Methods of treating PAH with combination of RALINEPAG with other agents

Abstract

The present disclosure includes a combination of rainepag and a cGMP-increasing agent or a prostaglandin, such as riociguat, treprostinil, or iloprost, for use in the treatment of PAH. The disclosed combination therapies provide advantages such as increased efficacy, increased safety, reduced dose and / or frequency of rainepag and / or riociguat, reduced dose and / or frequency of rainepag and / or treprostinil, and reduced dose and / or frequency of rainepag and / or iloprost. In some embodiments, the clinical effectiveness of reduced dose combinations is added or synergistic as compared to that provided by corresponding rainepag, riociguat, treprostinil, and / or iloprost monotherapy.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of November 10, 2017, application number 201780082981.0, and invention name “Method for treating PAH using a combination of RALINEPAG and other agents” (the application date of the corresponding PCT application is November 10, 2017, and the application number is PCT / US2017 / 061116).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 420,515, filed on November 10, 2016, and U.S. Provisional Application No. 62 / 530,533, filed on July 10, 2017, the contents of both U.S. Provisional Applications are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention relates to therapeutic methods and combinations of ralinepag (also known as APD811) and a cAMP-raising agent or a cGMP-raising agent (e.g., a soluble guanylate cyclase (sGC) stimulator such as riociguat, a combination of ralinepag and a prostaglandin (e.g., treprostinil), or a combination of ralinepag and a prostacyclin receptor agonist, which can be used to treat pulmonary arterial hypertension (PAH); idiopathic PAH; familial PAH; and PAH associated with collagen vascular disease, congenital heart disease, portal hypertension, HIV infection, drug or toxin ingestion, hereditary hemorrhagic telangiectasia, splenectomy, pulmonary veno-occlusive disease ( PAH associated with pulmonary PVOD or pulmonary capillary hemangioma (PCH); PAH with significant venous or capillary involvement; platelet aggregation; coronary artery disease; myocardial infarction; transient ischemic attack; angina pectoris; stroke; ischemia-reperfusion injury; restenosis; atrial fibrillation; blood clot formation in individuals undergoing angioplasty or coronary artery bypass graft surgery or in individuals with atrial fibrillation; atherothrombosis; asthma or its symptoms; a diabetes-related condition such as diabetic peripheral neuropathy, diabetic nephropathy, or diabetic retinopathy; glaucoma or another eye disease with abnormal intraocular pressure; hypertension; inflammation; psoriasis; psoriatic arthritis; rheumatoid arthritis; Crohn's disease; transplant rejection; multiple sclerosis; systemic lupus erythematosus (SLE); ulcerative colitis; atherosclerosis; acne; type 1 diabetes; type 2 diabetes; sepsis; and chronic obstructive pulmonary disease (COPD). Background Art

[0005] Pulmonary arterial hypertension (PAH) is a highly proliferative inflammatory vascular remodeling disease that leads to right heart failure and death. The pulmonary pharmacology of prostacyclins (epoprostenol) remains an area of ​​considerable interest due to the use of epoprostenol and its stable mimetics in the treatment of PAH. The prostaglandin compounds iloprost (intravenous, inhaled) and treprostinil (subcutaneous, intravenous, inhaled) are widely used to treat the disease. In view of the complications caused by the above-mentioned delivery routes, successful oral therapies are being sought for use early in the disease process, where the efficacy and / or effect of such compounds are improved (McLaughlin et al., 2009; Clapp and Gurung, 2015).

[0006] Oral beraprost (which contains several isomers of beraprost) is licensed only in Japan and South Korea (Vachiery, 2011) and appears to have limited efficacy in the clinic (Barst et al., 2003). Oral treprostinil has only recently been approved by the FDA, but it is unclear whether oral formulations will approach the clinical efficacy seen with subcutaneous or intravenous administration (Tapson et al., 2013). Selexipag (NS-304) is an oral non-prostaglandin IP receptor agonist (Skoro-Sajer and Lang, 2014; Sitbon et al., 2015). It is a non-prostaglandin diphenylpyrazine derivative, and its active metabolite MRE-269 (also known as ACT-333679) has been reported to be a highly selective IP receptor agonist (Kuwano et al., 2007). MRE-269 binds potently to the human IP receptor (K i =20 nM), while selexipag has no significant effect on the receptor (K i =260 nM) has a much lower affinity; however, both have a much lower affinity for other prostaglandin receptors (K i ≥2.6 μM) had almost no binding affinity.

[0007] In normal human pulmonary artery smooth muscle cells (PASMCs), IP receptors appear to be the primary mediator of the antiproliferative response to treprostinil and iloprost through the generation of cyclic AMP (cAMP) (Wharton et al., 2000; Clapp et al., 2002; Falcetti et al., 2010). In contrast, in human PASMCs isolated from IPAH patients, neither IP receptors nor cAMP appear to be the primary mediators supporting the antiproliferative effects of treprostinil and iloprost, at least at the concentrations studied (Falcetti et al., 2010). Thus, there is no clear indication in the scientific literature as to how effective drugs that are selective for IP receptors would be in diseases where IP receptor expression is low, as in patients with PAH (Lai et al., 2008; Falcetti et al., 2010).

[0008] Ralinepag (2-(((1r,4r)-4-(((4-chlorophenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)methoxy)acetic acid, also known as APD811) is an orally available non-prostaglandin prostacyclin (IP) receptor agonist used to treat various conditions as described herein, such as vasospastic diseases such as PAH. Ralinepag is disclosed in U.S. Patent Publication No. 2011 / 0053958, which is incorporated herein by reference in its entirety for all purposes.

[0009] Ralinepag is currently being evaluated as an oral monotherapy for the treatment of conditions such as PAH. In a recent clinical trial, patients were titrated to ralinepag in order to determine a tolerable dose. Potential approaches to administering ralinepag include administering it at an initial low dose once or twice daily, then increasing the dose over a period of time (usually weeks) by stepwise increasing the ralinepag dose and / or dosing frequency until the highest tolerable daily dose is reached.

[0010] The need for dose increases can delay delivery of the desired maximum tolerable therapeutic dose to the patient, thereby delaying potential clinical benefit. In addition, the need to administer multiple daily doses is inconvenient and may reduce patient compliance. Therefore, it is desirable to provide methods for administering ralinepag in which dose titration is reduced or eliminated, and / or the need for multiple daily doses is reduced or eliminated. In addition, it is desirable to provide methods for enhancing the therapeutic effect of ralinepag and / or co-administered compounds while minimizing side effects, for example by achieving clinical efficacy at lower doses of one or more compounds. Such methods will provide significant clinical benefits, as well as improve patient compliance. The present disclosure provides improved methods for treating and administering ralinepag, particularly for treating PAH and related conditions. Summary of the invention

[0011] In its various embodiments, the present invention is directed to a method of reducing cell proliferation (e.g., of pulmonary artery smooth muscle cells (PASMC)) in a patient in need thereof, comprising administering a therapeutically effective amount of ralinepag, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in combination with a therapeutically effective amount of one or more cGMP elevating agents, cAMP elevating agents, prostaglandins or prostacyclin receptor agonists.

[0012] In various embodiments, the one or more cGMP or cAMP raising agents are soluble guanylate cyclase modulators.

[0013] In various embodiments, ralinepag is administered in combination with one or more cGMP-elevating agents, such as any of the cGMP-elevating agents disclosed herein.

[0014] In various embodiments, ralinepag is administered in combination with one or more cAMP-elevating agents, such as any of the cAMP-elevating agents disclosed herein.

[0015] In various embodiments, ralinepag is administered in combination with a prostaglandin, such as any of the prostaglandins disclosed herein.

[0016] In various embodiments, co-administration of ralinepag with one or more cGMP elevating agents, cAMP elevating agents, prostaglandins, or prostacyclin receptor agonists does not require titration of, for example, ralinepag at the beginning of the co-administration method. In various embodiments, co-administration of ralinepag with one or more cGMP elevating agents, cAMP elevating agents, prostaglandins, or prostacyclin receptor agonists does not require titration of, for example, one or more cGMP elevating agents, cAMP elevating agents, prostaglandins, or prostacyclin receptor agonists. For example, in some embodiments, co-administration of ralinepag with riociguat does not require titration of ralinepag. In some embodiments, co-administration of ralinepag with riociguat does not require titration of riociguat.

[0017] In various embodiments, co-administration of ralinepag with one or more cGMP raising agents, cAMP raising agents, prostaglandins, or prostacyclin receptor agonists does not require as many steps as a standard titration regimen for one or both co-administered agents (e.g., a titration regimen provided on the product label for monotherapy). In various embodiments, co-administration of ralinepag with one or more cGMP raising agents, cAMP raising agents, prostaglandins, or prostacyclin receptor agonists allows for a higher initial dose than a standard titration regimen for one or both co-administered agents (e.g., a titration regimen provided on the product label for monotherapy).

[0018] In various embodiments, the daily dose of ralinepag in combination with the one or more cGMP raising agents, cAMP raising agents, or prostaglandins is at least about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.11 mg, 0.12 mg, 0.13 mg, 0.14 mg, 0.15 mg, 0.16 mg, 0.17 mg, 0.18 mg, 0.19 mg, 0.2 mg, 0.21 mg, 0.22 mg, 0.23 mg, 0.24 mg, 0.25 mg, 0.26 mg, 0.27 mg, 0.28 mg, 0.29 mg, or 0.3 mg (dose equivalent) less than the equivalent therapeutic amount of ralinepag in the absence of co-administration of the one or more cGMP raising agents, cAMP raising agents, or prostaglandins.

[0019] In various embodiments, co-administration of ralinepag with one or more cGMP raising agents, cAMP raising agents, prostaglandins or prostacyclin receptor agonists provides a reduced daily dose of ralinepag and / or a cGMP raising agent, cAMP raising agent, prostaglandin or prostacyclin receptor agonist relative to a daily dose of ralinepag and / or a cGMP raising agent, cAMP raising agent, prostaglandin or prostacyclin receptor agonist that provides an equivalent clinical effect.

[0020] In various embodiments, ralinepag is co-administered with one or more of: riociguat, vericiguat, ataciguat, nelociguat, lificiguat, IW-1701, IW-1973, IWP-051, IWP-121, IWP-427, IWP-953, BAY-60-2770, A-344905; A-350619, A-778935, BI-684067, BI-703704, BAY-41-2272, and BAY-41-8543.

[0021] In various embodiments, the amount of ralinepag is at or about 0.02, 0.025, 0.04, 0.05, 0.06, 0.075, 0.08, 0.1, 0.12, 0.125, 0.14, 0.15, 0.16, 0.175, 0.18, 0.2, 0.22, 0.24, 0.25, 0.26, 0.275, 0.28, 0.3, 0.32, 0.325, 0.34, 0.35, 0.36, In some embodiments, the amount of ralinepag is between or about 0.05-0.6 mg, or about 0.05-0.75 mg. In some embodiments, the amount of ralinepag is between or about 0.05-0.75 mg.

[0022] In various embodiments, the amount of ralinepag is at or about 0.02, 0.025, 0.04, 0.05, 0.06, 0.075, 0.08, 0.1, 0.12, 0.125, 0.14, 0.15, 0.16, 0.175, 0.18, 0.2, 0.22, 0.24, 0.25, 0.26, 0.275, 0.28, 0.3, 0.32, 0.325, 0.34, 0.35, 0.36 , 0.375, 0.38, 0.4, 0.42, 0.44, 0.45, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975 or 1.0 mg, or a range of any two of these amounts. For example, in various embodiments, the amount of ralinepag is or is about 0.05-0.6 mg per day.

[0023] In various embodiments, the amount of ralinepag is or is about 0.02, 0.025, 0.04, 0.05, 0.06, 0.075, 0.08, 0.1, 0.12, 0.125, 0.14, 0.15, 0.16, 0.175, 0.18, 0.2, 0.22, 0.24, 0.25, 0.26, 0.275, 0.28, 0.3, 0.32, 0.325, 0.34, 0.35, 0.36, 0.375 In some embodiments, the amount of ralinepag is about 0.05-0.6 mg QD, or about 0.1 mg QD.

[0024] In various embodiments, the amount of ralinepag is at or about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.075, 0.08, 0.09, 0.1, 0.11, 0.12, 0.125, 0.13, 0.14, 0.15, 0.16, 0.17, 0.175, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.275, 0.28, 0.29, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, or 0.5 mg twice daily (BID), or a range of any two of these amounts. For example, in various embodiments, the amount of ralinepag is at or about 0.05-0.3 mg BID.

[0025] In various embodiments, the amount of ralinepag is or is about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.075, 0.08, 0.09 or 0.1 mg every other day.

[0026] In various embodiments, the amount of riociguat is between or about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 mg, or a range of any two of these amounts. For example, in various embodiments, the amount of riociguat is between or about 0.5 and 2.5 mg.

[0027] In various embodiments, the amount of riociguat is or is about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 mg per day, or a range of any two of these amounts. For example, in various embodiments, the amount of riociguat is or is about 0.5 to 2.5 mg per day.

[0028] In various embodiments, the amount of riociguat is or is about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 mg once daily (QD), or a range of any two such amounts. For example, in various embodiments, the amount of riociguat is or is about 0.5 to 2.5 mg once daily. In various embodiments, the riociguat is administered twice daily. In various embodiments, the riociguat is administered three times daily.

[0029] In various embodiments, the amount of riociguat is or is about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25 or 2.5 mg twice daily (BID), or a range of any two of these amounts.

[0030] In various embodiments, the amount of riociguat is 0.5, 1, 1.5, 2, or 2.5 mg three times daily (TID), or a range of any two such amounts.

[0031] In various embodiments, the amount of riociguat is less than or equal to 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 mg.

[0032] In various embodiments, riociguat is in an inhaled form. In various embodiments, riociguat is in an intravenous form. In various embodiments, riociguat is in an oral form.

[0033] In various embodiments, the amount of Treprostinil is between or about 0.1, 0.125, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 0.25, 0.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 6, 7, 8, 9, or 10 mg, or a range of any two of these amounts. For example, in various embodiments, the amount of Treprostinil is between or about 0.125-5 mg.

[0034] In various embodiments, the amount of Treprostinil is at or about 1, 2.5, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 17.5, 18, 19, 20 or 25 mg / ml, or a range of any two of these amounts. In various embodiments, the amount of Treprostinil is 1-10 mg / ml in an intravenous subcutaneous form.

[0035] In various embodiments, the amount of Treprostinil is at or about 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9 or 1 mg / ml, or a range of any two of such amounts. In various embodiments, the amount of Treprostinil in inhaled form is 0.6 mg / ml.

[0036] In various embodiments, the amount of Treprostinil is less than or equal to 0.1, 0.125, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 0.25, 0.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 mg. In various embodiments, the amount of Treprostinil is less than or equal to 1, 2.5, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 mg / ml. In various embodiments, the amount of Treprostinil is less than or equal to 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, or 0.6 mg / ml.

[0037] In various embodiments, Treprostinil is in an inhaled form. In some embodiments, Treprostinil is in an injectable form. In various embodiments, Treprostinil is in an intravenous form. In various embodiments, Treprostinil is in an intravenous subcutaneous form. In various embodiments, Treprostinil is in an oral form. In various embodiments, Treprostinil is in a transdermal form. In various embodiments, Treprostinil is a transdermal patch. In various embodiments, Treprostinil is administered once daily. In various embodiments, Treprostinil is administered twice daily. In various embodiments, Treprostinil is administered three times daily.

[0038] In some embodiments, the therapeutically effective amount of ralinepag is a starting dose. In some embodiments, the therapeutically effective amount of ralinepag is a maximum tolerated dose. In some embodiments, the therapeutically effective amount of ralinepag is a maximum dose. In some embodiments, the therapeutically effective amount of ralinepag is a maximum tolerated dose. In some embodiments, the therapeutically effective amount of ralinepag is a maintenance dose.

[0039] In some embodiments, the starting dose is for a patient. In some embodiments, the starting dose is for a patient population. In some embodiments, the maximum tolerated dose is for a patient. In some embodiments, the maximum tolerated dose is for a patient population. In some embodiments, the maximum dose is for a patient. In some embodiments, the maximum dose is for a patient population. In some embodiments, the maximum tolerated dose is for a patient. In some embodiments, the maximum tolerated dose is for a patient population. In some embodiments, the maintenance dose is for a patient. In some embodiments, the maintenance dose is for a patient population.

[0040] In some embodiments, the starting dose of ralinepag is selected from or is selected from about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.075, 0.08, 0.09, or 0.1 mg per day. In some embodiments, the starting dose of ralinepag is 0.01 mg per day. In some embodiments, the starting dose of ralinepag is 0.02 mg per day. In some embodiments, the starting dose of ralinepag is 0.05 mg per day.

[0041] In some embodiments, the dose of ralinepag is increased at weekly intervals by 0.05 mg per day, up to a maximum tolerated dose of 0.8 mg per day.

[0042] In some embodiments, the dose of ralinepag is increased at weekly intervals. In some embodiments, the dose of ralinepag is increased at every two month intervals.

[0043] In some embodiments, the dose increase of ralinepag is selected from an amount of 0.02 mg, 0.05 mg, and 0.75 mg and 0.1 mg per day.

[0044] In some embodiments, the dosage of ralinepag is increased at weekly intervals by an amount selected from 0.02 mg, 0.05 mg, and 0.75 mg and 0.1 mg per day.

[0045] In some embodiments, the maximum tolerated dose of ralinepag is selected from or is selected from about 0.4 mg, 0.45 mg, 0.5 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.6 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.75 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.8 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.4 to 1.0 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.6 to 1.0 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.6 to 0.8 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.65 to 1.0 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.65 to 0.8 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is greater than 0.4 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is greater than 0.6 mg per day.

[0046] In some embodiments, the maximum dose of ralinepag is selected from or is selected from about 0.4 mg, 0.45 mg, 0.5 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day. In some embodiments, the maximum dose of ralinepag is 0.6 mg per day. In some embodiments, the maximum dose of ralinepag is 0.75 mg per day. In some embodiments, the maximum dose of ralinepag is 0.8 mg per day. In some embodiments, the maximum dose of ralinepag is 0.4 to 1.0 mg per day. In some embodiments, the maximum dose of ralinepag is 0.6 to 1.0 mg per day. In some embodiments, the maximum dose of ralinepag is 0.6 to 0.8 mg per day. In some embodiments, the maximum dose of ralinepag is 0.65 to 1.0 mg per day. In some embodiments, the maximum dose of ralinepag is 0.65 to 0.8 mg per day. In some embodiments, the maximum dose of ralinepag is greater than 0.4 mg per day. In some embodiments, the maximum dose of ralinepag is greater than 0.6 mg per day.

[0047] In some embodiments, the maximum tolerated dose of ralinepag is selected from or is selected from about 0.4 mg, 0.45 mg, 0.5 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.6 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.75 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.8 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.75 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.8 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.4 to 1.0 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.6 to 1.0 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.6 to 0.8 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.65 to 1.0 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is 0.65 to 0.8 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is greater than 0.4 mg per day. In some embodiments, the maximum tolerated dose of ralinepag is greater than 0.6 mg per day.

[0048] In some embodiments, the maximum dose of ralinepag in the dosage form is selected from or is selected from about 0.4 mg, 0.45 mg, 0.5 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day. In some embodiments, the maximum dose of ralinepag in the dosage form is 0.6 mg. In some embodiments, the maximum dose of ralinepag in the dosage form is 0.75 mg. In some embodiments, the maximum dose of ralinepag in the dosage form is 0.8 mg. In some embodiments, the maximum dose of ralinepag is 0.4 to 1.0 mg per day. In some embodiments, the maximum dose of ralinepag is 0.6 to 1.0 mg per day. In some embodiments, the maximum dose of ralinepag is 0.6 to 0.8 mg per day. In some embodiments, the maximum dose of ralinepag is 0.65 to 1.0 mg per day. In some embodiments, the maximum dose of ralinepag is 0.65 to 0.8 mg per day. In some embodiments, the maximum dose of ralinepag is greater than 0.4 mg per day. In some embodiments, the maximum dose of ralinepag is greater than 0.6 mg per day.

[0049] In some embodiments, the maintenance dose of ralinepag is selected from or is selected from about 0.01 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.16 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day. In some embodiments, the maintenance dose of ralinepag is 0.4 to 1.0 mg per day. In some embodiments, the maintenance dose of ralinepag is 0.6 to 1.0 mg per day. In some embodiments, the maintenance dose of ralinepag is 0.6 to 0.8 mg per day. In some embodiments, the maintenance dose of ralinepag is 0.65 to 1.0 mg per day. In some embodiments, the maintenance dose of ralinepag is 0.65 to 0.8 mg per day. In some embodiments, the maintenance dose of ralinepag is determined by tolerability. In some embodiments, the maintenance dose of ralinepag is greater than 0.4 mg per day. In some embodiments, the maintenance dose of ralinepag is greater than 0.6 mg per day.

[0050] In some embodiments, in patients receiving an intolerant dose of ralinepag, the dose of ralinepag is reduced to the previously tolerated dose. In some embodiments, the previously tolerated dose is the patient's maximum tolerated dose.

[0051] In some embodiments, the amount of ralinepag is adjusted to account for differences in bioequivalence between the immediate release form and the extended release form. For example, in some embodiments, a 0.8 mg extended release dosage form of ralinepag sustained release dosage form is provided to be equivalent to two 0.3 mg immediate release dosage forms of ralinepag, wherein the extended release dosage form is less than 100% bioequivalent to the immediate release dosage form.

[0052] In some embodiments, the therapeutically effective amount is suitable for administration once daily. In some embodiments, the therapeutically effective amount is suitable for administration twice daily. In some embodiments, the therapeutically effective amount is administered once daily. In some embodiments, the therapeutically effective amount is administered twice daily.

[0053] In various embodiments, ralinepag is titrated. In various embodiments, riociguat is titrated. In various embodiments, both ralinepag and riociguat are titrated. In some embodiments, the dosage is determined in accordance with product labeling approved by a regulatory agency (e.g., U.S. FDA, see, Label) titration of riociguat, which is incorporated herein by reference.

[0054] In various embodiments, ralinepag is titrated. In various embodiments, treprostinil is titrated. In various embodiments, both ralinepag and treprostinil are titrated. In some embodiments, the titration is performed according to product labeling approved by a regulatory agency (e.g., U.S. FDA, see Label) for titrating treprostinil, which is incorporated herein by reference.

[0055] In various embodiments, ralinepag is co-administered with one or more of treprostinil, iloprost, cisaprost, and epoprostenol.

[0056] In various embodiments, the co-administration methods disclosed herein can be used to treat PAH, such as idiopathic PAH; hereditary PAH; familial PAH; PAH associated with collagen vascular disease, congenital heart disease, congenital heart disease with repaired shunt, portal hypertension, connective tissue disease, HIV infection, drug or toxin ingestion, hereditary hemorrhagic telangiectasia, splenectomy, pulmonary veno-occlusive disease (PVOD) or pulmonary capillary hemangioma (PCH); and PAH with significant venous or capillary involvement. In some embodiments, the co-administration methods disclosed herein can be used to treat human subjects with symptomatic PAH. In some embodiments, the co-administration methods disclosed herein can be used to treat human subjects with PAH, WHO functional class I. In some embodiments, the co-administration methods disclosed herein can be used to treat human subjects with PAH, WHO functional class II. In some embodiments, the co-administration methods disclosed herein can be used to treat human subjects with PAH, WHO functional class III. In some embodiments, the co-administration methods disclosed herein can be used to treat human subjects with PAH, WHO functional class IV. In some embodiments, the co-administration methods disclosed herein can be used to treat human subjects with PAH, WHO Group I. In certain embodiments, the co-administration methods disclosed herein can be used to treat PAH patients with WHO functional class II-III symptoms. In various embodiments, the co-administration methods disclosed herein can be used to treat chronic thromboembolic pulmonary hypertension (CTEPH). In various embodiments, the co-administration methods disclosed herein can be used to treat persistent / recurrent CTEPH (WHO Group 4) after surgical treatment. In various embodiments, the co-administration methods disclosed herein can be used to treat inoperable CTEPH to improve exercise capacity and / or WHO functional class. In various embodiments, the co-administration methods disclosed herein can be used to treat PAH (WHO Group 1) to improve exercise capacity. In various embodiments, the co-administration methods disclosed herein can be used to improve WHO functional class and / or delay clinical deterioration. In various embodiments, the co-administration methods disclosed herein can be used to delay disease progression and / or reduce the risk of hospitalization due to PAH. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figures 1A-1FThe antiproliferative effect of ralinepag in combination with 100 nM riociguat is shown. Growth-arrested cells were incubated in human smooth muscle basal medium (SMBM) containing 9% FBS ± 0.1% DMSO, FBS plus ralinepag and DMSO in the absence or presence of 100 nM riociguat and SMBM for 96 hours (time control). Cell proliferation was normalized to the growth response induced by FBS and DMSO, which was taken as the FBS and DMSO response minus the time control (= 100% growth at 4 days). The growth response induced in the presence of ralinepag and solvent ± riociguat is expressed as the % change in cell proliferation relative to the FBS response alone. The data were fit using the variable slope sigmoidal curve fitting program in GraphPad, and the parameters of each fit are shown. Data are from 5 independent patient cell isolates. * Compared with ralinepag alone, * = P < 0.05; two-way ANOVA with Bonferroni post hoc test.

[0058] Figure 2 It is shown that ralinepag is a more potent inhibitor of serum-induced proliferation in human PASMCs from PAH patients in the presence of riociguat. Mean antiproliferative effects of increasing doses of ralinepag in the absence (A) and presence of 100 nM riociguat (B). Human PASMCs were grown in 9% serum (FBS) and 0.1% DMSO for 4 days ± drug. Cell proliferation was normalized to the growth response induced by FBS and DMSO, which was taken as the FBS response minus the time control (= 100% growth at 4 days). The growth response induced in the presence of ralinepag and solvent ± riociguat is presented as the % change in cell proliferation relative to the FBS response alone. The figure also shows the effect of riociguat (100 nM) in the presence of growth medium containing solvent. * = P < 0.05, *** = P < 0.001 compared to control (FBS and DMSO); one-way ANOVA with Bonferroni post hoc test (n = 5).

[0059] Figures 3A-3FThe antiproliferative effect of ralinepag in combination with 100 nM sildenafil is shown. Growth-arrested cells were incubated in human smooth muscle basal medium (SMBM) containing 9% FBS ± 0.1% DMSO, FBS plus ralinepag and DMSO for 96 hours (time control) in the absence and presence of 100 nM sildenafil or SMBM alone. Cell proliferation was normalized to the growth response induced by FBS alone, which was taken as the FBS response minus the time control (= 100% growth at 4 days). The growth response induced in the presence of ralinepag plus solvent is shown as the % change in cell proliferation relative to the FBS response alone. The data were fitted using the variable slope sigmoidal curve fitting program in GraphPad, and the parameters of each fit are displayed. The data are from 5 independent patient cell isolates.

[0060] Figure 4 The effect of sildenafil on the antiproliferative response of ralinepag in human PASMCs from PAH patients is shown. Mean antiproliferative effect of increasing doses of ralinepag in the absence (A) and presence of 100 nM sildenafil (B). Human PASMCs were grown in 9% serum (FBS) and 0.1% DMSO for 4 days ± drug. Cell proliferation was normalized to the growth response induced by FBS alone, which was taken as the FBS response minus the time control (= 100% growth at 4 days). The growth response induced in the presence of ralinepag and solvent ± sildenafil is expressed as the % change in cell proliferation relative to the FBS response alone. The figure shows the effect of sildenafil (Sild; 100 nM) in the presence of growth medium containing solvent. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 compared to control (FBS and DMSO); one-way ANOVA with Bonferroni post hoc test (n = 5).

[0061] Figures 5A-5F The antiproliferative effect of ralinepag in combination with 100 nM treprostinil is shown. Growth-arrested cells were incubated in human smooth muscle basal medium (SMBM) containing 9% FBS ± 0.1% DMSO, FBS plus ralinepag and DMSO for 96 hours (time control) in the absence and presence of 100 nM treprostinil or SMBM. Cell proliferation was normalized to the growth response induced by FBS alone, which was taken as the FBS response minus the time control (= 100% growth at 4 days). The growth response induced in the presence of ralinepag plus solvent is shown as the % change in cell proliferation relative to the FBS response alone. The data were fitted using the variable slope sigmoidal curve fitting program in GraphPad, and the parameters of each fit are displayed. The data are from 5 independent patient cell isolates.

[0062] Figure 6 It is shown that ralinepag is a more potent inhibitor of serum-induced proliferation in human PASMCs from PAH patients in the presence of treprostinil. Average antiproliferative effects of increasing doses of ralinepag in the absence (A) and presence of 100 nM treprostinil (B). Human PASMCs were grown in 9% serum (FBS) and 0.1% DMSO for 4 days ± drug. Cell proliferation was normalized to the growth response induced by FBS alone, which was taken as the FBS response minus the time control (= 100% growth at 4 days). The growth response induced in the presence of ralinepag and solvent ± treprostinil is presented as the % change in cell proliferation relative to the FBS response alone. The figure also shows the effect of treprostinil (Trep; 100 nM) in the presence of growth medium containing solvent. *=P<0.05, **=P<0.01, ***=P<0.001 compared to control (FBS and DMSO); one-way ANOVA with Bonferroni post hoc test (n=5).

[0063] Figures 7A-7F The antiproliferative effect of ralinepag combined with 100nM macitentan is shown. In the absence of and only in the presence of 100nM macitentan or SMBM, growth-arrested cells were incubated in human smooth muscle basal medium (SMBM) containing 9% FBS ± 0.1% DMSO, FBS plus ralinepag and DMSO for 96 hours (time control). Cell proliferation is normalized to the growth response induced only by FBS, which is used as FBS reaction minus time control (= 100% growth during 4 days). The growth response induced in the presence of ralinepag plus solvent is shown as the % change of cell proliferation relative to the independent FBS reaction. The variable slope S-shaped curve fitting program in GraphPad is used to fit the data, and the parameters of each fit are displayed. Data are from 5 independent patient cell isolates.

[0064] Figure 8The effect of macitentan on the antiproliferative response of ralinepag in human PASMCs from PAH patients is shown. The average antiproliferative effect of increasing doses of ralinepag in the absence of 100nM macitentan (A) and in the presence of 100nM macitentan (B). Human PASMCs were grown in 9% serum (FBS) and 0.1% DMSO for 4 days ± drug. Cell proliferation was normalized to the growth response induced by FBS alone, which was used as the FBS response minus the time control (=100% growth at 4 days). The growth response induced in the presence of ralinepag and solvent ± macitentan is expressed as the % change in cell proliferation relative to the FBS response alone. The figure shows the effect of macitentan (Maci; 100nM) in the presence of growth medium containing solvent. *=P<0.05, **=P<0.01, ***=P<0.001 compared to control (FBS and DMSO); one-way ANOVA with Bonferroni post hoc test (n=5).

[0065] Figures 9A-9F The antiproliferative effect of ralinepag in combination with 100 nM bosentan is shown. Growth-arrested cells were incubated in human smooth muscle basal medium (SMBM) containing 9% FBS ± 0.1% DMSO, FBS plus ralinepag and DMSO for 96 hours (time control) in the absence and presence of 100 nM bosentan or SMBM. Cell proliferation was normalized to the growth response induced by FBS alone, which was used as the FBS response minus the time control (= 100% growth at 4 days). The growth response induced in the presence of ralinepag plus solvent was shown as the % change in cell proliferation relative to the FBS response alone. The variable slope sigmoidal curve fitting program in GraphPad was used to fit the data, and the parameters of each fit are displayed. The data are from 5 independent patient cell isolates.

[0066] Fig.10The effect of bosentan on the antiproliferative response of ralinepag in human PASMCs from PAH patients is shown. Mean antiproliferative effect of increasing doses of ralinepag in the absence (A) and presence of 100 nM bosentan (B). Human PASMCs were grown in 9% serum (FBS) and 0.1% DMSO for 4 days ± drug. Cell proliferation was normalized to the growth response induced by FBS alone, which was taken as the FBS response minus the time control (= 100% growth at 4 days). The growth response induced in the presence of ralinepag and solvent ± bosentan is presented as the % change in cell proliferation relative to the FBS response alone. The figure shows the effect of bosentan (Bos; 100 nM) in the presence of growth medium containing solvent. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 compared to control (FBS and DMSO); one-way ANOVA with Bonferroni post hoc test (n = 5).

[0067] Figures 11A-11E The effect of PAH therapy combined with ralinepag on cell proliferation in human PASMC cells is shown. Comparisons were made in cells from the same PAH patient and passage, and the cells had been grown in 9% serum (FBS) and 0.1% DMSO for 4 days and treated with ralinepag ± riociguat (A), sildenafil (B), treprostinil (C), macitentan (D) or bosentan (E). Cell proliferation was normalized to the growth response induced by FBS plus solvent. Fitting was performed using the variable slope sigmoidal curve fitting program in Graph Pad, and the data are shown as the mean ± SEM in the fit, and the parameters of each fit are shown. * = P < 0.05 compared to ralinepag alone; one-way ANOVA with Bonferroni post hoc test (n = 5).

[0068] Fig.12 Comparison of different PAH drugs in combination with ralinepag on cell proliferation of human PASMCs from PAH patients is shown. Data are shown as mean ± SEM and are taken from Figures 1, 3, 5, 7, and 9. The concentration-dependent effect of ralinepag on serum-induced growth was compared when the drug was combined with 100 nM of bosentan, macitentan, sildenafil, treprostinil, or riociguat. * = P < 0.05, ** = P < 0.01 compared with ralinepag; two-way ANOVA with Bonferroni post hoc test. DETAILED DESCRIPTION

[0069] The present disclosure provides improved methods for administering ralinepag to patients in need thereof, particularly to patients with various forms of PAH, such as idiopathic PAH; familial PAH; PAH associated with collagen vascular disease, congenital heart disease, portal hypertension, HIV infection, drug or toxin ingestion, hereditary hemorrhagic telangiectasia, splenectomy, pulmonary veno-occlusive disease (PVOD), or pulmonary capillary hemangioma (PCH); and PAH with significant venous or capillary involvement. The methods of the present disclosure are also useful for treating other conditions such as platelet aggregation; coronary artery disease; myocardial infarction; transient ischemic attack; angina; stroke; ischemia-reperfusion injury; restenosis; atrial fibrillation; blood clot formation in individuals undergoing angioplasty or coronary artery bypass graft surgery or in individuals with atrial fibrillation; atherothrombosis; asthma or its symptoms; a diabetes-related condition such as diabetic peripheral neuropathy, diabetic nephropathy, or diabetic retinopathy; glaucoma or another eye disease with abnormal intraocular pressure; hypertension; inflammation; psoriasis; psoriatic arthritis; rheumatoid arthritis; Crohn's disease; transplant rejection; multiple sclerosis; systemic lupus erythematosus (SLE); ulcerative colitis; atherosclerosis; acne; type 1 diabetes; type 2 diabetes; sepsis; and chronic obstructive pulmonary disease (COPD).

[0070] Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive pulmonary vascular lesions leading to right ventricular hypertrophy. If not treated, right heart failure will occur. Compared with normal controls, the prostacyclin content in PAH patients is lower, and the prostacyclin has vasodilation and antiproliferative effects on the pulmonary vascular system. Prostacyclin or prostacyclin analogs, for example, exogenous administration of prostaglandins such as treprostinil, iloprost and beraprost has been used to treat PAH. However, prostacyclin, treprostinil and iloprost are not orally active and must be administered intravenously. Although beraprost is orally active, it has not yet been approved in Europe and the U.S.

[0071] Selexipag (2-{4-[(5,6-diphenylpyrazin-2-yl)(propan-2-yl)amino]butoxy}-N-(methylsulfonyl)acetamide) and its free carboxylic acid active metabolite MRE-269 are also known for the treatment of PAH. However, these drugs are expensive and have relatively short half-lives in vivo of approximately 8, 3-4, and 0.5 hours, respectively (Kuwano et al., 2007; Clapp and Gurung, 2015). In contrast, ralinepag has a relatively long plasma half-life regardless of dose. Therefore, ralinepag has the potential to provide improved therapeutic effects in the treatment of PAH (and related conditions as described herein) compared to known therapeutic agents.

[0072] A general description of the synthesis of ralinepag and its pharmaceutically acceptable salts, hydrates, polymorphs, and solvates, as well as pharmaceutical formulations and methods of treatment are disclosed, for example, in U.S. Application No. 12 / 933,196 (published as US2011 / 0053958; issued as US 8,895,776). Methods of treatment, including methods of titrating ralinepag, are disclosed in PCT / US2015 / 056824 (published as WO 2016 / 065103). The latter application describes in detail various methods for optimizing dosage for patients in need of treatment with ralinepag. In various embodiments, the initial dose of ralinepag is equivalent to 0.01 mg of the free acid form (referred to as a 0.01 mg equivalent dose) and is administered once daily (QD) or twice daily (BID). If this dose is tolerated, the dosing frequency is increased in the second week (e.g., from QD to BID dosing) and / or the amount of ralinepag is increased, e.g., from an initial dose equivalent to 0.01 mg BID to a BID dose of 0.3 mg ralinepag in weekly increments of, e.g., 0.01 mg (dose equivalents).

[0073] As used herein, a patient is said to "tolerate" a dose of a compound such as ralinepag if administration of that dose to the patient does not result in an unacceptable adverse event or combination of adverse events. Those skilled in the art will appreciate that tolerability is subjective, and that an amount that is tolerable to one patient may be intolerable to another. Generally, tolerability reflects a subjective balance between the clinical benefit of a dose relative to any adverse event. As used herein, an "adverse event" refers to an undesirable or unwanted clinical symptom associated with treatment with the compound. With respect to ralinepag, typical adverse events include headache, nausea, vomiting, jaw pain, flushing, abnormal pulse rate, abnormal QT interval, sitting systolic blood pressure greater than about 160 mmHg, sitting diastolic blood pressure greater than about 100 mmHg, systolic blood pressure less than about 90 mmHg, abdominal pain, epistaxis, muscle aches, feeling warm, palpitations, dizziness, itching, diarrhea, chest tightness, joint pain, tingling or prickling skin sensations, chest pain, chest discomfort, erythema, or any combination of the foregoing.

[0074] "Optimized dose" or "optimal dose" refers to a therapeutic dose, generally the highest therapeutic dose of a compound (or a pharmaceutically acceptable salt, solvate, hydrate, etc. thereof) that can bring the greatest desired clinical benefit to the patient while minimizing intolerable side effects (e.g., adverse events). Those skilled in the art will recognize that the optimal dose may vary from patient to patient, or even vary over time for a particular patient.

[0075] The need to titrate ralinepag to minimize side effects and determine an optimized or optimal dose can be time consuming. For example, in many embodiments, two to up to about nine weeks of titration (i.e., gradually increasing dosing doses and / or frequencies, typically on a weekly basis) may be required to determine the optimal dose of ralinepag. During this titration period, patients may remain on a suboptimal dose for a considerable period of time, which is undesirable. Given the severity of PAH, it is highly desirable to achieve the optimal dose as quickly as possible. In addition, in order to improve patient compliance, it is desirable to administer ralinepag on a QD regimen rather than a BID dosing regimen.

[0076] As described herein, the co-administration of ralinepag and cGMP or cAMP elevating agents such as riociguat, or the co-administration of ralinepag and prostacyclin receptor agonists or prostaglandins such as treprostinil or iloprost provides unexpected advantages. Specifically, these combinations provide substantial improvements. In some embodiments, the improvement allows better efficacy compared to any monotherapy. In some embodiments, the improvement allows the reduction of the dosage of ralinepag and / or cAMP elevating agents, cGMP elevating agents, prostacyclin receptor agonists or prostaglandins. Alternatively, or in addition, because such a combination provides substantial improvements, the best clinical results can be obtained at lower doses of ralinepag and / or cAMP elevating agents, cGMP elevating agents, prostacyclin receptor agonists or prostaglandins (compared to respective monotherapy), thereby providing the clinical benefits of a monotherapy treatment method, but with reduced side effects. Alternatively, the combination of the methods of the present invention can provide improved clinical benefits and reduced side effects.

[0077] The desired clinical benefit can be measured by any clinical index suitable for or used to measure patient improvement in the art. For example, according to the American Thoracic Society Six-Minute Walk Test Guidelines disclosed in Am.J.Respir.Crit.Care Med. Vol. 166, p.111-117, 2002; DO1:10.1164 / rccm.166 / 1 / 111, the clinical benefit can be, for example, an increase of at least 40 meters measured by the "Six-Minute Walk Test" (6MWT). "Equivalent" or substantially similar clinical benefit means a clinical benefit measured by a conventional clinical index, such as measured by the 6MWT, when comparing ralinepag and at least one cAMP elevating agent, cGMP elevating agent, prostacyclin receptor agonist or prostaglandin combination therapy described herein, compared to patients who experience similar levels of adverse events or side effects (similar physiological characteristics, clinical manifestations and responses to ralinepag), the index provides substantially the same results in otherwise identical patients or the same patient.

[0078] In some embodiments, compared to ralinepag monotherapy using the same or higher dose of ralinepag, ralinepag and at least one cAMP elevating agent, cGMP elevating agent, prostacyclin receptor agonist or prostaglandin co-administered as described herein provide an improvement in clinical benefit. For example, compared to the same daily dose of an otherwise identical patient administered with ralinepag monotherapy (i.e., without co-administered cAMP elevating agent, cGMP elevating agent, prostacyclin receptor agonist or prostaglandin), a combination therapy as described herein can show an increase in clinical benefit using a 6MWT index of at least about 5 m, at least about 10 m, at least about 15 m, at least about 20 m, at least about 25 m or at least about 30 m.

[0079] Similarly, as described herein, co-administering ralinepag with a cAMP-raising agent, a cGMP-raising agent, a prostacyclin receptor agonist, or a prostaglandin, the dosage of such a cAMP-raising agent, a cGMP-raising agent, a prostacyclin receptor agonist, or a prostaglandin can be reduced relative to the amount required to achieve an equivalent therapeutic effect in an otherwise identical patient who is administered such a cAMP-raising agent, a cGMP-raising agent, a prostacyclin, or a prostaglandin as a monotherapy (i.e., without ralinepag). For example, as discussed herein, the dosage of the corresponding cAMP-raising agent, cGMP-raising agent, a prostacyclin receptor agonist, or a prostaglandin can be reduced by about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the dosage required in the absence of ralinepag, while achieving a similar level of clinical efficacy, for example as measured by the 6MWT and / or or side effect levels. Alternatively, the combination of ralinepag and a cAMP-raising agent, cGMP-raising agent, prostacyclin receptor agonist, or prostaglandin provides an improvement in efficacy, e.g., as measured by a 6MWT (at least about 5 m, at least about 10 m, at least about 15 m, at least about 20 m, at least about 25 m, or at least about 30 m), compared to patients administered similar levels of the cAMP-raising agent, cGMP-raising agent, prostaglandin, or prostacyclin receptor agonist without ralinepag.

[0080] In one embodiment, when ralinepag is co-administered with riociguat, prostacyclin, treprostinil, or iloprost (or other prostaglandins), a significantly lower dose of ralinepag can be administered in the combination therapy compared to the amount required to achieve the same or substantially similar clinical benefit in an otherwise identical patient (or the same patient) with ralinepag monotherapy. That is, the combination of ralinepag and riociguat, prostacyclin, treprostinil, or iloprost acts synergistically, such that the clinical effect of ralinepag is potentiated or enhanced by the co-administration of riociguat, prostacyclin, treprostinil, or iloprost. For example, the daily dose of ralinepag required in the combination therapy described herein may be at least about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.225 mg, 0.25 mg, or 0.275 mg or 0.3 mg lower than the daily dose of ralinepag required to achieve the same or substantially the same clinical benefit in an otherwise identical patient (or the same patient) as a monotherapy. The daily dose may be provided according to a QD or BID dosing regimen.

[0081] In other embodiments, when ralinepag is co-administered with riociguat, prostacyclin, treprostinil, or iloprost (or other prostaglandins), the same dose of ralinepag can be administered in the combination therapy as can be administered in ralinepag monotherapy. However, in the combination therapy, a significantly improved clinical benefit is provided, e.g., with a substantially similar side effect profile (i.e., comparing the combination therapy to the monotherapy in otherwise identical patients or the same patient) as measured by the 6MWT (at least about 5 m, at least about 10 m, at least about 15 m, at least about 20 m, at least about 25 m, or at least about 30 m).

[0082] In other embodiments, an equivalent or substantially similar clinical benefit is provided by a combination of a significantly lower dose of ralinepag and a reduced dose of riociguat, prostacyclin, treprostinil, or iloprost (or other prostaglandins). For example, the daily dose of ralinepag required in the combination therapy described herein may be at least about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.225 mg, 0.25 mg, or 0.275 mg or 0.3 mg lower than the dose of ralinepag required to achieve the same or substantially the same clinical benefit in an otherwise identical patient (or the same patient) as a monotherapy. The daily dose of riociguat, prostacyclin, treprostinil or iloprost (or other prostaglandins) can be reduced by about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% relative to the dose required to achieve the same or substantially the same clinical benefit in otherwise identical patients (or the same patient) with monotherapy without ralinepag. For example, the daily dose of riociguat required in the combination therapy described herein can be at least about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.25 mg, 1.5 mg, 1.75 mg or 2 mg lower than the dose of riociguat required for equivalent clinical benefit and / or adverse event profile. The daily dose can be provided according to a QD or BID dosing regimen.

[0083] In some embodiments, a therapeutically effective amount of ralinepag is administered simultaneously with one or more cAMP elevating agents, cGMP elevating agents, prostacyclin receptor agonists or prostaglandins, so that the therapeutically effective amount of ralinepag and one or more cAMP elevating agents, cGMP elevating agents, prostacyclin receptor agonists or prostaglandins are present in the patient's body and exert their therapeutic effects simultaneously. Ralinepag can be administered before, simultaneously or after the cAMP elevating agent, cGMP elevating agent, prostacyclin receptor agonist or prostaglandin. If ralinepag and cAMP elevating agent, cGMP elevating agent, prostacyclin receptor agonist or prostaglandin are administered simultaneously, they can be administered in different dosage forms, respectively, or in some embodiments, in a combined dosage form. When administered in a separate dosage form, ralinepag can be administered orally in the form of a tablet or capsule, and the cAMP elevating agent, cGMP elevating agent, prostacyclin receptor agonist or prostaglandin can be administered orally (if an oral dosage form is available or possible) or as an intravenous or subcutaneous dosage form. In some embodiments, ralinepag can also be administered as an injectable or intravenous dosage form.

[0084] In one embodiment, ralinepag is co-administered with riociguat. In another embodiment, ralinepag is co-administered with treprostinil. In another embodiment, ralinepag is co-administered with iloprost. In yet another embodiment, ralinepag is co-administered with prostacyclin. In another embodiment, ralinepag is co-administered with riociguat and treprostinil. In another embodiment, ralinepag is co-administered with riociguat and iloprost.

[0085] Ralinepag is a highly selective IP receptor agonist. In normal human pulmonary artery smooth muscle cells (PASMCs), IP receptors appear to be the primary mediator of the antiproliferative response to treprostinil and iloprost through the generation of cyclic AMP (Wharton et al., 2000; Clapp et al., 2002; Falcetti et al., 2010). In contrast, in human PASMCs isolated from patients with idiopathic pulmonary arterial hypertension (IPAH), neither IP receptors nor cyclic AMP appear to be the primary mediators supporting the antiproliferative effects of treprostinil and iloprost, at least at the concentrations studied (Falcetti et al., 2010).

[0086] The functional role of ralinepag in pulmonary arteries or cells from normal tissue or microvessels from PAH patients was previously unknown, and therefore the pharmacological behavior of such selective IP agonists in the pulmonary circulation was unpredictable. Therefore, ralinepag was evaluated with other prostacyclin drugs, which are expected to have different effects through activation of other targets, including other prostaglandin receptors (Clapp and Gurung, 2015), to determine how it compares in terms of its ability to generate cAMP and inhibit smooth muscle proliferation in PASMCs isolated from PAH patients.

[0087] The present invention provides embodiments including but not limited to the following:

[0088] 1. A method of reducing cell proliferation in a patient in need thereof, comprising:

[0089] A therapeutically effective amount of ralinepag, or a pharmaceutically acceptable salt, hydrate or solvate thereof, is administered in combination with a therapeutically effective amount of one or more cGMP elevating agents, cAMP elevating agents or prostaglandins.

[0090] 2. The method of embodiment 1, wherein the one or more cGMP or cAMP elevating agents are soluble guanylate cyclase (sGC) stimulators.

[0091] 3. The method of embodiment 1 or 2, wherein the ralpagp is administered in combination with one or more cGMP elevating agents.

[0092] 4. The method of embodiment 1 or 2, wherein the ralinepag is administered in combination with one or more cAMP elevating agents.

[0093] 5. The method of embodiment 1, wherein the ralinepag is administered in combination with a prostaglandin.

[0094] 6. The method of embodiment 5, wherein the ralinepag is administered in combination with a synthetic prostacyclin analog.

[0095] 7. The method of any one of embodiments 1-6, wherein the dose of ralinepag does not require standard titration at the start of the method.

[0096] 8. A method as described in any of embodiments 1-7, wherein the therapeutically effective amount of ralinepag in combination with the one or more cGMP raising agents, cAMP raising agents, or prostaglandins is less than the equivalent therapeutically effective amount of ralinepag in the absence of co-administration of the one or more cGMP raising agents, cAMP raising agents, or prostaglandins.

[0097] 9. A method as described in embodiment 8, wherein the daily dose of ralinepag in combination with the one or more cGMP raisers, cAMP raisers or prostaglandins is at least about 0.01 mg (dose equivalent) lower than the equivalent therapeutic amount of ralinepag in the absence of co-administration of the one or more cGMP raisers, cAMP raisers or prostaglandins.

[0098] 10. The method of embodiment 8, wherein the daily dose of ralinepag in combination with the one or more cGMP elevating agents, cAMP elevating agents, or prostaglandins is at least about 0.05 mg (dose equivalent) lower than the equivalent therapeutic amount of ralinepag in the absence of co-administration of the one or more cGMP elevating agents, cAMP elevating agents, or prostaglandins.

[0099] 11. A method as described in embodiment 8, wherein the daily dose of ralinepag in combination with the one or more cGMP elevating agents, cAMP elevating agents or prostaglandins is at least about 0.1 mg (dose equivalent) lower than the equivalent therapeutic amount of ralinepag in the absence of co-administration of the one or more cGMP elevating agents, cAMP elevating agents or prostaglandins.

[0100] 12. The method of any one of embodiments 1-11, wherein the therapeutically effective amount of the one or more cGMP raising agents, cAMP raising agents, or prostaglandins is less than the equivalent therapeutically effective amount of the one or more cGMP raising agents in the absence of co-administration of ralinepag.

[0101] 13. The method of embodiment 12, wherein the therapeutically effective amount of the one or more cGMP raising agents, cAMP raising agents, or prostaglandins is at least about 1% less than the equivalent therapeutically effective amount of the one or more cGMP raising agents in the absence of co-administration of ralinepag.

[0102] 14. The method of embodiment 12, wherein the therapeutically effective amount of the one or more cGMP raising agents, cAMP raising agents, or prostaglandins is at least about 10% less than the equivalent therapeutically effective amount of the one or more cGMP raising agents in the absence of co-administration of ralinepag.

[0103] 15. A method as described in any of embodiments 1-14, wherein the frequency of administering a therapeutically effective amount of ralinepag in combination with one or more cGMP elevating agents, cAMP elevating agents, or prostaglandins is less than the frequency of administering an equivalent therapeutically effective amount of ralinepag without co-administering one or more cGMP elevating agents, cAMP elevating agents, or prostaglandins.

[0104] 16. The method of embodiment 15, wherein the therapeutically effective amount of ralinepag is administered once daily in combination with one or more cGMP elevating agents, cAMP elevating agents, or prostaglandins.

[0105] 17. A method as described in any of embodiments 1-16, wherein the frequency of administering a therapeutically effective amount of the one or more cGMP raising agents, cAMP raising agents, or prostaglandins in combination with ralinepag is less than the frequency of administering an equivalent therapeutically effective amount of the one or more cGMP raising agents, cAMP raising agents, or prostaglandins without co-administration of ralinepag.

[0106] 18. The method of embodiment 17, wherein the therapeutically effective amount of the one or more cGMP elevating agents, cAMP elevating agents, or prostaglandins in combination with ralinepag is administered once daily.

[0107] 19. The method of any one of embodiments 1-18, wherein the one or more cGMP raising agents are selected from riociguat, viliciguat, ataciguat, nelociguat, riviciguat, IW-1701, IW-1973, IWP-051, IWP-121, IWP-427, IWP-953, BAY-60-2770, A-344905, A-350619, A-778935, BI-684067, BI-703704, BAY-41-2272, and BAY-41-8543.

[0108] 20. The method of embodiment 1, wherein the cGMP elevating agent is riociguat.

[0109] 21. The method of embodiment 1, wherein the one or more prostaglandins is treprostinil.

[0110] 22. The method of embodiment 1, wherein the one or more prostaglandins is iloprost.

[0111] 23. The method of any one of embodiments 1-22, wherein the method comprises co-administering ralinepag and riociguat.

[0112] 24. The method of embodiment 23, wherein the amount of riociguat is selected from 0.5, 1, 1.5, 2 or 2.5 mg.

[0113] 25. The method of embodiment 24, wherein the amount of riociguat is administered once daily.

[0114] 26. The method of embodiment 24, wherein the amount of riociguat is administered twice daily.

[0115] 27. The method of embodiment 24, wherein the amount of riociguat is administered three times daily.

[0116] 28. The method of any one of embodiments 23 to 27, wherein the riociguat is titrated.

[0117] 29. The method of any one of embodiments 1-22, wherein the method comprises co-administering ralinepag and treprostinil.

[0118] 30. The method of any one of embodiments 1-22, wherein the method comprises co-administering ralinepag and iloprost.

[0119] 31. The method of any one of embodiments 1-22, wherein the method comprises co-administering ralinepag and a synthetic prostacyclin analog.

[0120] 32. The method of any one of embodiments 1-31, wherein the patient in need thereof is receiving treatment selected from the group consisting of pulmonary arterial hypertension (PAH), idiopathic PAH; familial PAH; PAH associated with collagen vascular disease, congenital heart disease, portal hypertension, HIV infection, drug or toxin ingestion, hereditary hemorrhagic telangiectasia, splenectomy, pulmonary veno-occlusive disease (PVOD), or pulmonary capillary hemangioma (PCH); PAH with significant venous or capillary involvement; and chronic thromboembolic pulmonary hypertension (CTEPH).

[0121] 33. The method of any one of embodiments 1-31, wherein the patient in need thereof is receiving treatment for PAH.

[0122] 34. The method of any one of embodiments 1-31, wherein the method of reducing cell proliferation is a method of treating PAH.

[0123] 35. The method of any one of embodiments 1-31, wherein the method of reducing cell proliferation is a method of treating CTEPH.

[0124] 36. The method of any one of embodiments 1-35, wherein the amount of ralinepag is selected from 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58 or 0.6 mg.

[0125] 37. The method of any one of embodiments 1-35, wherein the amount of ralinepag is selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.3 mg.

[0126] 38. The method of any one of embodiments 1-35, wherein the amount of ralinepag is selected from 0.01 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.16 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg and 1.0 mg per day.

[0127] 39. The method of any one of embodiments 1-35, wherein the amount of ralinepag is a starting dose selected from 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.075, 0.08, 0.09 and 0.1 mg per day.

[0128] 40. The method of any one of embodiments 1-35, wherein the amount of ralinepag is a maximum tolerated dose selected from 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day.

[0129] 41. The method of any one of embodiments 1-35, wherein the amount of ralinepag is selected from a maximum dose of 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day.

[0130] 42. The method of any one of embodiments 1-35, wherein the amount of ralinepag is a maximum tolerated dose selected from 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day.

[0131] 43. The method of any one of embodiments 1-35, wherein the amount of ralpagag is a maintenance dose selected from 0.01 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.16 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg per day.

[0132] 44. The method of any one of embodiments 36 to 43, wherein the amount of ralinepag is administered once daily.

[0133] 45. The method of any one of embodiments 36 to 43, wherein the amount of ralinepag is administered twice daily.

[0134] 46. ​​The method of any one of embodiments 1 to 45, wherein the ralinepag is titrated.

[0135] 47. A method of treating pulmonary arterial hypertension comprising administering ralinepag and riociguat to an individual in need thereof.

[0136] 48. The method of embodiment 47, wherein the amount of riociguat is selected from about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75 and 5 mg.

[0137] 49. A method of treating pulmonary arterial hypertension comprising administering ralinepag and treprostinil to an individual in need thereof.

[0138] 50. The method of embodiment 49, wherein the amount of Treprostinil is selected from 0.1, 0.125, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 0.25, 0.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75 and 5 mg.

[0139] 51. The method of embodiment 49, wherein the amount of Treprostinil is selected from 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 2.5, 3, 4, 5, 6, 7, 7.5, 8, 9 and 10 mg / ml.

[0140] 52. The method of any one of embodiments 47-51, wherein the amount of ralinepag is selected from about 0.01 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.16 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg.

[0141] Example

[0142] The effects of ralinepag in combination with an endothelin-1 receptor antagonist (ERA), a phosphodiesterase type 5 (PDE 5) inhibitor, or a cAMP / cGMP elevating agent (e.g., a soluble guanylate cyclase activator) were evaluated.

[0143] Lung tissue from hypertensive and normal patients

[0144] Lung tissue samples were obtained from patients with Group 1 PAH or Group 3 PAH (PAH associated with lung disease) who received a transplant after treatment failure or who had died. For control samples, donor lungs found to be unsuitable for transplantation or lungs resected for suspected malignancy were used for control samples (Benyahia et al., 2013).

[0145] The distal PASMC primary cell line from PAH patients was derived from transplanted lungs as previously described (Falcetti et al., 2010; Bubb et al., 2014). These cells have an abnormal proliferative capacity when grown in culture (Zhang et al., 2007; Falcetti et al., 2010). Frozen cells were revived and grown in human smooth muscle basal medium-2 (SMBM; Lonza, Slough, UK) supplemented with 9% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2. After reaching confluence, the cells were washed with phosphate-buffered saline (PBS) and treated with trypsin-EDTA for further passages. Only cells between passages 3 and 10 were used for experiments.

[0146] Cyclic AMP assay

[0147] Human PASMC from PAH patients were grown to 70-80% confluence in 12-well plates in DMEM / F12 containing 9% fetal bovine serum (FBS), penicillin (50 U / ml) / streptomycin (50 μg / ml). To assess the time course of cAMP elevation, cells were stimulated with 100 nM ralinepag for different times (0.5, 1, 2, 4, 8, 24, 48 hours). This dose of ralinepag was chosen based on its proximity to the EC for cAMP generation in preliminary experiments. 50 In other experiments, IP receptor agonists were added over the full concentration range (0.1-10,000 nM) for the indicated times (60 min) in the absence and presence of 1 μM of the IP receptor antagonist RO-1138452. Antagonists were pre-added for 30 min and maintained throughout.

[0148] To extract cyclic AMP, the culture medium was aspirated and the PASMC cells were incubated on ice in 0.1M HCl for 20 minutes, then centrifuged at 1000g for 10 minutes at 4°C. The protein concentration in the supernatant was determined using a Bradford-based protein assay (BCA, Novagen, EmD Chemicals, CA, USA). Intracellular cyclic AMP was measured using a competitive enzyme immunoassay 96-well plate kit (ADI-900-163, Enzo Life Sciences, Exeter, UK) and assayed according to the manufacturer's instructions. Each sample was run in duplicate, and the data were expressed as pmol of cAMP per mg of total protein for each individual sample. The basal level was subtracted from each agonist concentration data point. In view of the large differences in cAMP generation from different patient samples (about 15 times), the data were also normalized to the peak cyclic AMP response (designated as 100%) in each cell isolate.

[0149] Cell proliferation assay

[0150] Comparative concentration-dependent effects of IP receptor agonists on cell proliferation were evaluated in human PASMCs derived from PAH patients. Cells were plated at 1 × 10 4 Cells were plated at a density of 10 cells / ml in 96-well plates and grown in a humidified CO2 incubator in human smooth muscle basal medium-2 (SMBM; Lonza) containing 9% FBS and penicillin-streptomycin (Pen / Strep; 50 units / ml) at 37°C. After 24 hours, cell growth was stopped by incubation in fresh medium without added serum for 48 hours. The medium was then removed and replaced with human SMBM containing or not containing 9% serum with 0.1% DMSO in the absence and presence of IP receptor agonists (ralinepag, iloprost, treprostinil or MRE-269), and cells were treated with IP receptor agonists for 4 days in a concentration range (10-12-10-5M). The response was directly compared in cells containing the IP receptor antagonist RO-1138452 (1 μM), which was added to the cells 30-60 minutes before the addition of the IP agonist and maintained throughout the experiment. Cells incubated without the addition of serum for the same period of time (4 days) served as a time control.

[0151] Cell proliferation was obtained using the MTS Proliferation Kit (Promega), a colorimetric method for determining viable cell number based on the cleavage of the tetrazolium salt MTS into formazan by cellular mitochondrial dehydrogenases. The increase in cell number leads to the formation of The amount of dye is increased proportionally, which is quantified by measuring the absorbance of the dye solution at 490 nm. In each case, background absorbance is corrected by subtracting the mean absorbance of the "no cell" control wells from all other absorbance values.

[0152] Cell proliferation was then normalized to the growth response induced by FBS alone, which was taken as the FBS response minus the time control (= 100% growth at 4 days). Growth responses induced in the presence of vehicle ± drug are presented as % change in cell proliferation relative to the FBS response alone in all graphs.

[0153] Materials, reagents, and equipment

[0154] Human smooth muscle basal medium-2 (Lonza, Slough, UK; Cat No. CC-3181) DMEM / F12 (Life Technologies, Paisley, UK; Cat No 11320-074)

[0155] South American fetal bovine serum (FBS; Invitrogen, Cat No 10270106)

[0156] Penicillin-Streptomycin Pen / Strep (5000 units / ml; Life Technologies, Paisley, UK; Cat No 15070-063)

[0157] Sterile Ca2+ / Mg2+ free phosphate buffered saline (PBS; Life Technologies, Cat No 10010-056)

[0158] Sterile trypsin / EDTA solution (0.05%; Life Technologies, Cat No 25300-054)

[0159] Sterile dimethyl sulfoxide (DMSO; Sigma-Aldrich, Cat No, D2650)

[0160] MRE-269 ([4[(5,6-diphenylpyrazinyl)(1-methylethyl)amino]butoxy]acetic acid (CAY10010412), iloprost (50:50 R / S isomer; CAY 18215), treprostinil (CAY10162) and RO-1138452 (IP receptor antagonist; CAY 10441), riociguat (Cat No 2644-5), sildenafil citrate (Cat No 2872-10), macitentan (Cat No M009) and treprostinil (CAY10162) were purchased from Cambridge Bioscience, UK (distributor of Cayman Chemical Co). Bosentan (Cat No 11731) was purchased from Cayman Chemicals Company (USA).

[0161] Iloprost was dissolved in methyl acetate, which was blown off and replaced with DMSO to give a 10 mM stock solution. All other stock solutions were made in DMSO (treprostinil, RO-1138452, ralinepag, MRE-269) to a final concentration of 10 mM. Drugs were serially diluted in growth medium and the solvent concentration in each well was kept constant at 0.11% regardless of the concentration of reagent added.

[0162] Competitive enzyme immunoassay 96-well plate cAMP kit (ADI-900-163, Enzo Life Sciences, Exeter, UK)

[0163] Cell proliferation assay kit (MTS, Promega, UK, Cat No G5421)

[0164] BCA (bicinchoninic acid) protein assay kit (Cat No. 71285-3; Novagen, Merck Millipore, Nottingham, UK)

[0165] Galaxy R CO2 cell culture incubator (WolfLabs Ltd, York UK)

[0166] Tecan Genios microplate reader (Tecan Group Ltd, Germany) OpsysMRTM microplate reader (Dynex Technology, Chantilly, VA, USA) Data and statistical analysis

[0167] All data are expressed as the mean ± standard error of the mean (SEM) of at least 5 observations. Agonist logarithmic concentration curves were constructed and fitted using the nonlinear fitting program in GraphPad Prism 4 or 6 (San Diego, CA, USA). The 50% maximal response (E max ) is expressed as negative log(pEC 50 ) and calculated average EC 50 Statistical analysis was performed using one-way or two-way ANOVA with post hoc correction as indicated in the figure legends. P values ​​< 0.05 were considered statistically significant.

[0168] Example 1: Antiproliferative Effects of Ralinepag in Combination with Cyclic GMP and Cyclic AMP Elevators or Endothelin-1 Antagonists

[0169] The antiproliferative effects of ralinepag in human pulmonary artery smooth muscle cells (PASMCs) from PAH patients were compared with other prostacyclin mimetics and in combination with endothelin-1 receptor antagonists (ERAs), phosphodiesterase type 5 (PDE5) inhibitors, or soluble guanylate cyclase (sGC) activators.

[0170] Ralinepag and MRE-269 behaved as selective IP receptor agonists in cyclic AMP and cell proliferation assays in human PASMCs from PAH patients, with ralinepag producing 2-fold more cyclic AMP and 10-fold more antiproliferative effects. Ralinepag and MRE-269 produced weaker maximal effects in cyclic AMP and cell proliferation assays compared with treprostinil. Treprostinil and iloprost exhibited inhibition of cell proliferation through an IP-independent mechanism.

[0171] Antiproliferative effect of ralinepag combined with riociguat

[0172] Figure 1 shows the concentration-dependent antiproliferative effect of ralinepag in the absence and presence of 100 nM riociguat in human PASMC grown in 9% serum and 0.1% DMSO for 4 days. In four of the five cell isolates, when riociguat was combined with ralinepag, cell growth was more inhibited than when ralinepag was alone. From the average data, overall, ralinepag was more effective in the presence of riociguat over the entire concentration range (0.01-10,000 nM), and was significantly more effective at 10 nM and above (P<0.05, two-way ANOVA with Bonferroni post hoc correction).

[0173] Riociguat alone (100 nM) caused a significant inhibition of cell proliferation (-15%) compared to FBS and DMSO. At 100 nM, ralinepag inhibited growth by 28% ( Figure 2 A), which is almost twice the inhibition induced by the same dose of riociguat ( Figure 2 B). However, in the presence of 100 nM riociguat, significant cell growth inhibition occurred at ralinepag at a 100-fold lower dose (0.01 nM). This suggests that the agents act on different pathways to enhance the inhibition of riociguat proliferation through cGMP (Lang et al., 2012) and enhance the inhibition of ralinepag proliferation through the IP receptor and cAMP generation (the IP receptor antagonist RO1138452 completely inhibited proliferation in the experiments provided herein). The enhancement occurred at subnanomolar concentrations of ralinepag. At higher doses of ralinepag, the inhibition of cell growth appeared to be more than predicted by the additive effect.

[0174] Riociguat has been shown to induce apoptosis and inhibit the proliferation of pulmonary arterial cells associated with upregulation of soluble guanylate expression and increased cyclic GMP production (Lang et al., 2012). Therefore, the enhancement of ralinepag's effect by riociguat may be due to elevated cyclic AMP levels in response to cyclic GMP-dependent inhibition of PDE3, an isoform known to regulate cAMP production induced by IP receptor agonists (Knebel et al., 2013). Previous experiments have shown that riociguat combined with treprostinil is the most effective antiproliferative combination compared to PDE5 inhibitors or ERAs in human PASMCs from PAH patients (Patel et al., 2014). However, if additive, the combined effect is lower than predicted. The two agents may act on different pathways (cyclic GMP and IP receptor / cAMP pathways) with some overlap in growth inhibition mechanisms. For example, inhibition of cell growth by inhibiting the calcium-dependent phosphatase, calcineurin, can be achieved by cyclic GMP and cyclic AMP (Jabr et al., 2007; Lu et al., 2013).

[0175] Antiproliferative effect of ralinepag combined with sildenafil

[0176] Figure 3 shows the concentration-dependent antiproliferative effect of ralinepag in the absence and presence of 100 nM sildenafil in human PASMC grown for 4 days in 9% serum and 0.1% DMSO. In four of the five cell isolates, cell growth was more inhibited when sildenafil was combined with ralinepag compared to ralinepag alone. Ralinepag was significantly more effective in the presence of sildenafil across the entire concentration range (0.01-10,000 nM) (P < 0.001, two-way ANOVA), although Bonferroni post hoc analysis did not find significance for a single drug dose. Figure 4 The average antiproliferative effect of ralinepag in the absence and presence of 100 nM sildenafil is shown compared to the response to 9% serum and 0.1% DMSO alone. Unlike riociguat, sildenafil (100 nM) did not significantly inhibit serum-induced growth. However, when combined with ralinepag, cell growth was further inhibited compared to ralinepag alone. At 1 nM ralinepag, growth inhibition increased from 14% to 27% in the presence of sildenafil, although the magnitude of these changes was smaller at the highest dose tested (10 μM) (cell growth inhibition was 56% instead of 50%).

[0177] The potentiation of sildenafil was found to be less than that observed with riociguat. This may be due to riociguat being a direct activator of guanylate cyclase, thereby producing a greater rise in cyclic GMP than sildenafil, which would be expected to increase basal cyclic GMP levels. In addition, in three of the five cell isolates, the antiproliferative effect of the drug combination gradually weakened at higher doses of ralinepag, which is consistent with some overlap in the downstream mechanisms of PDE5 inhibitors and IP receptor agonists. In fact, sildenafil inhibits cell proliferation in part by activating PPARγ (Wang et al., 2013), which is a known mechanism that underlies the antiproliferative effect of treprostinil in these lung smooth muscle cells (Falcetti et al., 2010). It is likely that the doses used in these experiments were subtherapeutic for sildenafil, as the upper plasma level of this drug in patients is 1500nM (Burgess et al., 2008). However, it is much higher than the K of PDE5 inhibition reported for sildenafil. i , which is reported to be 1-3 nM (Ballard et al., 1998). At 100 nM, sildenafil may also inhibit PDE1 and PDE6 activity (K iThe inhibitory effects of ralinepag and treprostinil on the proliferation of treprostinil and ralinepag were 40 nM and 10 nM, respectively, so its therapeutic mode of action may not be entirely related to the inhibition of PDE5 (Bischoff, 2004).

[0178] Figure 5 shows the antiproliferative effect of ralinepag in the absence and presence of 100 nM treprostinil in human PASMC grown for 4 days in 9% serum and 0.1% DMSO. In four of the five cell isolates, cell growth was more inhibited when treprostinil was combined with ralinepag than when ralinepag alone was used. From the average data, overall ralinepag appeared to be more effective in the presence of treprostinil over the entire concentration range (P<0.001, two-way ANOVA), although treprostinil only significantly enhanced the highest dose (10 μM) of ralinepag in post hoc testing (P<0.05, two-way ANOVA with Bonferroni correction).

[0179] Figure 6 The average antiproliferative effect of ralinepag in the absence and presence of 100 nM treprostinil is shown compared to the response to 9% serum and 0.1% DMSO alone. Treprostinil significantly inhibited growth at 100 nM compared to serum and DMSO alone. When combined with ralinepag, significant cell growth inhibition occurred at 10-fold lower doses (0.1 nM) of ralinepag. At the highest dose tested (10 μM), ralinepag produced 69% cell growth inhibition, compared to 50% in the absence of treprostinil. This stronger inhibition of serum-induced cell proliferation can be explained in part by the degree of inhibition induced by treprostinil alone, and suggests that both agents inhibit cell proliferation through different pathways.

[0180] The enhanced ralinepag response by treprostinil may be surprising given that both agents are IP agonists and would likely activate the same pathway that inhibits cell proliferation. Treprostinil is also a strong activator of EP2 and DP1 receptors (reviewed in Clapp and Gurung, 2015), which could explain the greater response to ralinepag in the presence of treprostinil. In the experiments presented herein, it was observed that the antiproliferative responses to treprostinil were only weakly inhibited by an IP receptor antagonist, whereas those to ralinepag were completely inhibited. Antiproliferative Effects of Ralinepag in Combination with Endothelin-1 Receptor Antagonist (ERA)

[0181] Fig. 7 shows the antiproliferative effect of ralinepag in the absence and presence of 100nM macitentan in human PASMC grown for 4 days in 9% serum and 0.1% DMSO. The response to ralinepag was only slightly affected when macitentan (100nM) was combined, and in two of the five cell isolates, there was no enhanced antiproliferative effect in any concentration of ralinepag studied. In the other three cell isolates, there was a greater inhibitory trend to cell growth, which was more obvious in lower doses of ralinepag when combined with 100nM macitentan. However, overall, from the average data, ralinepag was not significantly more effective in the presence of macitentan compared to ralinepag alone.

[0182] Figure 8 The average antiproliferative effect of ralinepag in the absence and presence of 100 nM macitentan is shown compared to the response to 9% serum and 0.1% DMSO alone. Macitentan (100 nM) alone caused a significant inhibition of cell proliferation (~12%) compared to serum and DMSO. In addition, when combined with macitentan, ralinepag inhibited cell proliferation at a dose (0.1 nM) 10 times lower than that of ralinepag alone. At higher concentrations of ralinepag (>10 nM), the effects converged, making the response to ralinepag in the absence or presence of macitentan similar.

[0183] The modes of action of these two drugs can overlap and therefore provide no real additional benefit when combined. Macitentan is a mixed ET-1 antagonist that inhibits binding to both ETA and ETB receptors with an average IC 50 The values ​​were 0.5 nM and 391 nM, respectively (Iglarz et al., 2014). Therefore, at the concentrations used, macitentan will mainly inhibit ETA receptors, which are known to contribute to ET-1-induced proliferation of distal human PASMC cells (Zamora et al., 1993; Davie et al., 2002). On the other hand, prostacyclin analogs inhibit the release of ET-1 in human distal PASMCs induced by serum or transforming growth factor β (TGF-β), and this mechanism is partly assumed to constitute the basis for the antiproliferative effect of prostacyclin analogs in normal human PASMCs (Wort et al., 2001; Davie et al., 2002).

[0184] Figure 9 shows the antiproliferative effects of ralinepag in the absence and presence of 100 nM bosentan in human PASMCs grown in 9% serum and 0.1% DMSO for 4 days. Bosentan did not enhance the antiproliferative response to ralinepag in four of the five cell isolates compared to the other PAH drugs, and in one cell isolate, it actually reduced its response. Overall, from the averaged data, ralinepag was not more effective in the presence of bosentan across the concentration range. Figure 4 The average antiproliferative effect of ralinepag in the absence and presence of 100 nM bosentan is shown compared to the response to 9% serum and 0.1% DMSO alone. Unlike macitentan, bosentan (100 nM) does not cause significant inhibition of cell proliferation compared to serum and DMSO alone. In addition, when combined with ralinepag, bosentan does not enhance the inhibition of cell proliferation compared to ralinepag alone, nor does it reduce the concentration (1 nM) where ralinepag begins to significantly inhibit cell proliferation as observed with all other agents (riociguat, treprostinil, and macitentan). Therefore, bosentan appears to have a smaller inhibitory effect on cell proliferation than macitentan, which may be related to its lower potency for ETA receptors and / or differential receptor ET-1 receptor selectivity. Although bosentan is a mixed ET-1 receptor antagonist similar to macitentan, its potency and selectivity ratio for ETA and ETB receptors differ from its receptor kinetics. Bosentan is 10-fold less potent than macitentan, with a Ki of 4 nM for the ETA receptor, but the selectivity ratio for ETA / ETB is only about 20 (Davie et al., 2009), compared to 780 for macitentan (Iglarz et al., 2014).

[0185] Comparison of different PAH drugs in combination with ralinepag

[0186] Combining cGMP-elevating agents and treprostinil with ralinepag appeared to provide a greater antiproliferative effect, whereas ETRA did not significantly enhance the effect of ralinepag on cell proliferation (Figure 11). At 10 nM ralinepag, growth was inhibited by 45% when combined with riociguat, whereas in the presence of bosentan, growth was inhibited by only 21% ( Fig.12 ). However, the potentiation effect of sildenafil was smaller compared with riociguat and, to a lesser extent, treprostinil (even though there was a significant drug interaction, it was not significantly different with post hoc tests). The antiproliferative effects of ralinepag in the absence and presence of different PAH therapies were summarized, with the EC of each individual fit being obtained. 50 and EMax Values ​​(expressed as mean ± SEM using the data contained in Figures 1, 3, 5, 7 and 9 in Table 1). Table 1 shows that the potency of ralinepag was approximately 2.5-5 times greater in the presence of riociguat and sildenafil, respectively (EC 50 =10 nM and 5 nM). Overall, ralinepag produced significantly greater maximal responses in the presence of riociguat or treprostinil, but not in combination with other PAH therapies. Table 2 presents the EC values ​​obtained from fitting the average data. 50 and E Max The EC values ​​of ralinepag were analyzed (Figure 11). 50 Slightly higher than the individual fitted EC 50 (44 nM vs. 25 nM), although the potency was still enhanced 8-fold in the presence of riociguat and sildenafil. Ralinepag still produced a significantly greater maximal response in the presence of riociguat or treprostinil, but not in the presence of other PAH therapies.

[0187] Table 1. Mean antiproliferative effects of ralinepag in combination with other PAH drugs (extrapolated log EC from individual fits) 50 and Emax)

[0188]

[0189] Table 2. Average antiproliferative effects of ralinepag in combination with other PAH drugs (extrapolated log EC from average fits) 50 and Emax)

[0190]

[0191] Example 2: Clinical Trial

[0192] Methods: A 22-week randomized, double-blind, placebo-controlled study was conducted with a dose titration period of up to 9 weeks. Sixty-one patients were randomized 2:1 to ralinepag and placebo. Right heart catheter (RHC) measurements were obtained before day 1 and week 22 of the study dose titration period. The following values ​​were obtained and recorded: pulmonary artery pressure (PAP) (systolic, diastolic, and mean), heart rate (HR), right atrial pressure (RAP), pulmonary capillary wedge pressure (PCWP), right ventricular pressure (RVP), and cardiac output (CO), pulmonary vascular resistance (PVR), arterial and mixed venous oxygen saturation (FiO2) (if applicable). Systemic vascular resistance (SVR) was estimated from blood pressure measurements. All patients received background PAH treatment with endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, or soluble guanylate cyclase activators, either alone or in combination.

[0193] The primary efficacy endpoint of the study was the change from baseline in PVR after 22 weeks of treatment. Other analyses included the change from baseline in 6MWD after 22 weeks of treatment, hemodynamics, and safety and tolerability. Ralinepag was administered as capsules in 0.01, 0.02, 0.03, 0.04, and 0.10 mg dose strengths.

[0194] The starting dose of ralinepag is 0.01 mg twice daily. The dose of ralinepag is titrated according to patient tolerance. If the initial dose is tolerated (0.01 mg twice daily), the dose is increased once a week in the following manner: 0.02 mg twice daily, 0.03 mg twice daily, 0.04 mg twice daily, 0.06 mg twice daily, 0.08 mg, 0.1 mg twice daily, 0.2 mg twice daily, and 0.3 mg twice daily. The dose is optionally increased to the maximum possible total daily dose of 0.6 mg (0.3 mg twice daily) pending tolerance. If the dose is not tolerated, ralinepag is optionally reduced to the previous dose level. If the initial dose of 0.01 mg twice daily is not tolerated, the dose is optionally reduced to 0.01 mg once daily.

[0195] Compared to placebo, ralinepag achieved the primary endpoint of a statistically significant change from baseline in pulmonary vascular resistance (PVR). Ralinepag also demonstrated a numerical improvement in 6-minute walk distance (6MWD). Adverse events observed in this study were consistent with other prostacyclin therapies used to treat PAH. The maintenance dose distribution for patients treated with ralinepag was as follows: 0.02 mg (n=1), 0.03 mg (n=1), 0.04 mg (n=0), 0.06 mg (n=3), 0.08 mg (n=3), 0.12 mg (n=5), 0.16 mg (n=4), 0.2 mg (n=6), 0.4 mg (n=12), and 0.6 mg (n=5).

[0196] All documents cited herein, including patent and non-patent literature, are hereby incorporated by reference in their entirety for all purposes.

[0197] References

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Claims

1. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) or chronic thromboembolic pulmonary hypertension (CTEPH), wherein the medicament is formulated for administration in combination with one or more cGMP elevating agents, cAMP elevating agents or prostaglandins.

2. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) or chronic thromboembolic pulmonary hypertension (CTEPH) in combination with one or more cGMP elevating agents, cAMP elevating agents or prostaglandins.

3. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in combination with one or more cGMP elevating agents, cAMP elevating agents or prostaglandins in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) or chronic thromboembolic pulmonary hypertension (CTEPH).

4. The use according to claim 1 or 2 or 3, wherein the one or more cGMP or cAMP raising agents are soluble guanylate cyclase (sGC) stimulators.

5. The use of claim 1 or 2 or 3, wherein the ralpagp is formulated for administration in combination with one or more cGMP-elevating agents.

6. The use of claim 1 or 2 or 3, wherein the ralinepag is formulated for administration in combination with one or more cAMP elevating agents.

7. The use of claim 1 or 2 or 3, wherein the ralinepag is formulated for administration in combination with a prostaglandin.

8. The use of any one of claims 1-3, wherein the dose of ralinepag does not require standard titration at the start of the treatment.

9. The use of any one of claims 1-3, wherein the one or more cGMP raising agents, cAMP raising agents or prostaglandins are administered once daily in combination with ralinepag.

10. The method of any one of claims 1 to 3, wherein the one or more cGMP elevating agents are selected from the group consisting of viliciguat, ataciguat, nelociguat, riviciguat, IW-1701, IW-1973, IWP-051, IWP-121, IWP-427, IWP-953, BAY-60-2770, A-344905, A-350619, A-778935, BI-684067, BI-703704, BAY-41-2272, and BAY-41-8543.

11. The use according to any one of claims 1 to 3, wherein the cGMP elevating agent is a PDE-5 inhibitor.

12. The use according to any one of claims 1 to 3, wherein the one or more prostaglandins is treprostinil.

13. The use of any one of claims 1-3, wherein the one or more prostaglandins is iloprost.

14. The use of claim 11, wherein the ralinepag and the PDE-5 inhibitor are formulated for co-administration.

15. The use of any one of claims 1-3, wherein the medicament is formulated for co-administration of ralinepag and treprostinil.

16. The use of any one of claims 1-3, wherein the medicament is formulated for co-administration of ralinepag and iloprost.

17. The use of any one of claims 1-3, wherein the medicament is formulated for co-administration of ralinepag and a synthetic prostacyclin analog.

18. The use of any one of claims 1-3, wherein the PAH is selected from the treatment of the following conditions: idiopathic PAH; familial PAH; PAH associated with collagen vascular disease, congenital heart disease, portal hypertension, HIV infection, drug or toxin ingestion, hereditary hemorrhagic telangiectasia, splenectomy, pulmonary veno-occlusive disease (PVOD) or pulmonary capillary hemangioma (PCH); and PAH with significant venous or capillary involvement.

19. The use according to any one of claims 1 to 3, wherein the medicament is for the treatment of PAH.

20. The use according to any one of claims 1 to 3, wherein the medicament is for treating CTEPH.

21. The use of any one of claims 1-3, wherein the medicament is formulated to contain the ralinepag in an amount selected from 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58 and 0.6 mg.

22. The use of any one of claims 1-3, wherein the medicament is formulated to contain the ralinepag in an amount selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 and 0.3 mg.

23. The use of any one of claims 1-3, wherein the medicament is formulated to contain the ralinepag in an amount selected from 0.01 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.16 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg and 1.0 mg per day.

24. The use of any one of claims 1 to 3, wherein the medicament is formulated for once-daily administration of the ralinepag.

25. The use of any one of claims 1 to 3, wherein the medicament is formulated for twice daily administration of the ralinepag.

26. The use of any one of claims 1 to 3, wherein the medicament is formulated for titration of the ralinepag.

27. Use of Ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for treating pulmonary arterial hypertension (PAH), wherein the medicament is formulated for administration in combination with Treprostinil.

28. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) in combination with treprostinil.

29. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in combination with treprostinil for the preparation of a medicament for treating pulmonary arterial hypertension (PAH).

30. The use of any one of claims 27-29, wherein the pharmaceutical formulation comprises an amount of Treprostinil selected from the group consisting of 0.1, 0.125, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 0.25, 0.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, and 5 mg.

31. The use of any one of claims 27-29, wherein the medicament is formulated to contain an amount of Treprostinil selected from 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 2.5, 3, 4, 5, 6, 7, 7.5, 8, 9 and 10 mg / ml.

32. The use of any one of claims 27-29, wherein the medicament is formulated to contain an amount of ralinepag selected from about 0.01 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.16 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg.

33. Use of Ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for treating pulmonary arterial hypertension (PAH), wherein the medicament is formulated for administration in combination with a PDE-5 inhibitor.

34. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) in combination with a PDE-5 inhibitor.

35. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in combination with a PDE-5 inhibitor for the preparation of a medicament for treating pulmonary arterial hypertension (PAH).

36. The use according to any one of claims 33 to 35, wherein the PDE-5 inhibitor is sildenafil.

37. The use of any one of claims 33-35, wherein the medicament is formulated to contain an amount of ralinepag selected from about 0.01 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.16 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg.

38. The use of any one of claims 33-35, wherein the medicament is formulated to contain ralinepag in an amount selected from 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg and 1.0 mg per day.

39. Use of Ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for treating pulmonary arterial hypertension (PAH), wherein the medicament is formulated for administration in combination with an endothelin-1 receptor antagonist.

40. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for treating pulmonary arterial hypertension (PAH) in combination with an endothelin-1 receptor antagonist.

41. Use of ralinepag or a pharmaceutically acceptable salt, hydrate or solvate thereof in combination with an endothelin-1 receptor antagonist in the preparation of a medicament for treating pulmonary arterial hypertension (PAH).

42. The use according to any one of claims 39 to 41, wherein the endothelin-1 receptor antagonist is macitentan.

43. The use according to any one of claims 39 to 41, wherein the endothelin-1 receptor antagonist is bosentan.

44. The use of any one of claims 39-41, wherein the medicament is formulated to contain an amount of ralinepag selected from about 0.01 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.16 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, and 1.0 mg.

45. The use of any one of claims 39-41, wherein the medicament is formulated to contain ralinepag in an amount selected from 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg and 1.0 mg per day.

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