Combination therapy for the treatment of pulmonary hypertension

Abstract

A combination therapy for treating pulmonary hypertension (e.g., PAH, PH-ILD) includes a dose of treprostinil comprising greater than 10 micrograms of treprostinil and a dose of an ActRIIA fusion protein. The dose of treprostinil and the dose of ActRIIA fusion protein may be administered concurrently or sequentially. Further methods for treating pulmonary hypertension include administering a therapeutically effective amount of ActRIIA fusion protein to a subject who has received a composition containing treprostinil, or administering a composition containing treprostinil to a subject who has received a therapeutically effective amount of ActRIIA fusion protein. The treprostinil may be administered as a dry7 powder or liposomal composition.

Description

TITLECombination Therapy for the Treatment of Pulmonary HypertensionCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No.63 / 721,016, filed November 15, 2024, all of which is incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure provides an improvement to the treatment of pulmonary hypertension, a condition that deteriorates the lives of many thousands of patients toward an untimely death. The present disclosure, in some embodiments, provides a combination therapy comprising one or more treatments for pulmonary' hypertension administered sequentially or concurrently with an ActRIIA fusion protein to a subject in need thereof. In some embodiments, the present disclosure provides a combination therapy for the treatment of pulmonary' hypertension that includes an inhalable treprostinil formulation that is administered concurrently or sequentially with an ActRIIA fusion protein to a subject in need thereof. In some embodiments, the inhalable treprostinil formulation is a dry powder.BACKGROUND

[0003] Pulmonary hypertension (PH) is a condition characterized by high blood pressure in the pulmonary vessels. PH has several underlying causes. The World Health Organization (WHO) guidelines classify PH into five groups.• Group 1 : Pulmonary arterial hypertension (PAH),• Group 2: PH due to left heart disease,• Group 3: PH due to lung disease, e.g., chronic obstructive pulmonary disease (COPD) and pulmonary hypertension associated with interstitial lung disease (PH- ILD),• Group 4: PH due to chronic pulmonary' obstructions, e.g., blood clots in the lungs, and• Group 5: PH due to unknown causes.DBl / 153257679.1 1

[0004] PH initially presents as exertional dyspnea, lethargy, and fatigue and is often confused for other disease states. This confusion often leads to delayed diagnosis and treatment. As PH progresses, additional symptoms appear. Depending on the type of PH, these symptoms may include right ventricular failure, exertional chest pain (i.e., angina), exertional syncope, and peripheral edema. Diagnostic tools to identify PH as well as the WHO Group include right heart catheterization, echocardiogram, blood tests, x-rays, and computerized tomography (CT), among other things. Following diagnosis treatment is recommended to lower pulmonary pressures and treat symptoms. Although no cure exists for PH, treatment of PH is directed at improving hemodynamic measures and qualify of life.

[0005] PH can limit a patient’s functionality and qualify of life. These limitations may progress over the course of the disease. The New York Heart Association (NYHA) heart failure guidelines have been applied to PH to describe the severity of the disease state. Using this guideline, he severity may be classified as follows:• NYHA Class I: Subjects with no limitation of activities; they suffer no symptoms from ordinary activities.• NYHA Class II: Subjects with slight, mild limitation of activity; they are comfortable with rest or with mild exertion.• NYHA Class III: Subjects with marked limitation of activity: they are only comfortable at rest.• NYHA Class IV: Subjects who should be at complete rest, confined to bed or chair; any physical activity brings on discomfort and symptoms occur at rest.

[0006] WHO has also developed a PH-specific functional classification system that mirrors the NYHA guidelines. Under the WHO guidelines, severity is classified as follows:• WHO Class I: Subjects have no sy mptoms and no limitation of phy sical activity, • WHO Class II: Subjects have mild symptoms and slight limitation during ordinary physical activity,• WHO Class III: Subjects have marked symptoms with significant limitation during less-than-ordinary activity, and• WHO Class IV : Subjects have symptoms at rest and are unable to perform any physical activity without discomfort.

[0007] PAH and PH-ILD are two types of PH. PAH is classified as WHO Group 1. PH- ILD is classified as WHO Group 3.DBl / 153257679.1 2

[0008] PAH affects approximately 15 out of every' one million individuals. There are approximately 1.000 new cases of PAH diagnosed in the United States each year. The mean age at diagnosis is between 50 and 65 years of age, although the disorder may present much earlier in childhood or even infancy. While gender-based prevalence estimates for PAH are variable, estimates for the overall prevalence of pulmonary' hypertension (PH) in females is approximately twice that of males. PAH has several phenotypes including: idiopathic, heritable, associated with other conditions, PAH with specific features, and persistent pulmonary hypertension of the newborn to name a few.

[0009] Pulmonary arterial hypertension is a serious, progressive and life-threatening disease of the pulmonary vasculature, characterized by profound vasoconstriction and an abnormal proliferation of smooth muscle cells in the walls of the pulmonary arteries. Severe constriction of the blood vessels in the lungs leads to very' high pulmonary arterial pressures. These high pressures make it difficult for the heart to pump blood through the lungs to be oxygenated. Subjects with PAH suffer from extreme shortness of breath as the heart struggles to pump against these high pressures. Subjects with PAH typically develop significant increases in pulmonary vascular resistance (PVR) and sustained elevations in pulmonary' artery' pressure (PAP), which ultimately lead to right ventricular failure and death. Subjects diagnosed with PAH have a poor prognosis and equally compromised quality’ of life, with a mean life expectancy of 2 to 5 years from the time of diagnosis if untreated.

[0010] A variety of factors contribute to the pathogenesis of pulmonary' hypertension including proliferation of pulmonary cells which can contribute to vascular remodeling (i.e., hyperplasia). For example, pulmonary vascular remodeling occurs primarily by proliferation of arterial endothelial cells and smooth muscle cells of subjects with pulmonary hypertension. Overexpression of various cytokines is believed to promote pulmonary' hypertension. Further, it has been found that pulmonary' hypertension may rise from the hyperproliferation of pulmonary arterial smooth cells and pulmonary endothelial cells. Still further, advanced PAH may be characterized by muscularization of distal pulmonary arterioles, concentric intimal thickening, and obstruction of the vascular lumen by proliferating endothelial cells. Pietra et al., J. Am. Coll. Cardiol., 43:255-325 (2004).

[0011] Interstitial lung disease (ILD) is caused by inflammation and scarring of the lung tissue, fibrosis, making the lungs stiff and inefficient for oxygen exchange. These changes progress over the course of the disease leading to chronic hypoxia and increased lung DBl / 153257679.1 3pressures. There are over two-hundred lung disorders falling under ILD including COPD and PH-ILD. Approximately 3.5 to 15% of subjects with early stage ILD have PH.Approximately 30 to 50% of subjects having advanced stage ILD have PH. PH-ILD subjects are sicker and have a poorer prognosis than subjects with ILD alone. PH-ILD have a two-fold risk of death compared to PAH subjects.SUMMARY OF THE INVENTION

[0012] The present disclosure, in some embodiments, provide methods and compositions for treating pulmonary hypertension (e.g., PAH, PH-ILD). In some embodiments, certain methods comprise administering a therapeutically effective dose of one or more ActRIIA fusion proteins (e.g., sotatercept) to a human subject that is receiving or has received a background therapy for treating pulmonary hypertension. In some embodiments, the subject is a mammal. In other embodiments, the subject is human. In particular embodiments, the subject is a human suspected of having or diagnosed as having one or more forms of pulmonary hypertension. In some such embodiments, the background therapy comprises administration of a therapeutically effective dose of treprostinil, treprostinil prodrugs, or a pharmaceutically acceptable salt of any one thereof. In some embodiments, certain methods comprise administering treprostinil to a subject who has received a background therapy for treating pulmonary' hypertension. In some such embodiments, the background therapy comprises administration of a therapeutically effective dose of one or more ActRIIA fusion proteins (e.g., sotatercept). The treprostinil. treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof, in some embodiments, is administered via a dry powder inhaler. In some embodiments the treprostinil, treprostinil prodrugs, or pharmaceutically acceptable salt of any one thereof, is administered via a nebulizer.

[0013] In some embodiments, a method for treating pulmonary hypertension (e.g., PAH, PH-ILD) comprises administering to a subject a formulation of treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof, and one or more ActRIIA fusion proteins (e.g., sotatercept). In some cases, the present disclosure may also treat other indications under the pulmonary disease states.

[0014] In some embodiments, a method for treating pulmonary' hypertension (e.g., PAH, PH-ILD) comprises administering to a subject an inhalation dry powder formulation of treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof, and one or more ActRIIA fusion proteins (e.g., sotatercept).DBl / 153257679.1 4

[0015] In some embodiments, a method of treating pulmonary hypertension according to the present disclosure comprises administering to a subject in need thereof, an inhalation treatment for pulmonary arterial hypertension, comprising a dose of greater than 25 micrograms of treprostinil, and a fusion protein comprising an ActRIIA polypeptide domain comprising the amino acid sequence of SEQ ID NO: 2, an Fc domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and a linker domain. In other embodiments, the linker domain is positioned between the ActRIIA polypeptide domain and the Fc domain. In particular embodiments, the linker domain comprises TGGG (SEQ ID NO: 4). In particular embodiments, the ActRIIA polypeptide domain comprises SEQ ID NO: 11.

[0016] In some embodiments, the inhalation treatment dose comprises 100 micrograms to 350 micrograms of treprostinil. In other embodiments, the inhalation treatment dose comprises 25 micrograms to 220 micrograms of treprostinil enclosed in one or more dosage units. In other embodiments, the inhalation treatment dose comprises 50 micrograms to 110 micrograms of treprostinil enclosed in one or more dosage units. In particular embodiments, the inhalation treatment dose comprises 100 micrograms to 220 micrograms of treprostinil enclosed in one or more dosage units. In some embodiments, the inhalation treatment dose comprises 2.5 mg to 15 mg of dry powder particles. In other embodiments, the inhalation treatment dose comprises greater than or equal to 15 mg of dry powder particles.

[0017] The inhalation treatment dose may be enclosed as dry powder particles in one or more dosage units and administered to the subject via a dry powder inhaler configured to receive the one or more dosage units. For example, in some embodiments, the one or more dosage units may include capsules, cartridges, or other containment devices that may be opened by the dry powder inhaler to release the dry powder particles. In some embodiments, the dose of dry powder particles is packaged in a closeable container having a cap. In other embodiments, the one or more dosage units comprises one or more capsules. In some embodiments, each of the one or more capsules contains an amount of the dry powder particles that is configured to be administered to the subject over one to two breaths using the dry powder inhaler. In other embodiments, the dry7powder particles comprise about 0.5% by weight treprostinil. treprostinil prodrugs, or the pharmaceutically acceptable salt of any one thereof, and wherein each of the one or more capsules contains 25 micrograms to 220 micrograms treprostinil, treprostinil prodrug, or a pharmaceutically' acceptable salt of any oneDBl / 153257679.1 5thereof. In other embodiments, the dry powder particles comprise about 1% by weight treprostinil, treprostinil prodrugs, or the pharmaceutically acceptable salt of any one thereof, and wherein each of the one or more capsules contains 25 micrograms to 220 micrograms treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof.

[0018] In some embodiments, the subject receives greater than about 25 micrograms treprostinil per breath. In other embodiments, the dry powder composition comprises less than or equal to about 12.5 mg of dry powder per breath. In other embodiments, the subject inhales between 100 micrograms and 300 micrograms treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof, through four to eight breaths over a day. In other embodiments, the subject inhales between 100 micrograms and 1600 micrograms treprostinil, a treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof through four to twenty-four breaths per day. In some embodiments, about 600 micrograms treprostinil is inhaled by the subject in eight breaths or less per day. In other embodiments, about 1200 micrograms treprostinil is inhaled by the subject in sixteen breaths or less per day. In other embodiments, about 1600 micrograms treprostinil is inhaled by the subject in twenty-four breaths or less per day. In other embodiments, the subject inhales the dry powder inhalation treatment four times a day. In other embodiments, the dry powder inhalation treatment comprises between about 100 micrograms treprostinil and about 1600 micrograms treprostinil and is administered to a subject over a day through four to twenty- four breaths.

[0019] In some embodiments, the dry powder particles have a mass median aerodynamic diameter (MMAD) from 1 micrometer to 5 micrometers. In other embodiments, the dry powder particles have a MMAD of 3 micrometers or less. In some embodiments, the dry powder particles are formed having a predetermined size range. In other embodiments, the dry powder particles comprise particles that are substantially uniform in shape.

[0020] In some embodiments, the dry powder inhalation treatment comprises molded dry particles comprising a non-reducing sugar, a wetting agent, a hydrophobicity modifying agent, a pH modifying agent and a buffer. In other embodiments, the dry powder composition comprises molded dry particles comprising by percent solids about 0.581 percent treprostinil sodium, about 92.32 percent trehalose, about 2.19 percent polysorbate 80, about 4.39 percent L-leucine, about 0.26 percent sodium citrate, and about 0.25 percent sodium chloride. In other embodiments, the dry powder particles comprise about 0.53% by DBl / 153257679.1 6weight treprostinil or the pharmaceutically acceptable salt thereof. In other embodiments, the dry powder particles comprise about 1.06% by weight treprostinil or the pharmaceutically acceptable salt thereof. In other embodiments, the dry powder particles are dried to less than 5 percent water content. In other embodiments, the dry powder particles are dried to less than 2 percent water content.

[0021] In some embodiments, the fusion protein is administered at a dose between 0.1 mg / kg and 2.0 mg / kg. In particular embodiments, the fusion protein is administered at a dose of 0.3 mg / kg. In particular embodiments, the fusion protein is administered at a dose of 0.7 mg / kg. In some embodiments, the fusion protein is administered at a first dose of betw een 0.1 mg / kg and 1.0 mg / kg of said fusion protein for a first period of time, and a second dose of between 0.1 mg / kg and 1.0 mg / kg of said fusion protein is subsequently administered for a second period of time. In particular embodiments, the first period of time is at least 3 weeks. In particular embodiments, the second period of time is at least 3 weeks. In particular embodiments, the second period of time is at least 21 weeks. In particular embodiments, the second period of time is at least 45 weeks. In particular embodiments, the second period of time exceeds the first period of time. In some embodiments, the second dose exceeds the first dose. In other embodiments, the first dose is in the range of about 0.2 mg / kg to about 0.4 mg / kg followed by a second dose in the range of about 0.5 mg / kg to about 0.8 mg / kg. In other embodiments, the first dose is about 0.3 mg / kg followed by a second dose of about 0.7 mg / kg. In other embodiments, the fusion protein is administered using subcutaneous injection.

[0022] In some embodiments, the fusion protein is formulated into a pharmaceutical composition. In other embodiments, the fusion protein is administered to the subject on a schedule selected from the group consisting of every week, every tw o w eeks, every three weeks, and every four weeks. In other embodiments, the fusion protein is administered to the subject every 3 weeks. In other embodiments, the fusion protein is administered to the subject every 4 weeks.

[0023] In other embodiments, the method decreases the pulmonary arterial pressure by at least 3 mmHg. In other embodiments, the method decreases the pulmonary arterial pressure by at least 7 mmHg. In other embodiments, the method increases the subject's 6-minute walk distance. In other embodiments, the method increases the subject's 6-minute walk distance by at least 50 meters. In other embodiments, the method decreases ventricle hypertrophy in DBl / 153257679.1 7the subject. In other embodiments, the method decreases smooth muscle hypertrophy in the subject. In other embodiments, the method decreases pulmonary arteriole muscularity in the subject. In other embodiments, the method decreases pulmonary vascular resistance in the subject. In other embodiments, the method decreases pulmonary vascular resistance in the subject by at least 20%.

[0024] In some embodiments, the subject has pulmonary arterial hypertension and has Functional Class II or Class III pulmonary hypertension in accordance with the NYHA's functional classification system for pulmonary' hypertension.

[0025] In other embodiments, the method delays clinical worsening of pulmonary arterial hypertension. In other embodiments, the method delays clinical worsening of pulmonary arterial hypertension in accordance with the NYHA's functional classification system for pulmonary hypertension. In other embodiments, the method delays clinical worsening of pulmonary hypertension by delaying the progression from Functional Class II to Class III as recognized by the NYHA.

[0026] In some embodiments, the inhalation treatment (containing treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof) and the fusion protein (e.g., sotatercept) are administered sequentially. In some embodiments, the inhalation treatment is administered after the fusion protein. In other embodiments, the fusion protein is administered after the inhalation treatment. In other embodiments, the inhalation treatment and the fusion protein are administered sequentially within about 12 hours, about 24 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, or about 3 weeks of each other. In some embodiments, the inhalation treatment and the fusion protein are administered concurrently. In some embodiments, the inhalation treatment is administered daily, every other day, even.' third day, weekly, every other week, every three weeks, or monthly. In some embodiments, the fusion protein is administered daily, every other day, every third day, weekly, every other week, every three weeks, or monthly. In particular embodiments, the fusion protein is administered every' three weeks.

[0027] In some embodiments, the pulmonary hypertension is pulmonary arterial hypertension (PAH). In other embodiments, the pulmonary7hypertension is pulmonary’ hypertension caused by interstitial lung disease (PH-ILD).DBl / 153257679.1 8

[0028] In some embodiments, a pharmaceutical kit for treating pulmonary hypertension according to the present disclosure comprises an inhalation treatment for pulmonary arterial hypertension, comprising an inhalation treatment dose comprising greater than 25 micrograms of treprostinil enclosed in one or more dosage units, and a fusion protein comprising an ActRIIA polypeptide domain comprising the amino acid sequence of SEQ ID NO: 2, an Fc domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and a linker domain. In other embodiments, the linker domain is positioned between the ActRIIA polypeptide domain and the Fc domain. In other embodiments, the linker domain comprises TGGG (SEQ ID NO: 4). In particular embodiments, the ActRIIA polypeptide domain comprises SEQ ID NO: 11.

[0029] In some embodiments, the one or more dosage units include a total predetermined amount of the treprostinil, treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof in the range of 25 micrograms to 350 micrograms. In other embodiments, the pharmaceutical kit comprises a dry’ powder inhaler comprising a body configured to receive the one or more dosage units, and a mouthpiece that is moveable with respect to the body to an aligned position, wherein the dry powder particles may be inhaled by the subject through the mouthpiece when the mouthpiece is in the aligned position. In other embodiments, the mouthpiece is rotatable with respect to the body. In other embodiments, the dry powder inhaler comprises a moveable component configured to open the one or more dosage units to allow release of the dry powder particles from the one or more dosage units.

[0030] In some embodiments, each of the one or more dosage units contains treprostinil, treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof in an amount of 25 micrograms to 220 micrograms. In other embodiments, each of the one or more dosage units contains 2.5 mg to 30 mg of the dry powder particles. In some embodiments, each of the one or more dosage units contains greater than or equal to 15 mg of the dry powder particles. In some embodiments, the dry’ powder particles comprise about 0.53% by weight treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof. In other embodiments, the dry powder particles comprise about 1.06% by weight treprostinil, treprostinil prodrug, or pharmaceutically' acceptable salt of any one thereof.

[0031] In some embodiments, the dry powder particles have a mass median aerodynamic diameter (MMAD) between 1 micrometer and 5 micrometers. In other embodiments, the dry powder particles have a MMAD of 3 micrometers or less. In some embodiments, the dry' DBl / 153257679.1 9powder particles are dried to less than 5 percent water content. In some embodiments, the dose of dry powder particles is packaged in a closeable container having a cap. In some embodiments, the one or more dosage units comprises one or more capsules. In some embodiments, each of the one or more capsules contains an amount of the dry powder particles that is configured to be administered to the subject over one to two breaths using the dry powder inhaler. In particular embodiments, the dry powder particles are formed having a predetermined size range. In other embodiments, the dry powder particles comprise particles that are substantially uniform in shape.

[0032] In some embodiments, the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof becomes pharmaceutically available in less than 10 seconds upon delivery to the subject via inhalation. In other embodiments, the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof becomes pharmaceutically available in less than 5 seconds upon delivery to the subject via inhalation. In other embodiments, the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof becomes pharmaceutically available in less than 1 second upon deliver}7to the subject via inhalation.

[0033] In some embodiments, the pharmaceutical kit further comprises a second dosage unit receivable in the body of the dry powder inhaler, the second dosage unit containing a second predetermined amount of the dry powder particles. In other embodiments, each of the dosage unit and the second dosage unit contains from about 10 micrograms to about 220 micrograms treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof. In other embodiments, the dosage units contain the same predetermined amount of the dry powder particles. In other embodiments, the dosage units contain a different predetermined amount of the dry powder particles.

[0034] In some embodiments, the kit comprises one or more containers containing the fusion protein. In other embodiments, the kit comprises at least two containers containing the fusion protein. In other embodiments, the at least two containers can contain the same or different amounts of the fusion protein. In some embodiments, the containers comprise between 25 mg to 60 mg of the fusion protein. In other embodiments, at least one of the containers contains 60 mg of fusion protein. In other embodiments, at least one of the containers contains 45 mg of fusion protein. In other embodiments, at least one of the containers contains 30 mg of fusion protein. In some embodiments, at least one of the DBl / 153257679.1 10containers contains 25 mg of fusion protein. In other embodiments, a first container contains 45 mg of fusion protein and a second container contains 60 mg of fusion protein. In other embodiments, a first container contains 30 mg of fusion protein and a second container contains 60 mg of fusion protein. In other embodiments, a first container contains 45 mg of fusion protein and a second container contains 45 mg of fusion protein. In other embodiments, a first container contains 30 mg of fusion protein, a second container contains 45 mg of fusion protein, and a third container contains 60 mg of fusion protein. In other embodiments, a first container contains 25 mg of fusion protein, a second container contains 45 mg of fusion protein, and a third container contains 60 mg of fusion protein. In some embodiments, the containers are refrigerated at 2-8° C.

[0035] In some embodiments, the fusion protein is reconstituted into a sterile injectable solution. In other embodiments, the sterile injectable solution comprises sterile water for injection. In other embodiments, the sterile injectable solution is administered parenterally. In other embodiments, the sterile injectable solution is administered via subcutaneous injection. In other embodiments, the sterile injectable solution is administered via intradermal injection. In other embodiments, the sterile injectable solution is administered via intramuscular injection. In other embodiments, the sterile injectable solution is administered via intravenous injection. In other embodiments, the sterile injectable solution is self-administered. In other embodiments, the sterile injectable solution comprises a therapeutically effective dose. In other embodiments, the therapeutically effective dose comprises a subject weight-based dose. In other embodiments, the sterile injectable solution is administered ever}' 3 weeks. In other embodiments, the sterile injectable solution is administered every 4 weeks.

[0036] In some embodiments, a kit is used to treat pulmonary arterial hypertension (PAH). In other embodiments, a kit is used to treat pulmonary hypertension caused by interstitial lung disease (PH-ILD).

[0037] In some embodiments, a pharmaceutical kit for treating pulmonary hypertension according to the present disclosure comprises a dose of liposomes comprising greater than 30 micrograms of treprostinil enclosed in one or more dosage units, and a fusion protein comprising an ActRIIA polypeptide domain comprising the amino acid sequence of SEQ ID NO: 2, an Fc domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and a linker domain. In other embodiments, the linker domain is positioned DBl / 153257679.1 11between the ActRIIA polypeptide domain and the Fc domain. In other embodiments, the linker domain comprises TGGG (SEQ ID NO: 4). In particular embodiments, the ActRIIA polypeptide domain comprises SEQ ID NO: 11.BRIEF DESCRIPTION OF THE FIGURES

[0038] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention can be embodied in different forms and thus should not be construed as being limited to the illustrated embodiments set forth herein.

[0039] Figure 1 shows a three-dimensional rendering of a pollen particle according to an embodiment of the present invention.

[0040] Figure 2 is an SEM image showing pollen-shaped particles according to an embodiment of the present invention.

[0041] Figure 3 shows the canonical human ActRIIA precursor protein sequence (SEQ ID NO: 1).

[0042] Figure 4 shows the processed (mature) extracellular human ActRIIA polypeptide sequence (SEQ ID NO: 2).

[0043] Figure 5 shows the native amino acid sequence that may be used for the Fc portion of human IgGl (SEQ ID NO: 3).

[0044] Figure 6 shows the amino acid sequence of a preferred fusion protein (SEQ ID NO: 11).DETAILED DESCRIPTION OF EMBODIMENTS

[0045] It has been found that certain activin signaling inhibitors may be useful in treating PH (e.g., PAH), including certain activin receptor type 2a (ActRIIA) fusion proteins. One such example is sotatercept (available under the brand name WINREVAIR™) which is a fusion protein comprised of the Fc domain of human IgG linked to the extracellular domain DBl / 153257679.1 12of human ActRIIA. Without wishing to be bound by theory, it is believed that some such ActRIIA fusion proteins, e.g., sotatercept, may act as a ligand trap for selected TGF-P superfamily members, and inhibition of these ligands may rebalance pulmonary vascular homeostasis toward growth-inhibiting and proapoptotic signaling. This, in turn, may help alleviate inflammation in the vessel walls and restoration of vessel patency.Combination Therapy

[0046] ActRIIA fusion proteins (e.g., sotatercept) may be particularly effective for use in treating pulmonary hypertension in subjects that are concurrently receiving or have already received a background therapy for pulmonary hypertension (e.g., PAH). In some embodiments, the subject is a mammal. In other embodiments, the subject is human. In particular embodiments, the subject is a human suspected of having or diagnosed as having one or more forms of pulmonary hypertension.

[0047] Some embodiments of the present disclosure provide methods and therapies for treating pulmonary' hypertension (e.g., PAH or PH-ILD) that includes administration of a therapeutically effective dose of one or more ActRIIA fusion proteins (e.g., sotatercept) concurrently or sequentially with one or more other treatments for pulmonary hypertension. The one or more other treatments may include, in some embodiments, certain drugs that have been approved to treat PH, for example, endothelin receptor antagonists (ERAs), phosphodiesterase type 5 (PDE5) inhibitors, soluble guanylate cyclase stimulators, prostacyclin receptor agonists, and prostacyclin analogs. For example, in some embodiments, the one or more treatments for administration concurrently or sequentially with one or more ActRIIA fusion proteins (e.g., sotatercept) and their route of administration can include one or more of:• ERAs: bosentan (oral), ambrisentan (oral); and macitentan (oral);• PDE5 inhibitors: sildenafd (oral, intravenous (IV)) and tadalafil (oral);• Soluble Guanylate Cyclase (sGC) Stimulators: riociguat (oral);• Prostacyclin Receptor Agonists: selexipag (oral); and / or• Prostacyclin Analogs: epoprostenol (IV), iloprost (inhaled), and treprostinil (oral, subcutaneous IV and inhaled).

[0048] In some embodiments, the one or more treatments for administration concurrently or sequentially with one or more ActRIIA fusion proteins includes administration of DBl / 153257679.1 13treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof. In some embodiments, the one or more treatments for administration concurrently or sequentially with one or more ActRIIA fusion proteins includes administration of treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof, and administration of one or more of endothelin receptor antagonists (ERAs), phosphodiesterase type 5 (PDE5) inhibitors, soluble guanylate cyclase stimulators, prostacyclin receptor agonists, or other prostacyclin analogs. In some embodiments, the ActRIIA fusion protein may have an amino acid sequence comprising SEQ ID NO: 11. In some embodiments, the ActRIIA fusion protein is sotatercept.

[0049] Treprostinil is a chemically stable tricyclic benzidine prostanoid with vasodilator properties that is capable of reducing pulmonary vasoconstriction with minimal effects on systemic blood pressure. Treprostinil is a synthetic analog of prostacyclin (PGh) and the IUPAC name for treprostinil is (2-[[(lR,2R,3aS,9aS)-2-hydroxy-l-[(3S)-3-hydroxyoctyl]- 2,3,3a,4,9,9a-hexahydro-IH-cyclopenta[g]naphthalen-5-yl]oxy]acetic acid).

[0050] Treprostinil has been approved for the treatment of PAH under the trade names REMODULIN" (United Therapeutics Corporation; subcutaneous or IV infusion), TYVASO" (United Therapeutics Corporation: inhaled via ultrasonic, pulsed nebulization delivery device), TYVASO DPI® (United Therapeutics Corporation; inhaled via dry powder inhalation device) and ORENITRAM (United Therapeutics Corporation; oral tablet).TYVASO and TYVASO DPI have also been approved for the treatment of PH-ILD. A combination therapy according to some embodiments of the present disclosure includes or consists of administering the one or more ActRIIA fusion proteins (e.g., sotatercept) together or sequentially with an administration of treprostinil, a treprostinil prodrug, or a pharmaceutically acceptable salt of treprostinil (e.g., treprostinil sodium). The treprostinil, a treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof may be administered through one or more of the routes of administration previously mentioned (e.g., inhaled via a delivery device, oral administration, subcutaneous administration), and may be administered prior to, after, or concurrently with the administration of the one or more ActRIIA fusion proteins. In some embodiments, the treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof is included in an inhalable dry powder drug product that is administered to the subject via a dry powder inhaler. In still further embodiments, the treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of anyDBl / 153257679.1 14one thereof is administered via inhalable liposomes. In some particular embodiments, the ActRIIA fusion protein is administered to a subject who has previously received an administration of treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any¬ one thereof. In some embodiments, in addition to administration of treprostinil, treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof, a combination therapyaccording to the present disclosure may further include administration of one or more of endothelin receptor antagonists (ERAs), phosphodiesterase type 5 (PDE5) inhibitors, soluble guanylate cyclase stimulators, prostacyclin receptor agonists, or other prostacyclin analogs that are useful for treating PH.

[0051] Unless specified otherwise, the use of the term “treprostinil” alone in the description of embodiments herein shall be understood to encompass treprostinil free acid, and pharmaceutically acceptable salts of treprostinil, e.g., treprostinil sodium. In other embodiments, amphiphilic treprostinil may be particularly useful for loading into liposomes. In some embodiments, amphiphilic treprostinil may contain, for example, at least one functional group selected from the group consisting of a carboxyl group (-COOH) and a hydroxyl group (-OH), which is mostly soluble without forming insoluble crystals, precipitates or gels and is stable in alkaline solution. Treprostinil may also contain one or more functional groups in addition to the carboxylic functionality, although the presence of such functional group should not significantly alter the acidity of treprostinil from that of its nonfunctionalized counterpart. According to some embodiments, the amphiphilic treprostinil may be biologically active in its protonated form or any salt forms thereof. A salt of an amphiphilic treprostinil may be accompanied by any pharmaceutically acceptable counterion which is in an aqueous soluble form.

[0052] “Prodrug” is intended to describe a compound that may be converted under physiological conditions or by enzymatic or non-enzymatic hydrolysis, for example, to provide a biologically active compound described herein. Thus, the term “prodrug” refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, but is converted in vivo to an active compound, for example, by enzymatic or non-enzymatic hydrolysis. The prodrug compound often offers the advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, e.g., Bundgaard, H., Design of Prodrugs (1985) (Elsevier,DBl / 153257679.1 15Amsterdam). The term “prodrug” is also intended to include any covalently bonded carriers, which release the active compound in vivo when administered to a subject.

[0053] The phrase “treprostinil prodrug” as used herein refers to any derivative of treprostinil that converts in whole or in part to treprostinil in vivo following administration. The treprostinil prodrug may have reduced affinity for one or more of the IP. DP or EP receptors locally at the site of injection as compared to treprostinil. In some embodiments, a “treprostinil prodrug” can be a treprostinil derivative with one or more hydroxyl groups of the treprostinil structure modified to have reduced affinity for one or more of the IP, DP or EP receptors as compared to treprostinil, but which can be converted in vivo into active treprostinil following subcutaneous administration and subsequent diffusion into the blood. In some embodiments, the treprostinil prodrug is completely or substantially converted in vivo to treprostinil outside the subcutaneous space, such as in the bloodstream. Preferred treprostinil prodrug include amide, carbonate, or carbamate esters of treprostinil. In embodiments treprostinil is derivatized at one or more hydroxyl groups, including the hydroxyl group of the carboxyl group. In other embodiments, the carboxyl group is derivatized either separately or in conjunction with one or more hydroxyl groups. In some embodiments, the treprostinil prodrug has greater than 50%, 75%, 85%, 90%, 95%, or 98% conversion to treprostinil in vivo following administration. In some embodiments, this conversion takes place in 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hour, or 3 hours following administration. Treprostinil prodrug include pharmaceutically acceptable salts of such prodrugs.INHALATION POWDER DRUG PRODUCT

[0054] A combination therapy according to some embodiments of the present disclosure comprises administering the one or more ActRIIA fusion proteins (e.g., sotatercept) together or sequentially with an administration of treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of treprostinil (e.g., treprostinil sodium), where the treprostinil, treprostinil prodrug, or a pharmaceutically acceptable salt of treprostinil (e.g., treprostinil sodium) is administered as an inhalation powder drug product. The inhalation powder drug product may include inhalable particles containing treprostinil and one or more excipients that may be administered to the subject via a dry powder inhaler. In some such embodiments, each particle includes treprostinil and the one or more excipients.DBl / 153257679.1 16

[0055] The inhalation powder drug product according to certain aspects of the present disclosure provides a dry powder dosage form of treprostinil and excipients, where the treprostinil and excipients are formed into inhalable particles as described in U.S. Pat. Nos.10,898,494, 11,712,442, 11,744,836, 11,744,835, 11,660,304, all of which are incorporated by reference in their entirety herein. In some embodiments, the inhalable particles are filled into a capsule, for example, a hydroxypropyl methylcellulose (HPMC) capsule (size 3), cartridge, or other container configured for use with a dry powder inhaler for administration to the subject. In some embodiments, the dry powder is a treprostinil / excipient matrix from which particles of precise size and shape are formed according to the methods herein. In one example, the particles of the dry powder comprise a shape corresponding generally to a rounded triangular shape having a volume, where the inner portion of the rounded triangular shape, in size, fits a 1 micrometer equilateral triangle (otherwise referred to as being pollenshaped). A three-dimensional rendering of such a particle shape is depicted in Figure 1. In another embodiment, the pollen-shape may be trefoil-shaped with an inscribed circle diameter of 1 micrometer, and a prescribed thickness of a value or range between 0.5 and 1 micrometer, or more preferred 0.7 micrometer. In addition, certain embodiments of the present drug product include particles having 0.5% treprostinil in dose levels ranging from 25 micrograms to 150 micrograms treprostinil (e.g., 25 micrograms, 50 micrograms, 75 micrograms, 100 micrograms, 125 micrograms and 150 micrograms treprostinil). In further embodiments, a drug product according to the present disclosure may provide dose levels ranging from 150 micrograms to greater than 300 micrograms treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof (e.g., 175 micrograms, 200 micrograms, 225 micrograms, 250 micrograms, 275 micrograms, 300 micrograms, 325 micrograms, 350 micrograms treprostinil, 375 micrograms treprostinil, 400 micrograms treprostinil). In some embodiments, the treprostinil is included in the particles as a salt of treprostinil (e.g., treprostinil sodium). In further embodiments, a drug product according to the present disclosure may provide dose levels of 50 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms. 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil loaded into capsules for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 75 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms. 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil loaded into capsules for deliver}' to a subject in a dry powder. In further embodiments, a drug product according to DBl / 153257679.1 17the present disclosure may provide dose levels of 100 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil loaded into capsules for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 125 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms. 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil loaded into capsules for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 150 micrograms treprostinil plus or minus 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 % or 1 % treprostinil loaded into capsules for deli ven’ to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 175 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil loaded into capsules for deliver}’ to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 200 micrograms treprostinil plus or minus 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 % or 1 % micrograms treprostinil loaded into capsules for delivery’ to a subject in a dry’ powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 225 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms. 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil loaded into capsules for delivery' to a subject in a dry' powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 250 micrograms treprostinil plus or minus 10 %, 9 %, 8 %. 7 %, 6 %, 5 %. 4 %, 3 %, 2 % or 1 % treprostinil loaded into capsules for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may' provide dose levels of 275 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms. 2 micrograms or 1 microgram treprostinil loaded into capsules for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 300 micrograms treprostinil plus or minus 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 % or 1 % treprostinil loaded into capsules for delivery to a subject in a dry' powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 325 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 DBl / 153257679.1 18micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil loaded into capsules for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 350 micrograms treprostinil plus or minus 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 % or 1 % treprostinil loaded into capsules for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 375 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil loaded into capsules for delivery7to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 400 micrograms treprostinil plus or minus 10 %. 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 % or 1 % treprostinil loaded into capsules for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide dose levels of 300 micrograms treprostinil plus or minus 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 % or 1 % treprostinil loaded into capsules for deliver}7to a subject in a dry powder.

[0056] According to the present disclosure, the particles remain stable for long periods of time at relatively low humidity7conditions. In some embodiments, the present disclosure provides dry powder particles packaged under sealed conditions that remain stable for more than 3 months at 40 degrees Celsius and 75 percent relative humidity.

[0057] The present disclosure, in some embodiments, also provides a dry7formulation of treprostinil, which upon delivery to a subject via the inhaled route, becomes soluble and pharmaceutically available in less than 10 seconds. In some embodiments, the dry formulation composition becomes soluble and pharmaceutically available in less than 5 seconds. In some embodiments, the dry formulation composition becomes soluble and pharmaceutically available in less than 2 seconds. In some embodiments, the dry formulation composition becomes soluble and pharmaceutically available in about 1 second. In some embodiments, the dry formulation composition becomes soluble and pharmaceutically available in less than 1 second. In some embodiments, the dry formulation composition becomes soluble and pharmaceutically available in less than about 0.5 seconds. Furthermore, the excipients in the dry particle formulation of the present disclosure maintain pH and saltDBl / 153257679.1 19gradient during processing such that the active agent remains in a state to become soluble in the lung conditions of a user.

[0058] An exemplary formulation (also referred to herein as “LIQ861”), particle composition, particle geometry, packaging, device, delivery, stability, dose, and a description ofthe use are described in U.S. Pat. Nos. 10,898,494, 11,712.442, 11,744,836, 11,744,835, 11,660,304, 11,826,327 all of which are herein incorporated by reference in their entirety. Methods and materials that may be used for fabricating the particles according to embodiments of the present disclosure are further described and disclosed in issued patents and co-pending patent applications, each of which is incorporated herein by reference in its entirety: U.S. Pat. Nos. 8,518,316; 8,444,907; 8,420,124; 8.268,446; 8.263,129; 8,158.728; 8,128,393; 7,976,759; 9,214,590; 9,205,594; 8,662,878; 8,439,666;, 8, 128, 393;8, 992,992; 9,545,737; and U.S. Pat. Application Publications Nos. 2013-0228950, 2013-0011618, 2013- 0256354, 2010-0003291, and 2009-0165320.

[0059] In some embodiments, a formulation according to the present disclosure includes a drug substance (e.g., Treprostinil, Treprostinil Sodium, treprostinil palmitil) together with one or more excipients. In some embodiments, the one or more excipients may include a bulking agent, a wetting agent, a hydrophobicity modifier, a pH modifier, a buffer component, or combinations thereof. Examples of such formulations according to certain specific embodiments are provided in the tables below.DBl / 153257679.1 20LIQ861 Drug Product-Intermediate Description for Active (LIQ861) Formulations (dihydrate form calculations)LIQ861 Drug Product-Intermediate Description for Active (LIQ861) Formulations (anhydrous form calculations)DBl / 153257679.1 21Inhalation Particle Drug Product

[0060] The inhalation drug particle product, in some embodiments, includes or consists of a dry powder dosage form of treprostinil and excipients that may be filled into, for example, a HPMC capsule (size 3) or other container for use with an inhaler (e.g., dry powder inhaler). The dry’ powder, in some embodiments, is a treprostinil / excipient matrix from which particles of precise size (e.g., 1 pm) and shape (e.g., ‘'pollen-shaped’’). The ‘'pollen-shaped” particles may also be described as trefoil-shaped, with an inscribed circle diameter of 1 pm, and a thickness of 0.7 pm. A three-dimensional rendering of such a particle shape is depicted in Figure 1. LIQ861 comprised drug product capsule strengths of 25 micrograms,50 micrograms, and 75 micrograms treprostinil used in the first clinical study to investigate planned dose levels of 25 micrograms, 50 micrograms, 75 micrograms, 100 micrograms, 125 micrograms and 150 micrograms treprostinil. The 100 microgram, 125 microgram and 150 microgram doses may be made up of a combination of lower dose capsules. In further embodiments, a drug product according to the present disclosure may provide capsules with dose levels of lOOmicrograms, 125 micrograms, 150 micrograms, 175 micrograms, 200 micrograms, 225 micrograms, 250 micrograms, 275 micrograms, 300 micrograms, 325 micrograms, or 350 micrograms treprostinil. In further embodiments, a drug product according to the present disclosure may provide capsules with dose levels of 25 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil for delivery' to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide capsules with dose levels of 50 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms. 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil for delivery' to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide capsules with dose levels of 75 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms. 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil for delivery' to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide capsules with dose levels of 100 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms. 2 micrograms or 1 microgram treprostinil for delivery' to a subject in a dry powder. In further embodiments, a drug product DBl / 153257679.1 22according to the present disclosure may provide capsules with dose levels of 125 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms. 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil for del i \ ery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide capsules with dose levels of 150 micrograms treprostinil plus or minus 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 % or 1 % treprostinil for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide capsules with dose levels of 175 micrograms treprostinil plus or minus 10 micrograms, 9 micrograms, 8 micrograms, 7 micrograms, 6 micrograms, 5 micrograms, 4 micrograms, 3 micrograms, 2 micrograms or 1 microgram treprostinil for delivery to a subject in a dry powder. In further embodiments, a drug product according to the present disclosure may provide capsules with dose levels of 200 micrograms treprostinil plus or minus 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 % or 1 % treprostinil for delivery' to a subject in a dry' powder. A summary' of the LIQ861 formulation, including powder composition, particle geometry’, and a description of the dosing unit according to certain exemplary embodiments follows.LIQ861 Drug Product-Intermediate Description for Active (LIQ861) Formulations (dihydrate)DBl / 153257679.1 23Inhalation Drug Product Dosing Unit Description*Excipients only (no treprostinil). Abbreviations: HPMC, hydroxypropyl methylcellulose

[0061] According to some embodiments of the present disclosure, drug particles are provided that include a composition having a target dose of 15 - 90 pg of delivered treprostinil to the subject (current TYVASO® label is 18-54 pg). In some embodiments of the present disclosure the dose of treprostinil provided to the subject can be, for example, 100 micrograms, 125 micrograms or 150 micrograms. In some embodiments of the present disclosure the dose of treprostinil provided to the subject, for example, can contain about 100 micrograms, about 125 micrograms, about 150 micrograms or about 175 micrograms. In some embodiments, each dose contains greater than or equal to 200 micrograms of treprostinil. In some embodiments, each dose contains greater than or equal to 225 micrograms of treprostinil. In some embodiments, each dose contains greater than or equal to 250 micrograms of treprostinil. In some embodiments, each dose contains greater than or equal to 275 micrograms of treprostinil. In some embodiments, each dose contains greater than or equal to 300 micrograms of treprostinil. In some embodiments, each dose contains from about 1 micrograms to about 15 micrograms, 15 micrograms to about 20 micrograms, 20 micrograms to about 25 micrograms, 25 micrograms to about 30 micrograms, about 30 micrograms to about 35 micrograms, about 35 micrograms to about 40 micrograms, about 40 micrograms to about 45 micrograms, about 45 micrograms to about 50 micrograms, about 50 micrograms to about 55 micrograms, about 55 micrograms to about 60 micrograms, about 60 micrograms to about 65 micrograms, about 65 micrograms to about 70 micrograms, about 70 micrograms to about 75 micrograms, about 75 micrograms to about 80 micrograms, about 80 micrograms to about 85 micrograms, about 85 micrograms to about 90 micrograms, about 90 micrograms to about 95 micrograms, about 95 micrograms to about 100 micrograms, or about 100 micrograms to about 105 micrograms of treprostinil. In some embodiments, each DBl / 153257679.1 24dose contains from about 100 micrograms to about 110 micrograms, 110 micrograms to about 120 micrograms, 120 micrograms to about 130 micrograms, 130 micrograms to about 140 micrograms, about 140 micrograms to about 150 micrograms, about 150 micrograms to about 160 micrograms, about 160 micrograms to about 170 micrograms, about 170 micrograms to about 180 micrograms, about 180 micrograms to about 190 micrograms, about 190 micrograms to about 200 micrograms, about 200 micrograms to about 210 micrograms, about 210 micrograms to about 220 micrograms, about 220 micrograms to about 230 micrograms, about 230 micrograms to about 240 micrograms, about 240 micrograms to about 250 micrograms, about 250 micrograms to about 260 micrograms, about 260 micrograms to about 270 micrograms, about 270 micrograms to about 280 micrograms, about 280 micrograms to about 290 micrograms, about 290 micrograms to about 300 micrograms, about 300 micrograms to about 310 micrograms, about 310 micrograms to about 320 micrograms, about 320 micrograms to about 330 micrograms, about 330 micrograms to about 340 micrograms, or about 340 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 25 micrograms to about 400 micrograms of treprostinil. In some embodiments, each dose contains from about 25 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 25 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 50 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 175 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 200 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 225 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 250 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 275 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 50 micrograms to about 75 micrograms of treprostinil. In some embodiments, each dose contains from about 50 micrograms to about 100 micrograms of treprostinil. In some embodiments, each dose contains from about 50 micrograms to about 150 micrograms of treprostinil. In some DBl / 153257679.1 25embodiments, each dose contains from about 75 micrograms to about 100 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 125 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 150 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 175 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 200 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 225 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 250 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 275 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 75 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 125 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 150 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 175 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 200 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 225 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 250 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 275 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 100 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 150 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 175 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 200 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 225 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 250 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 275 micrograms of treprostinil. In some embodiments, each dose DBl / 153257679.1 26contains from about 125 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 125 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 175 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 200 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 225 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 250 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 275 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 150 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 175 micrograms to about 200 micrograms of treprostinil. In some embodiments, each dose contains from about 175 micrograms to about 225 micrograms of treprostinil. In some embodiments, each dose contains from about 175 micrograms to about 250 micrograms of treprostinil. In some embodiments, each dose contains from about 175 micrograms to about 275 micrograms of treprostinil. In some embodiments, each dose contains from about 175 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 175 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 175 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 200 micrograms to about 225 micrograms of treprostinil. In some embodiments, each dose contains from about 200 micrograms to about 250 micrograms of treprostinil. In some embodiments, each dose contains from about 200 micrograms to about 275 micrograms of treprostinil. In some embodiments, each dose contains from about 200 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 200 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 200 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 225 micrograms to about 250 micrograms of treprostinil. In some embodiments, each dose contains from about 225 micrograms to about 275 micrograms of treprostinil. In some embodiments, each dose contains from about 225 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 225 DBl / 153257679.1 27micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 225 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 250 micrograms to about 275 micrograms of treprostinil. In some embodiments, each dose contains from about 250 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 250 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 250 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 275 micrograms to about 300 micrograms of treprostinil. In some embodiments, each dose contains from about 275 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 275 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 300 micrograms to about 325 micrograms of treprostinil. In some embodiments, each dose contains from about 300 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 325 micrograms to about 350 micrograms of treprostinil. In some embodiments, each dose contains from about 350 micrograms to about 375 micrograms of treprostinil. In some embodiments, each dose contains from about 375 micrograms to about 400 micrograms of treprostinil. In some embodiments, each dose contains about 26.5 micrograms, about 53 micrograms, about 79.5 micrograms, about 106 micrograms, about 132.5 micrograms, about 159 micrograms, about 185.5 micrograms, about 212 micrograms, about 238.5 micrograms, about 265 micrograms, about 291.5 micrograms, about 318 micrograms, about 344.5 micrograms, about 371 micrograms, or about 397.5 micrograms of treprostinil.

[0062] In some embodiments, a subject may be provided with one. two, three, four, or more doses of treprostinil per day. In some embodiments, a subject may be provided up to one, two, three, or four doses of treprostinil per day. Each dose of treprostinil may be contained in a single capsule according to some embodiments, for example, a HPMC capsule (size 3). In other embodiments, a dose of treprostinil may be made up of a combination of lower dose capsules. Under the present disclosure, the dry powder particles provide significantly higher dose levels to be safely administered than currently marketed nebulized products such as TYVASO, such as for example, up to 100 micrograms of treprostinil per dosing, up to 125 micrograms of treprostinil per dosing and up to 150 micrograms of treprostinil per dosing. The higher levels of dosing were surprisingly demonstrated in the first clinical trial of LIQ861. In alternative embodiments, a subject may be provided with four DBl / 153257679.1 28doses of treprostinil per day to match the currently marketed nebulized product treatment cycle however the drug dose per treatment cycle under the present disclosure provides significantly higher dose levels to be achieved, such as for example, up to 200 micrograms of treprostinil per dosing and up to 300 micrograms of treprostinil per dosing as surprisingly demonstrated in pre-clinical toxicology studies using LIQ861.Excipients

[0063] According to some embodiments of the present disclosure, the dryTpowder (anhydrous) is comprised of particles that include, for example, the following excipients: trehalose, polysorbate 80, L-leucine, sodium citrate, and sodium chloride. In some embodiments, the ratio of treprostinil sodium and excipients is 0.581:92.32:2.19:4.39:0.26:0.25 (wt:wt solids) treprostinil sodium:trehalose:polysorbate 80:leucine:sodium citrate: sodium chloride. A summary of the function, quantity, and compendial status of these excipients is provided herein.

[0064] The excipients were selected based upon the following functional requirements for the formulation:• Trehalose Dihydrate: Trehalose comprises the bulk of the particle and was selected because it is a non-reducing sugar with a high glass transition temperature. Trehalose is an example of a non-reducing sugar (as opposed to lactose, which is a reducing sugar) that can be used in the present disclosure. Trehalose is more chemically compatible with compounds containing primary amines, such as leucine.• Ultra-Pure Polysorbate 80 (Ultra-Pure Tween 80): Polysorbate 80 is added as a processing aide / wetting agent to facilitate particle manufacturing. In some embodiments, Polysorbate 80 is a particle processing aide and enables fdm generation during particle manufacture by decreasing dewetting, leading to uniform particle morphology.• L-leucine: Leucine is added as a hydrophobicity and surface modifier to reduce the hygroscopicity of the particle and improve aerosol efficiency. L-leucine is an example of a formulation additive to reduce hygroscopicity to improve stability of the final drug product powder.

[0065] Sodium chloride and sodium citrate: Sodium citrate and sodium chloride are used to buffer the stock solution used in the manufacturing process and to help control acidity in DBl / 153257679.1 29the particle. Sodium chloride and sodium citrate are examples of buffers that help maintain pH and control ionization / acidity of the formulation. In some embodiments of the present disclosure, pH is maintained between about pH 6.0 and 7.2.In addition to the active pharmaceutical ingredient (e.g., treprostinil) the present drug particle comprises a bulking agent, wetting agent, hydrophobicity modifier, pH modifier and buffer. In some embodiments, the present drug particle comprises, along with the active ingredients, a bulking agent, hydrophobicity controlling agent, and a pH controlling agent.

[0066] According to another embodiment of the present disclosure, LIQ861 contains six ingredients as follows: treprostinil sodium: trehalose dihydrate:leucine:polysorbate 80:sodium citrate dihydrate:sodium chloride at ratios of 0.53:92.97:4:2:0.27:0.23. At an example treprostinil dose level of 100 pg / day of the present disclosure drug particles, a subject would receive the following daily excipient doses:• 18.6 mg of trehalose dihydrate. Assuming a subject weighs 60 kg and has a lung mass of 1000 g, this is equivalent to 310 pg / kg and 18.6 pg / g of lung.• 0.4 mg of polysorbate 80. Assuming a subject weighs 60 kg and has a lung mass of 1000 g. this is equivalent to 6.7 pg / kg and 0.4 pg / g of lung.• 0.8 mg of leucine. Assuming a subject weighs 60 kg and has a lung mass of 1000 g, this is equivalent to 13.3 pg / kg and 0.8 pg / g of lung.• 0.05 mg of sodium citrate and 0.05 mg of sodium chloride. Assuming a subject weighs 60 kg and has a lung mass of 1000 g, this is equivalent to 0.83 pg / kg for each compound and 0.05 pg / g of lung for each compound.Physiochemical and Biological Properties

[0067] The ‘"pollen-shaped" LIQ861 particles according to certain embodiments have an aerodynamic size to enable efficient delivery to the pulmonary arterioles (1 < MMAD < 5 pm) with a high fine particle fraction (FPF) to limit oropharyngeal deposition. A scanning electron microscopy (SEM) image of the “pollen-shaped” feature is provided in Figure 2. Example aerosol data for the active particles are also provided in the table below.

[0068] During the development of the LIQ861 formulation, the applicants tested other possible particle shapes and sizes (e.g., 1.5 pm donut, 3.0 pm donut). Based upon these studies, the applicants observed that the “pollen-shaped” feature resulted in a greater FPF,DBl / 153257679.1 30reduced MMAD, acceptable emitted dose (ED), and dose uniformity characteristics when compared to other features both with and without treprostinil.DBl / 153257679.1 31Representative Aerosol Data (NGI) for Active ParticlesAbbreviations: NGI, Next Generation Impactor™, MSP Corp.; MMAD, mass median aerodynamic diameter; GSD, geometric standard deviation; ED, emitted dose; FPF, fine particle fraction; wt, weight.Manufacture

[0069] In an embodiment, the particles are made by molding the materials intended to make up the particles in mold cavities.

[0070] In some embodiments, the molds can be polymer-based molds and the mold cavities can be formed into any desired shape and dimension. Uniquely, as the particles are formed in the cavities of the mold, the particles are highly uniform with respect to shape, size, and composition. Due to the consistency among the physical and compositional makeup of the particles of the present compositions, the compositions of the present disclosure provide highly uniform release rates and dosing ranges. Methods and materials that may be used for fabricating the particles according to embodiments of the present disclosure are further described and disclosed in issued patents and co-pending patent applications, each of which is incorporated herein by reference in its entirety: U.S. Pat. Nos. 8,518,316; 8,444,907; 8,420,124; 8,268,446; 8,263,129; 8,158,728; 8,128,393; 7,976,759, 9,214,590, 9,205,594, 8,662,878, 8.439,666, 8,128.393, 8,992,992, 9,545,737; and U.S. Pat. Application Publications Nos. 2013-0228950, 2013-0011618, 2013-0256354. 2010-0003291. 2009- 0165320.Batch-to-Batch Uniformity of Drug Particles

[0071] In some embodiments, the particle uniformity from batch-to-batch provides the present disclosure with an unexpected and exceptional advantage over the prior art. In certain embodiments, the uniformity' within any given batch is unexpected and exceptionallyDBl / 153257679.1 32advantageous over the prior art. The present disclosure includes highly conserved batch uniformity as shown in the following data. See the table below.Uniformity : Sample aerosol data (NGI) for active particlesIn the example shown, fine particle fraction remained within plus / minus 1 percent within a single batch run when sampled at the beginning, middle, and end of the runOther Excipients

[0072] In some alternative embodiments, treprostinil may be included in inhalable dry' powder particles having an excipient matrix that includes or consists essentially of fumaryl diketopiperazine (FDKP). FDKP. bis-3,6(4-fumarylaminobutyl)-2.5-diketopiperazine. is a fumaramide derivative of diketopiperazine, which has shown to be highly soluble at pH values above 6. In some embodiments, particles including FDKP may have a crystalline structure. In other embodiments, particles including FDKP may have an amorphous structure.

[0073] In some such embodiments, particles including FDKP and treprostinil may be formed by mixing a solution of treprostinil (e.g., treprostinil in ethyl alcohol) and a suspension of FDKP microcrystalline particles which is then spray dried to form inhalable powder particles. Methods and materials that may be used for fabricating the particles according to embodiments of the present disclosure are further described and disclosed in issued patents, each of which is incorporated herein by reference in its entirety7: U.S. Pat. Nos.10,130.685; U.S. 10,421,729; and U.S. 10,772,883; U.S. Pat. Pub. Nos. 2017 / 0216538, and 2019 / 0321290.

[0074] In some embodiments, the inhalable dry7pow der particles are contained in singledose cartridges. In other embodiments, the single-dose cartridges comprise about 1% treprostinil. In other embodiments, the single-dose cartridges comprise about 16 micrograms, about 32 micrograms, about 48 micrograms, or about 64 micrograms of treprostinil. In other DBl / 153257679.1 33embodiments, the single-dose cartridges comprise treprostinil adsorbed onto carrier particles consisting of fumaryl diketopiperazine (FDKP). In particular embodiments, the single-dose cartridge comprises about 1.6 mg, about 3.2 mg, about 4.8 mg, or about 6.4 mg of dry powder particles. In some embodiments, the inhalable dry powder particles are administered via inhalation. In other embodiments, the inhalable dry powder particles are administered at least once a day. at least twice a day, at least three times a day, or at least four times a day.LIPOSOMAL TREPROSTINIL COMPOSITION

[0075] In some embodiments, a combination therapy according to some embodiments of the present disclosure comprises administering the one or more ActRIlA fusion proteins (e.g.. sotatercept) together or sequentially with an administration of treprostinil or amphiphilic treprostinil, wherein the treprostinil is administered in a pharmaceutical composition including one or more liposomes containing the treprostinil or amphiphilic treprostinil. In some embodiments, the pharmaceutical composition further includes an external medium, and the one or more liposomes are suspended in the external medium. In some embodiments, the pharmaceutical composition including the one or more liposomes may be administered to the subject via inhalation. In some such embodiments, the pharmaceutical composition including the one or more liposomes may be administered to the subject via a nebulizer, e.g., a vibrating mesh nebulizer.

[0076] In some particular embodiments, one or more ActRIlA fusion proteins (e.g., sotatercept) are administered concurrently or sequentially with a pharmaceutical composition containing one or more liposomes suspended in an external medium, each liposome comprising: (a) an external lipid bilayer including at least one vesicle-forming phospholipid; and (b) an internal aqueous medium, comprising treprostinil or amphiphilic treprostinil and a salt to provide a pH gradient between the internal aqueous medium and the external medium (hereafter "pH gradient salt”). In some embodiments, the weight ratio of treprostinil or amphiphilic treprostinil to the at least one vesicle-forming phospholipid (i.e., Treprostinil / phospholipid, T / P ratio) is equal to or higher than about 0.035. In some embodiments, the liposome is formulated such that about less than 60% of the treprostinil or amphiphilic treprostinil is released within 2 hours after the administration of the pharmaceutical composition. In some embodiments, the liposome is formulated such that more than 80% of the treprostinil or amphiphilic treprostinil is released more than 2 hours to about 72 hours after the administration of the pharmaceutical composition.DBl / 153257679.1 34

[0077] In an exemplary embodiment, the liposomes are suspended in an external medium and the pH of the external medium is above the pKaof treprostinil. In another exemplar)’ embodiment, pH of the internal aqueous medium is at least 0.1 unit or at least 1 unit higher than the pH of the external medium. In yet another embodiment, the encapsulation efficiency of treprostinil is above about 80%, about 85%, about 90% or about 95%.

[0078] In some embodiments, the treprostinil-to-phospholipid ratio is equal to or higher than about 0.035, about 0.036, about 0.037, about 0.038, about 0.039, about 0.04, about 0.041, about 0.042, about 0.043, about 0.044, about 0.045, about 0.046, about 0.047, about 0.048, about 0.049, about 0.05, about 0.051. about 0.052, about 0.053, about 0.054, about 0.055, about 0.056, about 0.057. about 0.058, about 0.059 or about 0.06.

[0079] In some embodiments, the pharmaceutical composition reduces the burst release of treprostinil in the upper respiratory' track (about less than 60% of the treprostinil or amphiphilic treprostinil is released within 2 hours after the administration), including the oral cavity, the nasophary nx and the larynx above the vocal cords. As a result, the side effects of the treprostinil in the upper respiratory track, such as cough, throat irritation, phary ngeal pain, epistaxis, hemoptysis and wheezing, are reduced compared to that of a pharmaceutical composition wherein the treprostinil-to-phospholipid ratio is lower than about 0.035. In yet another exemplary embodiment, the pharmaceutical composition extends the release of treprostinil or amphiphilic treprostinil from more than 2 hours to at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours at least 16 hours, at least 24 hours, at least 48 hours or at least 72 hours after the administration of the pharmaceutical composition (i.e., more than 80% of the treprostinil is released more than 2 hours to about 72 hours after the administration) and reduces the dosing frequency.Liposomal components

[0080] The term “liposome” as used herein refers to microscopic vesicles or particles made up of one or more lipid bilayers enclosing an internal aqueous medium. To form liposomes, the presence of at least one “vesicle-forming lipid” is needed, which is an amphipathic lipid capable of either forming or being incorporated into a lipid bilayer. Any suitable vesicleforming lipid may be used to form the lipid bilayer constituting the liposomes. Vesicleforming lipid includes, but not limited to, phospholipids such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA),DBl / 153257679.1 35phosphatidylethanolamine (PE) or phosphatidylserine (PS), and charged lipids, such as a positively charge lipid or a negatively charged lipid.

[0081] The lipid bilayer of the liposome includes at least one vesicle-forming lipid and may include a sterol, which is selected from the group consisting of cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, and any combination thereof, but is not limited thereto. In an exemplary embodiment, the sterol is cholesterol.

[0082] In some embodiments, the vesicle-forming lipid is a mixture of a first phospholipid and a second phospholipid. In certain embodiments, the first phospholipid is phosphatidylcholine (PC), which is selected from the group consisting of hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoyl phosphatidylcholine (DPPC), distearyloyl phosphatidylcholine (DSPC), diarachidoyl phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC), oleoyl palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), dipetroselinoyl phosphatidylcholine, palmitoylelaidoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine, dilauroyl phosphatidylcholine (DLPC), diundecanoyl phosphatidylcholine, didecanoyl phosphatidylcholine, dinonanoyl phosphatidylcholine, and any combination thereof. In some embodiments, the second phospholipid is a polyethylene glycol modified phospholipid, containing a polyethylene glycol having a molecular weight of about 500 to about 10,000 daltons, such as 1,2- distearoly-677-glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] (DSPE-PEG2000). a negatively charged phospholipid, such as distearyloyl phosphatidylglycerol (DSPG). Dipalmitoylphosphatidylglycerol (DPPG) or dimyristoylphosphatidylglycerol (DMPG) or (DOPG). In an exemplary embodiment, the mole percent of the first phospholipid: cholesterol: the second phospholipid is 50-70: 20-45: 0.1-10, 50-70: 20-45: 0.5-8 or 55-65:25-40: 1-6.

[0083] In other embodiments, the vesicle-forming lipids are a mixture of a first phospholipid and a charged lipid. In an exemplary embodiment, vesicle-forming lipids are a mixture of a first phospholipid, a second phospholipid and a charged lipid. The charged lipid, includes stearylamine, 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), N4-Cholesteryl-Spermine (GL67), dimethyldioctadecylammonium (DDAB), l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), ethy lphosphocholine (ethyl PC) or combination DBl / 153257679.1 36thereof. In another exemplary embodiment, the mole percent of the first phospholipid: cholesterol: charged lipid is 50-70: 20-45: 0.1-10, 50-70: 20-45: 0.5-8 or 55-65:25-40: 1-6.

[0084] In some embodiments, the liposome includes an external lipid bilayer that includes or consists of HSPC, cholesterol, and DSPG. In some embodiments, the liposome includes an external lipid bilayer that includes or consists of HSPC, cholesterol, and DSPE-PEG2000. In an embodiment, the mole % of HSPC, cholesterol, and DSPG in the lipid bilayer is 50-70: 20-45: 0.1-10, 50-70: 20-45: 0.1-5 or 55-65:25-40:0.5-8. In one example embodiment, the mole % of HSPC, cholesterol, and DSPG in the lipid bilayer is about 59:40: 1.5. In another embodiment, the mole % of HSPC, cholesterol and DSPE-PEG2000 in the lipid bilayer is 50- 70: 20-45: 0.1-10, 50-70: 20-45: 0.1-5 or 55-65:25-40:0.5-8. In another embodiment, the mole % of DPPC:Chol:DSPE-PEG2000 in the lipid bilayer is 50-70: 20-45: 0.5-8, 50-70: 20- 45: 0.1-5 or 55-65:25-40:1-6.

[0085] In some embodiments, the lipid bilayer of the liposomes may also include at least one vesicle-forming lipid and a surfactant, which can be a non-ionic surfactant, a cationic surfactant or a zwitterionic surfactant. A non-ionic surfactant has no formally charged groups in its head. A cationic surfactant carries a net positive charge in its head. A zwitterion surfactant is electrically neutral but carries formal positive and negative charges on different atoms.

[0086] Non limiting examples of non-ionic surfactant include non-ionic water soluble mono-, di-, and tri-glycerides; non-ionic water soluble mono- and di-fatty acid esters of polyethyelene glycol; non-ionic water soluble sorbitan fatty acid esters (e.g. sorbitan monooleates such as TWEEN 20 (polyoxyethylene 20 sorbitan monooleate), SPAN 80; non- ionic water soluble triblock copolymers (e.g., poly(ethyleneoxide) / poly- (propyleneoxide) / poly(ethyleneoxide) triblock copolymers such as POLOXAMER406 (PLURONIC F-127), or derivatives thereof.

[0087] Non-limiting examples of cationic surfactant include dimethyldialkylammonium bromide or dodecyltrimethylammonium bromide.

[0088] Non limiting examples of zwitterionic surfactant include 3-(N,N-dimethyl palmitylammonio)-propanesulfonate.DBl / 153257679.1 37

[0089] In some embodiments, the liposomes are substantially free of detergent or an ionophore, which is a compound capable of facilitating the transport of H+or OH’ across the liposome membrane.

[0090] A solvent for dissolving a vesicle-forming lipid for the preparation of liposomes can be used, for example, methanol, ethanol, ether, and combinations thereof. Optionally, the solvent can be removed by a supercritical fluid later, and is preferably used in a minimum amount so as to decrease the time for performing an organic solvent removing step.

[0091] According to certain embodiments, the liposomes are prepared in a medium containing a salt to provide a pH gradient between the internal aqueous medium and the external medium of the liposomeWhen the vesicle-forming lipid is in contact with a medium containing the pH gradient salt, a liposome suspension is formed.

[0092] In some embodiments, the liposome in the suspension is subjected to size reduction. A liposome's size is typically referred to as its diameter. Liposome size reduction can be accomplished by a number of methods, such as extrusion, sonication, homogenization techniques or milling techniques, which are well know n and can be performed by persons skilled in the art. Extrusion includes passing liposomes, under pressure, one or more times through filters having defined pore sizes. The filters are generally made of polycarbonate, but can also be made of any durable material which does not interact with the liposomes and which is sufficiently strong to allow extrusion under sufficient pressure. The size of the liposomes can be reduced by sonication, which employs sonic energy to disrupt or shear liposomes that will spontaneously reform into smaller liposomes. For example, sonication can be conducted by immersing a glass tube containing the liposome suspension into the sonic epicenter produced in a bath-type sonicator, or a probe ty pe sonicator may be used in which the sonic energy is generated by vibration of a titanium probe in direct contact with the liposome suspension. In the present disclosure, the liposomes generally have a diameter of about 50 nm to 500 nm, such as about 500 nm or less, about 400 nm or less, about 300 nm or less, about 200 nm or less or about 100 nm or less.

[0093] After sizing, the concentration of the pH gradient salt in the external medium is adjusted to provide a pH gradient between the internal aqueous medium and the external medium, which can be carried out by a number of ways, for example, by exchanging the external medium with a suitable buffer lacking the pH gradient salts such as citric acid bufferDBl / 153257679.1 38(J hCfil hO) and phosphoric acid buffer (H3PO4), by methods such as diafiltration, dialysis, ultrafiltration, or tangential flow filtration.

[0094] The pH gradient salt provides a lower outside and a higher inside pH gradient between the external medium and the internal aqueous medium of the liposomes. In one embodiment, the pH of the internal aqueous medium is at least 0.1 unit higher than the pH of the external medium. In another embodiment, the pH of the internal aqueous medium is at least 1 unit higher than the pH of the external medium. In yet another embodiment, the pH of the internal aqueous medium is about 7, 8, 9 or 10 and the pH of the external medium is less than 7, less than 6, less than 5, less than 4, less than 3. about 3-7, about 3.5-6.5, or about 4-6. In yet another exemplary embodiment, the pH of the external medium is above the pKaof treprostinil.

[0095] The prepared liposome can be stored for substantial periods of time prior to treprostinil or amphiphilic treprostinil loading and administration to a subject. For example, liposomes can be stored at refrigerated conditions for substantial periods of time prior to treprostinil loading. Alternatively, liposomes can be dehydrated, stored, and subsequently rehydrated and loaded with treprostinil, prior to administration. Liposomes may also be dehydrated after being loaded with treprostinil. Dehydration can be performed by a number of methods available and known in the art. In some embodiments, liposomes are dehydrated using standard freeze-drying apparatus i.e. dehydration under low pressure conditions. Also, liposomes can be frozen e.g. using liquid nitrogen. Saccharides can be added to the liposomal environment, e.g.. to the buffer containing the liposomes, prior to dehydration, to ensure stability and integrity of the liposome during dehydration. Examples of saccharides include but are not limited to maltose, lactose, sucrose, trehalose, dextrose, sorbitol, mannitol, xylitol, or a combination thereof.

[0096] A liposome suspension having a T / P ratio of equal to or higher than about 0.035 as described above are ready for treprostinil or amphiphilic treprostinil loading. Typically, treprostinil or amphiphilic treprostinil is added to the external medium of the liposome and the resultant suspension is incubated, allowing diffusion of treprostinil or amphiphilic treprostinil into the internal aqueous medium of the liposome and until a desired loading concentration and encapsulation efficiency (the percentage of the intemal / encapsulated amount of treprostinil relative to the total amount of treprostinil in the composition) is achieved.DBl / 153257679.1 39Association between treprostinil to phospholipid ratio and controlled release profile

[0097] According to some embodiments, a pH gradient salt is used to provide a pH gradient between the intra- and extra-liposomal compartments and allow the loading of treprostinil into the internal aqueous medium, and not entrapped within the liposomal membrane or associated with external surface of the lipid bilayer.

[0098] Non limiting examples of pH gradient salt include a weak acid salt (such as carboxylic acid salt or bicarbonate salt) or an amino acid (such as a polar amino acid).

[0099] Bicarbonate salt may refer to a pharmaceutically acceptable salt compound including a bicarbonate anion and a cationic component. In one embodiment, the cationic component of the salt compound is a metal. Non-limiting examples of the metal include a Group IA or IIA metal, such as potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), cesium (Cs), and lithium (Li) or a metal other than Group 1A or IIA metal, such as ferrous iron (Fe) and nickel (Ni). Examples of bicarbonate salt include, but not limited to, potassium bicarbonate, sodium bicarbonate, calcium bicarbonate, magnesium bicarbonate, cesium bicarbonate, lithium bicarbonate, nickel bicarbonate, ferrous iron bicarbonate or any combination thereof. In some embodiments, the bicarbonate salt is sodium bicarbonate.

[0100] Carboxylic acid salt includes, but not limited to, formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate or a combination thereof. In one exemplary embodiment, the acetate is sodium acetate, calcium acetate, or a combination thereof.

[0101] In one embodiment, the pH gradient salt is a polar amino acid, including a neutral polar amino acid (Tyrosine, Asparagine, Glutamine, Cysteine, Serine, Threonine), a basic polar amino acid (Arginine, Lysine, Histidine), an acidic polar amino acid (Aspartate, Glutamate) or a combination thereof. The following table shows the classification of 20 amino acids:DBl / 153257679.1 40

[0102] In certain embodiments, the pH gradient salt is not phosphate. In an exemplar}' embodiment, the pH gradient salt is bicarbonate. In another exemplary embodiment, the pH gradient salt is acetate.

[0103] In some embodiments, a counter ion is selected to accompany the pH gradient salt to maintain a stable pH gradient such that the interaction between the counter ion with the pH gradient salt together achieve the optimal effect of the pH gradient salt, i.e., encapsulating a high concentration of treprostinil in the internal aqueous medium of the liposome and / or extending the release rate of the same from the liposome. It will be appreciated that after the pH gradient is established, excess counter ions within the liposome provide a wealth of hydroxide and these counter ions alone are membrane-impermeant. Treprostinil in its neutral form can permeate the lipid bilayer under the incubation conditions during liposome loading, and de-protonates in response to the counter ions. The de-protonated treprostinil does not readily permeate across the liposome bilayer. In certain embodiments, the counter ion may be an ion of alkali metals.

[0104] In certain embodiment, the internal aqueous medium is substantially free of precipitates, cry stals or gels. The internal aqueous medium is considered to be substantially free of precipitates, crystals or gels if no such precipitates, crystals or gels are visible on electron microscopy with at least 5000x, 8000x, lOOOOx, 12000x, 15000x or 20000x magnification.

[0105] The pharmaceutical compositions of the present disclosure having a specific range of a treprostinil-to-phospholipid ratio reduces the burst release of the encapsulated treprostinil or amphiphilic treprostinil and hence reduce the side effect of the treprostinil or amphiphilic treprostinil. Furthermore, sufficient amount of treprostinil or amphiphilic treprostinil for a desired therapeutic effect is released from the pharmaceutical composition and the release profile is extended compared to that of the pharmaceutical composition with the treprostinil- to-phospholipid ratio falling outside the specific range of this disclosure. This claimedDBl / 153257679.1 41treprostinil-to-phospholipid ratio unexpectedly extends the release of the entrapped treprostinil or amphiphilic treprostinil from the liposomal composition while reducing the side effects of treprostinil or amphiphilic treprostinil, without the use of a detergent or an ionophore (such as calcium ionophore) for the release profile described herein.

[0106] As used herein, the term “burst release” refers to rapid and / or somewhat uncontrolled release of treprostinil or amphiphilic treprostinil from the pharmaceutical composition within 2 hours of administration of the pharmaceutical composition. In certain embodiments, less than about 62%, about 61%, about 60%, about 59%, about 58%, about 57%, about 56%. about 55%, about 54%, about 53%, about 52%, about 51%, about 50%, about 49%. about 48%, about 47%, about 46%, about 45%. about 44%, about 43%, about 42%, about 41%, about 40%, about 39% or about 38% of the encapsulated treprostinil or amphiphilic treprostinil is released within about 2 hours of drug administration.

[0107] As used herein, the term “extended release” can be used interchangeably with “controlled release”, “delayed release”, “modified release”, “prolonged release”, “programmed release”, “time release”, “rate controlled” or “sustained release” refers, in the present context, to the release of encapsulated treprostinil or amphiphilic treprostinil during a period of more than 2 hours to about 72 hours after the administration of the pharmaceutical composition. In some embodiments, less than 60% of the encapsulated treprostinil or amphiphilic treprostinil is released within 2 hours of administration and a total of more than 80% of the encapsulated treprostinil or amphiphilic treprostinil is released by 72 hours after the administration of the pharmaceutical composition.

[0108] In certain embodiments, the pharmaceutical composition has a release profile wherein less than about 60%, 55%, 50%, 45%, 40% or 35% by weight of the entrapped treprostinil or amphiphilic treprostinil is released within 2 hours from the time of drug administration. In certain embodiments, the pharmaceutical composition has a release profile wherein more than about 90%, 85%, 80%, 75%, 70%, 65%, 60% or 55% by weight of the entrapped treprostinil or amphiphilic treprostinil is released from the liposome, more than 2 hours to 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, or 72 hours from the time of drug administration.Preparation of Treprostinil Liposomal CompositionDBl / 153257679.1 42

[0109] In some embodiments, a liposomal colloidal suspension may be prepared using ethanol injection technique. In one example, all lipid ingredients including a first phospholipid (e.g., HSPC), cholesterol and a second phospholipid (e.g., DSPE-PEG2000, DSPG) at a molar ratio of 3:2:0.075 were dissolved in 2.86 mL of ethanol solution at approximately 60°C. The resultant lipid solution was then injected into 17.14 mL of sodium bicarbonate solution (400 mM; pH 8.5) and was stirred at 60°C for liposome hydration. The mixture was extruded 6 to 10 times through polycarbonate membranes with specific pore sizes (0.2 micrometer and / or 0.1 micrometer, respectively), to obtain a suspension of liposomes having a mean particle size around 100 nm to 200 nm and a poly dispersity index (PDI) of <0.2. Subsequently, the suspension of liposomes was dialyzed with a tangential flow filtration system against 50 mM of sodium citrate buffer (pH 5.5) to form a transmembrane pH gradient between the internal aqueous medium of the liposome and the external medium (i.e., a higher inside and lower outside pH gradient). The suspension of liposomes with transmembrane pH gradient was stored at 4°C before drug loading process.

[0110] Treprostinil (purchased from Cayman Chemical, USA) was dissolved in 50 mM of sodium citrate aqueous solution, then added into the suspension of liposomes at a given treprostinil-to-phospholipid ratio and incubated at 40°C for 30 min. The resultant mixture was adjusted with sodium citrate buffer (pH 5.5) to obtain a liposomal treprostinil composition having a pH of 5.5 and a phospholipid concentration of 8.59 mg / mL.Example Liposomal Treprostinil Composition

[0111] A treprostinil liposomal composition for administration according to certain exemplary embodiments include the following components:DBl / 153257679.1 43qs = quantum satis (quantity sufficient for);'Trisodium citrate dihydrate and citric acid monohydrate are buffer ingredients to control the pH and are not required to be proportional.Administration of Liposomal Treprostinil Composition

[0112] In some embodiments, the liposomal treprostinil compositions described herein may be administered to the subject via inhalation. In some such embodiments, the liposomal treprostinil compositions may be administered via a vibrating-mesh nebulizer, for example, a breath-activated vibrating-mesh nebulizer. In some embodiments, the vibrating-mesh nebulizer is configured to aerosolize the liposomal treprostinil composition to produce aerosol particles having a MMAD of about 3.0 micrometers to about 5.0 micrometers. In some embodiments, the liposomal treprostinil composition may be contained in one or more ampoules, vials bottles, or other suitable containers that in turn may be used to fill the medicine chamber of the vibrating-mesh nebulizer for administration to the subject.

[0113] In some embodiments, the liposomal treprostinil compositions may be administered once a day, twice a day (BID), three times a day, or four times per day (QID) to a subject via inhalation. In some embodiments, the liposomal treprostinil compositions is administered concurrently or sequentially with administration of the one or more the ActRIIA fusion proteins (e.g., sotatercept). In some embodiments, for example, the liposomal treprostinil composition is administered at a dose of about 40 microgram treprostinil to about 400 microgram treprostinil, twice a day. In some embodiments, the dosing may be titrated over time such that the dose of liposomal treprostinil compositions administered to a subject is gradually increased until a predetermined maximum dose is reached or until adverse reactions are encountered. In some embodiments, the doses may be increased at regular intervals, for example, every day, every two days, every' three days, every' four days, every five days, every' six days, every 7 days (weekly), every two weeks, every’ three weeks, or every four weeks. For example, the dosing scheme according to some embodiments may be as follows:DBl / 153257679.1 44

[0114] In some embodiments, the administration of the one or more the ActRIIA fusion proteins (e.g., sotatercept) occurs after administration of at least one dose of the liposomal treprostinil composition. In some embodiments, the administration of the one or more the ActRIIA fusion proteins (e.g., sotatercept) occurs before administration of at least one dose of the liposomal treprostinil composition. In some embodiments, the administration of the one or more the ActRIIA fusion proteins (e.g., sotatercept) occurs concurrently with the administration of at least one dose of the liposomal treprostinil composition. In some embodiments, where the dose of the liposomal treprostinil composition is titrated, the administration of the one or more the ActRIIA fusion proteins (e.g., sotatercept) occurs after or concurrently with the administration of the highest dose of the liposomal treprostinil composition.TREPROSTINIL PRODRUGS DB1 / 153257679.1 45

[0115] In some embodiments, a combination therapy according to some embodiments of the present disclosure comprises administering the one or more ActRIIA fusion proteins (e.g., sotatercept) together or sequentially with an administration of treprostinil, where the treprostinil is a treprostinil prodrug.

[0116] In some embodiments, the treprostinil prodrug is a compound of Formula (I)

[0117] wherein R1is an optionally substituted, unbranched C2-2salkyl, or an optionally substituted, branched C2-2salkyl. In some embodiments, R1is tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl. In other embodiments, the tresprostinil prodrug is formulated as a dry powder composition comprising (a) from about 0.1 wt % to about 3 wt % of a compound of Formula (I) or an enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof.; (b) from about 0.01 wt % to about 3 wt % of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), (c) from about 10 wt % to about 50 wt % of leucine, and the balance being (d) a sugar selected from the group consisting of trehalose and mannitol; wherein the entirety of (a), (b), (c), and (d) is 100 wt %. In particular embodiments. R1is linear tetradecyl, linear pentadecyl, linear heptadecyl, linear octadecyl, or linear hexadecyl.

[0118] In some embodiments, the treprostinil prodrug is a compound of Formula (II), i.e. treprostinil palmitil wherein R1 is substituted with a straight chain, non-substituted hexadecyl alkane.DBl / 153257679.1 46

[0119] In some embodiments, the treprostinil prodrug is administered by aerosolizing a dry powder composition comprising treprostinil. In other embodiments, the aerosolized dry powder composition is administered to the lungs of the subject via inhalation. In particular embodiments, the aerosolized dry powder composition is administered at least once-a-day. twice-a-day, or three-times-a-day. In preferred embodiments, the aerosolized dry powder composition is administered once-a-day, twice-a-day, or three-times-a-day.ACTRIIA FUSION PROTEIN

[0120] In certain aspects, the disclosure relates to methods of treating pulmonary hypertension comprising administering to a subject in need thereof a therapeutically effective amount of an ActRIIA polypeptide. As described previously, in some embodiments the ActRIIA fusion protein is administered to a subject that is currently being treated with or has previously received treprostinil or other treatment for PH. In some embodiments the ActRIIA fusion protein is administered to a subject that is currently being treated with or has previously received treprostinil, where the treprostinil is or was administered to the subject via a nebulizer. In some embodiments the ActRIIA fusion protein is administered to a subject that is currently being treated with or has previously received treprostinil, where the treprostinil is or was administered via inhaled particles (e.g., LIQ861 formulation described above). In some embodiments the ActRIIA fusion protein is administered to a subject that is currently being treated with or has previously received treprostinil where the treprostinil is or was administered via inhalable liposomes. In embodiments, the treprostinil is administered as treprostinil, a pharmaceutically acceptable salt, or a treprostinil prodrug.

[0121] In some embodiments, the ActRIIA polypeptide comprises an amino acid sequence that is at least 70% (e.g.. at least 70%. 75%. 80%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DBl / 153257679.1 47of Figure 3 (SEQ ID NO: 1). In some embodiments, the ActRIIA polypeptide comprises an amino acid sequence that is at least 70% (e.g.. at least 70%. 75%. 80%. 85%. 86%. 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of Figure 4 (SEQ ID NO: 2). In some embodiments, the ActRIIA polypeptide is a fusion protein comprising an ActRIIA domain and one or more poly peptide domains heterologous to ActRIIA. In some embodiments, the ActRIIA polypeptide is a fusion protein comprising an Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is an Fc domain of an IgGl immunoglobulin. In some embodiments, the ActRIIA fusion protein comprises and Fc domain at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. or 100%) identical to the amino acid sequence of Figure 5 (SEQ ID NO: 3). In some embodiments, the ActRIIA fusion protein further comprises a linker domain positioned between the ActRIIA polypeptide domain and the one or more heterologous domains (e g., an Fc immunoglobulin domain). In some embodiments, the linker domain is selected from the group consisting of: TGGG (SEQ ID NO: 4), TGGGG (SEQ ID NO: 5), SGGGG (SEQ ID NO: 6). GGGGS (SEQ ID NO: 7). GGG (SEQ ID NO: 8). GGGG (SEQ ID NO: 9), and SGGG (SEQ ID NO: 10). In particular embodiments, the ActRIIA polypeptide comprises SEQ ID NO: 11.

[0122] The term “sequence similarity,” in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

[0123] “Percent (%) sequence identity” with respect to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. ForDBl / 153257679.1 48purposes herein, however, % amino acid (nucleic acid) sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0124] In certain aspects, ActRIIA polypeptides may be fusion proteins. For example, in some embodiments, an ActRIIA polypeptide may be a fusion protein comprising an ActRIIA polypeptide domain and one or more heterologous (non- ActRIIA) polypeptide domains. In some embodiments, an ActRIIA polypeptide may be a fusion protein that has, as one domain, an amino acid sequence derived from an ActRIIA polypeptide and one or more heterologous domains that provide a desirable property, such as improved pharmacokinetics, easier purification, targeting to particular tissues, etc. For example, a domain of a fusion protein may enhance one or more of in vivo stability , in vivo half-life, uptake / administration, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and / or purification. Optionally, an ActRIIA polypeptide domain of a fusion protein is connected directly (fused) to one or more heterologous polypeptide domains or an intervening sequence, such as a linker, may be positioned between the amino acid sequence of the ActRIIA polypeptide and the amino acid sequence of the one or more heterologous domains. In certain embodiments, an ActRIIA fusion protein comprises a relatively unstructured linker positioned between the heterologous domain and the ActRIIA domain. This unstructured linker may correspond to the roughly 15 amino acid unstructured region at the C-terminal end of the extracellular domain of ActRIIA, or it may be an artificial sequence of between 3 and 15. 20. 30, 50 or more amino acids that are relatively free of secondary structure. A linker may be rich in glycine and / or proline residues and may, for example, contain repeating sequences of threonine / serine and glycines. An example of a linker includes, but is not limited to, the sequence TGGG (SEQ ID NO: 4). In some embodiments, ActRIIA fusion proteins may comprise a constant domain of an immunoglobulin, including, for example, the Fc portion of an immunoglobulin. For example, an amino acid sequence that is derived from an Fc domain of an IgG (IgGl, IgG2, IgG3, or IgG4), IgA (IgAl or IgA2), DBl / 153257679.1 49IgE, or IgM immunoglobulin. For example, an Fc portion of an immunoglobulin domain may comprise, consist essentially of, or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3. Such immunoglobulin domains may comprise one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that confer an altered Fc activity, e.g., decrease of one or more Fc effector functions.

[0125] In certain aspects, the disclosure relates ActRIIA polypeptides and uses thereof (e.g., of treating, preventing, or reducing the progression rate and / or severity of pulmonary' hypertension or one or more complications of pulmonary hypertension) and / or an interstitial lung disease (e.g., idiopathic pulmonary fibrosis). As used herein, the term “ActRIIA” refers to a family of activin receptor type IIA (ActRIIA) proteins from any species and variants derived from such ActRIIA proteins by mutagenesis or other modification. Reference to ActRIIA herein is understood to be a reference to any one of the currently identified forms. Members of the ActRIIA family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity.

[0126] The term “ActRIIA polypeptide” includes polypeptides comprising any naturally occurring polypeptide of an ActRIIA family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ActRIIA polypeptides are provided throughout the present disclosure as well as in International Patent Application Publication Nos. WO 2006 / 012627 and WO 2007 / 062188. which are incorporated herein by reference in their entirety.Numbering of amino acids for all ActRIIA-related polypeptides described herein is based on the numbering of the human ActRIIA precursor protein sequence provided in Figure 3, unless specifically designated otherwise.

[0127] The processed (mature) extracellular human ActRIIA polypeptide sequence is as provided in Figure 4 (SEQ ID NO: 2).

[0128] ActRIIA is well-conserved among vertebrates, with large stretches of the extracellular domain completely conserved. Many of the ligands that bind to ActRIIA are also highly conserved. Accordingly, from these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ActRIIA-DBl / 153257679.1 50ligand binding activities as well as to predict amino acid positions that are likely to be tolerant to substitution without significantly altering normal ActRIIA-ligand binding activities. Therefore, an active, human ActRIIA variant polypeptide useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from the sequence of another vertebrate ActRIIA, or may include a residue that is similar to that in the human or other vertebrate sequences.

[0129] Moreover, as discussed above, ActRIIA polypeptides have been characterized in the art in terms of structural / functional characteristics, particularly with respect to ligand binding, such as references: Attisano et al. (1992) Cell 68(l):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal 22(7): 1555-1566; as well as U.S. Pat. Nos. 7,709,605, 7,612,041, and 7,842,663. In addition to the teachings herein, these references provide amply guidance for how to generate ActRIIA variants that retain one or more desired activities (e.g., ligand-binding activity).

[0130] Preferably, ActRIIA polypeptides are soluble (e.g., an extracellular domain of ActRIIA). In some embodiments, ActRIIA polypeptides inhibit (e.g., Smad signaling) of one or more GDF / BMP ligands [e.g., GDF11, GDF8, activin (activin A, activin B, activin AB. activin C, activin E) BMP6, GDF3, BMP15, and / or BMP10], In some embodiments, ActRIIA polypeptides bind to one or more GDF / BMP ligands [e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E) BMP6, GDF3, BMP15, and / or BMP 10], In some embodiments. ActRIIA polypeptides comprise, consist, or consist essentially of an amino acid sequence that is at least 70%. 75%. 80%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of Figure 4 (SEQ ID NO: 2).

[0131] In certain aspects, ActRIIA polypeptides of the present disclosure contain one or more modifications that are capable of “stabilizing” the polypeptides. By “stabilizing” is meant anything that increases the in vitro half-life, serum half-life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect of the agent. For example, such modifications enhance the shelf-life of the polypeptides, enhance circulatory half-life of the polypeptides, and / or reduce proteolytic degradation of the polypeptides. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an DBl / 153257679.1 51ActRIIA polypeptide domain and a stabilizer domain), modifications of a glycosylation site (including, for example, addition of a glycosylation site to a polypeptide of the disclosure), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from a polypeptide of the disclosure). As used herein, the term “stabilizer domain” not only refers to a fusion domain (e.g., an immunoglobulin Fc domain) as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinaceous moiety, such as polyethylene glycol. In certain preferred embodiments, an ActRIIA polypeptide is fused with a heterologous domain that stabilizes the polypeptide (a “stabilizer” domain), preferably a heterologous domain that increases stability of the polypeptide in vivo. Fusions with a constant domain of an immunoglobulin (e.g., a Fc domain) are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties.

[0132] An example of a native amino acid sequence that may be used for the Fc portion of human IgGl (GIFc) is shown in Figure 5. In part, the disclosure provides polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence in Figure 5 (SEQ ID NO: 3).

[0133] Optionally, the IgGl Fc domain has one or more mutations at residues such as Asp- 265, lysine 322, and Asn-434. In certain cases, the mutant IgGl Fc domain having one or more of these mutations (e.g., Asp-265 mutation) has reduced ability of binding to the Fey receptor relative to a wild-type Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g.. Asn-434 mutation) has increased ability of binding to the WIC class I-related Fc-receptor (FcRN) relative to a wild-type IgGl Fc domain.

[0134] It is understood that different elements of the fusion proteins (e.g., immunoglobulin Fc fusion proteins) may be arranged in any manner that is consistent with desired functionality. For example, an ActRIIA polypeptide domain may be placed C-terminal to a heterologous domain, or alternatively, a heterologous domain may be placed C-terminal to an ActRIIA polypeptide domain. The ActRIIA polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains.DBl / 153257679.1 52

[0135] For example, an ActRIIA receptor fusion protein may comprise an amino acid sequence as set forth in the formula A-B-C. The B portion corresponds to an ActRIIA polypeptide domain. The A and C portions may be independently zero, one, or more than one amino acid, and both the A and C portions when present are heterologous to B. The A and / or C portions may be attached to the B portion via a linker sequence. A linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues) or glycine and proline residues and may, for example, contain a single sequence of threonine / serine and glycines or repeating sequences of threonine / serine and / or glycines, e.g., TGGG (SEQ ID NO: 4), TGGGG (SEQ ID NO: 5), SGGGG (SEQ ID NO: 6), GGGGS (SEQ ID NO: 7), GGG (SEQ ID NO: 8), GGGG (SEQ ID NO: 9), and SGGG (SEQ ID NO: 10) singlets, or repeats. In certain embodiments, an ActRIIA fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a leader (signal) sequence, B consists of an ActRIIA polypeptide domain, and C is a polypeptide portion that enhances one or more of in vivo stability', in vivo half-life, uptake / administration, tissue localization or distribution, formation of protein complexes, and / or purification. In certain embodiments, an ActRIIA fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a TPA leader sequence, B consists of an ActRIIA receptor polypeptide domain, and C is an immunoglobulin Fc domain. A preferred fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 11.Pharmaceutical Compositions of Fusion Protein

[0136] The therapeutic agents described herein (e.g., an ActRIIA fusion protein and treprostinil) may be formulated into pharmaceutical compositions. Pharmaceutical compositions for use in accordance with the present disclosure may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Such formulations will generally be substantially pyrogen-free, in compliance with most regulatory requirements.

[0137] In certain embodiments, the therapeutic methods of the disclosure include administering the composition systemically, or locally as an implant or device. When administered, the therapeutic composition for use in this disclosure is in a substantially pyrogen-free, or pyrogen-free, physiologically acceptable form. Therapeutically useful agents other than an ActRIIA fusion protein and an inhalation powder comprising treprostinil whichDBl / 153257679.1 53may also optionally be included in the composition as described above, may be administered simultaneously or sequentially with the subject compounds in the methods disclosed herein.

[0138] Typically, protein therapeutic agents disclosed herein will be administered parentally, and particularly intravenously or subcutaneously . Pharmaceutical compositions suitable for parenteral administration may comprise an ActRIIA fusion protein or treprostinil in combination with one or more pharmaceutically acceptable stenle isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0139] The compositions and formulations may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0140] Further, the composition may be encapsulated or injected in a form for deli ven' to a target tissue site. In certain embodiments, compositions of the present disclosure may include a matrix capable of delivering one or more therapeutic compounds (e.g., an ActRIIA fusion protein and treprostinil) to a target tissue site, providing a structure for the developing tissue and optimally capable of being resorbed into the body. For example, the matrix may provide slow release of the ActRIIA fusion protein or treprostinil. Such matrices may be formed of materials presently in use for other implanted medical applications.

[0141] The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the subject compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate,DBl / 153257679.1 54tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.

[0142] In certain embodiments, methods of the invention can be administered orally, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient. An agent may also be administered as a bolus, electuary or paste.

[0143] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds of the present disclosure may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as. for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatinDBl / 153257679.1 55capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0144] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

[0145] Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0146] The compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chlonde, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0147] It is understood that the dosage regimen will be determined by the attending healthcare provider considering various factors which modify the action of the subj ect compounds of the disclosure. The various factors include, but are not limited to, the subject's age, sex, and diet, the severity disease, time of administration, and other clinical factors. Optionally, the dosage may vary with the type of matrix used in the reconstitution and the types of compounds in the composition. The addition of other known growth factors to the final composition, may also affect the dosage. Progress can be monitored by periodicDBl / 153257679.1 56assessment of bone growth and / or repair, for example, X-rays (including DEXA), histomorphometric determinations, and tetracycline labeling.

[0148] A targeted delivery system for an ActRIIA fusion protein is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see e.g., Fraley, et al., Trends Biochem. Sci.. 6:77, 1981). Methods for efficient gene transfer using a liposome vehicle, are known in the art, see e g., Mannino, et al., Biotechniques, 6:682, 1988. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0149] Examples of lipids useful in liposome production include those discussed above. Additional components useful with proteins include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides, and illustrative phospholipids such as egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and di stearoylphosphatidylcholine. The targeting of liposomes is also possible based on, for example, organ-specificity, cellspecificity. and organelle-specificity and is known in the art.

[0150] The disclosure provides formulations that may be varied to include acids and bases to adjust the pH; and buffering agents to keep the pH within a narrow range.Methods of Treatment

[0151] In part, the present disclosure relates to methods of treating pulmonary hypertension (e.g., pulmonary arterial hypertension) comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an effective amount of treprostinil (e.g., a dry inhalation powder comprising treprostinil, nebulized treprostinil composition, treprostinil prodrug and / or a liposomal treprostinil composition as discussed above). In some embodiments, the disclosure contemplates methods of treating one or more DBl / 153257679.1 57complications of pulmonary hypertension (e.g., smooth muscle and / or endothelial cell proliferation in the pulmonary artery, angiogenesis in the pulmonary artery, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis) comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an inhalation treatment comprising treprostinil and / or a liposomal treprostinil composition. In some embodiments, the disclosure contemplates methods of preventing one or more complications of pulmonary hypertension comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an ry inhalation treatment comprising treprostinil and / or a liposomal treprostinil composition. In some embodiments, the disclosure contemplates methods of reducing the progression rate of pulmonary hypertension comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an inhalation treatment comprising treprostinil and / or a liposomal treprostinil composition. In some embodiments, the disclosure contemplates methods of reducing the progression rate of one or more complications of pulmonary hypertension comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an inhalationtreatment comprising treprostinil and / or a liposomal treprostinil composition. In some embodiments, the disclosure contemplates methods of reducing the severity of pulmonary hypertension comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an inhalationtreatment comprising treprostinil and / or a liposomal treprostinil composition. In some embodiments, the disclosure contemplates methods of reducing the severity of one or more complications of pulmonary' hypertension comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an inhalation treatment comprising treprostinil and / or a liposomal treprostinil composition. To prevent a disorder or condition can refer to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0152] In some embodiments, the present disclosure relates to methods of treating an interstitial lung disease (e.g., idiopathic pulmonary' fibrosis) comprising administering to a subject in need thereof an effective amount of any of an ActRIIA fusion protein and an inhalation treatment comprising treprostinil and / or a liposomal treprostinil composition disclosed herein. In some embodiments, the interstitial lung disease is pulmonary' fibrosis. In DBl / 153257679.1 58some embodiments, the interstitial lung disease is caused by any one of the following: silicosis, asbestosis, berylliosis, hypersensitivity pneumonitis, drug use (e.g., antibiotics, chemotherapeutic drugs, antiarrhythmic agents, statins), systemic sclerosis, polymyositis, dermatomyositis, systemic lupus erythematosus, rheumatoid arthritis, an infection (e.g., atypical pneumonia, pneumocystis pneumonia, tuberculosis, Chlamydia trachomatis, and / or respiratory syncytial virus), lymphangitic carcinomatosis, cigarette smoking, or developmental disorders. In some embodiments, the interstitial lung disease is idiopathic (e.g., sarcoidosis, idiopathic pulmonary fibrosis, Hamman-Rich syndrome, and / or antisynthetase syndrome). In particular embodiments, the interstitial lung disease is idiopathic pulmonary fibrosis.

[0153] Treating can include amelioration or elimination of the condition once it has been established. In either case, prevention or treatment may be discerned in the diagnosis provided by a physician or other health care provider and the intended result of administration of the therapeutic agent.

[0154] In general, treatment or prevention of a disease or condition as described in the present disclosure is achieved by administering an ActRIIA fusion protein and am inhalation treatment comprising treprostinil in an effective amount. An effective amount of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent of the present disclosure may vary7according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

[0155] The terms ‘‘subject,’' an “individual,” or a “subject” are interchangeable throughout the specification and generally refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the subject is a mammal. In other embodiments, the subject is human. In particular embodiments, the subject is a human suspected of having or diagnosed as having one or more forms of pulmonary hypertension.DBl / 153257679.1 59

[0156] In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the progression rate and / or severity of one or more complications of pulmonary hypertension) comprising administering to a subject in need thereof an effective amount an ActRIIA fusion protein and an inhalation treatment comprising treprostinil. In some embodiments, the method relates to pulmonary hypertension subjects that have pulmonary arterial hypertension. In some embodiments, the method relates pulmonary hypertension subjects that have pulmonary hypertension with left heart disease. In some embodiments, the method relates to pulmonary hypertension subjects that have lung disease and / or hypoxemia. In some embodiments, the method relates to pulmonary hypertension subjects that have chronic thrombotic and / or embolic disease. In some embodiments, the method relates to pulmonary hypertension subjects that have sarcoidosis, histiocytosis X, or lymphangiomatosis and compression of pulmonary vessels.

[0157] In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and / or severity of pulmonary hypertension (e.g., treating, preventing, or reducing the progression rate and / or severity of one or more complications of pulmonary hypertension) comprising administering to a subject in need thereof an effective amount an ActRIIA fusion protein and n inhalation treatment comprising treprostinil, wherein the subject has resting pulmonary arterial pressure (PAP) of at least 20 mm Hg (e.g., 20, 25, 30, 35, 40, 45, or 50 mm Hg). In some embodiments, the method relates to subjects having a resting PAP of at least 20 mm Hg. In some embodiments, the method relates to subjects having a resting PAP of at least 25 mm Hg. In some embodiments, the method relates to subjects having a resting PAP of at least 30 mm Hg. In some embodiments, the method relates to subjects having a resting PAP of at least 35 mm Hg. In some embodiments, the method relates to subjects having a resting PAP of at least 40 mm Hg. In some embodiments, the method relates to subjects having a resting PAP of at least 45 mm Hg. In some embodiments, the method relates to subjects having a resting PAP of at least 50 mm Hg.

[0158] In some embodiments, the disclosure relates to methods of adjusting one or more hemodynamic parameters in the PH subject toward a more normal level (e.g., normal as compared to healthy people of similar age and sex), comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and n inhalation treatment comprising treprostinil. In some embodiments, the method relates to reducing PAP. In someDBl / 153257679.1 60embodiments, the method relates to reducing the subject's PAP by at least 3 mmHg. In certain embodiments, the method relates to reducing the subject's PAP by at least 5 mmHg. In certain embodiments, the method relates to reducing the subject's PAP by at least 7 mmHg. In certain embodiments, the method relates to reducing the subject's PAP by at least 10 mmHg. In certain embodiments, the method relates to reducing the subject's PAP by at least 12 mmHg. In certain embodiments, the method relates to reducing the subject's PAP by at least 15 mmHg. In certain embodiments, the method relates to reducing the subject's PAP by at least 20 mmHg. In certain embodiments, the method relates to reducing the subject's PAP by at least 25 mmHg. In some embodiments, the method relates to reducing pulmonary vascular resistance (PVR). In some embodiments, the method relate to increasing pulmonary capillary' yvedge pressure (PCWP). In some embodiments, the method relate to increasing left ventricular end-diastolic pressure (LVEDP).

[0159] In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and / or severity’ of one or more complications of pulmonary hypertension comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an inhalation treatment comprising treprostinil and / or a liposomal treprostinil composition. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity’ of cell proliferation in the pulmonary artery of a pulmonary hypertension subject. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity of smooth muscle and / or endothelial cells proliferation in the pulmonary- artery- of a pulmonary hypertension subject. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity of angiogenesis in the pulmonary artery of a pulmonary’ hypertension subject. In some embodiments, the method relates to increasing physical activity of a subject having pulmonary hypertension. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity of dyspnea in a pulmonary hypertension subject. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity' of chest pain in a pulmonary hypertension subject. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity of fatigue in a pulmonary’ hypertension subject. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity of pulmonary’ fibrosis in a pulmonary hypertension subject. In some embodiments, the method relates to treating, preventing, or reducing the progression rate DBl / 153257679.1 61and / or severity of fibrosis in a pulmonary hypertension subject. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity of pulmonary vascular remodeling in a pulmonary hypertension subject. In some embodiments, the method relates to treating, preventing, or reducing the progression rate and / or severity of right ventricular hy pertrophy in a pulmonary' hypertension subject.

[0160] In certain aspects, the disclosure relates to methods of increasing exercise capacity in a subject having pulmonary hypertension comprising administering to a subject in need thereof an effective amount of an ActRIIA fusion protein and an inhalation treatment comprising treprostinil and / or a liposomal treprostinil composition. Any suitable measure of exercise capacity can be used. For example, exercise capacity in a 6-minute walk test (6 MWT), which measures how far the subject can walk in 6 minutes, i.e., the 6-minute walk distance (6 MWD), is frequently used to assess pulmonary hypertension severity and disease progression. The Borg dyspnea index (BDI) is a numerical scale for assessing perceived dyspnea (breathing discomfort). It measures the degree of breathlessness, for example, after completion of the 6 MWT, where a BDI of 0 indicates no breathlessness and 10 indicates maximum breathlessness. In some embodiments, the method relates to increasing 6 MWD by at least 10 meters in the subject having pulmonary hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 20 meters in the subject having pulmonary’ hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 30 meters in the subject having pulmonary hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 40 meters in the subject having pulmonary' hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 50 meters in the subject having pulmonary hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 60 meters in the subject having pulmonary hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 70 meters in the subject having pulmonary hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 80 meters in the subject having pulmonary hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 90 meters in the subject having pulmonary hypertension. In some embodiments, the method relates to increasing 6 MWD by at least 100 meters in the subject having pulmonary' hypertension. In some embodiments, the method relates to lowering BDI by at least 0.5 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by' at least 1 index points in the subject having pulmonaryDBl / 153257679.1 62hypertension. In some embodiments, the method relates to lowering BDI by at least 1.5 index points in the subject having pulmonary’ hypertension. In some embodiments, the method relates to lowering BDI by at least 2 index points in the subject having pulmonary hypertension. In some embodiments, the method relate to lowering BDI by’ at least 2.5 index points in the subject having pulmonary' hypertension. In some embodiments, the method relates to lowering BDI by at least 3 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 3.5 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 4 index points in the subject having pulmonary’ hypertension. In some embodiments, the method relates to lowering BDI by at least 4.5 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 5 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 5.5 index points in the subject having pulmonary' hypertension. In some embodiments, the method relates to lowering BDI by at least 6 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 6.5 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 7 index points in the subject having pulmonary’ hypertension. In some embodiments, the method relates to lowering BDI by at least 7.5 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 8 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 8.5 index points in the subject having pulmonary' hypertension. In some embodiments, the method relates to lowering BDI by at least 9 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 9.5 index points in the subject having pulmonary hypertension. In some embodiments, the method relates to lowering BDI by at least 3 index points in the subject having pulmonary’ hypertension. In some embodiments, the method relates to lowering BDI by 10 index points in the subject having pulmonary hypertension.

[0161] In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and / or severity of pulmonary hypertension (e g., treating, preventing, or reducing the progression rate and / or severity of one or more complications of pulmonary hypertension) comprising administering to a subject in need thereof an effective DBl / 153257679.1 63amount of an ActRIIA fusion protein and an inhalation treatment comprising treprostinil and / or a liposomal treprostinil composition, wherein the subject has Class I, Class II. Class III, or Class IV pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to a subject that has Class I pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to a subject that has Class II pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to a subject that has Class III pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to a subject that has Class IV pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to preventing or delaying subject progression from Class I pulmonary hypertension to Class II pulmonary' hypertension as recognized by the WHO. In some embodiments, the method relates to promoting or increasing subject regression from Class II pulmonary hypertension to Class I pulmonary- hypertension as recognized by the WHO. In some embodiments, the method relates to a subject that has Class III pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to preventing or delaying subject progression from Class II pulmonary hypertension to Class III pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to promoting or increasing subject regression from Class III pulmonary hypertension to Class II pulmonary' hypertension as recognized by the WHO. In some embodiments, the method relates to promoting or increasing subject regression from Class III pulmonary hypertension to Class I pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to a subject that has Class IV pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to preventing or delaying subject progression from Class III pulmonary hypertension to Class IV pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to promoting or increasing subject regression from Class IV pulmonary hypertension to Class III pulmonary hypertension as recognized by the WHO. In some embodiments, the method relates to promoting or increasing subject regression from Class IV pulmonary hypertension to Class II pulmonary’ hypertension as recognized by the WHO. In some embodiments, the method relates to promoting or increasing subject regression from Class IV pulmonary hypertension to Class I pulmonary hypertension as recognized by the WHO.

[0162] In some embodiments, administration of the ActRIIA polypeptide decreases pulmonary arterial pressure in the subject. In some embodiments, administration of the DBl / 153257679.1 64ActRIIA polypeptide decreases pulmonary arterial pressure in the subject by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%. 50%. 55%. 60%. 65%. 70%. 75%. or at least 80%). In some embodiments, administration of the ActRIIA polypeptide decreases ventricle hypertrophy in the subject. In some embodiments, administration of the ActRIIA polypeptide decreases ventricle hypertrophy in the subject by at least 10% (e g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%. 55%. 60%. 65%. 70%. 75%. or at least 80%). In some embodiments, administration of the ActRIIA polypeptide decreases smooth muscle hypertrophy in the subject. In some embodiments, administration of the ActRIIA polypeptide decreases smooth muscle hypertrophy in the subject by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the ActRIIA polypeptide decreases pulmonary arteriole muscularity in the subject. In some embodiments, administration of the ActRIIA polypeptide decreases pulmonary arteriole muscularity in the subject by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the ActRIIA polypeptide decreases pulmonary vascular resistance in the subject. In some embodiments, administration of the ActRIIA polypeptide decreases pulmonary vascular resistance in the subject by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, administration of the ActRIIA polypeptide decreases pulmonary vascular resistance in the subject by at least 25-30%.

[0163] In some embodiments, the subject has a 6-minute walk distance from 150 to 400 meters. In some embodiments, the method increases the subject's 6-minute walk distance by at least 10 meters (e.g., at least 10. 20. 30. 40, 50, 60, 70, 80, 90, 100. 125, 150, 175. 200, 250, 300, or more than 400 meters). In some embodiments, the subject has a blood hemoglobin level from >8 and <15 g / dl. In some embodiments, the method delays clinical worsening of pulmonary arterial hypertension. In some embodiments, the method delays clinical worsening of pulmonary hypertension in accordance with the NYHA's functional classification system for pulmonary hypertension. In some embodiments, the method reduces the risk of hospitalization for one or more complications associated with pulmonary arterial hypertension. In some embodiments, the ActRIIA polypeptides binds to one or more ligands selected from the group consisting of: activin A, activin B, GDF11, GDF8, BMP10, and BMP6.DBl / 153257679.1 65

[0164] In some embodiments, the subject has pulmonary arterial hypertension and has Functional Class II or Class III pulmonary hypertension in accordance with the NYHA's functional classification system for pulmonary hypertension. In some embodiments, the subject has pulmonary arterial hypertension that is classified as one or more subtypes selected from the group consisting of: idiopathic or heritable pulmonary arterial hypertension, drug- and / or toxin-induced pulmonary hypertension, pulmonary hypertension associated with connective tissue disease, and pulmonary hypertension associated with congenital systemic- to-pulmonary shunts at least 1 year following shunt repair. In some embodiments, the subject has been treated with one or more vasodilators.Dosing Regimen for Administration of Treprostinil and ActRIIA Fusion Protein

[0165] According to certain embodiments, methods of treating pulmonary hypertension in a subject include administering to the subject at least one dose of treprostinil and at least one dose of an ActRIIA fusion protein (e.g.. sotatercept). More particularly, in some embodiments, a method of treating pulmonary hypertension in a subject includes administering to the subject at least one dose of a composition containing treprostinil via inhalation and at least one dose of an ActRIIA fusion protein. In some embodiments, the dose of the ActRIIA fusion protein is administered concurrently with the at least one dose of the treprostinil composition. In some embodiments, the dose of the ActRIIA fusion protein is administered sequentially with the at least one dose of the treprostinil composition. In some embodiments, the dose of the ActRIIA fusion protein is administered after administration of at least one dose of the treprostinil compositionto the subject. In some embodiments, the dose of the ActRIIA fusion protein is administered at least one day after administration of at least one dose of the treprostinil compositionto the subject. In other embodiments, the dose of the ActRIIA fusion protein is administered on the same day as administration of at least one dose of the treprostinil compositionto the subject. In still other embodiments, the dose of the ActRIIA fusion protein is administered before administration of at least one dose of the treprostinil composition to the subject. In some embodiments, the treprostinil composition may be self-administered by the subject as dry powder particles via a dry powder inhaler. In some embodiments, the treprostinil composition is administered once a day, twice a day, three times a day, or four times a day. In some embodiments, the ActRIIA fusion protein may be administered to the subject via injection (e.g., subcutaneous injection, intradermal injection, intramuscular injection, or intravenous injection).DBl / 153257679.1 66

[0166] According to further embodiments, a method of treating pulmonary hypertension in a subject includes administering to the subject at least one dose of a liposomal treprostinil composition via inhalation and at least one dose of an ActRIIA fusion protein. In some embodiments, the dose of the ActRIIA fusion protein is administered concurrently with the at least one dose of liposomal treprostinil composition. In some embodiments, the dose of the ActRIIA fusion protein is administered sequentially with the at least one dose of liposomal treprostinil composition. In some embodiments, the dose of the ActRIIA fusion protein is administered after administration of at least one dose of liposomal treprostinil composition to the subject. In some embodiments, the dose of the ActRIIA fusion protein is administered at least one day after administration of at least one dose of liposomal treprostinil composition to the subject. In other embodiments, the dose of the ActRIIA fusion protein is administered on the same day as administration of at least one dose of liposomal treprostinil composition to the subject. In still other embodiments, the dose of the ActRIIA fusion protein is administered before administration of at least one dose of liposomal treprostinil composition to the subject. In some embodiments, the liposomal treprostinil composition may be selfadministered by the subject via a nebulizer, e.g., a breath-actuated vibrating-mesh nebulizer. In some embodiments, the liposomal treprostinil composition may be self-administered by the subject via dry pow der inhaler. In some embodiments, the liposomal treprostinil composition is administered once a day, twice a day, three times a day, or four times a day. In some embodiments, the ActRIIA fusion protein may be administered to the subject via injection (e.g., subcutaneous injection, intradermal injection, intramuscular injection, or intravenous injection).

[0167] Example dosing amounts and frequencies of administration are provided in the table belowcDBl / 153257679.1 67

[0168] In some embodiments, the treprostinil dosing (whether via dry powder particles, liposomal composition, or other formulation) may be titrated over time such that the dose of treprostinil administered to a subject is gradually increased until a predetermined maximum dose is reached or until adverse reactions are encountered. In some embodiments, the doses may be increased at regular intervals, for example, every7day, every7two days, every three days, every four days, every five days, every7six days, every77 days (weekly), every two weeks, every three weeks, or every four weeks until the maximum dose is reached. The doses may be increased linearly over time or non-linearly in other embodiments. In some embodiments, the dose is increased, for example, from a starting dose ranging from 25 micrograms to 40 micrograms treprostinil per dose to a maximum dose ranging from 100 micrograms to 400 micrograms treprostinil per dose.

[0169] In some embodiments, where the dose of the treprostinil is titrated, the administration of the one or more the ActRIIA fusion proteins (e.g., sotatercept) occurs after or concurrently with the administration of the highest dose of the treprostinil. In other embodiments, the administration of the one or more the ActRIIA fusion proteins (e.g., sotatercept) occurs before the administration of the highest dose of the treprostinil. In some embodiments, the administration of the one or more the ActRIIA fusion proteins (e.g., sotatercept) occurs after at least one dose of the treprostinil but before the administration of the highest dose of the treprostinil.

[0170] In still further embodiments, the one or more the ActRIIA fusion proteins (e.g., sotatercept) and treprostinil (whether via dry7powder particles, liposomal composition, or other formulation) may be used in further combination with other PH treatments, e.g., endothelin receptor antagonists (ERAs), phosphodiesterase type 5 (PDE5) inhibitors, soluble guanylate cyclase stimulators, prostacyclin receptor agonists, and other prostacyclin analogs, as described previously. For example, one or more of: bosentan (oral), ambrisentan (oral), sildenafil (oral. IV), tadalafil (oral), riociguat (oral), selexipag (oral), epoprostenol (IV), and / or iloprost (inhaled). These other drugs may be administered before, concurrently with, or after administration of the one or more the ActRIIA fusion proteins.

[0171] The term “concurrent” or “concurrently” means at or about the same time. The terms “concurrent” and “concurrently” are synonymous with “simultaneous” and “simultaneously.” Concurrent administration of a combination therapy7means administration of two or more treatments at or about the same time. Typically, concurrent administration DBl / 153257679.1 68with two or more treatments (e.g., an inhalation treatment and a fusion protein) occurs between 0 and 10 days. Typically, concurrent administration occurs no longer than about 7 days apart, such as about 5 days, preferably no later than about 3 days, such as within 24 hours, such as within about 8 hours or less. Commonly, concurrent administration of two or more treatments occurs within about 2 hours or less, such that the first and at least second treatments are administered within a period of 2 hours, a period of 1 hour, or within about 30 minutes, or about 10 minutes. In some instances, concurrent administration is performed at the same time, e.g., in one or more administrations.

[0172] It will be appreciated by those skilled in the art that changes could be made to the exemplary embodiments shown and described above without departing from the broad inventive concepts thereof. It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways.

[0173] Unless specifically set forth herein, the terms “a”, "an" and “the” are not limited to one element but instead should be read as meaning “at least one”. Further, unless specifically set forth herein, a disclosed or claimed method should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the steps may be varied.DBl / 153257679.1 69

Claims

1. We claim:

1. A method of treating pulmonary7hypertension comprising:3.administering to a subject in need thereof:4.one or more doses of dry powder particles, each dose of the dry powder particles comprising greater than 10 micrograms of treprostinil, a treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof; and5.a fusion protein comprising an ActRIIA polypeptide domain comprising the amino acid sequence of SEQ ID NO: 2, an Fc domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and a linker domain.

2. The method of claim 1, wherein the linker domain is positioned between the ActRIIA polypeptide domain and the Fc domain.

3. The method of claim 1 or 2, wherein the linker domain comprises TGGG (SEQ ID NO: 4).

4. The method of any one of claims 1 to 3, wherein the ActRIIA polypeptide domain comprises SEQ ID NO: 11.

5. The method of any one of claims 1 to 4, wherein each dose of the dry powder particles comprises 10 micrograms to 400 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

6. The method of any one of claims 1 to 4, wherein each dose of the dry powder particles comprises 25 micrograms to 220 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

7. The method of any one of claims 1 to 4, wherein each dose of the dry powder particles comprises 25 micrograms to 110 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

8. The method of any one of claims 1 to 4, wherein each dose of the dry powder particles comprises 100 micrograms to 220 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

9. The method of any one of claims 1 to 8, wherein each dose of the dry powder particles comprises 2.5 mg to 20 mg of the dry powder particles.14.DBl / 153257679.1 70 10. The method of any one of claims 1 to 8, wherein each dose of the dry powder particles comprises greater than or equal to 20 mg of the dry powder particles.

11. The method of claim 5, wherein each dose of the dry powder particles comprises 16 micrograms of the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

12. The method of claim 5, wherein each dose of the dry powder particles comprises 26.5 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

13. The method of claim 5, wherein each dose of the dry pow der particles comprises 32 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

14. The method of claim 5, wherein each dose of the dry' powder particles comprises 48 micrograms of the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

15. The method of claim 5, wherein each dose of the dry powder particles comprises 51 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

16. The method of claim 5, wherein each dose of the dry powder particles comprises 53 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

17. The method of claim 5, wherein each dose of the dry' powder particles comprises 64 micrograms of the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

18. The method of claim 5, wherein each dose of the dry powder particles comprises 79.5 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

19. The method of claim 5, yvherein each dose of the dry pow der particles comprises 80 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.24.DBl / 153257679.1 71 20. The method of claim 5, wherein each dose of the dry' powder particles comprises 102 micrograms of the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

21. The method of claim 5, wherein each dose of the dry powder particles comprises 106 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

22. The method of claim 5, wherein each dose of the dry powder particles comprises 132.5 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

23. The method of claim 5, wherein each dose of the dry' powder particles comprises 153 micrograms of the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

24. The method of claim 5, wherein each dose of the dry powder particles comprises 159 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

25. The method of claim 5, yvherein each dose of the dry powder particles comprises 185.5 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

26. The method of claim 5, wherein each dose of the dry' pow der particles comprises 204 micrograms of the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

27. The method of claim 5, wherein each dose of the dry powder particles comprises 212 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

28. The method of any one of claims 1 to 27, wherein each dose of the dry powder particles is administered to the subject via a dry powder inhaler.

29. The method of any one of claims 1 to 28, wherein each dose of the dry poyver particles, prior to administration to the subject, are enclosed in one or more dosage units.

30. The method of claim 29, yvherein each of the one or more dosage units contains an amount of the dry- powder particles that is configured to be administered to the subject over one to two breaths using a dry powder inhaler.35.DBl / 153257679.1 72 31. The method of any one of claims 29 or 30. wherein the dry powder particles comprise about 0.5% by weight treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof, and wherein each of the one or more dosage units contains 25 micrograms to 220 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

32. The method of any one of claims 29 or 30, wherein the dry powder particles comprise about 1% by weight treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof, and wherein each of the one or more dosage units contains 25 micrograms to 220 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

33. The method of any one of claims 28 to 32, wherein the subject receives greater than about 12.5 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof per breath via the dry powder inhaler.

34. The method of any one of claims 28 to 33, wherein the subject receives less than or equal to about 30 mg of dry powder per breath.

35. The method of any one of claims 28 to 34, wherein the one or more doses of the dry powder particles contains between 25 micrograms and 400 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one and is administered through eight breaths over a day via the dry powder inhaler.

36. The method of any one of claims 28 to 34, wherein one or more doses of the dry powder particles contains between 100 micrograms and 1600 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof and is administered through four to twenty -four breaths over a day via the dry powder inhaler.

37. The method of any one of claims 1 to 36, wherein the dry powder particles have a mass median aerodynamic diameter from 1 micrometer to 5 micrometers.

38. The method of any one of claims 1 to 36, wherein the dry powder particles have a mass median aerodynamic diameter from 1 micrometer to 3 micrometers.

39. The method of any one of claims 1 to 38, wherein the dry powder particles have a predetermined size range.

40. The method of any one of claims 1 to 39, wherein the dry powder particles comprise particles that are substantially uniform in shape.44.DBl / 153257679.1 73 41. The method of any one of claims 1 to 40, wherein each particle of the dry powder particles comprises the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof and an excipient matrix.

42. The method of any one of claims 1 to 41, wherein the dry particles comprise a nonreducing sugar, a wetting agent, a hydrophobicity modifying agent, a pH modifying agent and a buffer.

43. The method of any one of claims 1 to 42, wherein the dry powder particles comprise by percent solids about 0.581 percent treprostinil sodium, about 92.32 percent trehalose, about 2.19 percent polysorbate 80. about 4.39 percent L-leucine, about 0.26 percent sodium citrate, and about 0.25 percent sodium chloride.

44. The method of any one of claims 1 to 43, wherein the dry powder particles comprise about 0.53% by weight treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof.

45. The method of any one of claims 1 to 44, wherein the dry powder particles comprise about 1.06% by weight treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof.

46. The method of any one of claims 1 to 45, wherein the dry7powder particles have less than 5 percent by weight water content.

47. The method of any one of claims 1 to 46, wherein the dry powder particles have less than 2 percent by weight water content.

48. The method of claim 41, wherein the excipient matrix comprises fumaryl diketopiperazine.

49. The method of claim 48, wherein the dry powder particles have a crystalline structure.

50. The method of claim 48, wherein the dry7powder particles have an amorphous structure.

51. The method of any one of claims 48 to 50. wherein the excipient matrix is configured to dissolve at pH values above 6.

52. The method of any one of claims 1 to 51, wherein the fusion protein is administered at a dose between 0.1 mg / kg and 2.0 mg / kg.56.DBl / 153257679.1 74 53. The method of any one of claims 1 to 52, wherein the fusion protein is administered at a dose of 0.3 mg / kg.

54. The method of any one of claims 1 to 52, wherein the fusion protein is administered at a dose of 0.7 mg / kg.

55. The method of any one of claims 1 to 54, wherein the fusion protein is administered at a first dose of between 0.1 mg / kg and 1.0 mg / kg of said fusion protein for a first period of time, and a second dose of between 0.1 mg / kg and 1.0 mg / kg of said fusion protein is subsequently administered for a second period of time, wherein the second dose is different from the first dose.

56. The method of claim 53, wherein the first period of time is at least 3 weeks.

57. The method of any one of claims 55 or 56, wherein the second period of time is at least 3 weeks.

58. The method of any one of claims 55 to 57, wherein the second period of time is at least 21 weeks.

59. The method of any one of claims 55 to 58, wherein the second period of time is at least 45 weeks.

60. The method of any one of claims 55 to 59. wherein the second period of time exceeds the first period of time.

61. The method of any one of claims 55 to 60. wherein the second dose exceeds the first dose.

62. The method of any one of claims 55 to 61, wherein the first dose is in the range of about 0.2 mg / kg to about 0.4 mg / kg followed by a second dose in the range of about 0.5 mg / kg to about 0.8 mg / kg.

63. The method of any one of claims 55 to 62, wherein the first dose is about 0.3 mg / kg followed by a second dose of about 0.7 mg / kg.

64. The method of any one of claims 1 to 63, wherein the fusion protein is administered using subcutaneous injection.

65. The method of any one of claims 1 to 64, wherein the fusion protein is formulated into a pharmaceutical composition.69.DBl / 153257679.1 75 66. The method of any one of claims 1 to 65, wherein the fusion protein is administered to the subject on a schedule selected from the group consisting of: every week, every two weeks, every three weeks, and every four weeks.

67. The method of any one of claims 1 to 66, wherein the fusion protein is administered to the subject every 3 weeks.

68. The method of any one of claims 1 to 66, wherein the fusion protein is administered to the subject every 4 weeks.

69. The method of any one of claims 1 to 68, wherein the method decreases pulmonary' arterial pressure in the subject.

70. The method of any one of claims 1 to 69, wherein the method decreases the pulmonary' arterial pressure by at least 3 mmHg.

71. The method of any one of claims 1 to 70, wherein the method decreases the pulmonary arterial pressure by at least 7 mmHg.

72. The method of any one of claims 1 to 71, wherein the method increases the subject’s 6-minute walk distance.

73. The method of any one of claims 1 to 72, wherein the method increases the subject’s 6-minute walk distance by at least 50 meters.

74. The method of any one of claims 1 to 73, wherein the method decreases ventricle hypertrophy in the subject.

75. The method of any one of claims 1 to 74, wherein the method decreases smooth muscle hypertrophy in the subject.

76. The method of any one of claims 1 to 75, wherein the method decreases pulmonary arteriole muscularity in the subject.

77. The method of any one of claims 1 to 76, wherein the method decreases pulmonary vascular resistance in the subject.

78. The method of any one of claims 1 to 77, wherein the method decreases pulmonary vascular resistance in the subject by at least 20%.

79. The method of any one of claims 1 to 78, wherein the subject has Functional Class II or Class III pulmonary7hypertension in accordance with the NYHA’s functional classification system for pulmonary' hypertension.83.DBl / 153257679.1 76 80. The method of any one of claims 1 to 79, wherein the method delays clinical worsening of pulmonary arterial hypertension.

81. The method of any one of claims 1 to 80, wherein the method delays clinical worsening of pulmonary arterial hypertension in accordance with the NYHA's functional classification system for pulmonary hypertension.

82. The method of any one of claims 1 to 81, wherein the method delays clinical worsening of pulmonary hypertension by delaying the progression from Functional Class II to Class III as recognized by the NYHA.

83. The method of any one of claims 1 to 82, wherein the inhalation treatment and the fusion protein are administered sequentially.

84. The method of any one of claims 1 to 83, wherein the inhalation treatment is administered after the fusion protein.

85. The method of any one of claims 1 to 84, wherein the fusion protein is administered after the inhalation treatment.

86. The method of any one of claims 1 to 85, wherein the inhalation treatment and the fusion protein are administered sequentially within about 12 hours, about 24 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, or about 3 weeks of each other.

87. The method of any one of claims 1 to 86, wherein the inhalation treatment and the fusion protein are administered concurrently or simultaneously.

88. The method of any one of claims 1 to 87, wherein the inhalation treatment is administered daily, every other day, every third day, weekly, every other week, every' three weeks, or monthly.

89. The method of any one of claims 1 to 88, wherein the fusion protein is administered daily, every other day, every third day, weekly, every other week, every three weeks, or monthly.

90. The method of any one of claims 1 to 89, wherein the fusion protein is administered every three weeks.

91. The method of any one of claims 1 to 90, wherein the pulmonary hypertension is pulmonary arterial hypertension (PAH).95.DBl / 153257679.1 77 92. The method of any one of claims 1 to 90, wherein the pulmonary hypertension is pulmonary hypertension caused by interstitial lung disease (PH-ILD).

93. A pharmaceutical kit for treating pulmonary hypertension comprising:97.an inhalation treatment for pulmonary arterial hypertension, comprising a dose of dry¬ powder particles comprising greater than 25 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof enclosed in one or more dosage units; and98.a fusion protein comprising an ActRIIA polypeptide domain comprising the amino acid sequence of SEQ ID NO: 2, an Fc domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and a linker domain.

94. The pharmaceutical kit of claim 93, wherein the linker domain is positioned between the ActRIIA polypeptide domain and the Fc domain.

95. The pharmaceutical kit of claim 93 or 94, wherein the linker domain comprises TGGG (SEQ ID NO: 4).

96. The pharmaceutical kit of any one of claims 93 to 95, wherein the ActRIIA polypeptide domain comprises SEQ ID NO: 11.

97. The pharmaceutical kit of any one of claims 93 to 96, wherein the one or more dosage units include a total predetermined amount of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof in the range of 25 micrograms to 400 micrograms.

98. The pharmaceutical kit of any one of claims 93 to 97, comprising a dry powder inhaler comprising a body configured to receive the one or more dosage units, and a mouthpiece that is moveable with respect to the body to an aligned position, wherein the drypowder particles may be inhaled by a subject through the mouthpiece when the mouthpiece is in the aligned position.

99. The pharmaceutical kit of claim 98, wherein the mouthpiece is rotatable with respect to the body.

100. The pharmaceutical kit of claim 98 or 99, wherein the dry powder inhaler comprises a moveable component configured to open the one or more dosage units to allow release of the dry- powder particles from the one or more dosage units.106.DBl / 153257679.1 78 101. The pharmaceutical kit of any one of claims 93 to 100, wherein each of the one or more dosage units contains the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof in an amount of 25 micrograms to 220 micrograms.

102. The pharmaceutical kit of any one of claims 93 to 101, wherein each of the one or more dosage units contains 2.5 mg to 20 mg of the dry powder particles.

103. The pharmaceutical kit of any one of claims 93 to 101, wherein each of the one or more dosage units contains greater than or equal to 20 mg of the dry powder particles.

104. The pharmaceutical kit of any one of claims 93 to 103, wherein the dry powder particles comprise about 0.53% by weight treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof.

105. The pharmaceutical kit of any one of claims 93 to 103, wherein the dry powder particles comprise about 1.06% by weight treprostinil, treprostinil prodrug, or pharmaceutically acceptable salt of any one thereof.

106. The pharmaceutical kit of any one of claims 93 to 105, wherein the dry powder particles have a mass median aerodynamic diameter between 1 micrometer and 5 micrometers.

107. The pharmaceutical kit of any one of claims 93 to 105, wherein the dry powder particles have a mass median aerodynamic diameter of 3 micrometers or less.

108. The pharmaceutical kit of any one of claims 93 to 107, wherein the dry powder particles have less than 5 percent water content.

109. The pharmaceutical kit of any one of claims 93 to 108, wherein the dose of dry powder particles is packaged in a closeable container having a cap.

110. The pharmaceutical kit of any one of claims 93 to 109, wherein the one or more dosage units comprises one or more capsules.

111. The pharmaceutical kit of any one of claims 93 to 110, wherein the dry powder particles have a predetermined size range.

112. The pharmaceutical kit of any one of claims 93 to 111, wherein the dry powder particles comprise particles that are substantially uniform in shape.116.DBl / 153257679.1 79 113. The pharmaceutical kit of any one of claims 93 to 112, wherein the treprostinil, a treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof becomes pharmaceutically available in less than 10 seconds upon delivery to the subject via inhalation.

114. The pharmaceutical kit of any one of claims 93 to 113, wherein the treprostinil. a treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof becomes pharmaceutically available in less than 5 seconds upon delivery' to the subject via inhalation.

115. The pharmaceutical kit of any one of claims 93 to 114, wherein the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof becomes pharmaceutically available in less than 1 second upon delivery to the subject via inhalation.

116. The pharmaceutical kit of any one of claims 98 tol 15, wherein each of the one or more capsules contains an amount of the dry’ powder particles that is configured to be administered to the subject over one to two breaths using the dry powder inhaler.

117. The pharmaceutical kit of any one of claims 98 to 116, further comprising a second dosage unit receivable in the body of the dry' powder inhaler, wherein the second dosage unit contains a second predetermined amount of the dry powder particles.

118. The pharmaceutical kit of claim 117, wherein each of the dosage unit and the second dosage unit contains from about 10 micrograms to about 220 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof.

119. The pharmaceutical kit of any one of claims 93 to 118, wherein the kit comprises at least a first container containing the fusion protein.

120. The pharmaceutical kit of any one of claims 93 to 119, wherein the kit comprises at least the first container and a second container containing the fusion protein.

121. The pharmaceutical kit of any one of claims 93 to 120, wherein the at least first and second container contain the same or different amounts of the fusion protein.

122. The pharmaceutical kit of any one of claims 119 to 121. wherein the at least first and second containers each comprise between 25 mg to 60 mg of the fusion protein.

123. The pharmaceutical kit of any one of claims 119 to 122, wherein at least one of the at least first and second containers contains 60 mg of fusion protein.

124. The pharmaceutical kit of any one of claim 119 to 122, wherein at least one of the at least first and second containers contains 45 mg of fusion protein.128.DBl / 153257679.1 80 125. The pharmaceutical kit of any one of claim 119 to 122, wherein at least one of the at least first and second containers contains 30 mg of fusion protein.

126. The pharmaceutical kit of any one of claim 119 to 122, wherein at least one of the at least first and second containers contains 25 mg of fusion protein.

127. The pharmaceutical kit of any one of claim 119 to 122, wherein the first container contains 45 mg of fusion protein and the second container contains 60 mg of fusion protein.

128. The pharmaceutical kit of any one of claim 119 to 122, wherein the first container contains 30 mg of fusion protein and the second container contains 60 mg of fusion protein.

129. The pharmaceutical kit of any one of claim 119 to 122, wherein the first container contains 45 mg of fusion protein and the second container contains 45 mg of fusion protein.

130. The pharmaceutical kit of any one of claim 119 to 122, wherein the first container contains 30 mg of fusion protein, the second container contains 45 mg of fusion protein, and a third container contains 60 mg of fusion protein.

131. The pharmaceutical kit of any one of claim 119 to 122, wherein the first container contains 25 mg of fusion protein, the second container contains 45 mg of fusion protein, and a third container contains 60 mg of fusion protein.

132. The pharmaceutical kit of any one of claims 119 to 128. wherein the containers are refrigerated at 2-8° C.

133. The pharmaceutical kit of any one of claims 93 to 129, wherein the fusion protein is reconstituted into a sterile injectable solution.

134. The pharmaceutical kit of claim 133, wherein the sterile injectable solution comprises sterile water for injection.

135. The pharmaceutical kit of claim 133 or 134, wherein the sterile injectable solution is administered parenterally.

136. The pharmaceutical kit of any one of claims 133 to 135, wherein the sterile injectable solution is administered via subcutaneous injection.

137. The pharmaceutical kit of any one of claims 133 to 135, wherein the sterile injectable solution is administered via intradermal injection.

138. The pharmaceutical kit of any one of claims 133 to 135, wherein the sterile injectable solution is administered via intramuscular injection.142.DBl / 153257679.1 81 139. The pharmaceutical kit of any one of claims 133 to 135, wherein the sterile injectable solution is administered via intravenous injection.

140. The pharmaceutical kit of any one of claims 133 to 139. wherein the sterile injectable solution is self-administered.

141. The pharmaceutical kit of any one of claims 133 to 140. wherein the sterile injectable solution comprises a therapeutically effective dose.

142. The pharmaceutical kit of claim 141, wherein the therapeutically effective dose comprises a subject weight-based dose.

143. The pharmaceutical kit of any one of claims 133 to 142, wherein the sterile injectable solution is administered every 3 weeks.

144. The pharmaceutical kit of any one of claims 133 to 142, wherein the sterile injectable solution is administered every 4 weeks.

145. The pharmaceutical kit of any one of claims 93-144, wherein the kit is used to treat pulmonary hypertension.

146. The pharmaceutical kit of any one of claims 93 to 145, wherein the kit is used to treat pulmonary arterial hypertension (PAH).

147. The pharmaceutical kit of any one of claims 93 to 145, wherein the kit is used to treat pulmonary hypertension caused by interstitial lung disease (PH-ILD).

148. A method of treating pulmonary7hypertension comprising:152.administering to a subject in need thereof a fusion protein comprising an ActRIIA polypeptide domain comprising the amino acid sequence of SEQ ID NO: 2, an Fc domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

3. and a linker domain, wherein the subject received one or more doses of treprostinil, a treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof prior to administration of the fusion protein.

149. The method of claim 148, wherein the linker domain is positioned between the ActRIIA polypeptide domain and the Fc domain.

150. The method of claim 148 or 149, wherein the linker domain comprises TGGG (SEQ ID NO: 4).155.DBl / 153257679.1 82 151. The method of any one of claims 148 to 150, wherein the ActRIIA polypeptide domain comprises SEQ ID NO: 11.

152. The method of any one of claims 148 to 151, wherein the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof is administered via a soft mist inhaler or a nebulizer.

153. The method of any one of claims 152, wherein the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof is administered by a nebulizer.

154. The method of claims 148 to 151, wherein the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof is administered to the subject via a dry powder inhaler.

155. The method of claim 154, wherein a treatment comprises a dose of dry powder particles comprising greater than 25 micrograms of the treprostinil. the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof enclosed in one or more dosage units.

156. The method of claim 154 or 155, wherein the inhalation treatment comprises between about 400 micrograms and about 1600 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof administered to a subject over a day through four to twenty-four breaths.

157. The method of any one of claims 154 to 156, wherein the dry powder particles have a mass median aerodynamic diameter from 1 micrometer to 5 micrometers.

158. The method of any one of claims 154 to 157, wherein the dry powder particles have a mass median aerodynamic diameter from 1 micrometer to 3 micrometers.

159. The method of any one of claims 154 to 158, wherein the dry powder particles have a predetermined size range.

160. The method of any one of claims 154 to 159, wherein the dry powder particles comprise particles that are substantially uniform in shape.

161. The method of any one of claims 148 to 154, wherein the treprostinil is administered via an aerosolized liposomal composition.166.DBl / 153257679.1 83 162. The method of claim 1 1, wherein the liposomal composition comprises one or more liposomes suspended in an external medium.

163. The method of claim 162, wherein the one or more liposomes each comprise:168.(a) a lipid bilayer comprising at least one vesicle-forming phospholipid; and169.(b) an internal aqueous medium encapsulated by the lipid bilayer comprising:170.(1) treprostinil, and171.(2) a pH gradient salt;172.wherein a weight ratio of treprostinil to the at least one vesicle-forming phospholipid is equal to or higher than about 0.035.

164. The method of claim 163, wherein the lipid bilayer further comprises a sterol selected from the group consisting of: cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, and any combination thereof.

165. The method of claim 164, wherein the at least one vesicle-forming phospholipid comprises a first phospholipid and a second phospholipid.

166. The method of claim 165, wherein the first phospholipid is selected from the group consisting of: HSPC, DSPC, DPPC, DMPC, and any combination thereof, and the second phospholipid is selected from the group consisting of: DSPG. DPPG, DMPG, PEG-DSPE. and any combination thereof.

167. The method of claim 166, wherein the lipid bilayer comprises about 50-70 mole % of the first phospholipid, 20-45 mole % of the sterol, and 0.1-10 mole % of the second phospholipid.

168. The method of claim 167, wherein the first phospholipid is HSPC, the sterol is cholesterol, and the second phospholipid is DSPG.

169. The method of any one of claims 163 to 168, wherein the pH gradient salt comprises a bicarbonate salt.

170. The method of any one of claims 163 to 169, wherein the one or more liposomes have a mean particle size of about 100 nm to about 200 nm and a poly dispersity index of less than 0.2.

171. The method of any one of claims 148 to 170, wherein the fusion protein is administered at a dose between 0.1 mg / kg and 2.0 mg / kg.181.DBl / 153257679.1 84 172. The method of any one of claims 148 to 171, wherein the fusion protein is administered at a first dose of between 0.1 mg / kg and 1.0 mg / kg of said fusion protein for a first period of time, and a second dose of between 0.1 mg / kg and 1.0 mg / kg of said fusion protein is subsequently administered for a second period of time.

173. The method of any one of claims 148 to 172, wherein the fusion protein is administered using subcutaneous injection.

174. The method of any one of claims 148 to 173, wherein the fusion protein is formulated into a pharmaceutical composition.

175. The method of any one of claims 148 to 174, wherein the fusion protein is administered daily, every other day, every third day, weekly, every other week, every three weeks, or monthly.

176. The method of any one of claims 148 to 175, wherein the fusion protein is administered every three weeks.

177. The method of any one of claims 148 to 176, wherein the pulmonary hypertension is pulmonary arterial hypertension (PAH).

178. The method of any one of claims 148 to 177, wherein the pulmonary hypertension is pulmonary hypertension caused by interstitial lung disease (PH-ILD).

179. A method of treating pulmonary hypertension comprising administering treprostinil, a treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof to a subject in need thereof, wherein the subject received prior to administration of the treprostinil, a treprostinil prodrug, or a pharmaceutically acceptable salt of any one thereof, one or more doses of a fusion protein comprising an ActRIIA polypeptide domain comprising the amino acid sequence of SEQ ID NO: 2, an Fc domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and a linker domain.

180. The method of claim 179, wherein the linker domain is positioned between the ActRIIA polypeptide domain and the Fc domain.

181. The method of claim 179 or 180, wherein the linker domain comprises TGGG (SEQ ID NO: 4).

182. The method of any one of claims 179 to 181, wherein the ActRIIA polypeptide domain comprises SEQ ID NO: 11.192.DBl / 153257679.1 85 183. The method of any one of claims 179 to 182, wherein the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof is administered via a soft mist inhaler or a nebulizer.

184. The method of claim 183, wherein the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof is administered via a nebulizer.

185. The method of claims 179 to 182, wherein the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof is administered to the subject via a dry' powder inhaler.

186. The method of claim 184, wherein a treatment comprises a dose of dry powder particles comprising greater than 25 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof enclosed in one or more dosage units.

187. The method of claim 184 or 185, wherein a treatment comprises between about 400 micrograms and about 1600 micrograms of the treprostinil, the treprostinil prodrug, or the pharmaceutically acceptable salt of any one thereof administered to a subject over a day through four to twent -four breaths.

188. The method of any one of claims 184 to 187, wherein the dry powder particles have a mass median aerodynamic diameter from 1 micrometer to 5 micrometers.

189. The method of any one of claims 184 to 188, wherein the dry powder particles have a mass median aerodynamic diameter from 1 micrometer to 3 micrometers.

190. The method of any one of claims 184 to 189, wherein the dry powder particles have a predetermined size range.

191. The method of any one of claims 184 to 190, wherein the dry powder particles comprise particles that are substantially uniform in shape.

192. The method of any one of claims 179 to 184, wherein the treprostinil is administered via an aerosolized liposomal composition.

193. The method of claim 192, wherein the liposomal composition comprises one or more liposomes suspended in an external medium.

194. The method of claim 193, wherein the one or more liposomes each comprise:204.(a) a lipid bilayer comprising at least one vesicle-forming phospholipid; and205.DBl / 153257679.1 86 (b) an internal aqueous medium encapsulated by the lipid bilayer comprising:206.(1) treprostinil, and207.(2) a pH gradient salt;208.wherein a weight ratio of treprostinil to the at least one vesicle-forming phospholipid is equal to or higher than about 0.035.

195. The method of claim 194, wherein the lipid bilayer further comprises a sterol selected from the group consisting of: cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, and any combination thereof.

196. The method of claim 195, wherein the at least one vesicle-forming phospholipid comprises a first phospholipid and a second phospholipid.

197. The method of claim 196, wherein the first phospholipid is selected from the group consisting of: HSPC, DSPC, DPPC, DMPC, and any combination thereof, and the second phospholipid is selected from the group consisting of: DSPG, DPPG, DMPG, PEG-DSPE, and any combination thereof.

198. The method of claim 195, wherein the lipid bilayer comprises about 50-70 mole % of the first phospholipid, 20-45 mole % of the sterol, and 0.1-10 mole % of the second phospholipid.

199. The method of claim 197, wherein the first phospholipid is HSPC, the sterol is cholesterol, and the second phospholipid is DSPG.

200. The method of any one of claims 194 to 199, wherein the pH gradient salt comprises a bicarbonate salt.

201. The method of any one of claims 194 to 200, wherein the one or more liposomes each have a mean particle size of about 100 nm to about 200 nm and a poly dispersity index of less than 0.2.

202. The method of any one of claims 179 to 201, wherein the fusion protein was administered at a dose between 0.1 mg / kg and 2.0 mg / kg.

203. The method of any one of claims 179 to 202, wherein the fusion protein is administered at a first dose of betw een 0.1 mg / kg and 1.0 mg / kg of said fusion protein for a first period of time, and a second dose of between 0.1 mg / kg and 1.0 mg / kg of said fusion protein is subsequently administered for a second period of time.218.DBl / 153257679.1 87 204. The method of any one of claims 179 to 203, wherein the fusion protein is administered using subcutaneous injection.

205. The method of any one of claims 179 to 204, wherein the fusion protein is formulated into a pharmaceutical composition.

206. The method of any one of claims 179 to 205, wherein the fusion protein is administered daily, every other day, every third day, weekly, every other week, every three weeks, or monthly.

207. The method of any one of claims 179 to 206, wherein the fusion protein is administered every' three weeks.

208. The method of any one of claims 179 to 207, wherein the pulmonary hypertension is pulmonary' arterial hypertension (PAH).

209. The method of any one of claims 179 to 207, wherein the pulmonary hypertension is pulmonary hypertension associated with interstitial lung disease (PH-ILD).

210. A method of treating pulmonary' hypertension comprising:225.administering to a subject in need thereof:226.one or more doses of dry powder particles, each dose of the dry powder particles comprising greater than 10 micrograms of treprostinil and an excipient matrix comprising fumaryl diketopiperazine; and227.a fusion protein comprising an ActRIIA polypeptide domain comprising SEQ ID NO: 11.

211. A method of treating pulmonary hypertension comprising:229.administering to a subject in need thereof:230.one or more doses of greater than 10 micrograms of treprostinil palmitil; and a fusion protein comprising an ActRIIA polypeptide comprising SEQ ID NO:

11.

212. A method of treating pulmonary hypertension comprising:231.administering to a subject in need thereof:232.one or more doses of an aerosolized liposomal composition comprising greater than 10 micrograms of treprostinil; and233.a fusion protein comprising an ActRIIA polypeptide domain comprising SEQ ID NO: 11.234.DBl / 153257679.1 88 213. A method of treating pulmonary hypertension comprising administering to a subject in need thereof:235.one or more doses of dry powder particles, each dose of the dry powder particles comprising greater than 10 micrograms of treprostinil, wherein the dry powder particles have a mass median aerodynamic diameter from 1 micrometer to 5 micrometers and are substantially uniform in shape; and236.a fusion protein comprising an ActRIIA polypeptide domain comprising SEQ ID NO: 11.

214. A method of treating pulmonary hypertension comprising:238.administering to a subject in need thereof:239.a fusion protein comprising an ActRIIA polypeptide comprising SEQ ID NO: 11 to a subject in need thereof; and240.one or more doses of dry powder particles, each dose of the dry powder particles comprising greater than 10 micrograms of treprostinil and an excipient matrix comprising fumaryl diketopiperazine; wherein administration of the dry powder particles is no more than one month after administration of the fusion protein.

215. A method of treating pulmonary hypertension comprising:242.administering to a subject in need thereof:243.one or more doses of greater than 10 micrograms of treprostinil palmitil; wherein the subject is administered the one or more doses of treprostinil palmitil less than 1 month after the subject was administered a fusion protein comprising an ActRIIA polypeptide domain comprising SEQ ID NO: 11.

216. A method of treating pulmonary hypertension comprising:245.administering to a subject in need thereof:246.one or more doses of an aerosolized liposomal composition comprising greater than 10 micrograms of treprostinil;247.wherein the subject is administered the one or more doses of the aerosolized liposomal composition less than 1 month after the subject was administered a fusion protein comprising an ActRIIA polypeptide domain comprising SEQ ID NO: 11.

217. A method of treating pulmonary hypertension comprising:249.administering to a subject in need thereof:250.DBl / 153257679.1 89 one or more doses of dry powder particles, each dose of the dry powder particles comprising greater than 10 micrograms of treprostinil, wherein the dry powder particles have a mass median aerodynamic diameter from 1 micrometer to 5 micrometers and are substantially uniform in shape;251.wherein the subject is administered the one or more doses of dry powder particles less than 1 month after the subject was administered a fusion protein comprising an ActRIIA polypeptide domain comprising SEQ ID NO: 11.

218. A method of treating pulmonary7hypertension comprising:253.administering to a subject in need thereof:254.a fusion protein comprising an ActRIIA polypeptide domain identical to SEQ ID NO: 11;255.wherein the subject is administered the fusion protein less than 1 month after the subject was administered one or more doses of dry7powder particles, each dose of the dry7powder particles comprising greater than 10 micrograms of treprostinil and an excipient matrix comprising fumaryl diketopiperazine.

219. A method of treating pulmonary hypertension comprising:257.administering to a subject in need thereof:258.a fusion protein comprising an ActRIIA polypeptide domain comprising SEQ ID NO: 11;259.wherein the subject is administered the fusion protein less than 1 month after the subject was administered one or more doses of greater than 10 micrograms of treprostinil palmitil.

220. A method of treating pulmonary hypertension comprising:261.administering to a subject in need thereof:262.a fusion protein comprising an ActRIIA poly peptide domain comprising SEQ ID NO: 11;263.wherein the subject is administered the fusion protein less than 1 month after the subject was administered one or more doses of an aerosolized liposomal composition comprising greater than 10 micrograms of treprostinil.

221. A method of treating pulmonary7hypertension comprising:265.administering to a subject in need thereof:266.DBl / 153257679.1 90 a fusion protein comprising an ActRIIA polypeptide domain comprising SEQ ID NO: 11;267.wherein the subject is administered the fusion protein less than 1 month after the subject was administered one or more doses of dry powder particles, each dose of the dry powder particles comprising greater than 10 micrograms of treprostinil, wherein the dry powder particles have a mass median aerodynamic diameter (MMAD) from 1 micrometer to 5 micrometers and are substantially uniform in shape.268.DBl / 153257679.1 91

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