daily dose of about 25 mg. In other examples, the subject has class 1 PH and mirabegron is administered to the subject at a daily dose of about 50 mg. In various examples, the subject has class 4 PH and mirabegron is administered to the subject at a daily dose of about 25 mg or about 50 mg. In some examples, the subject has class 4 PH and mirabegron is administered to the subject at a daily dose of about 25 mg. In other examples, the subject has class 4 PH and mirabegron is administered to the subject at a daily dose of about 50 mg.

Endothelin antagonist

In specific examples, the methods described herein further include the step of administering to the subject a therapeutically effective amount of an endothelin antagonist, such as an endothelin receptor antagonist.

Example 1 herein demonstrates that β3-adrenergic receptor agonists may be effective additions to endothelin receptor antagonists in the treatment of PH. In particular, this combination led to significantly improved RV-PA coupling efficiency . While additional beneficial effects of combination therapy with PDE5 inhibitors or sGC stimulators and an endothelin receptor antagonist are also shown in clinical studies, the unique effects of β3- adrenergic receptor agonist therapy, alone or combined with an endothelin receptor antagonist, in reducing RV fibrosis in PH indicate likely improved patient outcomes.

The term “endothelin antagonist refers to a compound or agent that broadly blocks or inhibits endothelin receptor-mediated signal transduction. The term “endothelin receptor antagonist refers to a compound or agent that selectively blocks or inactivates an endothelin receptor. As used herein, the term “selectively blocks or inactivates” refers to a compound that preferentially binds to and blocks or inactivates endothelin receptor with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the G protein- coupled receptor family. Compounds that selectively block or inactivate an endothelin receptor, but that may also block or inactivate other G protein-coupled receptors sub-types, as partial or full antagonists, are also contemplated. This may include any compound that can directly or indirectly block the signal transduction cascade related to the endothelin receptor and includes compounds acting directly (e.g., by binding) on endothelin, endothelin receptor type A (ETA) and/or endothelin receptor type B (ETB) proteins and that are able to prevent the interaction or binding between endothelin and its receptors). Endothelin receptor antagonists further include dual ETA and ETB receptor antagonists (i.e., ETA/ETB antagonists), selective ETA receptor antagonists or selective ETB receptor antagonists. The endothelin antagonist may also include compounds that inhibit the binding of endothelin to an endothelin receptor or inhibit endothelin downstream signalling. Such antagonists can further include endothelin inhibitors, such as compounds able to prevent the action of endothelin (ET-1) on its receptors ETA and ETB, inhibitors of endothelin formation and inhibitors of endothelin expression. Typically, an endothelin antagonist is a small organic molecule, an oligonucleotide, a polypeptide, an aptamer, an antibody or an intra-antibody.

Exemplary endothelin receptor antagonists include, but are not limited to, macitentan, bosentan, darusentan, sitaxsentan, tezosentan, ambrisentan, atrasentan, avosentan, clazosentan, zibotentan, edonentan, enrasentan, danusentan, A- 182086, A-192621, ABT-627, BMS193884, BQ-123, BQ-788, CI 1020, FR-139317, S-0139, CPU0213, J- 104132, SB-209670, TA-0201, TAK-044, TBC3711, YM-598, ZD-1611, ZD-4054 and pharmaceutically acceptable salts or solvates thereof.

Three main types of endothelin receptor antagonist exist: (1): selective ETA receptor antagonists (sitaxentan, ambrisentan, atrasentan, BQ-123, zibotentan), which affect endothelin A receptors; (2): dual antagonists (bosentan, macitentan, tezosentan), which affect both endothelin A and B receptors; and (3) selective ETB receptor antagonists (BQ-788 and A192621) which affect endothelin B receptors. The efficacy of β3 -adrenergic receptor agonist described herein in addition to bosentan, a dual antagonist of endothelin A and B receptor, indicates that β3-adrenergic receptor agonist may be effective in addition to the currently used selective ETA, selective ETB and/or dual ETA/ETB receptor antagonists for the treatment of PH.

In one example, the endothelin receptor antagonist is or comprises bosentan. Bosentan is an endothelin receptor antagonist, belonging to a class of highly substituted pyrimidine derivatives. Bosentan is marketed in its monohydrate form, chemically known as 4-tert-butyl- N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-[2,2']-bipyrim idin-4-yl]- benzenesulfonamide monohydrate, which is represented by the formula below.

The term “pharmaceutically acceptable salf’ refers to a salt of a compound, which possesses the desired pharmacological activity of the parent compound. Such salts include, for example: (1) acid addition salts, formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric add, and the like; or formed with organic acids, such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4- methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminium ion; or coordinates with an organic base, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like.

Formulation

Suitably, the various therapeutic agents and/or composition described herein are administered to a subject as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient. In this regard, any dosage form and route of administration, such as those provided herein, may be employed for providing a subject with the composition provided herein.

Accordingly, in one broad form, the present disclosure provides a composition or medicament comprising a β3-adrenergic receptor agonist, optionally one or more further therapeutic agents (e.g., an endothelin antagonist) and optionally a pharmaceutically acceptable carrier, diluent or excipient for use in the treatment and/or prevention of pre-capillary PH, and more particularly PAH, in a subject.

By “pharmaceutically-acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfides, organic acids such as acetates, propionates and malonates and pyrogen-free water.

A usefill reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference. Any safe route of administration may be employed for providing a patient with the composition of the present disclosure. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this pinpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

Compositions of the present disclosure suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre- determined amount of one or more therapeutic agents of the present disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in- water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the present disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such an amount as is pharmaceutically effective. The dose administered to a patient, in the context of the present disclosure, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

Suitably, the various therapeutic agents described herein, such as the β3-adrenergic receptor agonist (e.g., mirabegron) and the endothelin receptor antagonist, are to be administered in a repetitive or repeated manner at specific time intervals between doses so as to achieve a desired therapeutic effect. By way of example, one or more of the therapeutic agents, such as the β3-adrenergic receptor agonist, may be administered in a repetitive manner at dosing intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours (or any range therein).

It is envisaged that the β3-adrenergic agonist and/or the further therapeutic agent, such as an endothelin antagonist, can be formulated as discrete doses, such as in the form of a kit. Such a kit may further comprise a package insert comprising printed instractions for simultaneous, concurrent, sequential, successive, alternate or separate use of the inhibitors in the treatment and/or prevention of pre-capillary PH, as described herein, in a patient in need thereof. Accordingly, the aforementioned kits are suitably for use in a method of treating and/or preventing pre-capillary PH, as described herein.

Instructions supplied in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine - readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. The instructions relating to the use of the β3-adrenergic receptor agonist and optionally the further therapeutic agent (e.g., an endothelin antagonist), generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The kit may further comprise a description of selecting an individual suitable for treatment.

Alternatively, the β3-adrenergic agonist and/or the further therapeutic agent, such as an endothelin antagonist, can be formulated together in a composition that optionally includes a pharmaceutically acceptable carrier, excipient or diluent.

So that preferred embodiments of the present disclosure may be folly understood and put into practical effect, reference is made to the following non-limiting examples.

Examples

Example 1. Determining the effect of a β3-adrenergic agonist in mouse models of pulmonary arterial hypertension

Materials and Methods

Experimental Models of PAH and Treatments

Using adult male FVB/N mice (10-16 weeks old) with a body weight of 20-25 g (Australian BioResources, Moss Vale, NSW, Australia), we established two models of PAH: i) hypoxia model (Hx), where mice were exposed to chronic hypoxia (fraction of inspired Ch [FiCh]=0.10) for 3 weeks in a hypoxia chamber (BioSpherics, Parish, NY, USA), ii) Sugen hypoxia model (SUHx), where mice were exposed to chronic hypoxia (FiCh =0.10) for 3 weeks and received weekly subcutaneous injection of the vascular endothelial growth factor receptor inhibitor SU5416 (20 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) (total 3 injections, Figure 7) [7], In both models, mice were housed in the hypoxia chamber for an additional 2 weeks during which they were treated by daily gavage with riociguat (MedChemExpress, Monmouth Junction, NJ, USA) at a dose of 10 mg/kg/day (n= 10/model) [7], or sildenafil (MedChemExpress, Monmouth Junction, NJ, USA) at a dose of 50 mg/kg/day (n= 10/model) [7], or with the selective β3 AR agonist CL316243 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in saline via osmotic minipump (Alzet, Cupertino, CA, USA) at an infusion rate of 40 μg/kg/h (n= 10/model) [6], Additionally, one group of animals in either model received CL316243 via osmotic minipump (40 μg/kg/h) and daily gavage with bosentan (MedChemExpress, Monmouth Junction, NJ, USA) at a dose of 100 mg/kg/day for 2 weeks (n=10/model). Mice receiving vehicle and exposed to FiCh=0.1 or the ambient air for 5 weeks were used as control groups (n=20).

Echocardiography Transthoracic echocardiography was performed in lightly sedated mice (isoflurane 1%) with a Vevo 2100 high-resolution imaging system equipped with a 30 MHz transducer (VisualSonics, Toronto, ON, Canada). Pulmonary artery acceleration time (PAT), a non- invasive estimate of PA pressure [8], RV wall thickness, and left ventricular (LV) systolic and diastolic internal diameters and ejection fraction were measured [8], Analysis was performed on Vevo Lab software (VisualSonics, Toronto, ON, Canada) by an investigator blinded to the treatment assignments.

Invasive Haemodynamic Measurements

Pressure-volume loops were acquired in intubated and ventilated mice at a respiratory rate of 120 breathsmin" ¹ , a tidal volume of 10 mLkg" ¹ , FiOi 1.0 with isoflurane 1% to 1.5% [9, 10], Using an open chest approach via the RV apex, a Millar microtip catheter (PVR-1045) connected to an MPVS Ultra Single Segment Pressure-Volume Unit and PowerLab (ADInstruments, Colorado Springs, CO, USA) was introduced to the RV cavity, and RV pressure and volume were simultaneously measured [9-11], After baseline recordings, measurements were repeated during brief occlusion of inferior vena cava to alter the venous return. Parameters of RV function were derived from the baseline RV pressure-volume loops, including RV end-systolic pressure, cardiac output, ejection fraction, stroke work, relaxation factor (τ), and chamber compliance (end-diastolic elastance ^-1 ) [10], Pressure-volume area (PVA; an estimate of myocardial O ₂ consumption), stroke work, and ventricular mechanical efficiency ([stroke work.PVA ^-1 ]*100) were estimated based on the baseline RV pressurevolume relations [10], Load-independent indices of systolic function such as preload recruitable stroke work and end-systolic elastance (Ees) were derived from PV loops with vena cava occlusion. To assess RV afterload and RV-PA coupling efficiency, effective arterial elastance (Ea), and Ees/Ea were calculated. Total pulmonary vascular resistance (tPVR=RV end systolic pressure/cardiac output) was used to estimate the resistance of pulmonary vasculature [10], Analysis was performed on LabChart software (v8.1.16, ADInstruments, Colorado Springs, CO, USA) by an investigator blinded to the treatment assignments.

Assessment of RV Hypertrophy

After excision of the heart, the RV wall was separated from the LV wall and the interventricular septum. The ratio of the RV weight to the weight of left ventricle plus septum (i.e., Fulton index) was calculated as an index of RV hypertrophy [8], Morphometry of Pulmonary Microvasculature Lung sections (4 pm) were incubated with an antibody against a-smooth muscle actin (a-SMA) (Ab 124964, Abeam, Cambridge, UK) using the ImmPRESS Horse Anti-Rabbit IgG PLUS Polymer Kit Peroxidase (MP-7801, Vector laboratories, Burlingame, CA, USA) and stained with hematoxylin and eosin. Images were acquired by systematically traversing the total surface area of the tissue slice by Axio Scan.Zl slide scanner (Carl Zeiss Microscopy GmbH, Germany). Intraacinar blood vessels (≥15 and <50 pm in external diameter) containing a-SMA positive cells in their walls were counted [8],

RV Collagen Content Assay

RV was fixed in 2% paraformaldehyde and stained with Picrosirius red to determine interstitial collagen fractions, as previously described [7],

Immunohistochemistry for Proliferating Cell Nuclear Antigen

Immunohistochemical staining was performed in tissue sectioned at 4 mm using an antibody against a-SMA (Ab 124964, Abeam, Cambridge, UK). Proliferating cell nuclear antigen (PCNA) staining was performed with rat polyclonal anti-PCNA antibody (Ab252848, Abeam, Cambridge, UK) [7], Quantification of positive staining for PCNA colocalised with a- SMA was performed using ImagePro software (Media Cybernetics, Rockville, MD, USA) and presented as percentage of positive staining for both markers per view.

Western Blot

Protein fiom lung tissue was extracted by radioimmunoprecipitation assay buffer (Abl56034, Abeam, Cambridge, UK). Denatured proteins were resolved following the NuPAGE Bis-Tris Mini Gel Electrophoresis protocol (NP0336BOX, NuPAGE 4-12% Bis-Tris Protein Gels, Thermo Scientific, Rockford, IL, USA), and transferred onto nitrocellulose membranes and probed with primary antibodies overnight at 4°C (rabbit anti-sGCal antibody [Ab50358, Abeam, Cambridge, UK]; rabbit anti-sGCβ1 antibody [Abl54841, Abeam, Cambridge, UK]; rabbit anti-eNOS antibody [Abl99956, Abeam, Cambridge, UK]; muscle actin [Ab 136812, Abeam, Cambridge, UK]; and rabbit anti-β actin antibody [Abl 15777, Cambridge, UK]). Following incubation with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-mouse, Abeam, Cambridge, UK and Bio-red, CA, USA), blots were developed with an enhanced chemiluminescence kit (34075, Thermo Scientific, Rockford, IL, USA). The expression of proteins was quantified by densitometry as the ratio of target protein divided by β actin.

Immunodetection of eNOS Glutathionylation

Glutathionylation of eNOS was detected in the tissue homogenate by coimmunoprecipitation. First, eNOS was immunoprecipitated using an antibody against eNOS (AF950, R&D, Minneapolis, MN, USA)) and protein A/G plus agarose beads (SCZ-SC-2003, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The proteins bound to the collected beads were eluted in Laemmli buffer, subjected to SDS-PAGE, and probed with anti- glutathione antibody (#101-A-250, ViroGen, Watertown, MA, USA), as previously described [5].

High-Performance Liquid Chromatography Analysis of Dihydroethidium Oxidation Products

Immediately after harvesting from mice, a piece of lung was incubated in phosphate buffered saline containing the metal chelator diethylenetriaminepentaacetic acid (100 pmol/L) to minimise artificial oxidation, and dihydroethidium (DHE, 50 pmol/L) in 1.5 mL Eppendorf tubes (37°C, 30 minutes, in the dark), as previously described [5], Separation of the O ₂ .- dependent 2-hydroxy-ethidium (2-OH-E+) product from the nonspecific product ethidium (E+) was performed [12], using Vanquish UHPLC coupled to TSQ Altis Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, US). The quantified 2-OH-E+ levels were normalised to the DHE peak (to account for the DHE that has entered the tissue) and protein concentration (to account for the size of the tissue). β3 AR Expression in Human Lungs

We searched for the relative expression levels of β3 ARs in the RNA microarray analysis (Affymetrix) performed in the explanted lungs of patients with PAH (n=15) versus normal controls (n=ll) [13] on the NCBI Gene Expression Omnibus (accession number: GSE113439; httDS.//www.ncbi.n1m.nih.gov/geo/geo2r/?acc=GSEl 13439).

Statistics

Data are presented as mean ± S.D. Normality of data was assessed using Kolmogorov- Smirnov test. P values are from univariate ANOVA and post-hoc Tukey’s test, or Kruskal- Wallis test with Dunn’s multiple comparisons where appropriate. Unpaired Student’s t-test was used for comparison between two groups. Two-tailed tests were used. Statistical analysis was conducted using GraphPad Prism (version 9.0.0, San Diego, CA, US). Statistical significance was inferred at p <0.05.

Results

Echocardiographic Indices

In both Hx and SHx models, RV wall thickness increased in hypoxic mice treated with vehicle compared with the normoxic control mice (Table 1, Table 3). All p-values for differences between groups are reported in the Tables and the Figures. Treatment with CL316243, sildenafil or riociguat decreased the RV wall thickness compared with the hypoxic mice treated with vehicle. PAT decreased in hypoxic mice treated with vehicle, while treatment with CL316243, sildenafil or riociguat increased PAT in both models. Addition of bosentan to CL316243 further increased PAT in the SUHx model but not in the Hx model. There were no differences in the LV dimensions or ejection fraction between the normoxic and hypoxic groups in both models (Table 1, Table 3).

Invasive Right Ventricular Haemodynamics

Representative pressure-volume loops for normoxic mice treated with vehicle and hypoxic mice treated with vehicle or CL316243 are shown in Figure 1. RV systolic pressure was significantly increased in both models compared with the normoxic mice (Table 2, Table 4), confirming establishment of PAH. Treatment with CL316243, sildenafil or riociguat reduced RV systolic pressure by a similar magnitude compared with the mice treated with vehicle in both models. Combination of bosentan and CL316243 led to a further reduction in the RV systolic pressure compared with the mice treated with CL316243 in the SUHx model. RV stroke work and PVA increased in the hypoxic control mice compared with the normoxic controls. All treatments reduced both PVA and stroke work such that their ratio was not altered by therapies compared with the hypoxic control mice (Table 2).

In both models, RV afterload (Ea and tPVR) increased in the hypoxic mice treated with vehicle compared with the normoxic control mice (Table 2, Table 4), and treatment with CL316243, riocigaut or sildenafil reduced the RV afterioad compared with the hypoxic control mice. Combination of CL316243 and bosentan further reduced the RV afterioad compared with the mice treated with CL316243 in the SUHx model (Table 4).

To compensate for the increase in the RV afterioad, RV contractility, as measured by the preload independent contractility indices (Ees, and PRSW), increased in hypoxic mice in both models (Table 2, Table 4). The increase in the contractility was not adequate to effectively maintain RV-PA coupling efficiency in hypoxic mice treated with vehicle in both models, with the Ees/Ea ratio significantly dropping compared with the noromoxic controls (Figure 1, Table 4). Treatment with CL316243 (as monotherapy or with bosentan) or sildenafil in the Hx model, and all treatments in the SUHx model, significantly increased Ees/Ea ratio compared to control hypoxic mice, suggesting improved RV-PA coupling efficiency by treatments (Figure 1, Table 4). Consistent with the reduced RV afterioad and enhanced RV-PA coupling efficiency, the treatments increased cardiac output and RV ejection fraction (Table 2, Table 4).

All treatments resulted in reductions in the RV diastolic pressure in the Hx model (Table 2), while in the SUHx model, only treatment with bosentan plus CL316243 reduced the RV diastolic pressure compared with the control mice (Table 4). Of the indicators of RV diastolic function, chamber compliance but not T was significantly decreased in the control mice in both models (Table 2, Table 4). No significant differences in T or chamber compliance were observed between hypoxic mice treated with vehicle and active treatment groups.

Right Ventricular and Pulmonary Vascular Remodelling

The Fulton index increased in the vehicle in both models, indicating RV hypertrophy (Figure IB, Figure 8A). All treatments in both models resulted in attenuation of the RV hypertrophy, reflecting the improved matching of ventricular-vascular elastance. Collagen content in the RV was higher in the Hx mice treated with vehicle compared with the normoxic mice treated with vehicle (Figure 2A). Treatment with CL316243 as monotherapy or in combination with bosentan, and not riociguat or sildenafil, reduced the collagen content in the RV compared with the control hypoxic mice (Figure 2A).

In both models, there was an increase in the number of muscularised arteries in the pulmonary microvasculature in the hypoxic control mice treated with vehicle compared with the normoxic control mice (Figure 2A, Figure 8A). All treatments significantly decreased the number of muscularised arteries compared with vehicle in both models, however the reversal of the histological changes was partial.

Pulmonary Vascular Cell Proliferation

Immunoreactivity for PCNA in cells that were positive for a-SMA was significantly increased in the lungs of hypoxic mice compared with the healthy controls (Figure 3). The index of proliferation was significantly reduced in the hypoxic mice that received CL316243, sildenafil, riociguat, or CL316243 plus bosentan compared with the hypoxic mice treated with vehicle. eNOS Expression, eNOS Glutathionylation and Soluble Guanylyl Cyclase Expression

Expression levels of eNOS were higher in the lungs of hypoxic mice treated with vehicle or active treatments compared with the normoxic controls (Figure 4A). Treatment with CL316243, riociguat, sildenafil, or CL316243 plus bosentan did not change the eNOS expression levels compared with the hypoxic control mice. Glutathionylation of eNOS, a reversible oxidative modification that results in eNOS uncoupling [4], was significantly increased in the lungs of hypoxic mice treated with vehicle compared with the normoxic control mice, as assessed by co-immunopreciptation of eNOS and glutathionylated proteins normalised to eNOS expression (Figure 4B).

Treatment with CL316243 alone or in combination with bosentan decreased eNOS glutathionylation compared with the hypoxic controls, suggesting that these treatments led to ‘recoupling” of eNOS. Treatment with sildenafil or riociguat did not significantly change eNOS glutathionylation levels compared with the hypoxic control mice. Moreover, expression of sGCal decreased in the hypoxic control mice while expression of sGCβ1 was not affected

(Figure 4C). All treatments led to increased expression of sGCal in the lungs, but not sGCβ1, compared with the hypoxic mice treated with vehicle.

Oxidative Stress

Representative chromatograms from high-performance liquid chromatography of the

DHE oxidation products are shown in Figure 5A. There was a significant increase in O2.- levels in the lungs of hypoxic mice as measured by 2-OH-E+ levels, the specific product of

DHE oxidation by O2.- (Figure 5B). All treatments resulted in a reduction in the 2-0H-E+ levels, compared with the hypoxic control mice. p3 AR Receptor Expression in Human PAH

The expression levels of β3 ARs were significantly lower in the explanted lungs of patients with PAH compared with the normal controls (Figure 9).

Table 1. Echocardiographic Indices in the Hypoxia Model.

N=5-10 mice/group, *p<0.05 vs. normoxic controls; f p<0.05 vs. hypoxic controls. CL=CL316243, LV=left ventricle, LVEDD=left ventricular end-diastolic dimension,

LVESD=left ventricular end-systolic dimension, RV=right ventricle.

Table 2. Functional and Hemodynamic Parameters From the Right Ventricular

Pressure-Volume Loop Analysis in the Hypoxia Model.

N=5-8 mice per group. *p<0.05 vs. normoxic controls; t p<0.05 vs. hypoxic controls.

CL=CL316243; Ea=arterial elastance; Ees=end-systolic elastance; PRSW=preload-recruitable stroke work; PVA=pressure-volume area; RVDP=right ventricular diastolic pressure;

RVSP=right ventricular systolic pressure; tPVR=total pulmonary vascular resistance. Table 3. Echocardiographic Indices in the Sugen Hypoxia Model

Values are meansiSD. N=5-10 mice/group, *p<0.05 vs. normoxic control; t p<0.05 vs. hypoxic control. CL=CL316243, LV=left ventricle, LVEDD=left ventricular end-diastolic dimension, LVESD=left ventricular end-systolic dimension, RV=right ventricle.

Table 4. Invasive Hemodynamic Measurements in the Sugen Hypoxia Model

Values are means±SD. N=5-7 mice per group. *p<0.05 vs. normoxic control; t p<0.05 vs. hypoxic control.Values are means±SD. CL=CL316243; Ea=arterial elastance; Ees=end- systolic elastance; PRSW=preload recruitable stroke work; PVA=pressure-volume area;

RVDP=right ventricular diastolic pressure; RVSP=right ventricular systolic pressure; tPVR=total pulmonary vascular resistance.

Discussion

In the present Example, the present inventors demonstrate that: (1) chronic exposine to hypoxia (Hx model) or hypoxia with SU5416 (SUHx model) resulted in severe PAH, RV hypertrophy, reduced RV ejection fraction and cardiac output, and muscularisation of pulmonary microvasculature, which recapitulate the RV-PA findings in patients with severe PAH; (2) CL316243, a highly selective agonist of β3 AR, reduced RV systolic pressure and hypertrophy to a similar degree to riociguat and sildenafil, while addition of bosentan to CL316243 led to further reduction in the RV systolic pressure compared with CL316243 monotherapy, in the SUHx model; (3) CL316243 reduced RV afterioad and improved RV-PA coupling efficiency, and also improved RV ejection fraction and cardiac output, with the effects generally occurring to a similar magnitude to riociguat and sildenafil; (4) CL316243, akin to sildenafil and riociguat, decreased proliferation of a-SMA positive cells and attenuated pulmonary vascular remodelling; (5) CL316243 as monotherapy or combined with bosentan decreased oxidative stress and glutathionylation of eNOS, and led to up-regulation of sGCal expression; it thus potentiated the NO-dependent signalling in the lungs of hypoxic mice. These findings are summarised in the schematic Figure 6.

Clinical application of RV pressure-volume loop analysis, often with methodologic approximations, has shown that lower values of Ees/Ea (a quantitative measure of RV-PA coupling) predict mortality in PAH, while increased RV-PA coupling efficiency on PAH- specific treatment is associated with improved survival [14-16], Ees/Ea ratio markedly decreased in both models of PAH, compared with the normoxic control, which is consistent with maladaptation and RV failure after prolonged exposure (> 28 days) to increased afterioad in experimental PAH [17], CL316243, riociguat or sildenafil reduced Ea, a composite of PA resistance (static component measured by tPVR) and compliance (dynamic component, not measured in the present study) [14], The decrease in Ea was at least in part due to the reduced tPVR, which was a consequence of the decreased muscularisation of the pulmonary microvasculature resulting from these treatments.

The RV-PA uncoupling was partially corrected by CL316243, riociguat or sildenafil, in the SUHx model, as well as by CL316243, alone or with bosentan, or sildenafil in the Hx model. Since the RV energetic input (O2 consumption linearly correlating with PVA) and output (external work measured by stroke work) were both reduced by therapies without a change in their ratio (i.e., RV mechanical efficiency), the improved Ees/Ea ratio was likely due to homeometric adaptation to the reduced afterioad without direct inotropic effects on the RV [10], We excluded a contribution by LV to the improved RV pump function [16] by echocardiography, which revealed no changes in the LV systolic function in the hypoxic mice treated with active therapies compared with hypoxic controls.

RV fibrosis is an independent risk factor for poor prognosis in patients with PAH [18], Interestingly, only CL316243 as monotherapy or in combination with bosentan reduced RV fibrosis in the Hx model. This finding is akin to the reduction in the LV fibrosis induced by mechanical and neurohormonal stress, by β3 AR activation via a decrease in the paracrine release of profibrotic agents [19], Without being bound by any theory, it is believed that the reduction in the RV fibrosis in the present Example was a consequence of β3 AR activation in the RV. Despite treatments reducing RV hypertrophy and diastolic pressure, we did not detect significant effects on indices of diastolic function.

The increase in expression of eNOS in the lungs of hypoxic mice is consistent with previous reports of increased expression of eNOS in experimental PAH and in the plexiform lesions of patients with PAH [20], However, eNOS expression is not synonymous with NO generation. Indeed, we detected increased levels of eNOS glutathionylation in the lungs of hypoxic mice - a reversible oxidative modification of the reactive cysteine residues in the reductive domain of eNOS that leads to loss of NO generation by eNOS and instead, eNOS- related production of O2.- [4], While all treatments resulted in decreased levels of O2.-, only CL316243 (as single or combined therapy with bosentan) significantly decreased eNOS glutathionylation. This specific effect of CL316243 points to the spatial colocalisation of β3 ARs with eNOS-sGC-cGMP [5] and membrane sources of reactive oxygen species such as NADPH oxidase [6] in the caveolae-rich membrane rafts. These membrane rafts provide structural basis for microdomain-specific redox signalling. We have previously shown that CL316243 results in decreased NADPH oxidase-mediated O2.- generation and enhancement in colocalisation of the deglutathionylation enzyme glutaredoxine-1 with eNOS and other target proteins in the membrane rafts in endothelial cells [6] and cardiac myocytes [21], These mechanisms may also be responsible for the effects of CL316243 on redox stress and eNOS glutathionylation in the lungs of hypoxic mice.

The heterodimeric α/β sGC is the downstream target of NO in the canonical NO- dependent signalling [7], In accordance with a previous report [7], we detected a decrease in the expression of the sGCal but not β1 subunit in the lungs of hypoxic mice compared with the normoxic controls. Consistent with the effect of CL316243 to “recouple” eNOS, such that it produces NO rather than O2 ', expression of sGCal was increased by CL316243, to a similar degree to the direct sGC stimulator riociguat. These data suggest potentiation of the downstream NO signalling by CL316243.

The effect of CL316243 to recouple eNOS may confer theoretical advantages compared with the current PAH-specific therapies that target the downstream cellular effectors of NO. PDE5 inhibitors (sildenafil and tadalafil) competitively bind to the catalytic domain of PDE5 and prevent cGMP binding and hydrolysis [22], while riociguat binds to the reduced (heme-bound) form of sGC to activate sGC via two different mechanisms: stabilizing the NO- sGC complex in the presence of NO [7, 22], or activating sGC directly in the absence of NO by binding to a separate regulatory site [23], The efficacy of PDE5 inhibitors is dependent on the availability of cGMP, which in turn is dependent on bioavailability of NO [22], Since NO is progressively depleted as PAH worsens, efficacy of PDE5 inhibitors is diminished, so is the NO-dependent activity of sGC stimulators [22], The effect of β3 AR stimulation to recouple eNOS, combined with the resistance of β3 ARs to agonist-induced desensitisation in response to sustained stimulation [5], indicate that there is a low likelihood of tachyphylaxis to β3 AR agonists in PAH. Moreover, in contrast to PDE5 inhibitors and sGC stimulators that cause systemic hypotension, a side effect that can limit the dose up-titration in clinical practice [24], β3 AR agonists do not decrease the systemic arterial pressure [5], β3 AR agonists may thus prove to be suitable alternative therapies in patients in whom PDE5 inhibitors and sGC stimulators are inefficacious or poorly tolerated. Finally, additional beneficial effects of combined CL316243 and bosentan in experimental PAH shown in this Example suggest that β3 AR agonists may be effective additions to endothelin receptor antagonists.

Haemodynamic effects of β3 AR agonism in a porcine model of pulmonary venous banding, repheating pulmonary hypertension secondary to left heart ftiilure (group 2 pulmonary hypertension) have been reported [25], Nonetheless, both pathobiology and haemodynamic profile of PAH are fundamentally different compared with the group 2 pulmonary hypertension. Moreover, PAH-specific therapies are ineffective or even harmfill in heart ftiilure with reduced ejection fraction [26], In this regard, mirabegron, a selective β3 AR agonist that is approved for use in the overactive bladder syndrome, did not impact the LV ejection fraction in a small, randomised study in patients with heart ftiilure and reduced ejection fraction [27], The present study is the first to report beneficial haemodynamic effects of a selective β3 AR agonist in experimental PAH, suggesting β3 AR agonists may be beneficial for treatment of this distinct group of PAH patients. Clinical significance of the present findings may also be supported by the lower expression of β3 ARs in the lungs of patients with PAH compared with the normal controls. Although the causality of the decreased expression levels of β3 ARs in the development of human PAH cannot be inferred, based on the experimental data presented in this Example, it is plausible that potentiation of the downregulated β3 AR-dependent pathway by agonists may prove beneficial in human PAH.

In conclusion, despite advances in the medical treatment of PAH, mortality due to this condition remains high and novel therapeutic approaches are required.

Example 2. β3-Adrenergic Agonists in Pulmonary Hypertension (BEAT-PH) trial

The findings of Example 1 have provided the rationale for β3-Adrenergic Agonists in Pulmonary Hypertension (BEAT-PH) trial (ACTRN12620001349932, http://www.anzctr.org.au/), which is a proof-of-concept, acute haemodynamic study of mirabegron conducted in pre-capillary pulmonary hypertension.

Materials and Methods

The BEAT-PH trial compares the effect of the β3-adrenergic receptor agonist Mirabegron (at single 25 mg or 50 mg dose) with the effect of inhaled nitric oxide on pulmonary vascular resistance (PVR) in patients with PAH.

The BEAT-PH trial also 1) compares changes in mean pulmonary artery pressure (mean PAP) fiom baseline with Mirabegron to changes in mean PAP with inhaled nitric oxide, 2) correlates the PVR values to plasma levels of Mirabegron, and 3) once safety and tolerability of Mirabegron is established, Mirabegron is administered for 8 days to determine the short term administration effects on echocardiography parameters of PH.

Key inclusion criteria - Participants that fulfil all of the foliowine criteria:

Patients have a diagnosis of pre-capillary pulmonary hypertension:

• class 1 PH, or

• class 4 PH, such as, chronic pulmonary hypertension due to inoperable small distal pulmonary emboli.

The patient is:

• PH treatment naive, or

• on an endothelin receptor antagonist (ERA) (e.g., Bosentan, Macitentan or Ambrisentan) as monotherapy, or • on combination therapy with an ERA (e.g. , Bosentan, Macitentan or Ambrisentan) plus an agent in the NO pathway (e.g., Sildenafil, Tadalafil or Riociguat) when the NO- related therapy can be safely withheld in short-term, or

• C-Right heart catheterisation is clinically indicated.

Key Exclusion criteria - Participants should have none of the following criteria:

• Significant hepatic (transaminases or bilirubin x 3 above upper reference level) or renal impairment (GFR< 30 ml/min/1.73 m ² );

• Significant hypotension (systolic blood pressure < 80 mmHg) or hypertension (systolic >180 mmHg and/or diastolic blood pressure >110 mmHg);

• Right heart failure: systemic venous congestion and fluid retention (e.g. peripheral oedema, ascites);

• Left heart failure: LVEF <50% and clinical signs of left heart failure;

• Severe interstitial lung disease with forced vital capacity (FVC) < 70%;

• Congenital or drug-induced QT prolongation; or

• Pregnancy or breast feeding.

The total number of patients enrolled is N=5 for Part A (Mirabegron 25 mg) and N=6 for Part B (Mirabegron 50 mg). The design of the BEAT-PH trial is summarised in the schematic in Figure 10.

The BEAT-PH trial has been approved by the Human Research Ethics Committee of the Sydney Local Health District (X20-0143), is registered at http://www.anzctr.org.au/. ACTRN12620001349932.

Preliminary Results

Provided herein is data for a single patient for Part A (Mirabegron 25mg) as representative of the trial results. The patient was diagnosed with class 1 PH and being treated with the combination therapy of Ambrisentan and Sildenafil, with the Sildenafil being withheld for 2 days prior to administration of 25mg of mirabegron.

The single dose of 25mg Mirabegron in this patient reduced the PVR by a maximal value of 40.9% at 2 hours post oral administration of Mirabegron. In comparison, inhaled nitric oxide, which is the standard pulmonary vasodilator used in PH, induced only a 17% reduction in the PVR The change in the PVR appears to be mediated by a reduction in the mean pulmonary pressure (an absolute reduction 6 mmHg) and an increase in the cardiac output (an absolute increase of 0.7 L/min).

Since our murine data suggested that β3-adrenergic receptors lead to the release of nitric oxide, we measured the levels of exhaled nitric oxide pre- and post-administration of Mirabegron. Indeed, a low level of exhaled nitric oxide (5 ppm versus the normal value of 10 ppm) was subsequently raised to a maximum of 7 ppm after administration of Mirabegron, which is consistent with our murine data.

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