Prostacyclin compounds, compositions and methods of use thereof

Abstract

To provide prostacyclin compounds and compositions comprising the same.SOLUTION: Specifically, prostacyclin compounds comprising treprostinil covalently linked to a linear C5-C18 alkyl, branched C5-C18 alkyl, linear C2-C18 alkenyl, branched C3-C18 alkenyl, aryl, aryl-C1-C18 alkyl or an amino acid or a peptide (e.g., dipeptide, tripeptide, tetrapeptide) are described. The linkage, in one embodiment, is via a carbamate, amide or ester bond. Prostacyclin compounds provided herein can also include at least one hydrogen atom substituted with at least one deuterium atom. Methods for treating pulmonary hypertension (e.g., pulmonary arterial hypertension) and portopulmonary hypertension are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 042,123, filed August 26, 2014; U.S. Provisional Application No. 62 / 028,758, filed July 24, 2014; U.S. Provisional Application No. 61 / 950,967, filed March 11, 2014; U.S. Provisional Application No. 61 / 910,703, filed December 2, 2013; and U.S. Provisional Application No. 61 / 895,680, filed October 25, 2013, each of which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Pulmonary hypertension (PH) is characterized by abnormally high blood pressure in the pulmonary vasculature. It is a progressive, fatal disease that can lead to heart failure and occur in the pulmonary arteries, veins, or capillaries. Symptomatically, patients experience shortness of breath, dizziness, syncope, and other symptoms, all of which are worsened by exertion. There are many causes, which may be idiopathic with unknown origin, and can lead to hypertension in other systems, such as portopulmonary hypertension, in which a patient has both portal and pulmonary hypertension.

[0003] Pulmonary hypertension is classified into five groups by the World Health Organization (WHO). Group I is referred to as pulmonary arterial hypertension (PAH), which includes PAH with no known cause (idiopathic), hereditary PAH (i.e., familial PAH or FPAH), PAH caused by drugs or toxins, and PAH caused by conditions such as connective tissue disease, HIV infection, liver disease, and congenital heart disease. Group II pulmonary hypertension is characterized by pulmonary hypertension associated with left heart disease. Group III pulmonary hypertension is characterized by PH associated with lung diseases, such as chronic obstructive pulmonary disease and interstitial lung disease, and PH associated with sleep-related breathing disorders (e.g., sleep apnea). Group IV PH is PH caused by chronic thrombotic and / or embolic diseases, such as PH caused by blood clots or blood clotting disorders within the lungs. Group V includes PH caused by other disorders or conditions, such as blood disorders (e.g., polycythemia vera, essential thrombocythemia), systemic disorders (e.g., sarcoidosis, vasculitis), and metabolic disorders (e.g., thyroid disease, glycogen storage disease).

[0004] Pulmonary arterial hypertension (PAH) afflicts approximately 200,000 people worldwide, with approximately 30,000 to 40,000 of these patients in the United States. PAH patients experience narrowing of the pulmonary arteries, which leads to high pulmonary artery pressure and makes it difficult for the heart to pump blood to the lungs. Patients suffer from shortness of breath and fatigue, which often significantly limits their ability to perform physical activities.

[0005] The New York Heart Association (NYHA) The NYHA classifies patients into four functional classes used to grade the severity of their disease. Patients with NYHA Class I PAH have no limitations on physical activity because ordinary physical activity does not cause undue dyspnea or fatigue, chest pain, or near-syncope. Patients with Class I PAH do not require treatment. Patients with NYHA Class II PAH have mild limitations on physical activity. These patients are comfortable at rest, but ordinary physical activity causes undue dyspnea or fatigue, chest pain, or near-syncope. Patients with NYHA Class III PAH have severe limitations on physical activity. While comfortable at rest, patients with Class III PAH experience undue dyspnea or fatigue, chest pain, or near-syncope as a result of less than ordinary physical activity. Patients with NYHA Class IV PAH are completely unable to perform physical activity without symptoms. Patients with Class IV PAH may experience dyspnea and / or fatigue at rest, and physical activity increases their discomfort. Patients with class IV PAH often present with signs of right heart failure.

[0006] Patients with PAH are treated with endothelin receptor antagonists (ERAs), type 5 phosphodiesterase (PDE-5) inhibitors, guanylate cyclase stimulators, prostanoids (e.g., prostacyclin), or combinations thereof. ERAs include ambrisentan (abrisentan) (Letairis®), sitaxsentan, bosentan (Tracleer®), and macitentan (Opsumit®). PDE-5 inhibitors indicated for the treatment of PAH include sildenafil (Revatio®) and tadalafil (Adcirca®). Prostanoids indicated for the treatment of PAH include iloprost, epoprostenol, and treprostinil (Remodulin®, Tyvaso®). One approved guanylate cyclase stimulator is riociguat (Adempas®). Additionally, patients are often treated with combinations of the above compounds.

[0007] Portopulmonary hypertension, defined by the coexistence of portal hypertension and pulmonary hypertension, is a serious complication of liver disease. The diagnosis of portopulmonary hypertension is based on hemodynamic criteria: (1) portal hypertension and / or liver disease (clinical diagnosis - ascites / varices / splenomegaly), (2) mean pulmonary artery pressure >25 mmHg at rest, and (3) >240 dynes / cm. 5 (4) a pulmonary vascular resistance of <15 mmHg or a transpulmonary gradient of >12 mmHg. PPH is a serious complication of liver disease, present in 0.25-4% of patients suffering from cirrhosis. Today, PPH is a comorbidity in 4-6% of patients referred for liver transplantation. Summary of the Invention [Problem to be solved by the invention]

[0008] Although treatments for PAH and PPH exist, current prostacyclin therapy is associated with significant toxicity and tolerability issues and the need for inconvenient dosing schedules. The present invention overcomes these issues by providing compounds and treatment schedules that offer less toxicity, better tolerability, and more convenient dosing schedules. [Means for solving the problem]

[0009] In one aspect of the present invention, a compound of formula (I): [ka] (Wherein, R1 is NH, O or S; R2 is H, linear C5-C 18 Alkyl, branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl, branched C3-C 18 Alkenyl, aryl; aryl-C1-C 18alkyl; amino acid or peptide; R3 is H, OH, O-alkyl or O-alkenyl; R4 is an optionally substituted straight or branched C1-C 15 Alkyl or optionally substituted straight or branched C-C 15 alkenyl; and n is an integer from 0 to 5. or a pharmaceutically acceptable salt thereof, with the proviso that the prostacyclin compound is not treprostinil.

[0010] In another aspect of the present invention, a compound of formula (II): [ka] (Wherein R1 is NH, O or S; R2 is a linear or branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl or branched C3-C 18 Alkenyl, aryl, aryl-C1-C 18 alkyl, amino acid, or peptide; and n is an integer from 0 to 5. or a pharmaceutically acceptable salt thereof.

[0011] In one embodiment, compounds of formula (I) and / or (II) are provided in which one or more hydrogen atoms are replaced with deuterium. Thus, in one embodiment, the present invention relates to isotopic species of formula (I) and / or (II) in which one or more deuterium atoms are replaced. The isotopic species of formula (I) and / or (II) can be used to accurately determine the concentration of compounds of formula (I) and / or (II) in biological fluids and to determine the metabolic patterns of compounds of formula (I) and / or (II) and their isotopic species. The present invention further provides compositions comprising these deuterium isotopic species and methods for treating diseases and conditions as described herein.

[0012] In one embodiment of the present invention, there is provided a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof, wherein R1 is N and n is 1. In another embodiment, R2 is a linear C5-C 18 Alkyl or branched C5-C 18 In another embodiment, R2 is a linear C-C alkyl. 12 Alkyl or branched C6-C 12 It is alkyl.

[0013] Another embodiment of the present invention provides compounds of formula (I) or (II) wherein R1 is O and n is 1. In another embodiment, compounds of formula (I) or (II) are provided wherein R1 is S and n is 1. In yet another embodiment of the present invention, compounds of formula (I) or (II) are provided wherein R1 is N and n is 0.

[0014] Another embodiment of the present invention is a linear C5-C 18 In another embodiment, n is 0 or 1. In yet another embodiment, R1 is N or O. In yet another embodiment, R2 is a linear C6-C 16 In yet another embodiment, R1 is N and R2 is a linear C-C alkyl. 18 In yet another embodiment, R2 is a linear C6, C8, C 10 , C 12 or C 14 It is alkyl.

[0015] Another embodiment of the present invention is a compound in which R2 is a branched C5-C 18 Provided are prostacyclin compounds of Formula (I) or (II) or pharmaceutically acceptable salts thereof, wherein n is alkyl. In other embodiments, n is 0 or 1. In yet other embodiments, R is N or O. In yet other embodiments, the branched alkyl is hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl.

[0016] In yet another embodiment, R2 is a linear C5-C 18 Provided are prostacyclin compounds of Formula (I) or (II) or pharmaceutically acceptable salts thereof, which are alkenyl. In other embodiments, n is 0 or 1. In yet other embodiments, R is N or O. In yet other embodiments, the branched alkyl is hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl.

[0017] In yet another embodiment, R2 is a branched C5-C 18 Provided are prostacyclin compounds of Formula (I) or (II) or pharmaceutically acceptable salts thereof, which are alkenyl. In other embodiments, n is 0 or 1. In yet other embodiments, R is N or O. In still other embodiments, the branched alkenyl is pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, or octadecenyl.

[0018] In one embodiment, there is provided a prostacyclin compound or pharmaceutically acceptable salt of Formula (I) or (II), wherein R2 is a branched alkyl, which is a symmetrical branched alkyl or an asymmetrical branched alkyl. In one embodiment of Formula (I) or (II), R1 is O or N, and R2 is [ka] wherein m1 and m2 are independently integers selected from 1 to 9, and each occurrence of R' is independently H, linear or branched C1-C8 alkyl, or linear or branched C1-C8 alkenyl. When m1 and / or m2 are integers from 2 to 9, m1 / m2 at the end of the carbon chain is CH3, and the remaining m1 / m2 groups are CH2. In other embodiments, n is 0 or 1. In yet other embodiments, n is 1, R1 is O, and R2 is [ka] and the following compound: [ka] or a pharmaceutically acceptable salt thereof. In one embodiment, m1 and m2 are both 4. In another embodiment, m1 is 3 and m2 is 4. In yet another embodiment, n is 1.

[0019] In one embodiment, a compound of Formula (I) or (II) is provided, wherein R1 is O and R2 is [ka] In yet another embodiment of formula (I) or (II), R1 is O and R2 is [ka] is.

[0020] In one embodiment, a compound of Formula (I) or (II) is provided, wherein R1 is N and R2 is [ka] In yet another embodiment of formula (I) or (II), R1 is N and R2 is [ka] is.

[0021] In other embodiments, n is 1 and the following compound: [ka] (referred to herein as 5-nonanyl-treprostinil or 5C9-TR) is provided.

[0022] In one embodiment, prostacyclin compounds of the formulas provided herein having a branched alkyl or alkenyl at the R2 position (e.g., R2 of the formulas provided herein is 5-nonanyl, 3-heptyl, 4-heptyl, 4-octyl, 3-octyl, 2-octyl, 2-dimethyl-1-propyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, 3-pentyl) exhibit a slower conversion rate than prostacyclin compounds having a linear alcohol chain at the R2 position and have the additional advantage of high solubility.

[0023] Yet another embodiment of the present invention is a compound of formula (III): [ka] (wherein R1 and R2 are as defined above, R5 and R6 are independently H, optionally substituted straight or branched C1-C 15 Alkyl, optionally substituted straight or branched C-C 15 Alkenyl, (C=O)-optionally substituted straight or branched C1-C 15 Alkyl or (C=O)-optionally substituted straight or branched C-C 15 alkenyl) or a pharmaceutically acceptable salt thereof, with the proviso that the prostacyclin compound of formula (III) is not treprostinil.

[0024] Another aspect of the present invention relates to a prostacyclin composition comprising a prostacyclin compound of Formula (I), (II), or (III). In one embodiment, the prostacyclin composition comprises a prostacyclin compound of Formula (I), (II), or (III) and a hydrophobic additive. In another embodiment, the hydrophobic additive is a hydrocarbon, a terpene, or a hydrophobic lipid. In another embodiment, the hydrophobic additive is cholesteryl acetate, ethyl stearate, palmitate, myristate, palmityl palmitate, tocopherol acetate, a monoglyceride, a diglyceride, a triglyceride, such as palmitate, myristate, dodecanoate, decanoate, octanoate, or squalane. In yet another embodiment, the hydrophobic additive is squalane.

[0025] In another aspect of the present invention, a composition is provided comprising a prostacyclin compound of Formula (I), (II), or (III) and an amphiphilic substance. In one embodiment, the amphiphilic substance is a PEGylated lipid, surfactant, or block copolymer. In another embodiment, the prostacyclin composition comprises a prostacyclin compound of Formula (I), (II), or (III) and a PEGylated lipid. In another embodiment, the PEGylated lipid comprises PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG5000. In other embodiments, the lipid component of the PEGylated lipid is selected from the group consisting of dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphoethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dimyristoylglycerol (DMG), diphosphatidylglycerol (DPG), distearoylglycerol (DSG), and the like. Contains covalently bound PEG.

[0026] In another embodiment of the present invention, a composition is provided comprising a prostacyclin compound of formula (I), (II) or (III), a hydrophobic additive and an amphiphilic substance. In one embodiment, the amphiphilic substance is a PEGylated lipid, surfactant or block copolymer. In another embodiment, the hydrophobic additive is squalane. In another embodiment, the PEGylated lipid present in the composition comprises PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000 or PEG5000.

[0027] In another aspect of the present invention, methods for treating pulmonary hypertension (PH) are provided. The methods include treating Group I (PAH), Group II, Group III, Group IV, or Group V PH. In one embodiment, the method for treating PH includes treating pulmonary arterial hypertension (PAH) in a patient in need thereof. In one embodiment, the method for treating PAH includes administering to a patient in need thereof a prostacyclin compound of Formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof, or a composition comprising a prostacyclin compound of Formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof. In other embodiments, administration is via subcutaneous, oral, nasal, intravenous, or pulmonary routes of administration. For pulmonary administration, the compound of Formula (I), (II), or (III) or a composition comprising a prostacyclin compound of Formula (I), (II), or (III) is administered to the patient via a nebulizer, dry powder inhaler, or metered dose inhaler.

[0028] Another aspect of the present invention provides a method for treating portopulmonary hypertension (PPH) in a patient in need thereof. In one embodiment, the method for treating PPH comprises administering to a patient in need thereof a prostacyclin compound of Formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof, or a composition comprising a prostacyclin compound of Formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof. In other embodiments, administration is by subcutaneous, oral, nasal, intravenous, or pulmonary route. For pulmonary administration, the compound of Formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof, or a composition comprising a prostacyclin compound of Formula (I), (II), or (III) is administered to the patient via a nebulizer, dry powder inhaler, or metered-dose inhaler.

[0029] In one embodiment of the present invention, a method for treating PH, PAH, or PPH in a patient in need thereof is provided, comprising administering a prostacyclin compound of Formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof to the patient's lungs via a metered-dose inhaler containing a propellant. In other embodiments, the propellant is a fluorocarbon. In one embodiment, the compound of Formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof is administered to the lungs of a patient in need of PH, PAH, or PPH treatment via a metered-dose inhaler, and administration is once, twice, or three times daily. In embodiments in which the compound of Formula (I), (II), or (III) or a composition comprising a compound of Formula (I), (II), or (III) is administered orally, nasally, subcutaneously, intravenously, or to the lungs (e.g., via nebulization, dry powder inhaler, or metered-dose inhaler), administration to the patient is once or twice daily. In one embodiment, a compound of Formula (I), (II) or (III) or a composition comprising a compound of Formula (I), (II) or (III) is administered once daily to a patient in need of treatment by subcutaneous, intravenous, oral, nasal, or pulmonary administration by aerosolization using a nebulizer, dry powder inhaler, or metered dose inhaler.

[0030] In one embodiment, a patient treated for PH, PAH, or PPH with the compounds, compositions, and methods described herein experiences a reduced number of side effects or a reduced severity of side effects compared to the number of side effects or the severity of the side effects experienced by the patient when administered treprostinil. In one embodiment, the side effect is the patient's cough response, and the frequency and / or severity is reduced compared to the frequency and / or severity of the cough response experienced by the patient when administered treprostinil.

[0031] In another embodiment, the prostacyclin compound administered to a patient in need thereof by the pulmonary route by the methods of treating PH, PAH, or PPH described herein has a pulmonary elimination half-life (t ) that is shorter than that of treprostinil when the treprostinil is administered to a patient by the pulmonary route (e.g., by nebulization, dry powder inhaler, or metered dose inhaler). 1 / 2 ) compared to the longer pulmonary elimination half-life (t 1 / 2 ) is provided.

[0032] In another embodiment, the prostacyclin compound administered to a patient in need thereof by the methods of treating PH, PAH, or PPH described herein has a systemic elimination half-life (t 1 / 2 ) compared to the longer systemic half-life (t 1 / 2 In other embodiments, administration of the prostacyclin compound and treprostinil comprises subcutaneous or intravenous administration.

[0033] In another embodiment, the prostacyclin compound administered to a patient in need of PH, PAH, or PPH treatment has a respective lung or plasma C of treprostinil when administered to the patient. max Higher mean pulmonary C for treprostinil compared with max and / or lower plasma C max is provided to patients.

[0034] In another embodiment, the prostacyclin compound administered to a patient in need of PH (e.g., PAH) or PPH treatment is a prostacyclin compound that is more potent than the mean lung or plasma area under the curve (AUC) of treprostinil when administered to the patient. 0~t ) compared with a larger mean lung or plasma area under the curve (AUC 0~t In yet another embodiment, the prostacyclin compound administered to a patient in need thereof has a time to peak lung or plasma concentration (t) of treprostinil when treprostinil is administered to the patient. max ) compared to prostacyclin compounds and / or its metabolite treprostinil, resulting in a longer time to peak lung or plasma concentration (t max ) is provided. The present invention provides, for example, the following items. (Item 1) Formula (I): [ka] wherein R1 is NH, O, or S; R2 is a linear C5-C 18 Alkyl, branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl, branched C3-C 18 Alkenyl, aryl, aryl-C1-C 18 alkyl; amino acid or peptide; R3 is H, OH, optionally substituted linear or branched C1-C 15 Alkoxy, O-optionally substituted straight or branched C2-C 15 Alkenyl, O(C=O)-optionally substituted straight or branched C1-C 15 Alkyl or O(C=O)-Optionally substituted straight or branched C-C 15 is alkenyl; R4 is an optionally substituted straight or branched C1-C 15 Alkyl or optionally substituted straight or branched C-C 15 is alkenyl; n is an integer from 0 to 5. Prostacyclin compounds. (Item 2) Formula (II): [ka] wherein R1 is NH, O, or S; R2 is a linear C5-C 18 Alkyl, branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl, branched C3-C 18 Alkenyl, aryl, aryl-C1-C 18 alkyl; amino acid or peptide; n is an integer from 0 to 5. Prostacyclin compounds. (Item 3) R2 is a linear C5-C 18 Alkyl or branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl, branched C3-C 18 3. The prostacyclin compound according to item 1 or 2, wherein n is 0 or 1 and n is alkenyl. (Item 4) 3. The prostacyclin compound according to item 1 or 2, wherein R2 is an amino acid or a peptide containing 2 to 10 amino acids. (Item 5) 5. The prostacyclin compound according to any one of items 1 to 4, wherein R1 is N. (Item 6) 5. The prostacyclin compound according to any one of items 1 to 4, wherein R1 is O. (Item 7) 5. The prostacyclin compound according to any one of items 1 to 4, wherein R1 is S. (Item 8) 8. The prostacyclin compound according to any one of items 1 to 7, wherein n is 0. (Item 9) 8. The prostacyclin compound according to any one of items 1 to 7, wherein n is 1. (Item 10) R2 is linear C5~C 18 The prostacyclin compound according to any one of items 1 to 3 and 5 to 9, wherein the prostacyclin compound is alkyl. (Item 11) R2 is linear C5~C 18 The prostacyclin compound according to any one of items 1 to 3 and 5 to 9, which is alkenyl. (Item 12) R2 is branched C5~C 18 The prostacyclin compound according to any one of items 1 to 3 and 5 to 9, wherein the prostacyclin compound is alkyl. (Item 13) R2 is branched C5~C 18 The prostacyclin compound according to any one of items 1 to 3 and 5 to 9, which is alkenyl. (Item 14) R2 is aryl or aryl-C1-C 18 The prostacyclin compound according to any one of items 1 to 3 and 5 to 9, wherein the prostacyclin compound is alkyl. (Item 15) 15. The prostacyclin compound according to item 14, wherein R2 is aryl. (Item 16) The aryl is C5 to C 10 16. The prostacyclin compound according to item 15, which is aryl. (Item 17) 17. The prostacyclin compound of item 16, wherein said aryl is phenyl, naphthyl, thienyl, indolyl, or anthracenyl. (Item 18) 17. The prostacyclin compound according to item 15 or 16, wherein the aryl group comprises a fused ring structure. (Item 19) R2 is aryl-C1-C 1815. The prostacyclin compound according to item 14, wherein the prostacyclin compound is alkyl. (Item 20) R2 is aryl-C5~C 18 (Item 21) The prostacyclin compound according to Item 19, The aryl-C1 to C 18 Alkyl is C5-C 10 21. The prostacyclin compound according to item 19 or 20, which comprises an aryl. (Item 22) The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is pentyl. (Item 23) The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is hexyl. (Item 24) The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is heptyl. (Item 25) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is octyl. (Item 26) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is nonyl. (Item 27) The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is decyl. (Item 28) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is undecyl. (Item 29) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is dodecyl. (Item 30) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is tridecyl. (Item 31) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is tetradecyl. (Item 32) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is pentadecyl. (Item 33) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is hexadecyl. (Item 34) The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is heptadecyl. (Item 35) 11. The prostacyclin compound according to any one of items 1 to 3 and 5 to 10, wherein R2 is octadecyl. (Item 36) R2 is a linear C5 alkenyl, a linear C6 alkenyl, a linear C8 alkenyl, a linear C 10 Alkenyl, Linear C 12 Alkenyl, Linear C 14 Alkenyl, Linear C 16 Alkenyl or linear C 18 12. The prostacyclin compound according to any one of items 1 to 3, 5 to 9 and 11, which is alkenyl. (Item 37) 37. The prostacyclin compound according to any one of items 1 and 3-36, wherein R3 is OH. (Item 38) 37. The prostacyclin compound according to any one of items 1 and 3-36, wherein R3 is H. (Item 39) 37. The prostacyclin compound according to any one of items 1 and 3-36, wherein R4 is O-alkyl. (Item 40) n is 1, R1 is O, and R2 is a linear C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 41) n is 1, R1 is S, and R2 is a linear C5-C 183. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 42) n is 1, R1 is N, and R2 is a linear C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 43) n is 0, R1 is N, and R2 is a linear C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 44) R2 is a linear C5 alkyl, a linear C6 alkyl, a linear C8 alkyl, a linear C 10 Alkyl, linear C 12 Alkyl, linear C 14 Alkyl, linear C 16 Alkyl or linear C 18 44. The prostacyclin compound according to any one of items 40 to 43, wherein the prostacyclin compound is alkyl. (Item 45) n is 1, R1 is O, and R2 is a linear C 10 ~C 18 alkyl, R3 is OH, and R4 is hydroxyl-substituted C1-C 15 2. The prostacyclin compound according to item 1, wherein the prostacyclin compound is alkyl. (Item 46) n is 1, R1 is S, and R2 is a linear C5-C 18 alkyl, R3 is OH, and R4 is hydroxyl-substituted C1-C 15 2. The prostacyclin compound according to item 1, wherein the prostacyclin compound is alkyl. (Item 47) n is 1, R1 is N, and R2 is a linear C5-C 18 alkyl, R3 is OH, and R4 is hydroxyl-substituted C1-C 15 2. The prostacyclin compound according to item 1, wherein the prostacyclin compound is alkyl. (Item 48) n is 0, R1 is N, and R2 is a linear C5-C 18 alkyl, R3 is OH, and R4 is hydroxyl-substituted C1-C15 2. The prostacyclin compound according to item 1, wherein the prostacyclin compound is alkyl. (Item 49) R4 is hydroxyl-substituted C5-C 10 49. The prostacyclin compound according to any one of items 45 to 48, wherein R is alkyl and said hydroxyl is at the C2 position of the R group. (Item 50) n is 1, R1 is O, and R2 is a linear C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 51) n is 1, R1 is S, and R2 is a linear C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 52) n is 1, R1 is N, and R2 is a linear C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 53) n is 0, R1 is N, and R2 is a linear C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 54) R2 is a linear C5 alkyl, a linear C6 alkyl, a linear C8 alkyl, a linear C 10 Alkyl, linear C 12 Alkyl, linear C 14 Alkyl, linear C 16 Alkyl or linear C 18 54. The prostacyclin compound according to any one of items 50 to 53, wherein the prostacyclin compound is alkyl. (Item 55) n is 1, R1 is O, and R2 is a linear or branched C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 56) n is 1, R1 is S, and R2 is a linear or branched C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 57) n is 1, R1 is N, and R2 is a linear or branched C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 58) n is 0, R1 is N, and R2 is a linear or branched C5-C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 59) R2 is a linear or branched C5 alkyl, a linear C6 alkyl, a linear C8 alkyl, a linear or branched C 10 Alkyl, linear or branched C 12 Alkyl, linear or branched C 14 Alkyl, linear or branched C 16 Alkyl or linear or branched C 18 3. The prostacyclin compound according to item 1 or 2, wherein the prostacyclin compound is alkyl. (Item 60) n is 1, R1 is O, and R2 is a linear or branched C5-C 18 3. The prostacyclin compound according to item 1 or 2, which is alkenyl. (Item 61) n is 1, R1 is S, and R2 is a linear or branched C5-C 18 3. The prostacyclin compound according to item 1 or 2, which is alkenyl. (Item 62) n is 1, R1 is N, and R2 is a linear or branched C5-C 18 3. The prostacyclin compound according to item 1 or 2, which is alkenyl. (Item 63) n is 0, R1 is N, and R2 is a linear or branched C5-C 18 3. The prostacyclin compound according to item 1 or 2, which is alkenyl. (Item 64) R2 is a linear or branched C5 alkenyl, a linear C6 alkenyl, a linear C8 alkenyl, a linear or branched C 10Alkenyl, linear or branched C 12 Alkenyl, linear or branched C 14 Alkenyl, linear or branched C 16 Alkenyl or linear or branched C 18 The prostacyclin compound according to any one of items 1 to 3 and 5 to 9, which is alkenyl. (Item 65) 3. The prostacyclin compound according to item 1 or 2, wherein one or more hydrogen atoms are replaced with deuterium atoms. (Item 66) 3. The prostacyclin compound according to item 1 or 2, wherein R1 is O and R2 is symmetrically branched alkyl or asymmetrically branched alkyl. (Item 67) 67. The prostacyclin compound according to item 66, which is 5-nonanyl-treprostinil (5C9-TR). (Item 68) 3. The prostacyclin compound according to item 1 or 2, wherein the R2 moiety is a mixture of R and S isomers. (Item 69) 3. The prostacyclin compound according to item 1 or 2, wherein the R2 moiety is an R isomer or an S isomer. (Item 70) R2 [ka] 3. The prostacyclin compound according to item 1 or 2, wherein (Item 71) Formula (II) [ka] (wherein n is 1, R1 is NH, O or S, and R2 is [ka] [ka] is) Prostacyclin compounds. (Item 72) R1 is O and the compound has the following structure: [ka] 164. The prostacyclin compound according to item 163, having the formula: (Item 73) 3. The prostacyclin compound according to item 2, wherein n is 1, R1 is NH, O or S, and R2 is selected from the group consisting of 5-nonanyl, 4-heptyl, 4-octyl, 3-octyl, 2-dimethyl-1-propyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl and 3-pentyl. (Item 74) 74. The prostacyclin compound according to item 73, wherein R1 is O and R2 is 5-nonanyl. (Item 75) 74. The prostacyclin compound according to item 73, wherein R1 is O and R2 is 4-heptyl. (Item 76) 74. The prostacyclin compound according to item 73, wherein R1 is O and R2 is 4-octyl. (Item 77) 74. The prostacyclin compound according to item 73, wherein R1 is O and R2 is 3-octyl. (Item 78) 74. The prostacyclin compound according to item 73, wherein R1 is O and R2 is 2-dimethyl-1-propyl. (Item 79) 74. The prostacyclin compound according to item 73, wherein R1 is O and R2 is 3,3-dimethyl-1-butyl. (Item 80) 74. The prostacyclin compound according to item 73, wherein R1 is O and R2 is 2-ethyl-1-butyl. (Item 81) 74. The prostacyclin compound according to item 73, wherein R1 is O and R2 is 3-pentyl. (Item 82) Formula (Ia″), (Ib″), (Ic″) or (Id″) [ka] wherein R3 is OH; R2 is H, [ka] [ka] [ka] and linear C6-C 16 alkyl) Prostacyclin compounds by. (Item 83) Formula (III) [ka] (In the formula, R1 is NH, O or S; R2 is a linear C5-C 18 Alkyl, branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl, branched C3-C 18 Alkenyl, aryl, aryl-C1-C 18 alkyl; amino acid or peptide; R5 and R6 are independently H, optionally substituted straight or branched C1-C 15 Alkyl, optionally substituted straight or branched C-C 15Alkenyl, (C=O)-optionally substituted straight or branched C1-C 15 Alkyl or (C=O)-optionally substituted straight or branched C-C 15 alkenyl) A prostacyclin compound of formula (III) with the proviso that it is not treprostinil. (Item 84) R1 is NH and R2 is a linear C5-C 18 84. The prostacyclin compound according to item 83, wherein R is alkyl, R5 is H, and R6 is H. (Item 85) R1 is O and R2 is a linear C5-C 18 84. The prostacyclin compound according to item 83, wherein R is alkyl, R5 is H, and R6 is H. (Item 86) R2 is linear C6~C 12 85. The prostacyclin compound according to item 84, wherein the prostacyclin compound is alkyl. (Item 87) R2 is linear C 10 ~C 18 86. The prostacyclin compound according to item 85, wherein the prostacyclin compound is alkyl. (Item 88) R2 is linear C6~C 10 87. The prostacyclin compound according to item 86, which is alkyl. (Item 89) R1 is S and R2 is a linear C6-C 18 84. The prostacyclin compound according to item 83, wherein R is alkyl, R5 is H, and R6 is H. (Item 90) 90. A pharmaceutically acceptable salt of the prostacyclin compound according to any one of items 1 to 89. (Item 91) 91. A composition comprising the prostacyclin compound or a pharmaceutically acceptable salt thereof according to any one of items 1 to 90, and an amphiphilic substance. (Item 92) 92. The composition of claim 91, wherein the amphiphile is a PEGylated lipid, surfactant, fatty acid, or block copolymer. (Item 93) 93. The composition of claim 92, wherein the amphiphile is a surfactant. (Item 94) 94. The composition of claim 93, wherein the surfactant is nonionic. (Item 95) 92. The composition of claim 91, wherein the amphiphile is a fatty acid. (Item 96) 93. The composition of claim 92, wherein the block copolymer is PEO-PPO-PEO or PEO-poly(isoprene)-PEO. (Item 97) 93. The composition of claim 92, wherein the amphiphile is a PEGylated lipid. (Item 98) 98. The composition of claim 97, wherein the pegylated lipid comprises PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG5000. (Item 99) 99. The composition of claim 98, wherein the pegylated lipid comprises PEG1000. (Item 100) 99. The composition of claim 98, wherein the pegylated lipid comprises PEG2000. (Item 101) The composition according to any one of Items 97 to 100, wherein the lipid is cholesterol. (Item 102) 101. The composition according to any one of items 97 to 100, wherein the lipid is a phospholipid. (Item 103) 101. The composition of any one of items 97 to 100, wherein the lipid is distearoylphosphatidylethanolamine (DSPE). (Item 104) 101. The composition of any one of items 97 to 100, wherein the lipid is dimyristoylphosphoethanolamine (DMPE). (Item 105) 101. The composition of any one of items 97 to 100, wherein the lipid is distearoylglycerol (DSG). (Item 106) 98. The composition of claim 97, wherein the PEGylated lipid is cholesterol-PEG2000, DSPE-PEG1000, or DSG-PEG2000. (Item 107) 107. The composition of any one of items 91 to 106, further comprising a hydrophobic additive. (Item 108) Item 108. The composition according to item 107, wherein the hydrophobic additive is a hydrocarbon, a terpene or a hydrophobic lipid, an alkyl ester, a cholesteryl ester, a mono-, di-, or tri-alkyl-glyceride. (Item 109) Item 109. The composition according to item 107 or 108, wherein the hydrophobic additive is a hydrocarbon. (Item 110) Item 109. The composition according to item 107 or 108, wherein the hydrophobic additive is a terpene. (Item 111) Item 109. The composition according to item 107 or 108, wherein the hydrophobic additive is a hydrophobic lipid. (Item 112) Item 111. The composition according to item 110, wherein the terpene is squalane. (Item 113) 113. The composition according to any one of items 91 to 112, comprising a suspension of nanoparticles in an aqueous medium. (Item 114) 113. The composition of any one of items 91 to 112, formulated as a dry powder. (Item 115) 90. A dry powder composition comprising the prostacyclin compound or a pharmaceutically acceptable salt thereof according to any one of items 1 to 89. (Item 116) 90. A composition comprising the prostacyclin compound or a pharmaceutically acceptable salt thereof according to any one of items 1 to 89 and a propellant. (Item 117) 91. The composition of claim 90, wherein the propellant is a hydrofluoroalkane. (Item 118) A method for treating pulmonary hypertension (PH) in a patient in need thereof, comprising administering to the patient an effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof described in any one of items 1 to 89, or a prostacyclin composition described in any one of items 91 to 117. (Item 119) 119. The method of item 118, wherein the patient is a WHO group I PH patient. (Item 120) 119. The method of item 118, wherein the patient is a WHO group II PH patient. (Item 121) 119. The method of item 118, wherein the patient is a WHO group III PH patient. (Item 122) 119. The method of item 118, wherein the patient is a WHO group IV PH patient. (Item 123) 119. The method of item 118, wherein the patient is a WHO group V PH patient. (Item 124) 119. The method of claim 118, wherein the effective amount of a prostacyclin compound is administered to the patient's lungs. (Item 125) 124. The method according to any one of items 118 to 123, wherein the effective amount of the prostacyclin compound or prostacyclin composition is administered to the patient orally, intranasally, intravenously or subcutaneously. (Item 126) 125. The method of any one of items 118 to 124, wherein the effective amount of the prostacyclin compound is administered to the patient's lungs by a metered dose inhaler. (Item 127) 125. The method of claim 124, wherein the effective amount of the prostacyclin compound is administered to the patient's lungs by a dry powder inhaler. (Item 128) 125. The method of any one of items 118 to 124, wherein the effective amount of the prostacyclin compound or prostacyclin composition is administered to the patient's lungs by a nebulizer. (Item 129) 129. The method of any one of items 118-124 and 126-128, wherein administration of the effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof results in a decrease in the number of side effects experienced by the patient or a decrease in the severity of side effects experienced by the patient compared to the number of side effects or the severity of side effects experienced by the patient when administered treprostinil or iloprost. (Item 130) Item 126. The method of item 125, wherein administration of the effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof results in a decrease in the number of side effects experienced by the patient or a decrease in the severity of side effects experienced by the patient compared to the number of side effects or the severity of side effects experienced by the patient when administered treprostinil or iloprost. (Item 131) 130. The method of claim 129, wherein the reduction in the severity of a side effect is a reduction in the frequency or severity of a cough response. (Item 132) 132. The method according to any one of items 118 to 131, wherein the effective amount of the prostacyclin compound is administered once a day. (Item 133) 132. The method according to any one of items 118 to 131, wherein the effective amount of the prostacyclin compound is administered twice a day. (Item 134) 132. The method of any one of items 118 to 131, wherein the effective amount of the prostacyclin compound is administered three or more times a day. (Item 135) Item 129. The method of item 128, wherein the nebulizer is a vibrating mesh nebulizer. (Item 136) 90. A method for treating pulmonary arterial hypertension (PAH) in a patient in need thereof, comprising administering to the patient an effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof according to any one of items 1 to 89. (Item 137) 118. A method for treating pulmonary arterial hypertension (PAH) in a patient in need thereof, comprising administering to the patient an effective amount of a prostacyclin composition described in any one of items 91 to 117. (Item 138) 138. The method according to item 136 or 137, wherein the patient is a PAH patient of class I as classified by the New York Heart Association (NYHA). (Item 139) 138. The method according to item 136 or 137, wherein the patient is a PAH patient of class II as classified by the New York Heart Association (NYHA). (Item 140) 138. The method according to item 136 or 137, wherein the patient is a PAH patient of class III as classified by the New York Heart Association (NYHA). (Item 141) 138. The method according to item 136 or 137, wherein the patient is a PAH patient of class IV as classified by the New York Heart Association (NYHA). (Item 142) 138. The method of claim 136 or 137, wherein the effective amount of the prostacyclin compound is administered to the patient's lungs. (Item 143) 143. The method according to any one of items 136 to 142, wherein the effective amount of the prostacyclin compound or prostacyclin composition is administered to the patient orally, intranasally, intravenously or subcutaneously. (Item 144) Item 137. The method of item 136, wherein the effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof is administered to the patient's lungs by a metered dose inhaler. (Item 145) Item 137. The method of item 136, wherein the effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof is administered to the patient's lungs by a dry powder inhaler. (Item 146) 138. The method of claim 137, wherein the effective amount of the prostacyclin composition is administered to the patient's lungs by a nebulizer. (Item 147) 143. The method according to any one of items 136 to 142, wherein the effective amount of a prostacyclin compound, a pharmaceutically acceptable salt thereof, or a prostacyclin composition is administered to the lungs of the patient by a nebulizer. (Item 148) 148. The method of any one of items 136 to 147, wherein administration of the effective amount of a prostacyclin compound, a pharmaceutically acceptable salt thereof, or a prostacyclin composition results in a decrease in the number of side effects experienced by the patient or a decrease in the severity of side effects experienced by the patient compared to the number of side effects or the severity of side effects experienced by the patient when administered treprostinil or iloprost. (Item 149) 149. The method of claim 148, wherein the reduction in severity of a side effect is a reduction in the severity of a cough response. (Item 150) 149. The method according to any one of items 136 to 149, wherein the effective amount of the prostacyclin compound is administered once a day. (Item 151) 149. The method according to any one of items 136 to 149, wherein the effective amount of the prostacyclin compound is administered twice a day. (Item 152) 149. The method of any one of items 136 to 149, wherein the effective amount of the prostacyclin compound is administered three or more times daily. (Item 153) Item 148. The method of item 147, wherein the nebulizer is a vibrating mesh nebulizer. (Item 154) A method for treating chronic thromboembolic pulmonary hypertension in a patient in need thereof, comprising administering to the patient an effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof according to any one of items 1 to 89, or a prostacyclin composition according to any one of items 91 to 117. (Item 155) 90. A method of treating portopulmonary hypertension (PPH) in a patient in need thereof, comprising administering to the patient an effective amount of a prostacyclin compound according to any one of items 1 to 89, or a pharmaceutically acceptable salt thereof. (Item 156) 118. A method of treating portopulmonary hypertension (PPH) in a patient in need thereof, comprising administering to the patient an effective amount of a prostacyclin composition according to any one of paragraphs 91 to 117. (Item 157) 157. The method according to any one of items 154 to 156, wherein the effective amount of a prostacyclin compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to the lungs of the patient. (Item 158) 157. The method of any one of items 154 to 156, wherein the effective amount of a prostacyclin compound, a pharmaceutically acceptable salt thereof, or a prostacyclin composition is administered to the patient orally, intranasally, intravenously, or subcutaneously. (Item 159) 156. The method of claim 154 or 155, wherein the effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof is administered to the patient's lungs by a metered dose inhaler. (Item 160) 156. The method of claim 154 or 155, wherein the effective amount of a prostacyclin compound or a pharmaceutically acceptable salt thereof is administered to the patient's lungs by a dry powder inhaler. (Item 161) 157. The method of any one of items 154 to 156, wherein the effective amount of the prostacyclin composition is administered to the patient's lungs by a nebulizer. (Item 162) 157. The method according to any one of items 154 to 156, wherein the effective amount of a prostacyclin compound, a pharmaceutically acceptable salt thereof, or a prostacyclin composition is administered to the lungs of the patient by a nebulizer. (Item 163) 163. The method of any one of items 154 to 162, wherein administration of the effective amount of a prostacyclin compound, a pharmaceutically acceptable salt thereof, or a prostacyclin composition results in a decrease in the number of side effects experienced by the patient or a decrease in the severity of side effects experienced by the patient compared to the number of side effects or the severity of side effects experienced by the patient when administered treprostinil or iloprost. (Item 164) Item 164. The method of item 163, wherein the reduction in severity of side effects is a reduction in severity of cough response. (Item 165) 165. The method according to any one of items 154 to 164, wherein the effective amount of the prostacyclin compound or a pharmaceutically acceptable salt thereof is administered once a day. (Item 166) 165. The method according to any one of items 154 to 164, wherein the effective amount of the prostacyclin compound or a pharmaceutically acceptable salt thereof is administered twice a day. (Item 167) 165. The method according to any one of items 154 to 164, wherein the effective amount of the prostacyclin compound or a pharmaceutically acceptable salt thereof is administered three times a day. (Item 168) Item 163. The method of item 162, wherein the nebulizer is a vibrating mesh nebulizer. (Item 169) administration of the prostacyclin compound or a pharmaceutically acceptable salt thereof to the patient in need thereof, 0~t or plasma area under the curve AUC 0~t a greater mean area under the lung curve (AUC) of the prostacyclin compound and / or treprostinil compared to 0~t) or plasma area under the curve (AUC 0~t 169. The method according to any one of items 118 to 168, wherein (Item 170) administration of the prostacyclin compound or a pharmaceutically acceptable salt thereof to a patient in need thereof reduces the time to peak lung concentration or peak plasma concentration of treprostinil when treprostinil is administered to the patient; max a greater lung peak concentration or time to plasma peak concentration (t max 169. The method according to any one of items 118 to 168, wherein (Item 171) administration of the prostacyclin compound or a pharmaceutically acceptable salt thereof to a patient in need thereof is 1 / 2 a longer pulmonary elimination half-life (t 1 / 2 169. The method according to any one of items 118 to 168, wherein (Item 172) 118. The composition according to any one of items 91 to 117 in aerosolized form. (Item 173) Item 173. The composition according to item 172, wherein the MMAD of the aerosol particles is from about 1 μm to about 5 μm, or from about 1 μm to about 4 μm, or from about 1 μm to about 3 μm, or from about 1 μm to about 2 μm, as measured by an Anderson Cascade Impactor (ACI) or a Next Generation Impactor (NGI). (Item 174) 173. The composition of claim 172, wherein the MMAD of the aerosol particles is about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less, as measured by cascade impaction, e.g., ACI or NGI. (Item 175) 175. The composition of any one of items 172 to 174, wherein the FPF of the aerosol particles is greater than or equal to about 50% as measured by ACI or NGI, greater than or equal to about 60% as measured by ACI or NGI, or greater than or equal to about 70% as measured by ACI or NGI. (Item 176) 175. The composition of any one of items 172 to 174, wherein the FPF of the aerosol particles is about 50% to about 80%, about 50% to about 70%, or about 50% to about 60%, as measured by NGI or ACI. (Item 177) 163. The method of any one of items 128, 147 and 162, wherein the MMAD of the nebulized composition is from about 1 μm to about 5 μm, or from about 1 μm to about 4 μm, or from about 1 μm to about 3 μm or from about 1 μm to about 2 μm, as measured by ACI or NGI. (Item 178) 163. The method of any one of items 128, 147, and 162, wherein the FPF of the nebulized composition is greater than or equal to about 50% as measured by ACI or NGI, greater than or equal to about 60% as measured by ACI or NGI, or greater than or equal to about 70% as measured by ACI or NGI. (Item 179) 161. The method of any one of items 127, 145, 146 and 160, wherein the MMAD of the administered dry powder composition is from about 1 μm to about 10 μm, or from about 1 μm to about 9 μm, or from about 1 μm to about 8 μm, or from about 1 μm to about 7 μm, or from about 1 μm to about 6 μm, or from about 1 μm to about 5 μm, or from about 1 μm to about 4 μm, or from about 1 μm to about 3 μm or from about 1 μm to about 2 μm in diameter, as measured by NGI or ACI. (Item 180) 162. The method of any one of items 127, 145, 146 and 160-161, wherein the FPF of the administered dry powder is about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, or about 40% to about 50%, as measured by ACI or NGI. [Brief explanation of the drawings]

[0035] [Figure 1A] Figure 1A is a graph showing the rate of spontaneous hydrolysis of treprostinil compounds versus time (C3: propyl ester, C4: butyl ester, C5: pentyl ester, C6: hexyl ester, C8: octyl ester, and C10: decyl ester).

[0036] [Figure 1B] FIG. 1B is a graph showing the rate of esterase-mediated hydrolysis of the alkyl chain at various time points (15 min, 30 min, 60 min) for a treprostinil compound dissolved in aqueous buffer and a treprostinil composition containing a PEGylated lipid.

[0037] [Figure 2] Figure 2 is a graph of mean particle size for various treprostinil alkyl esters in formulations containing PEGylated lipids as a function of alkyl ester chain length. The alkyl chain is present on the carboxylic acid moiety of treprostinil. PD is polydispersity index.

[0038] [Figure 3A] Figures 3A, 3B, and 3C are graphs of the relative cAMP response vs. time of CHO-K1-P4 cells (2.5 x 10 cells / well) in response to 10 μM (Figure 3A), 1 μM (Figure 3B), or 0.1 μM (Figure 3C) treprostinil and treprostinil alkyl ester compositions (C6: hexyl ester, C8: octyl ester, C10: decyl ester). [Figure 3B] Figures 3A, 3B, and 3C are graphs of the relative cAMP response vs. time of CHO-K1-P4 cells (2.5 x 10 cells / well) in response to 10 μM (Figure 3A), 1 μM (Figure 3B), or 0.1 μM (Figure 3C) treprostinil and treprostinil alkyl ester compositions (C6: hexyl ester, C8: octyl ester, C10: decyl ester). [Figure 3C]Figures 3A, 3B, and 3C are graphs of the relative cAMP response vs. time of CHO-K1-P4 cells (2.5 x 10 cells / well) in response to 10 μM (Figure 3A), 1 μM (Figure 3B), or 0.1 μM (Figure 3C) treprostinil and treprostinil alkyl ester compositions (C6: hexyl ester, C8: octyl ester, C10: decyl ester).

[0039] [Figure 4] 4 is a graph of the relative cAMP response vs. time of CHO-K1-P4 cells (2.5×10 cells / well) in response to 5 μM treprostinil and treprostinil alkyl ester compositions (C6: hexyl ester, C8: octyl ester, C10: decyl ester, C12: dodecyl ester).

[0040] [Figure 5] FIG. 5 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) versus time in response to challenge with treprostinil at 5 μM and various treprostinil alkyl ester compounds.

[0041] [Figure 6] 6 is a graph of relative cAMP activity vs. time in CHO-K1-P4 cells (2.5×10 cells / well) in response to challenge with treprostinil and nebulized and non-nebulized treprostinil alkyl ester compositions, as measured by a modified GloSensor assay. "(N)" indicates the nebulized composition.

[0042] [Figure 7] FIG. 7 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) vs. free treprostinil at various dosages and time points.

[0043] [Figure 8]FIG. 8 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) versus T554 (C2-TR) treprostinil alkyl ester composition challenge at various dosages and time points.

[0044] [Figure 9] FIG. 9 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) versus T568 (C12-TR) treprostinil alkyl ester composition challenge at various dosages and time points.

[0045] [Figure 10] FIG. 10 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) versus T631 (C14-TR) treprostinil alkyl ester composition challenge at various dosages and time points.

[0046] [Figure 11] FIG. 11 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) versus T623 (C16-TR) treprostinil alkyl ester composition challenge at various dosages and time points.

[0047] [Figure 12] FIG. 12 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) versus treprostinil ethyl ester (C2) compound challenge at various dosages and time points.

[0048] [Figure 13] FIG. 13 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) versus treprostinil ethyl ester (C12) compound challenge at various dosages and time points.

[0049] [Figure 14]FIG. 14 is a graph of the relative cAMP response of CHO-K1-P4 cells (2.5×10 4 cells / well) vs. treprostinil ethyl ester (C2) composition at various dosages and time points.

[0050] [Figure 15A] Figure 15A is a graph of pulmonary artery pressure (expressed as a percent of the starting hypoxic value) versus time in response to animal challenge with phosphate-buffered saline (PBS), treprostinil, and prostacyclin compositions (T554 (C2) and T-568 (C12)). The target dose for treprostinil and prostacyclin alkyl ester was 76.8 nmol / kg; the achieved deposited dose could be 5x lower than these targets.

[0051] [Figure 15B] Figure 15B is a dot plot showing the effect of treprostinil and C2, C8, C10, and C12 treprostinil alkyl ester compositions on PAP (expressed as a percent of starting hypoxic values) in an acutely hypoxic rat model of PAH in vivo. Doses are target values; actual lung doses achieved may be approximately 5x lower.

[0052] [Figure 16] FIG. 16 is a graph of systemic arterial pressure (expressed as a percent of the starting hypoxic value) versus time in response to animal challenge with PBS, treprostinil, and treprostinil alkyl ester compositions (T554 (C2-TR) and T-568 (C12-TR)) in an in vivo acute hypoxic rat model of PAH. The vertical dotted lines represent the change in time increments on the x-axis. The target dose for treprostinil and prostacyclin alkyl ester was 76.8 nmol / kg; achieved adherence doses could be 5× lower than these target values.

[0053] [Figure 17]Figure 17 is a graph of in vivo heart rate (expressed as a percent of the starting hypoxic value) versus time in response to animal challenge with PBS, treprostinil, and treprostinil alkyl ester compositions (T554 (C2) and T-568 (C12)) in an in vivo acute hypoxic rat model of PAH. The vertical dotted lines represent the change in time increments on the x-axis. The target dose for treprostinil and prostacyclin alkyl ester was 76.8 nmol / kg; achieved adherence doses could be 5x lower than these target values.

[0054] [Figure 18] The top panel of Figure 18 is a graph of the relative cAMP response of CHO-K1 cells as a function of 5C9-TR (a 5-nonanyl-treprostinil alkyl ester composition) challenge at various doses and time points. The bottom panel of Figure 18 shows the EC50 of 5C9-TR over time, calculated from the cAMP response of CHO-K1 cells vs. 5C9-TR.

[0055] [Figure 19] The top panel of Figure 19 is a graph of the relative cAMP response of CHO-K1 cells vs. C14-TR (a C14 treprostinil alkyl ester composition) challenge at various doses and time points. The bottom panel of Figure 19 shows the EC50 of C14-TR over time, calculated from the cAMP response of CHO-K1 cells vs. C14-TR.

[0056] [Figure 20] The top panel of Figure 20 is a graph of the relative cAMP response of CHO-K1 cells vs. C16-TR (a C16 treprostinil alkyl ester composition) challenge at various doses and time points. The bottom panel of Figure 20 shows the EC50 of C16-TR over time, calculated from the cAMP response of CHO-K1 cells vs. C16-TR.

[0057] [Figure 21]Figure 21 is a graph of the relative cAMP response of CHO-K1 cells versus time in response to challenge with C12-TR, C14-TR, C16-TR, or 5-nonanyl-TR (5C9-TR) at 10 μM (top panel) or 5 μM (bottom panel).

[0058] [Figure 22] Figure 22 (top panel) is a graph of the relative cAMP response of CHO-K1 cells vs. T679 (C14-TR 45 mol%, squalane 45 mol%, chol-PEG2k 10%) treprostinil alkyl ester composition challenge at various dosages and time points. Figure 22 (bottom panel) shows the EC50 of T679 over time, calculated from the cAMP response of CHO-K1 cells vs. T679.

[0059] [Figure 23] Figure 23 is a graph of the relative cAMP response vs. time of CHO-K1 cells in response to challenge with treprostinil at 10 μM (top panel) or 5 μM (bottom panel), T631 (C14-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%), or T679 (C14-TR 45 mol%, squalane 45 mol%, chol-PEG2k 10 mol%).

[0060] [Figure 24] The top panel of Figure 24 is a graph of the relative cAMP response of CHO-K1 cells vs. T647 (C14-TR 90 mol%, chol-PEG2k 10 mol%) treprostinil alkyl ester composition challenge at various dosages and time points. The bottom panel of Figure 24 shows the EC50 of T647 over time, calculated from the cAMP response of CHO-K1 cells vs. T647-TR.

[0061] [Figure 25]Figure 25 is a graph of the relative cAMP response vs. time of CHO-K1 cells in response to challenge with treprostinil at 10 μM (top panel) or 5 μM (bottom panel), T631 (C14-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%), or T647 (C14-TR 90 mol%, chol-PEG2k 10 mol%).

[0062] [Figure 26] The top panel of Figure 26 is a graph of the relative cAMP response of CHO-K1 cells vs. T637 (C18-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%) treprostinil alkyl ester lipid nanoparticle composition challenge at various dosages and time points. The bottom panel of Figure 26 shows the EC50 of T637 over time, calculated from the cAMP response of CHO-K1 cells vs. T637-TR.

[0063] [Figure 27] Figure 27 shows the results of the 10 μM (upper panel) or 5 μM (lower panel) treprostinil, T555 (C8-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%), T556 (C10-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%), T568 (C12-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%), T631 (C14-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%), and T623 (C16-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%). 10 mol%) or T637 (C18-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%).

[0064] [Figure 28] FIG. 28 is a graph of the conversion rate (% of total) over time (hours) for linear (C8TR) versus branched (2-dimethyl-1-propanyl-TR, 3,3-dimethyl-1-butanyl-TR, 2-ethyl-1-butanyl-TR, 5-nonanyl-TR, or 3-pentanyl-TR) prostacyclin compounds.

[0065] [Figure 29] 29 is a graph showing the conversion of treprostinil compounds derivatized with various linear alkyl chains versus the conversion of treprostinil compounds derivatized with an octyl moiety (R2=C8), as measured after 1 hour of incubation with esterase.

[0066] [Figure 30] 30 is a graph showing the conversion of treprostinil compounds derivatized with various branched alkyl chains versus the conversion of treprostinil compounds derivatized with an octyl moiety (R2=C8). Conversions were measured after 1 hour of incubation with esterase.

[0067] [Figure 31] FIG. 31 is a schematic diagram of the Jaeger-NYU nose-only, directed-flow inhalation exposure system (CH Technologies, Westwood, NJ, www.onares.org) used for the 24-hour pharmacokinetic study.

[0068] [Figure 32] The left side of Figure 32 is a graph of treprostinil plasma levels (ng / mL) as a function of time for treprostinil and various inhaled treprostinil alkyl ester formulations. The right side of Figure 32 is a graph of treprostinil plasma levels (ng / mL) as a function of time for treprostinil and various inhaled treprostinil alkyl ester micelle formulations.

[0069] [Figure 33] Figure 33 is a graph of treprostinil and treprostinil alkyl ester concentrations in the lungs after administration of nebulized treprostinil solution or formulated treprostinil alkyl ester suspension. Lungs were collected 6 hours after administration. Treprostinil alkyl ester concentrations are presented as treprostinil equivalents on a molar basis.

[0070] [Figure 34] The top part of Figure 34 is a graph of treprostinil plasma levels (ng / mL) as a function of time in rats after nose-only inhalation of a nebulized treprostinil alkyl ester formulation. The bottom part of Figure 34 is a graph of treprostinil and treprostinil alkyl ester plasma levels (ng / mL) as a function of time in rats after nose-only inhalation of a nebulized treprostinil alkyl ester formulation.

[0071] [Figure 35] The top of Figure 35 is a graph of treprostinil plasma levels (ng / mL) as a function of time after nebulization (nose-only administration) of various concentrations of C16-TR formulations. The bottom of Figure 35 is a graph of treprostinil and C16-TR plasma levels (ng / mL) as a function of time after nebulization (nose-only administration) of various concentrations of C16-TR formulations.

[0072] [Figure 36] Figure 36 is a graph of the plasma concentration of treprostinil (ng / mL) in intubated dogs as a function of time after administration of treprostinil or a T623 lipid nanoparticle formulation (C16-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%).

[0073] [Figure 37]Figure 37 (left) is a graph of the conversion of treprostinil alkyl esters to treprostinil as a function of time for various treprostinil alkyl esters exposed to rat lung tissue homogenate. Figure 37 (right) is a graph of the conversion of C12-treprostinil to treprostinil as a function of time in rat, dog, and monkey lung tissue homogenates.

[0074] [Figure 38] Figure 38 is a graph of mean pulmonary artery pressure (mPAP) as a function of time in rats treated with PBS, treprostinil, T568 (C12-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%), or T623 (C16-TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, DOPC 10 mol%).

[0075] [Figure 39] Figure 39 (top) is a graph of mean systemic arterial pressure (mSAP) as a function of time in rats treated with PBS, treprostinil, T568, or T623. Figure 39 (bottom) is a graph of heart rate as a function of time in rats treated with PBS, treprostinil, T568, or T623.

[0076] [Figure 40] FIG. 40 is a graph of treprostinil plasma levels (ng / mL) as a function of time in rats following administration of free treprostinil, T568, or T623.

[0077] [Figure 41] FIG. 41 is a graph of treprostinil plasma levels (ng / mL) as a function of time in rats following administration of Composition T763i. DETAILED DESCRIPTION OF THE INVENTION

[0078] As used herein, the term "alkyl" refers to both straight-chain alkyls, where the alkyl chain length is designated by a numerical range, and branched alkyls, where branching points exist within the chain and the total number of carbon atoms in the chain is designated by a numerical range. In exemplary embodiments, "alkyl" refers to an alkyl chain, as defined above, containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbons (i.e., C6-C 16 refers to alkyl).

[0079] The term "alkenyl," as used herein, refers to a carbon chain containing one or more carbon-carbon double bonds.

[0080] As used herein, the term "aryl" refers to a cyclic hydrocarbon in which the ring is characterized by delocalized π-electrons (aromaticity) shared between the ring members, and the number of ring atoms is designated by a numerical range. In exemplary embodiments, "aryl" refers to a cyclic hydrocarbon as described above containing 6, 7, 8, 9, or 10 ring atoms (i.e., C6-C6). 10 Examples of aryl groups include, but are not limited to, benzene, naphthalene, tetralin, indene, and indane.

[0081] The term "alkoxy" as used herein refers to --O-(alkyl), where "alkyl" is defined above.

[0082] As used herein, the term "substituted" in reference to a moiety means that refers to another substituent attached to the moiety at any permissible position on the moiety. Unless otherwise specified, the moiety can be attached through a carbon, nitrogen, oxygen, sulfur, or any other permissible atom.

[0083] The term "amino acid" refers to both natural (genetically encoded) and unnatural (non-genetically encoded) amino acids and moieties thereof. Of the 20 natural amino acids, 19 have the general structure: [ka] where R is an amino acid side chain. The 20th amino acid, proline, is also within the scope of the present invention and has the following structure: [ka] Of the 20 naturally occurring amino acids, all but glycine are chiral, and both D- and L-amino acid isomers, as well as mixtures thereof, are acceptable for use in the prostacyclin compounds described herein. It is also noted that amino acid moieties are encompassed by the term "amino acid." For example, the amino acid moiety [ka] are encompassed by the term "amino acid."

[0084] Examples of unnatural amino acids that are acceptable for use in the present invention include β-alanine (β-Ala); 2,3-diaminopropionic acid (Dpr); nipecotic acid (Nip); pipecolic acid (Pip); ornithine (Om); citrulline (Cit); t-butylalanine (t-BuA); 2-tbutylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (PhG); cyclohexylalanine (ChA); norleucine (Nle); naphthylalanine (Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(3-F)); These include homothienylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH2)); N-methylvaline (MeVal); homocysteine ​​(hCys), homophenylalanine (hPhe); homoserine (hSer); hydroxyproline (Hyp); homoproline (hPro); and the corresponding D-enantiomers of each of the above. Other non-genetically encoded amino acid residues include 3-aminopropionic acid; 4-aminobutyric acid; isonipecotic acid (Inp); aza-pipecolic acid (azPip); aza-proline (azPro); α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); and N-methylglycine (MeGly).

[0085] A "peptide" is a polymer of amino acids (or portion thereof) linked by peptide bonds. Peptides for use in the present invention contain from about 2 to about 15 amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (or portion thereof).

[0086] As used herein, the term "salt" or "salts" encompasses pharmaceutically acceptable salts commonly used to form alkali metal salts of free acids and to form addition salts of free bases. The nature of the salt is not critical, provided it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Exemplary pharmaceutical salts are disclosed in Stahl, PH, Wermuth, CG (eds.), Handbook of Pharmaceutical Salts: Properties, Selection and Use; Verlag Helvetica Chimica Acta / Wiley-VCH: Zurich, 2002, the contents of which are incorporated herein by reference in their entirety. Specific, non-limiting examples of inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids include, but are not limited to, aliphatic, cyclic, aromatic, aryl aliphatic, and carboxylic and sulfonic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, stearic acid, salicylic acid, p-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, toluenesulfonic acid, 2-hydroxyethanesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, algenic, 3-hydroxybutyric acid, galactaric acid, or or heterocyclyls containing galacturonic acid. Suitable pharmaceutically acceptable salts of the free acid-containing compounds disclosed herein include, but are not limited to, metallic salts and organic salts. Exemplary metallic salts include, but are not limited to, suitable alkali metal (Group Ia) salts, alkaline earth metal (Group IIa) salts, and other physiologically acceptable metals. Such salts can be made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Exemplary organic salts can be made from primary amines, secondary amines, tertiary amines, and quaternary ammonium salts, such as tromethamine, diethylamine, tetra-N-methylammonium, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine.

[0087] In one aspect, the present invention provides a prostacyclin compound, such as a treprostinil derivative, or a composition comprising the same, which is effective when used in a once-daily, twice-daily, or three-times-daily dosing regimen, for example, for the treatment of pulmonary arterial hypertension or portopulmonary hypertension in a patient in need thereof. In one embodiment, the prostacyclin compound provided herein can be administered less frequently than treprostinil with comparable or greater efficacy. Furthermore, in one embodiment, the side effect profile of the compound provided herein is less adverse than that resulting from treprostinil administration. In one embodiment, these advantages allow for greater patient compliance. In one embodiment, treatment is carried out by pulmonary administration of one of the compounds provided herein, for example, via a nebulizer, dry powder inhaler, or metered-dose inhaler. In some embodiments, a composition comprising one of the compounds provided herein is administered to a patient in need of PH treatment via a nebulizer. In some embodiments, the compound described herein is suspended in a propellant and delivered to the patient via a metered-dose inhaler.

[0088] In one aspect of the invention described herein, a compound of formula (I): [ka] wherein R1 is NH, O, or S; R2 is H, linear C5-C 18 Alkyl, branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl, branched C3-C 18 Alkenyl, aryl, aryl-C1-C 18 alkyl; amino acid or peptide; R3 is H, OH, optionally substituted linear or branched C1-C 15 Alkoxy, O-optionally substituted linear or branched C2-C 15 Alkenyl , O—(C═O)—Optionally substituted linear or branched C1-C 15 Alkyl or O-(C=O)- optionally substituted linear or branched C2-C 15 is alkenyl; R4 is an optionally substituted straight or branched C1-C 15 Alkyl or optionally substituted straight or branched C-C 15 is alkenyl; n is an integer from 0 to 5. or a pharmaceutically acceptable salt thereof, with the proviso that the prostacyclin compound of formula (I) is not treprostinil.

[0089] In other embodiments, prostacyclin compounds of Formula (I) are provided wherein R3 is OH and n is 0 or 1. In still other embodiments, R4 is an optionally substituted straight or branched C1-C 15 In yet other embodiments, R is NH or O.

[0090] In one embodiment, R1 is NH, O, or S; and R2 is a linear C5-C 18 Alkyl, branched C5-C18 Alkyl, linear C2-C 18 Alkenyl, branched C3-C 18 alkenyl; R3 is H, OH or O-alkyl; R4 is an optionally substituted straight or branched C1-C 15 Alkyl or optionally substituted straight or branched C-C 15 Prostacyclin compounds of Formula (I) are provided wherein R1 is NH or O and R2 is a linear C5-C 18 Alkyl or branched C5-C 18 It is alkyl.

[0091] In one embodiment, R2 is aryl or aryl-C1-C 18 R3 is OH and n is 0 or 1. In still other embodiments, R4 is an optionally substituted straight or branched C1-C 15 It is alkyl.

[0092] In one embodiment, the present invention provides a compound of formula (Ia), (Ib), (Ic) or (Id): [ka] (Wherein, R2 is H, a linear or branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl or branched C3-C 18 alkenyl; R3 is H, OH, optionally substituted linear or branched C1-C 15 Alkoxy, O-optionally substituted straight or branched C2-C 15 Alkenyl, -O(C=O)- optionally substituted straight or branched C1-C 15 Alkyl or -O(C=O)- optionally substituted straight or branched C-C 15 alkenyl; R4 is [ka] , optionally substituted linear or branched C1-C 15 Alkyl or optionally substituted straight or branched C-C 15 alkenyl, and R5 is H, optionally substituted straight or branched C1-C 15 Alkyl, optionally substituted straight or branched C-C 15 Alkenyl, (C=O)-optionally substituted straight or branched C1-C 15 Alkyl or (C=O)-optionally substituted straight or branched C-C 15 alkenyl) or a pharmaceutically acceptable salt thereof. In another embodiment, R4 is [ka] with the proviso that the compound is not treprostinil, i.e., R2 and R5 cannot both be H.

[0093] In one embodiment of Formula (Ia), Formula (Ib), Formula (Ic) and Formula (Id), R2 is a linear or branched C5-C 18 In yet other embodiments, R2 is alkyl. [ka] or [ka] wherein m1 and m2 are each independently an integer selected from 1 to 9, and each occurrence of R' is independently H, a straight or branched C1-C8 alkyl, or a straight or branched C1-C8 alkenyl. In yet another embodiment, R2 is [ka] and m1 and m2 are both 4. In another embodiment, R2 is [ka] and m1 is 3 and m2 is 4, or m1 is 2 and m2 is 3.

[0094] When m1 and / or m2 are integers from 2 to 9, the m1 / m2 at the end of the carbon chain is CH3, and the remaining m1 / m2 groups are CH2.

[0095] In one embodiment of Formula (Ia), Formula (Ib), Formula (Ic) and Formula (Id), R2 is [ka] [ka] In other embodiments, R3 is OH and R4 is [ka] and R5 is H, optionally substituted linear or branched C1-C 15 Alkyl, optionally substituted straight or branched C-C 15 Alkenyl, (C=O)-optionally substituted straight or branched C1-C 15 Alkyl or (C=O)-optionally substituted straight or branched C-C 15 It is alkenyl.

[0096] In one embodiment of Formula (Ia), (Ib), (Ic) or (Id), R2 is H, R3 is OH and R4 is [ka] and R5 is [ka] or [ka] wherein m1 and m2 are each independently an integer selected from 1 to 9, and each occurrence of R' is independently H, a straight or branched C1-C8 alkyl, or a straight or branched C1-C8 alkenyl. When m1 and / or m2 are integers from 2 to 9, the m1 / m2 at the end of the carbon chain is CH3, and the remaining m1 / m2 groups are CH2.

[0097] In another embodiment, a compound of formula (Ia'), (Ib'), (Ic') or (Id'): [ka] (Wherein, R2 is H, a linear or branched C5-C 18 Alkyl or linear or branched C5-C 18 alkenyl; R4 is [ka] , optionally substituted linear or branched C1-C 15 Alkyl or optionally substituted straight or branched C-C 15 alkenyl, and R5 is H, optionally substituted straight or branched C1-C 15 Alkyl, optionally substituted straight or branched C-C 15 Alkenyl, (C=O)-optionally substituted straight or branched C1-C 15Alkyl or (C=O)-optionally substituted straight or branched C-C 15 alkenyl, provided that R2 and R5 cannot both be H. In one embodiment of formula (Ia'), formula (Ib'), formula (Ic') and formula (Id'), R4 is [ka] and R2 is [ka] or R5 is [ka] or [ka] wherein m1 and m2 are each independently an integer selected from 1 to 9, and each occurrence of R' is independently H, a straight or branched C1-C8 alkyl, or a straight or branched C1-C8 alkenyl. In yet another embodiment, R2 is [ka] [ka] is.

[0098] Yet another embodiment of the present invention is a compound of formula (Ia″), (Ib″), (Ic″) or (Id″): [ka] (In the formula, R2 is H, linear or branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl, branched C3-C 18 Alkenyl, aryl, aryl-C1-C 18 alkyl; amino acid or peptide; R3 is H, OH, optionally substituted linear or branched C1-C 15 Alkoxy, O-optionally substituted straight or branched C2-C 15 Alkenyl, O—(C═O)—optionally substituted straight or branched C1-C 15 Alkyl or O-(C=O)- optionally substituted linear or branched C2-C 15 is alkenyl; R5 is H, optionally substituted straight or branched C1-C 15 Alkyl, optionally substituted straight or branched C-C 15 Alkenyl, (C=O)-optionally substituted straight or branched C1-C 15 Alkyl or (C=O)-optionally substituted straight or branched C-C 15 alkenyl, provided that R2 and R5 cannot both be H. or a pharmaceutically acceptable salt thereof. In another embodiment, R3 is OH, and R2 is 5-nonanyl, 4-heptyl, 4-octyl, 3-octyl, 2-dimethyl-1-propyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, 3-pentyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl. In yet another embodiment, R2 is decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl. In yet another embodiment, R2 is linear alkyl.

[0099] One embodiment of the present invention is directed to compounds of formula (Ic), (Ic') and (Ic''). In another embodiment, R2 is a linear C5-C 18 Alkyl or branched C5-C 18 In yet another embodiment, R2 is a linear C-C alkyl. 18 Alkyl or branched C6-C 18 In yet another embodiment, R2 is a linear C-C alkyl. 14 Alkyl, for example, linear C6 alkyl, C8 alkyl, C 10 Alkyl, C 12 Alkyl or C 14 It is alkyl.

[0100] In one embodiment, R2 is a linear C5-C 18 R is OH and R is H. In another embodiment, compounds of formula (Ic") are provided wherein R is a linear C-C alkyl; R is OH and R is H. 18 In a further embodiment, compounds of formula (Ic") are provided wherein R2 is a linear C6-C alkyl, R3 is OH, and R5 is H. 16 R is OH and R is H. In yet another embodiment, compounds of formula (Ic") are provided wherein R is a linear C8-C 14 Compounds of formula (Ic'') are provided wherein R3 is OH and R5 is OH.

[0101] In one embodiment, R2 is a linear C5-C 18 R is OH and R is H. In another embodiment, compounds of formula (Ic") are provided wherein R is a branched C-C alkyl; R is OH and R is H. 18 In a further embodiment, compounds of formula (Ic") are provided wherein R2 is a branched C6-C alkyl; R3 is OH and R5 is H. 16 R is OH and R is H. In yet another embodiment, compounds of formula (Ic") are provided wherein R is a branched C8-C 14R3 is OH and R5 is H. Compounds of formula (Ic'') are provided wherein R3 is OH and R5 is H.

[0102] In yet other embodiments, compounds of Formulae (Ic), (Ic') and (Ic'') are administered to patients in need of PH treatment via a metered dose inhaler.

[0103] In yet another embodiment of Formula (Ia″), (Ib″), (Ic″) or (Id″), R3 is OH, R5 is H and R2 is [ka] and m1 and m2 are each independently an integer selected from 1 to 9. In yet another embodiment, R2 is [ka] [ka] is.

[0104] In yet another embodiment of formula (Ia″), (Ib″), (Ic″) or (Id″), R2 is H, R3 is OH and R5 is [ka] and m1 and m2 are each independently an integer selected from 1 to 9. In yet another embodiment, R2 is [ka] [ka] is.

[0105] In one embodiment, R2 is a linear or branched C5-C 18 Provided is a prostacyclin compound of formula (I), (Ia), (Ib), (Ic) or (Id), wherein R2 is alkyl. In other embodiments, R2 is 5-nonanyl, 4-heptanyl, 4-octanyl, 3-octanyl, 2-dimethyl-1-propanyl, 3,3-dimethyl-1-butanyl, 2-ethyl-1-butanyl, 3-pentanyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl.

[0106] In one embodiment, R2 is a linear or branched C5-C 18 In still other embodiments, R2 is a linear C5-C 18 In another embodiment, R2 is alkyl. [ka] wherein m1 and m2 are each independently an integer selected from 1 to 9, and each occurrence of R' is independently H, a straight or branched C1-C8 alkyl, or a straight or branched C1-C8 alkenyl. In yet another embodiment, R2 is [ka] [ka] is.

[0107] In another embodiment, R2 is a branched C5-C 18Prostacyclin compounds of formula (I), (Ia), (Ib), (Ic) or (Id) are provided, wherein R2 is alkyl. In other embodiments, R2 is 5-nonanyl, 4-heptyl, 4-octyl, 3-octyl, 2-dimethyl-1-propyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, 3-pentyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl.

[0108] In one embodiment of the present invention, the prostacyclin compound of the present invention has the following structure: [ka] wherein R1 is NH, O, or S. It has.

[0109] For example, R1 is O or N and the following compounds (5-nonanyl treprostinil (alkyl ester, 5C9-TR) or 5-nonanyl treprostinil (amide linked; 5C9-TR-A): [ka] One of the following will be provided.

[0110] In one embodiment, R2 is [ka] wherein m1 and m2 are each independently an integer selected from 1 to 9, and R', at each occurrence, is independently H, a straight-chained or branched C1-C8 alkyl, or a straight-chained or branched C1-C8 alkenyl.

[0111] When m1 and / or m2 are integers from 2 to 9, the m1 / m2 at the end of the carbon chain is CH3, and the remaining m1 / m2 groups are CH2.

[0112] In yet another embodiment, compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic'') or (Id'') are provided, wherein R2 is [ka] [ka] is.

[0113] The compounds provided herein can include symmetrically branched alkyl or asymmetrically branched alkyl as the R2 moiety. For example, R2 can be [ka] When m1 and m2 are different, R2 is an asymmetrical branched alkyl.

[0114] In another embodiment, a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic'') or (Id'') is provided, wherein R2 is [ka] wherein m1 is 2 and m2 is 3, m1 and m2 are each independently 4, or m1 and m2 are each independently 3.

[0115] In another embodiment, the prostacyclin compound comprises an asymmetric branched alkyl at the R2 position, such as 3-hexanyl (3C6), 2-heptanyl (2C7), 3-heptanyl (3C7), 2-octanyl (2C8), 3-octanyl (3C8), or 4-octanyl (4C8).

[0116] In another embodiment, there is provided a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id), wherein R2 is a branched alkyl selected from 2,2-diethyl-1-pentyl, 3-pentyl, 4-octyl, 5-nonanyl, 2-ethyl-1-butyl, 2-propyl-1-pentyl, 12-butyl-1-octyl, 2-dimethyl-1-propyl and 3,3-dimethyl-1-butyl.

[0117] In another embodiment, R2 is a linear or branched C5-C 18 Prostacyclin compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), or (Id') are provided, wherein R2 is a linear C5-C alkenyl selected from pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, or octadecenyl. 18 In another embodiment, R3 is OH. In another embodiment, R2 is a branched C5-C alkyl selected from pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, or octadecenyl. 18 In another embodiment, R3 is OH.

[0118] In one embodiment, a prostacyclin compound of formula (I), (Ia), (Ib), (Ic) or (Id) is provided, wherein R4 is [ka] In other embodiments, R4 is [ka] is.

[0119] In one embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) is provided, wherein R2 is a linear C5-C 18 alkyl, R3 is OH, and R4 is [ka] In other embodiments, R2 is 5-nonanyl, 4-heptyl, 4-octanyl, 3-octanyl, 2-dimethyl-1-propyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, 3-pentyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl.

[0120] In one embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) is provided, wherein R2 is hexyl, dodecyl, tetradecyl, hexadecyl, 5-nonanyl, 4-heptanyl, 4-octanyl, 3-octanyl, 2-dimethyl-1-propyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, 3-pentyl, R3 is OH, and R4 is [ka] is.

[0121] In one embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) is provided, wherein R2 is hexyl, R3 is OH, and R4 is [ka] is.

[0122] In one embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) is provided, wherein R2 is hexyl, R3 is OH, and R4 is [ka] is.

[0123] In another embodiment, prostacyclin compounds of formula (Ia″), (Ib″), (Ic″) or (Id″) are provided wherein R2 is hexyl, R3 is OH, and R4 is H. In another embodiment, the compound is a compound of formula (Ic″). In yet another embodiment, prostacyclin compounds of formula (Ia″), (Ib″), (Ic″) or (Id″) are provided wherein R2 is dodecyl, tetradecyl, pentadecyl, or hexadecyl, R3 is OH, and R4 is H. In another embodiment, the compound is a compound of formula (Ia″). In yet other embodiments, the compound is present in a lipid nanoparticle formulation, as described in more detail below.

[0124] In one embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof is provided, wherein R2 is heptyl, R3 is OH, and R4 is [ka] is.

[0125] In one embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof is provided, wherein R2 is octyl, R3 is OH, and R4 is [ka] is.

[0126] In one embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof is provided, wherein R2 is nonyl, R3 is OH, and R4 is [ka] is.

[0127] In another embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof is provided, wherein R2 is decyl, R3 is OH, and R4 is [ka] is.

[0128] In yet another embodiment, there is provided a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof, wherein R2 is undecyl, R3 is OH and R4 is [ka] is.

[0129] In yet another embodiment, there is provided a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof, wherein R2 is dodecyl, R3 is OH and R4 is [ka] is.

[0130] In one embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof is provided, wherein R2 is tridecyl, R3 is OH, and R4 is [ka] is.

[0131] In another embodiment, a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof is provided, wherein R2 is tetradecyl, R3 is OH, and R4 is [ka] is.

[0132] In yet another embodiment, there is provided a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic) or (Id) or a pharmaceutically acceptable salt thereof, wherein R2 is pentadecyl, R3 is OH, and R4 is [ka] is.

[0133] Another embodiment of the present invention is a compound wherein R2 is hexadecyl, R3 is OH, and R4 is [ka] or a pharmaceutically acceptable salt thereof.

[0134] Yet another embodiment of the present invention is a compound wherein R2 is heptadecyl, R3 is OH, and R4 is [ka] or a pharmaceutically acceptable salt thereof.

[0135] Yet another embodiment of the present invention is a compound wherein R2 is octadecyl, R3 is OH, and R4 is [ka] or a pharmaceutically acceptable salt thereof.

[0136] In one embodiment, compounds of Formula (I), (Ia), (Ib), (Ic), or (Id) or pharmaceutically acceptable salts thereof are provided, in which one or more hydrogen atoms have been replaced with deuterium. Accordingly, in one embodiment, the present invention relates to isotopic species of Formula (I), (Ia), (Ib), (Ic), or (Id) in which one or more deuterium atoms have been replaced. The isotopic species of Formula (I), (Ia), (Ib), (Ic), or (Id) can be used to accurately determine the concentration of compounds of Formula (I), (Ia), (Ib), (Ic), or (Id) in biological fluids and to determine the metabolic patterns of compounds of Formula (I), (Ia), (Ib), (Ic), or (Id) and their isotopic species. The present invention further provides compositions comprising these deuterium isotopic species and methods for treating diseases and conditions as described herein.

[0137] In another embodiment of the present invention, there is provided a prostacyclin compound of formula (II) or a pharmaceutically acceptable salt thereof: [ka] wherein R1 is NH, O, or S; R2 is a linear or branched C5-C 18Alkyl, linear C2-C 18 Alkenyl or branched C3-C 18 Alkenyl, aryl, aryl-C1-C 18 is an alkyl, amino acid, or peptide; n is an integer from 0 to 5. is provided.

[0138] In one embodiment, R1 is NH, O, or S; R2 is a linear or branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl or branched C3-C 18 Provided is a prostacyclin compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein n is alkenyl; and n is an integer from 0 to 5. In other embodiments, n is 1 and R is NH or O.

[0139] In one embodiment, the present invention provides a compound of formula (IIa), (IIb), (IIc) or (IId): [ka] [ka] (Wherein, R2 is a linear or branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl or branched C3-C 18 Alkenyl, aryl, aryl-C1-C 18 alkyl, amino acid or peptide) or a pharmaceutically acceptable salt thereof. In another embodiment, R2 is a linear or branched C5-C 18 Alkyl, linear C2-C 18 Alkenyl or branched C3-C 18Compounds of formula (IIa), (IIb), (IIc), or (IId) are provided, which are alkenyl. In one embodiment, compounds of formula (II), (IIa), (IIb), (IIc), or (IId) are provided, in which one or more hydrogen atoms are replaced with deuterium. Thus, in one embodiment, the present invention relates to isotopic species of formula (II), (IIa), (IIb), (IIc), or (IId) in which one or more deuterium atoms are replaced. The isotopic species of formula (II), (IIa), (IIb), (IIc), or (IId) can be used to accurately determine the concentration of compounds of formula (II), (IIa), (IIb), (IIc), or (IId) in biological fluids and to determine the metabolic patterns of compounds of formula (II), (IIa), (IIb), (IIc), or (IId) and their isotopic species. The present invention further provides compositions comprising these deuterium isotope species and methods of treating diseases and conditions such as those set forth herein.

[0140] In one embodiment, the prostacyclin derivative is a compound of formula (IIc): In another embodiment, R2 is a linear C5-C 18 Alkyl or branched C5-C 18 For example, in one embodiment, R2 is a linear C-C alkyl. 18 In another embodiment of Formula (IIc), R2 is a linear C-C alkyl. 10 In still other embodiments of Formula (IIc), R2 is hexyl, heptyl, or octyl.

[0141] Compounds of formula (IIa) and formula (IId) are shown in Tables A and B below. [Table A] [Table B]

[0142] Yet another embodiment of the present invention is a compound of formula (III): [ka] wherein R1 and R2 are defined as provided for formulas (I) and (II), and R5 and R6 are independently H, optionally substituted linear or branched C1-C 15 Alkyl, optionally substituted straight or branched C-C 15 Alkenyl, (C=O)-optionally substituted straight or branched C1-C 15 Alkyl or (C=O)-optionally substituted straight or branched C-C 15 alkenyl) or a pharmaceutically acceptable salt thereof, with the proviso that the prostacyclin compound of formula (III) is not treprostinil.

[0143] In one embodiment, the branched-chain prostacyclin compounds provided herein exhibit both higher solubility and slower enzymatic conversion to treprostinil relative to linear-derivatized prostacyclin compounds. In one embodiment, asymmetric branched-chain prostacyclin compounds are provided that are more stable than the corresponding symmetric branched-chain prostacyclin compounds.

[0144] In one embodiment, the present invention provides a prostacyclin compound comprising a chiral moiety at one or more of the R2, R5, and / or R6 positions. For example, in one embodiment, the moiety at the R2 position is a chiral moiety and comprises an R isomer, an S isomer, or a mixture thereof. The optical isomers at the R2, R5, and / or R6 positions can also be classified using the D / L nomenclature system. For example, when R2 is an amino acid or amino acid moiety, the amino acid or amino acid moiety can be a D isomer, an L isomer, or a mixture thereof.

[0145] In one embodiment, one or more of the R2, R5, and / or R6 moieties are the R or S isomer. In another embodiment, one or more of the R2, R5, and / or R6 moieties provided herein comprise a mixture of R and S moieties. As used herein, "R isomer" or "S isomer" refers to an enantiomerically pure isomer. An "enantiomerically pure isomer" has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% purity of the R- or S-isomer, or, when the D / L nomenclature is used, the D- or L-isomer. A racemate is a compound having a mixture of equal amounts of both enantiomers.

[0146] In another aspect of the present invention, the prostacyclin compounds described herein are provided in compositions for delivery to patients, for example, to treat pulmonary hypertension (PH). The compositions can include the compounds, pharmaceutically acceptable salts of the compounds, or combinations thereof. In one embodiment, the PH is pulmonary arterial hypertension (PAH). Prostacyclin compositions (so-called "lipid nanoparticle compositions") and formulations comprising prostacyclin, a cationic compound, and a surfactant are described in PCT Publication No. WO2014 / 085813, the disclosure of which is incorporated herein by reference in its entirety for all purposes. The compositions described in WO2014 / 085813 are acceptable for use with the prostacyclin derivative compounds provided herein.

[0147] In one embodiment, the composition comprises one of the prostacyclin compounds described herein, i.e., a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III), and an amphiphile. In one embodiment, when formulated together, the prostacyclin compound and the amphiphile form micro- or nanoparticles. In one embodiment, the amphiphile is a PEGylated lipid, surfactant, or block copolymer. In another embodiment, the prostacyclin compositions provided herein comprise two or more prostacyclin compounds described herein (e.g., compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) containing a deuterated compound) and an amphiphile (e.g., a PEGylated lipid, lipid, surfactant, or block copolymer). In one embodiment, when formulated together, the prostacyclin composition comprising a prostacyclin compound component and an amphiphile comprises a plurality of nanoparticles. In other embodiments, the average diameter of the plurality of nanoparticles is about 20 nm to about 700 nm, e.g., about 50 nm to about 500 nm, about 100 nm to about 600 nm, or about 100 nm to about 500 nm. When the amphiphile comprises a lipid, e.g., a PEGylated lipid, e.g., cholesterol-PEG or distearoylphosphatidylethanolamine-PEG (DSPE-PEG), the composition is said to comprise lipid nanoparticles.

[0148] In another embodiment, the prostacyclin composition comprises a prostacyclin compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) and a PEGylated lipid as the amphilphilic agent. In another embodiment, the PEGylated lipid The PEGylated lipid includes PEG400 to PEG5000. For example, in one embodiment, the PEGylated lipid includes PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG5000. In other embodiments, the lipid component of the PEGylated lipid includes cholesterol, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphoethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dimyristoylglycerol (DMG), diphosphatidylglycerol (DPG), or distearoylglycerol (DSG). In yet other embodiments, the PEGylated lipid is cholesterol-PEG2000 or DSPE-PEG2000.

[0149] Depending on its molecular weight (MW), PEG is also referred to in the art as polyethylene oxide (PEO) or polyoxyethylene (POE). PEGylated lipids can contain branched or unbranched PEG molecules and are not limited by a particular PEG MW.

[0150] For example, in one embodiment, the PEGylated lipid comprises a PEG molecule having a molecular weight of 300 g / mol, 400 g / mol, 500 g / mol, 1000 g / mol, 1500 g / mol, 2000 g / mol, 2500 g / mol, 3000 g / mol, 3500 g / mol, 4000 g / mol, 4500 g / mol, 5000 g / mol, or 10,000 g / mol. In one embodiment, the PEG has a MW of 1000 g / mol or 2000 g / mol.

[0151] The lipid component of the PEGylated lipid may have a net charge (e.g., cationic or anionic) or may be net neutral. The lipids used in the PEGylated lipid component of the present invention may be synthetic, semi-synthetic, or naturally occurring lipids, including phospholipids, sphingolipids, glycolipids, ceramides, tocopherols, sterols, fatty acids, or glycoproteins, such as albumin. In one embodiment, the lipid is a sterol. In another embodiment, the sterol is cholesterol. In another embodiment, the lipid is a phospholipid. Phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA). In one embodiment, the phospholipid is egg phospholipid, soybean phospholipid, or hydrogenated egg and soybean phospholipid. In one embodiment, the PEGylated lipid comprises a phospholipid. In other embodiments, the phospholipids contain ester bonds of fatty acids containing 12 to 26 carbon atoms at the 2- and 3-positions of the glycerol, and different head groups at the 1-position of the glycerol, including choline, glycerol, inositol, serine, ethanolamine, and the corresponding phosphatidic acids. The chains on these fatty acids may be saturated or unsaturated, and the phospholipids may be composed of fatty acids of different chain lengths and different degrees of unsaturation. In particular, in one embodiment, the PEGylated lipid in the prostacyclin composition provided herein contains distearoylphosphoethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphoethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dimyristoylglycerol (DMG), diphosphatidylglycerol (DPG), or distearoylglycerol (DSG).

[0152] Other examples of lipids for use in the compositions comprising the PEGylated lipids disclosed herein include dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylcholine (DMPG), ... PEGylated lipids include phospholipids such as palmitoylstearoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylethanolamine (DOPE), and mixed phospholipids such as palmitoylstearoylphosphatidylcholine (PSPC) and palmitoylstearoylphosphatidylglycerol (PSPG), triacylglycerols, diacylglycerols, ceramides, sphingosine, sphingomyelin, and single acylated phospholipids such as mono-oleoyl-phosphatidylethanolamine (MOPE). In another embodiment, the lipid portion of the PEGylated lipid is an ammonium salt of a fatty acid, a phospholipid, a glyceride, a phospholipid and glyceride, a sterol (e.g., cholesterol), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylcholine (PPE), or a mixture of phospholipids and glycerides. ) (PC), phosphatidylinositol (PI), phosphatidylserine (PS), or a combination thereof. In one embodiment, the fatty acid comprises a saturated or unsaturated fatty acid with a carbon chain length of 12 to 26 carbon atoms. Some specific examples include: myristylamine, palmitylamine, laurylamine, and stearylamine, dilauroylethylphosphocholine (DLEP), dimyristoylethylphosphocholine (DMEP), dipalmitoylethylphosphocholine (DPEP), and distearoylethylphosphocholine (DSEP), N-(2,3-di-(9(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA), and 1,2-bis(oleoyloxy)-3 The examples of sterols that can be used in the compositions provided herein include cholesterol and ergosterol.The examples of PG, PA, PI, PC and PS that can be used in the compositions provided herein include DMPG, DPPG, DSPG, DMPA, DPPA, DSPA, DMPI, DPPI, DSPI, DMPS, DPPS and DSPS, DSPC, DPPG, DMPC, DOPC, egg PC and soybean PC.

[0153] In one embodiment, the PEGylated lipid is cholesterol-PEG2000, DSPE-PEG1000, or DSG-PEG2000.

[0154] In another embodiment, the prostacyclin compositions provided herein comprise a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) and a hydrophobic excipient. In other embodiments, the compositions comprise an amphiphile, for example, a PEGylated lipid as described above.

[0155] In yet another embodiment, two or more prostacyclin compounds described herein (e.g., compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III)), an amphiphilic agent (e.g., a PEGylated lipid, lipid, surfactant or block copolymer), and a hydrophobic additive are provided in the composition.

[0156] In one embodiment, the prostacyclin composition comprises a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III) and a PEGylated lipid. In another embodiment, the prostacyclin composition comprises a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III) and a surfactant. In yet another embodiment, the prostacyclin composition comprises a prostacyclin compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III), a hydrophobic additive, and an amphiphile. In other embodiments, the amphiphile is a surfactant, a PEGylated lipid, or a block copolymer. In yet other embodiments, the amphiphile is a PEGylated lipid.

[0157] In one embodiment, the prostacyclin compound is present in the composition at 5 mol% to 99 mol%. In another embodiment, the prostacyclin compound is present in the composition at 40 mol% to 95 mol%. In another embodiment, the prostacyclin compound is present in the composition at 40 mol% to 60 mol%. In one embodiment, the prostacyclin compound is present in the composition at about 40 mol% or about 45 mol%.

[0158] When present in the composition, in one embodiment, the amphiphile, e.g., the PEGylated lipid, is present at 10 mol% to 30 mol%, e.g., 10 mol% to 20 mol% or 15 mol% to 25 mol%, hi yet another embodiment, the PEGylated lipid is present in the composition at about 10 mol% or 20 mol%.

[0159] When present in the composition, in one embodiment, the hydrophobic additive is present in the composition at 25 mol% to 50 mol%, e.g., 30 mol% to 50 mol%, 35 mol% to 45 mol%, hi yet another embodiment, the hydrophobic additive is present in the composition at about 40 mol% or about 45 mol%.

[0160] In one embodiment, the prostacyclin composition comprises a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) or a pharmaceutically acceptable salt thereof, as described herein, an amphiphile, and a hydrophobic excipient. In one embodiment, the hydrophobic excipient (e.g., an excipient that is at least partially hydrophobic) is a hydrocarbon, a terpene compound, or a hydrophobic lipid (e.g., tocopherol, tocopherol acetate, sterol, sterol ester, alkyl ester, vitamin A acetate, triglyceride, phospholipid). In one embodiment, the composition comprises a prostacyclin compound, such as a compound of Formula (I) or (II), an amphiphile, and a hydrocarbon. The hydrocarbon may be an aromatic, alkane, alkene, cycloalkane, or alkyne. In one embodiment, the hydrocarbon is an alkane (i.e., a saturated hydrocarbon). In another embodiment, the hydrocarbon is a C 15 ~C 50 In another embodiment, the hydrocarbon is a C 15 , C 20 , C 25 , C 30 , C 35 , C 40 , C 45 or C 50 In yet another embodiment, the hydrophobic additive is a C 15 ~C 25 Hydrocarbons, C 15 ~C 35 Hydrocarbons, C 15 ~C 45 Hydrocarbons, C 15 ~C 20 Hydrocarbons, C 20 ~C 25 Hydrocarbons, C 25 ~C 30 Hydrocarbons, C 30 ~C 35 Hydrocarbons, C 35 ~C 40 Hydrocarbons, C 40 ~C 45 Hydrocarbon or C 45 ~C50 It is a hydrocarbon.

[0161] In one embodiment, a composition is provided comprising a prostacyclin compound, an amphiphile, and a terpene compound (e.g., a hydrophobic additive). In another embodiment, the composition comprises a PEGylated lipid as the amphiphile. However, as noted above, block copolymers as well as surfactants can be used as the amphiphilic component of the composition. In one embodiment, the terpene compound (hydrophobic additive) is a hydrocarbon (e.g., isoprene, squalane, or squalene). In another embodiment, the terpene compound is a hemiterpene (C5H8), a monoterpene (C 10 H 16 ), sesquiterpenes (C 15 H 24 ), diterpenes (C 20 H 32 ) (e.g., cafestol, kahweol, cembrene, taxadiene), sesterterpenes (C 25 H 40 ), triterpenes (C 30 H 48 ), sesquaterpene (C 35 H 56 ), tetraterpenes (C 40 H 64 ), polyterpenes (e.g., polyisoprene with trans double bonds) or norisoprenoids (e.g., 3-oxo-α-ionol, 7,8-dihydroionone derivatives). In another embodiment, the terpene compound is selected from one of the compounds shown in Table 1 below. In one embodiment, the hydrophobic additive is squalane. [Table 1]

[0162] As provided above, in one embodiment, the compositions provided herein comprise a prostacyclin compound and one or more PEGylated lipids. In other embodiments, the compositions comprise a hydrophobic additive as described above. In one embodiment, the compositions provided herein comprise a prostacyclin compound of one of formulas (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III), a hydrophobic additive, and a PEGylated lipid. In other embodiments, the hydrophobic additive comprises a hydrocarbon, such as a terpene compound.

[0163] In one embodiment, the treprostinil derivative compositions provided herein comprise the ingredients shown in Table C below. [Table C]

[0164] The present invention also provides a method for treating a patient in need thereof with one of the prostacyclin compounds or compositions described herein. It should be understood that a reference to a prostacyclin compound in a treatment method includes the use of a pharmaceutically acceptable salt of the compound. Similarly, the administration of a prostacyclin composition comprising a prostacyclin compound includes the use of a pharmaceutically acceptable salt in the composition.

[0165] In one aspect, a method for treating pulmonary hypertension (PH) is provided. In one embodiment, the method comprises administering a compound provided herein, a pharmaceutically acceptable salt thereof, or a composition thereof to a patient in need thereof. In one embodiment, administration is pulmonary, for example, using a metered-dose inhaler (MDI), a dry powder inhaler (DPI), or a nebulizer. The World Health Organization (WHO) classifies PH into five groups. WHO Group I PH includes pulmonary arterial hypertension (PAH), idiopathic pulmonary arterial hypertension (IPAH), familial pulmonary arterial hypertension (FPAH), and pulmonary arterial hypertension associated with other diseases (APAH). For example, pulmonary arterial hypertension associated with collagen vascular disease (e.g., scleroderma), congenital shunts between the systemic and pulmonary circulation, portal hypertension, and / or HIV infection is included in Group I PH. In one embodiment, the method provided herein is for treating a WHO Group I PH patient in need thereof, such as a PAH patient, an IPAH patient, an FPAH patient, or an APAH patient. WHO Group II PH includes pulmonary hypertension associated with left heart disease, such as atrial or ventricular disease, or valvular disease (e.g., mitral valve stenosis). In one embodiment, the method provided herein is for treating a WHO Group II patient in need thereof. WHO Group III pulmonary hypertension is characterized by pulmonary hypertension associated with a lung disease, such as chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD), and / or hypoxemia. In one embodiment, the method provided herein is for treating a WHO Group III patient in need thereof. WHO Group IV pulmonary hypertension is pulmonary hypertension caused by chronic thrombotic and / or embolic disease. Group IV PH is also referred to as chronic thromboembolic pulmonary hypertension. Group IV PH patients experience vascular blockage or narrowing due to blood clots. In one embodiment, the methods provided herein are provided for treating WHO Group IV patients in need thereof. The WHO classifies Group V PH as a "miscellaneous" category, which includes: Included are PH caused by blood disorders (e.g., polycythemia vera, essential thrombocythemia), systemic disorders (e.g., sarcoidosis, vasculitis), and / or metabolic disorders (e.g., thyroid disease, glycogen storage disease). In one embodiment, the methods provided herein are provided for treating a WHO Group V patient in need thereof.

[0166] The methods provided herein can be used to treat patients with PH in WHO Group I (i.e., pulmonary arterial hypertension or PAH), Group II, Group III, Group IV, or Group V. In one embodiment of a method for treating PH, a method for treating pulmonary arterial hypertension (PAH) is provided. In another embodiment, a method for treating patients with chronic thromboembolic pulmonary hypertension is provided. In one embodiment, a method for treating PH (e.g., PAH) comprises administering an effective amount of one of the compounds described herein to a patient in need thereof by pulmonary (e.g., inhalation via an MDI, nebulizer, or dry powder inhaler), subcutaneous, oral, nasal, or intravenous route of administration. In one embodiment, administration is by inhalation via an MDI or nebulizer. In one embodiment, when compound delivery is via a nebulizer, the compound is provided to the patient as a composition, for example, a lipid nanoparticle composition as described above.

[0167] In another aspect of the present invention, a method for treating portopulmonary hypertension (PPH) is provided. In one embodiment, the method comprises administering an effective amount of one of the compounds described herein (or a pharmaceutically acceptable salt thereof) to a patient in need thereof by pulmonary (inhalation), subcutaneous, oral, nasal, or intravenous administration. In one embodiment, administration is by inhalation with an MDI or nebulizer. In one embodiment, when compound delivery is by nebulizer, the compound is provided to the patient as a composition, for example, as a lipid nanoparticle composition as described above.

[0168] Methods of administering treprostinil and its analogs for the treatment of pulmonary hypertension are described in U.S. Pat. Nos. 5,153,222, 6,521,212, 7,544,713, and U.S. Patent Application Publication No. 2010 / 0076083, the disclosures of each of which are incorporated by reference in their entirety for all purposes.

[0169] In one embodiment, the method of treating a patient for PH (e.g., PAH) or PPH includes administering to a patient in need thereof one of the prostacyclin compounds or compositions provided herein, e.g., those of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId ... The method includes administering a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III), or a pharmaceutically acceptable salt thereof, or a composition comprising a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III), or a pharmaceutically acceptable salt thereof. In one embodiment, the method of treating PH (e.g., PAH) or PPH includes administering to a patient in need thereof a prostacyclin compound or composition provided herein, e.g., a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III), or a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib' (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III), or a composition comprising a deuterated compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III). Routes of administration to the patient include pulmonary (inhalation), subcutaneous, oral, nasal, and intravenous.In one embodiment, administration of a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III) or a pharmaceutically acceptable salt thereof is by inhalation with an MDI or a nebulizer. In one embodiment, when compound delivery is by nebulizer, the compound is provided to the patient as a composition, for example, a lipid nanoparticle composition as described above.

[0170] In one embodiment, a method for treating PH, PAH, or PPH comprises administering to a patient in need thereof an effective amount of a prostacyclin compound or prostacyclin composition described herein. In other embodiments, the compound, or a pharmaceutically acceptable salt of the compound, is administered to the patient by pulmonary (inhalation), subcutaneous, oral, nasal, or intravenous routes of administration. In other embodiments, administration is by inhalation, and the prostacyclin compound or composition is administered by a nebulizer, dry powder inhaler, or MDI. In yet other embodiments, the prostacyclin composition or compositions comprise a prostacyclin compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III) or a deuterated version thereof or a pharmaceutically acceptable salt of the compound.

[0171] In one embodiment, administration of an effective amount of a prostacyclin compound or composition of the present invention for treating PH, PAH, or PPH by inhalation, oral, nasal, subcutaneous, or intravenous administration results in a reduction in the number of side effects or a decrease in the severity of one or more side effects (also referred to herein as "adverse events") compared to administration of an effective amount of treprostinil when administered by inhalation, oral, nasal, subcutaneous, or intravenous administration. For example, in one embodiment, when a patient with PH, PAH, or PPH is administered a prostacyclin compound or composition of the present invention by inhalation (e.g., by nebulization, dry powder inhaler, or metered dose inhaler), the inhaled administration of treprostinil to the patient The patient experiences a decreased severity and / or frequency of cough or a decreased cough response compared to the severity and / or frequency of cough or cough response induced by

[0172] In another embodiment, oral, nasal, intravenous, subcutaneous or inhaled administration of an effective amount of a prostacyclin compound or composition of the present invention results in a decrease in the severity of, or a decrease in the occurrence of, one or more of the following adverse events: headache, throat irritation / sore pharynx, nausea, flushing and / or syncope, compared to oral, nasal, subcutaneous, intravenous or inhaled administration of treprostinil.

[0173] In another embodiment, oral, intranasal, intravenous, subcutaneous, or inhaled administration of an effective amount of a prostacyclin compound or composition of the invention for the treatment of PH, PAH, or PPH results in a decrease in the severity of systemic adverse events or a decrease in the occurrence of systemic adverse events compared to oral, intranasal, subcutaneous, intravenous, or inhaled administration of treprostinil.

[0174] Without being bound by theory, this invention exhibited by patients The improved adverse event profile of the present prostacyclin compounds and compositions, compared to treprostinil, is believed to result in improved patient compliance.

[0175] In one embodiment, the prostacyclin compounds and compositions of the present invention are administered less frequently than currently approved therapies for PH, PAH (e.g., Tyvaso®, Remodulin®), or PPH, while still achieving a substantially similar or better therapeutic response. Routes of administration to a patient include pulmonary (inhalation), subcutaneous, oral, nasal, and intravenous. In one embodiment, the patient's therapeutic response is a decrease in pulmonary vascular resistance index (PVRI) from pre-treatment values, a decrease in mean pulmonary artery pressure from pre-treatment values, an increase in hypoxemia score from pre-treatment values, a decrease in oxygen saturation index from pre-treatment values, an improvement in right heart function compared to pre-treatment values, or an improvement in exercise capacity compared to pre-treatment values ​​(e.g., as measured by a 6-minute walk test). In one embodiment, the therapeutic response is an improvement of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to pre-treatment values. In another embodiment, the therapeutic response is about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 70%, about 20% to about 60%, or about 10% to about 50% improvement compared to pre-treatment levels.

[0176] Without being bound by theory, the less frequent administration of the compounds and compositions of the present invention allows for improved patient compliance compared to patient compliance administered different PH, PAH or PPH treatments (e.g., treprostinil-Tyvaso®, Remodulin®).

[0177] In one embodiment, a composition or compound of the present invention is administered to a patient in need of PH, PAH, or PPH treatment via a metered-dose inhaler (MDI). In one embodiment, the composition or compound is delivered via an MDI using a propellant, such as a chlorofluorocarbon (CFC) or fluorocarbon. In one embodiment, when delivery is via an MDI, the compound is not formulated as a lipid nanoparticle composition, but instead is directly suspended or dissolved in a propellant solution. In one embodiment, a patient is administered a prostacyclin compound or composition of the present invention once daily, twice daily, or three times daily. In one embodiment, administration is with food. In one embodiment, each administration comprises one to five doses (puffs) from the MDI, e.g., one dose (1 puff), two doses (2 puffs), three doses (3 puffs), four doses (4 puffs), or five doses (5 puffs). In one embodiment, the MDI is small and portable for the patient.

[0178] In another embodiment, the prostacyclin compound or prostacyclin composition is administered to a patient in need of PH, PAH, or PPH treatment by nebulizer. In one embodiment, administration is once daily, twice daily, three times daily, or every other day.

[0179] In one embodiment, a composition or compound of the present invention is administered to a patient in need of PH, PAH, or PPH treatment via a dry powder inhaler (DPI). In one embodiment, the patient is administered a prostacyclin compound or composition of the present invention once daily, twice daily, or three times daily. In one embodiment, administration is with food. In one embodiment, each administration comprises 1 to 5 doses (puffs) from the DPI, e.g., 1 dose (1 puff), 2 doses (2 puffs), 3 doses (3 puffs), 4 doses (4 puffs), or 5 doses (5 puffs). In one embodiment, the DPI is small and portable for the patient to carry.

[0180] In another embodiment, the prostacyclin compound administered to a patient in need thereof by the pulmonary route by the methods of treating PH, PAH, or PAH described herein has a pulmonary elimination half-life (t ) of treprostinil when administered by the pulmonary route (e.g., by nebulization, dry powder inhaler, or metered dose inhaler) to a patient in need of PH, PAH, or PPH treatment. 1 / 2 ) compared with the longer pulmonary elimination half-life (t 1 / 2 ) is provided.

[0181] In another embodiment, the prostacyclin compound administered to a patient in need thereof by the methods of treating PH, PAH, or PPH described herein has a systemic elimination half-life (t 1 / 2 ) compared to the longer systemic half-life (t 1 / 2 In other embodiments, administration of the prostacyclin compound and treprostinil comprises oral, nasal, subcutaneous, or intravenous administration.

[0182] In another embodiment, the prostacyclin compound administered to a patient in need of PH, PAH, or PPH treatment has a respective lung or plasma C of treprostinil when administered to the patient. max Higher mean pulmonary C for treprostinil compared with max and / or lower plasma C max In another embodiment, the administration of the prostacyclin compound and treprostinil comprises intravenous administration.

[0183] In another embodiment, the prostacyclin compound administered to a patient in need of PH, PAH, or PPH treatment is a prostacyclin compound that is effective in reducing the mean lung or plasma area under the curve (AUC) of treprostinil when administered to the patient. 0~t) compared with the mean lung or plasma area under the curve (AUC 0~t In yet another embodiment, the prostacyclin compound administered to a patient in need thereof is a prostacyclin compound that is administered to a patient in need thereof in a manner that is shorter than the time to peak lung or plasma concentration (t) of treprostinil when treprostinil is administered to the patient. max ) compared with treprostinil's longer time to peak lung or plasma concentration (t max ) is provided.

[0184] In another aspect of the present invention, a method for treating diseases, disorders or conditions other than PH, PAH or PPH is provided.US Patent No. 5,153,222, the entirety of which is incorporated herein by reference, describes the use of treprostinil for the treatment of pulmonary hypertension.Treprostinil is approved for intravenous and subcutaneous administration, the latter avoiding the possibility of septic events associated with continuous intravenous catheters.US Patent No. 6,521,212 and US Patent No. 6,756,033, the entirety of which is incorporated herein by reference, describe the administration of treprostinil by inhalation for the treatment of pulmonary hypertension, peripheral vascular disease and other diseases and conditions.US Patent No. 6,803,386, the entirety of which is incorporated herein by reference, discloses the administration of treprostinil for the treatment of cancers such as lung, liver, brain, pancreas, kidney, prostate, breast, colon and head and neck cancer. U.S. Patent Application Publication No. 2005 / 0165111, incorporated herein by reference in its entirety, discloses treprostinil treatment of ischemic lesions. U.S. Patent Application Publication No. 7,199,157, incorporated herein by reference in its entirety, discloses that treprostinil treatment improves renal function. U.S. Patent Application Publication No. 7,879,909, incorporated herein by reference in its entirety, discloses treprostinil treatment of neuropathic foot ulcers. U.S. Patent Application Publication No. 2008 / 0280986, incorporated herein by reference in its entirety, discloses treprostinil treatment of pulmonary fibrosis, interstitial lung disease, and asthma. U.S. Patent Application Publication No. 6,054,486, incorporated herein by reference in its entirety, discloses the treatment of peripheral vascular disease with treprostinil. U.S. Patent Application Publication No. 2009 / 0036465, the entire contents of which are incorporated herein by reference, discloses combination therapies including treprostinil. U.S. Patent Application Publication No. 2008 / 0200449 discloses the delivery of treprostinil using a metered dose inhaler.U.S. Patent Nos. 7,417,070, 7,384,978, and 7,544,713, each of which is incorporated herein by reference in its entirety, and U.S. Patent Application Publication Nos. 2007 / 0078095, 2005 / 0282901, and 2008 / 0249167, describe oral formulations of treprostinil and other prostacyclin analogs and their use for the treatment of various conditions. U.S. Patent Application Publication No. 2012 / 0004307, each of which is incorporated herein by reference, discloses the use of oral treprostinil for the treatment of Raynaud's phenomenon, systemic sclerosis, and digital ischemic lesions. Each of the above-mentioned indications can be treated with the compounds and compositions provided herein. Routes of administration to patients in need of treatment include pulmonary (inhalation), subcutaneous, oral, nasal, and intravenous.

[0185] Additionally, the following references are incorporated by reference in their entirety for all purposes for the practice of embodiments of the present invention: J. Org. Chem. 2004, 69, 1890-1 902 pages, Drug of the Future, 2001, Volume 26 (No. 4), Pages 364-374, U.S. Patent Nos. 5,153,222, 6,054,486, 6,521,212, 6,756,033, 6,803,386 and 7,199,157, U.S. Patent Application Publication Nos. 2005 / 0165111, 2005 / 0282903, 2008 / 0200449, 2008 / 0280986, 2009 / 0036465 and 2012 / 0010159.

[0186] In one embodiment, a method is provided for treating a patient in need thereof for congestive heart failure, peripheral vascular disease, asthma, severe intermittent claudication, immunosuppression, proliferative diseases, cancers such as lung, liver, brain, pancreatic, kidney, prostate, breast, colon and head and neck cancer, ischemic lesions, neuropathic foot ulcers and pulmonary fibrosis, renal function and / or interstitial lung disease. In one embodiment, the method comprises administering an effective amount of one of the prostacyclin compounds or compositions provided herein, e.g., a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III) or a deuterated version thereof, or a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic') (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III), or a composition comprising a deuterated compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) to the patient. In one embodiment, administration is by inhalation (e.g., with a nebulizer or metered-dose inhaler), subcutaneous, oral, nasal, or intravenous. In some embodiments, the pharmaceutical formulation can include one or more active ingredients in addition to treprostinil monohydrate.

[0187] In one embodiment, a method is provided for treating and / or preventing interstitial lung disease (e.g., pulmonary fibrosis) or asthma, or a condition associated with interstitial lung disease or asthma, in a patient in need of such treatment. In another embodiment, the method comprises administering to the patient an effective amount of one of the prostacyclin compounds or compositions provided herein, e.g., a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III) or a deuterated version thereof, or a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), The method comprises administering a composition comprising a compound of formula (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III), or a composition comprising a deuterated compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III). In one embodiment, the composition or compound is delivered by an MDI using a propellant, such as a chlorofluorocarbon (CFC) or a fluorocarbon. In one embodiment, the patient is administered a prostacyclin compound or composition of the present invention once daily, twice daily, or three times daily. In one embodiment, administration is with food. In one embodiment, each administration comprises 1 to 5 doses (puffs) from an MDI, for example, 1 dose (1 puff), 2 doses (2 puffs), 3 doses (3 puffs), 4 doses (4 puffs), or 5 doses (5 puffs). In one embodiment, the MDI is small and portable for the patient. In another embodiment, administration is oral, nasal, subcutaneous, or intravenous.In another embodiment, oral, nasal, intravenous, subcutaneous, or inhaled administration of an effective amount of a prostacyclin compound or composition of the invention for the treatment of interstitial lung disease (e.g., pulmonary fibrosis) or asthma, or a condition associated with interstitial lung disease or asthma, results in a decrease in the severity of systemic adverse events or a decrease in the occurrence of systemic adverse events compared to oral, nasal, subcutaneous, intravenous, or inhaled administration of treprostinil.

[0188] In one embodiment, provided is a method of treating an ischemic disease or condition, such as scleroderma, including systemic sclerosis, or Raynaud's phenomenon, in a patient in need of such treatment. In another embodiment, the method comprises administering an effective amount of one of the prostacyclin compounds or compositions provided herein, e.g., a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) or a deuterated version thereof, or a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic') (Ia), (Ib), (Ic), (Id), or (III), or a composition comprising a deuterated compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId), or (III) to the patient. In one embodiment, administration is by inhalation (e.g., using a nebulizer or metered dose inhaler), oral, nasal, subcutaneous, or intravenous administration. In another embodiment, oral, intranasal, intravenous, subcutaneous or inhalation administration of an effective amount of a prostacyclin compound or composition of the present invention for the treatment of an ischemic disease or condition, such as scleroderma, including systemic sclerosis, or Raynaud's phenomenon, results in a decrease in the severity of systemic adverse events or a decrease in the occurrence of systemic adverse events compared to oral, intranasal, subcutaneous, intravenous or inhalation administration of treprostinil.

[0189]

[0023] Prostacyclin compounds or compositions provided herein include compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III) or deuterated versions thereof, or compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), Compositions comprising a compound of formula (IId) or (III), or deuterated compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III), in one embodiment, are used to treat a patient for ischemic digital lesions, such as digital ulcers or necrotic lesions, or to ameliorate symptoms or impairments and / or reduce the number of symptoms and / or impairments associated with ischemic digital lesions. The term "ischemic digital lesion" refers to a lesion in the digits, i.e., toes or fingers, of a subject, such as a human. In one embodiment, the ischemic digital lesion may be caused by or associated with an ischemic disease or condition, such as scleroderma, including systemic sclerosis, or Raynaud's phenomenon. Symptoms that can be improved and / or reduced may be, for example, ischemic ulcers in the digital and / or pain associated with scleroderma. In some embodiments, administering a prostacyclin compound or composition provided herein, when administered to a patient in need of treatment, provides an improvement or reduction in one or more functional disorders associated with ischemic lesions in the digital. For example, in one embodiment, the prostacyclin compound or composition provided herein improves or reduces hand dysfunction, i.e., improves hand function in the treated patient. In one embodiment, administration is by inhalation (e.g., by nebulizer or metered-dose inhaler), oral, nasal, subcutaneous, or intravenous administration.In another embodiment, oral, intranasal, intravenous, subcutaneous or inhalation administration of an effective amount of a prostacyclin compound or composition of the present invention for the treatment of ischemic lesions of the digital results in a decrease in the severity of systemic adverse events or a decrease in the occurrence of systemic adverse events compared to oral, intranasal, subcutaneous, intravenous or inhalation administration of treprostinil.

[0190] In one embodiment, a method is provided for improving kidney function or treating kidney dysfunction or symptoms associated with kidney dysfunction in a patient in need thereof. In another embodiment, the method comprises administering to a patient in need thereof an effective amount of a prostacyclin compound or composition provided herein, e.g., a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) or a deuterated version thereof, or a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib' (Ic'), (Id'), (Ia'), (Ib'), (Ic'), (Id'), (II), (IIa), (IIb), (IIc), (IId) or (III), or a composition comprising a deuterated compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia'), (Ib'), (Ic'), (Id'), (II), (IIa), (IIb), (IIc), (IId) or (III). Specific symptoms related to reduced kidney function include, for example, abnormally little urination, high blood levels of creatinine and urea nitrogen, protein leakage in the urine, and / or pain. In one embodiment, administration is by inhalation (e.g., with a nebulizer or metered-dose inhaler), oral, nasal, subcutaneous, or intravenous administration. In another embodiment, oral, nasal, intravenous, subcutaneous, or inhalation administration of an effective amount of a prostacyclin compound or composition of the present invention for improving renal function or ameliorating symptoms related to renal dysfunction or dysfunction results in a decrease in the severity of systemic adverse events or a decrease in the occurrence of systemic adverse events compared to oral, nasal, intravenous, subcutaneous, or inhalation administration of treprostinil.

[0191] In one embodiment, a method of treating cardiovascular disease, including congestive heart failure, is provided. In one embodiment, the method comprises administering to a patient in need thereof a prostacyclin compound or composition provided herein, e.g., a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III) or a deuterated version thereof, or a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), administering a composition comprising a compound of formula (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III), or a composition comprising a deuterated compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III). In one embodiment, administration is by inhalation (e.g., with a nebulizer or metered dose inhaler), subcutaneous, oral, nasal, or intravenous administration.

[0192] In one embodiment, a method of treating peripheral vascular disease, including peripheral arterial occlusive disease and intermittent claudication, is provided. In one embodiment, the method includes administering to a patient in need thereof a prostacyclin compound or composition provided herein, e.g., a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) or a deuterated version thereof, or a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III), or a composition comprising a deuterated compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III). In addition to the prostacyclin compounds and compositions provided herein, other pharmacologically active substances known to be useful in treating peripheral vascular disease may be present in the formulations of the present invention. For example, a compound of the invention may be present together with trental, a substance known to increase red blood cell deformability, hi one embodiment, administration is by inhalation (e.g., with a nebulizer or metered dose inhaler), subcutaneous, oral, nasal, or intravenous administration.

[0193] In one embodiment, a method of treating and / or preventing neuropathic diabetic foot ulcers is provided. In one embodiment, the method comprises administering to a patient in need thereof a prostacyclin compound or composition provided herein, e.g., a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId), or (III) or a deuterated version thereof, or a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), administering a composition comprising a compound of formula (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III), or a composition comprising a deuterated compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia"), (Ib"), (Ic"), (Id"), (II), (IIa), (IIb), (IIc), (IId) or (III). In one embodiment, administration is by inhalation (e.g., with a nebulizer or metered dose inhaler), subcutaneous, oral, nasal, or intravenous administration. In addition to the prostacyclin compounds and compositions provided herein, other pharmacologically active substances known to be useful in treating and / or preventing foot ulcers in patients with diabetic neuropathy may be present in the formulations of the present invention. For example, the compounds of the present invention may be present together with analgesics, dressing changes, vasodilator medications, and topical or oral antibiotics to treat pain.

[0194] In one embodiment, administration of an effective amount of a prostacyclin compound or composition of the present invention by inhalation, subcutaneous, oral, nasal, or intravenous administration for the treatment of the various diseases and indications described throughout results in a reduced number of side effects or a reduced severity of one or more side effects (also referred to herein as "adverse events") compared to administration of an effective amount of treprostinil when administered by inhalation, subcutaneous, oral, nasal, or intravenous administration. For example, in one embodiment, a patient treated with the methods provided herein experiences a reduced severity and / or frequency of cough or a reduced cough response when administered a prostacyclin compound or composition of the present invention by inhalation (e.g., by nebulization, dry powder inhaler, or metered-dose inhaler) compared to the severity and / or frequency of cough or cough response induced by inhalation administration of treprostinil to the patient.

[0195] In another embodiment, the prostacyclin compound administered to a patient in need of treatment has a C value that is greater than or equal to the respective lung or plasma C values ​​of treprostinil when treprostinil is administered to the patient. max Higher mean pulmonary C for treprostinil compared with max and / or lower plasma C max In another embodiment, the administration of the prostacyclin compound and treprostinil comprises intravenous administration.

[0196] In another embodiment, the prostacyclin compound administered to a patient in need of treatment is a prostacyclin compound that is effective in reducing the mean lung or plasma area under the curve (AUC) of treprostinil when treprostinil is administered to the patient. 0~t ) compared with a larger mean lung or plasma area under the curve (AUC 0~t In yet another embodiment, the prostacyclin compound administered to a patient in need thereof has a time to peak lung or plasma concentration (t) of treprostinil when treprostinil is administered to the patient. max ) compared with a longer time to peak lung or plasma concentration (tmax ) is provided.

[0197] In one embodiment, a prostacyclin compound or composition provided herein, such as a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III) or a deuterated version thereof, or a composition comprising a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ia'), (Ib'), (Ic'), (Id'), (Ia''), (Ib''), (Ic''), (Id''), (II), (IIa), (IIb), (IIc), (IId) or (III) or a deuterated version thereof, is administered in combination with one or more additional active agents. In some embodiments, such one or more additional active agents can be administered together with the prostacyclin compounds or compositions provided herein using a metered-dose inhaler. In one embodiment, such one or more additional active agents can be administered separately, i.e., prior to or subsequent to the prostacyclin compounds or compositions provided herein. The specific additional active agent that can be administered in combination with treprostinil may depend on the specific disease or condition for which treprostinil is administered to treat or prevent. In some cases, the additional active agent can be a cardiovascular therapeutic agent, such as a COX-2 inhibitor, a RHO kinase inhibitor, a calcium channel blocker, a phosphodiesterase inhibitor, an endothelial antagonist, or an antiplatelet agent.

[0198] As provided above, the prostacyclin compounds and compositions of the present invention can be delivered to patients in need thereof via oral, nasal, pulmonary, intravenous, or subcutaneous routes. For the pulmonary route, the prostacyclin compounds and compositions of the present invention can be used in any dosage-dispensing device adapted for such administration. In one embodiment, the device is configured to ensure optimal metering accuracy and compatibility of its components, such as containers, valves, and actuators, with the formulation, and can be based on a mechanical pump system, such as a metered-dose inhaler, dry powder inhaler, soft-mist inhaler, or nebulizer. For example, pulmonary delivery devices include jet nebulizers, electronic nebulizers, soft-mist inhalers, and capsule-based dry powder inhalers.

[0199] For example, suitable propellants for MDI delivery can be selected from among gases such as fluorocarbons, chlorofluorocarbons (CFCs), hydrocarbons, hydrofluoroalkane propellants (e.g., HFA-134a and HFA-227), nitrogen and nitrous oxide, or mixtures thereof.

[0200] The inhalation delivery device may be a nebulizer, a dry powder inhaler, or a metered dose inhaler (MDI), or any other suitable inhalation delivery device known to those skilled in the art. The device may contain and be used to deliver a single dose of the prostacyclin composition, or the device may contain and be used to deliver multiple doses of the composition of the present invention.

[0201] A nebulizer-type inhalation delivery device can contain the composition of the present invention as a solution, usually an aqueous solution, or a suspension. For example, a prostacyclin compound or composition can be suspended in saline and loaded into an inhalation delivery device. To generate an atomized spray of the composition for inhalation, the nebulizer delivery device can be driven ultrasonically, by compressed air, by other gases, electronically, or mechanically (e.g., by a vibrating mesh or aperture plate). A vibrating mesh nebulizer generates a low-velocity aerosol of fine particles and sprays therapeutic solutions and suspensions at a faster rate than conventional jet or ultrasonic nebulizers. Therefore, a vibrating mesh nebulizer can shorten the duration of treatment compared with a jet or ultrasonic nebulizer. Acceptable vibrating mesh nebulizers for use in the methods described herein include Philips Respironics I-Neb®, Omron MicroAir, Nektar Aeroneb®, and Pari eFlow®.

[0202] The nebulizer can be designed to be portable and handheld and can have a self-contained electrical unit. The nebulizer device can include a nozzle with two simultaneous outlet channels of predetermined opening size through which the liquid formulation can be accelerated. This results in the impaction of the two streams and atomization of the formulation. The nebulizer can be mechanically actuated. A actuator can be used to force the liquid formulation through a multi-orifice nozzle of predetermined opening size to create an aerosol of the formulation for inhalation. In single dose nebulizer designs, a blister pack containing a single dose of the formulation can be used.

[0203] In the present invention, a nebulizer can be used to ensure that the particle sizing is optimal for depositing the particles within, for example, the lung membrane.

[0204] Once atomized, the atomized composition (also referred to as an "aerosolized composition") is in the form of aerosolized particles. An aerosolized composition may be, for example, a "mass median aerodynamic diameter" or "fine particle diameter" associated with the aerosolized composition. Aerosol particle size can be characterized by measuring the "fine particle fraction." The "mass median aerodynamic diameter" or "MMAD" is normalized with respect to the aerodynamic separation of aqua aerosol droplets and is measured using impactor measurements, e.g., Anderson Cascade Impactor (ACI) or Next Generation Impactor. In one embodiment, the gas flow rates are 28 liters / minute for ACI and 15 liters / minute for NGI.

[0205] "Geometric standard deviation" or "GSD" is a measure of the spread of the aerodynamic particle size distribution. A low GSD is characterized by a narrow droplet size distribution (uniformly sized droplets), which This is advantageous in directing the aerosol toward the respiratory system. In one embodiment, the nebulized compositions provided herein have an average droplet size of less than 5 μm or from about 1 μm to about 5 μm, and a GSD in the range of 1.0 to 2.2, or from about 1.0 to about 2.2, or from 1.5 to 2.2, or from about 1.5 to about 2.2.

[0206] As used herein, "fine particle fraction" or "FPF" refers to the fraction of aerosol having particles less than 5 μm in diameter as measured by cascade impaction. FPF is usually expressed as a percentage.

[0207] In one embodiment, the nebulized composition has a mass median aerodynamic diameter (MMAD) of about 1 μm to about 5 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm, or about 1 μm to about 2 μm, as measured by an Anderson Cascade Impactor (ACI) or Next Generation Impactor (NGI). In another embodiment, the MMAD of the nebulized composition is about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less, as measured by cascade impaction, e.g., by an ACI or NGI.

[0208] In one embodiment, the MMAD of the aerosol of the pharmaceutical composition is less than about 4.9 μm, less than about 4.5 μm, less than about 4.3 μm, less than about 4.2 μm, less than about 4.1 μm, less than about 4.0 μm, or less than about 3.5 μm, as measured by cascade impaction.

[0209] In one embodiment, the MMAD of the aerosol of the pharmaceutical composition is about 1.0 μm to about 5.0 μm, about 2.0 μm to about 4.5 μm, about 2.5 μm to about 4.0 μm, about 3.0 μm to about 4.0 μm, or about 3.5 μm to about 4.5 μm, as measured by cascade impaction (e.g., by ACI or NGI).

[0210] In one embodiment, the FPF of the aerosolized composition is greater than or equal to about 50% as measured by ACI or NGI, greater than or equal to about 60% as measured by ACI or NGI, or greater than or equal to about 70% as measured by ACI or NGI. In another embodiment, the FPF of the aerosolized composition is between about 50% and about 80%, or between about 50% and about 70%, or between about 50% and about 60%, as measured by NGI or ACI.

[0211] In one embodiment, a metered dose inhaler (MDI) is used as the inhalation delivery device for the compositions of the present invention. In another embodiment, the prostacyclin compound is suspended in a propellant (e.g., a hydrofluorocarbon) prior to loading into the MDI. The basic structure of an MDI includes a metering valve, an actuator, and a container. A propellant is used to expel the formulation from the device. The composition may consist of particles of a predetermined size suspended in a pressurized propellant liquid, or the composition may be in solution or suspension in a pressurized liquid propellant. The propellants used are primarily air-friendly hydrofluorocarbons (HFCs), such as 134a and 227. The inhalation system device can deliver a single dose, for example, via a blister pack, or it may be designed for multiple doses. The pressurized metered-dose inhaler of the inhalation system can be breath-activated to deliver a precise dose of the lipid-containing formulation. The formulation delivery can be microprocessor-programmed to occur at specific points in the inhalation cycle to ensure dosing accuracy. The MDI can be portable and handheld.

[0212] In one embodiment, a dry powder inhaler (DPI) is used as the inhalation delivery device for the compositions of the present invention. In one embodiment, the DPI generates particles having an MMAD of about 1 μm to about 10 μm, or about 1 μm to about 9 μm, or about 1 μm to about 8 μm, or about 1 μm to about 7 μm, or about 1 μm to about 6 μm, or about 1 μm to about 5 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm, or about 1 μm to about 2 μm in diameter, as measured by NGI or ACI. In another embodiment, the DPI produces particles having an MMAD of about 1 μm to about 10 μm, or about 2 μm to about 10 μm, or about 3 μm to about 10 μm, or about 4 μm to about 10 μm, or about 5 μm to about 10 μm, or about 6 μm to about 10 μm, or about 7 μm to about 10 μm, or about 8 μm to about 10 μm, or about 9 μm to about 10 μm, as measured by NGI or ACI.

[0213] In one embodiment, the MMAD of particles generated by the DPI is about 1 μm or less, about 9 μm or less, about 8 μm or less, about 7 μm or less, 6 μm or less, 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less, as measured by NGI or ACI.

[0214] In one embodiment, the MMAD of particles produced by the DPI is less than about 9.9 μm, less than about 9.5 μm, less than about 9.3 μm, less than about 9.2 μm, less than about 9.1 μm, less than about 9.0 μm, less than about 8.5 μm, less than about 8.3 μm, less than about 8.2 μm, less than about 8.1 μm, less than about 8.0 μm, less than about 7.5 μm, less than about 7.3 μm, less than about 7.2 μm as measured by NGI or ACI. less than about 7.1 μm, less than about 7.0 μm, less than about 6.5 μm, less than about 6.3 μm, less than about 6.2 μm, less than about 6.1 μm, less than about 6.0 μm, less than about 5.5 μm, less than about 5.3 μm, less than about 5.2 μm, less than about 5.1 μm, less than about 5.0 μm, less than about 4.5 μm, less than about 4.3 μm, less than about 4.2 μm, less than about 4.1 μm, less than about 4.0 μm or less than about 3.5 μm.

[0215] In one embodiment, the MMAD of the particles produced by the DPI is from about 1.0 μm to about 10.0 μm, from about 2.0 μm to about 9.5 μm, from about 2.5 μm to about 9.0 μm, from about 3.0 μm to about 9.0 μm, from about 3.5 μm to about 8.5 μm, or from about 4.0 μm to about 8.0 μm.

[0216] In one embodiment, the FPF of the prostacyclin microparticle composition produced by the DPI is greater than or equal to about 40% as measured by ACI or NGI, greater than or equal to about 50% as measured by ACI or NGI, greater than or equal to about 60% as measured by ACI or NGI, or greater than or equal to about 70% as measured by ACI or NGI. In another embodiment, the FPF of the aerosolized composition is about 40% to about 70%, or about 50% to about 70%, or about 40% to about 60%, as measured by NGI or ACI. [Example]

[0217] The present invention will be further illustrated by reference to the following examples, however, as with the embodiments described above, it should be noted that these examples are illustrative only and are not to be construed as limiting the scope of the invention in any way. Example 1 Synthesis of Treprostinil Alkyl Esters

[0218] Treprostinil compounds derivatized with alkyl groups at the carboxylic acid moiety were prepared. Specifically, treprostinil was derivatized at the carboxylic acid moiety with alkyl groups at C2, C3, C4, C5, C6, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, C60, C61, C62, C63, C64, C65, C76, C77, C78, ​​C79, C80, C81, C82, C83, C84, C85, C86, C87, C88, C89, C91, C92, C93, C94, C95, C96, C97, C98, C99, C91, C92, C93, C94, C95, C96, C97, C98, C99, C99, C100, C111, C112, C113, C114, C115, C116, C117, C118, C119, C120, C121, C122, C123, C124, C125, C136, 10 , C 12 , C 16 and C 18 Alkyl chain (i.e., in the following formula (A), R2 is C2, C3, C4, C5, C6, C8, C 10 , C 12 , C 16 or C 18 Treprostinil alkyl esters were derivatized with treprostinil (which is alkyl) to produce treprostinil alkyl esters with various ester chain lengths. Treprostinil can be synthesized, for example, by the methods disclosed in U.S. Patent Nos. 6,765,117 and 8,497,393. The synthesis of prostaglandin derivatives is described in U.S. Patent No. 4,668,814. The disclosures of U.S. Patent Nos. 6,765,117, 8,497,393 and 4,668,814 are each incorporated by reference in their entirety for all purposes. [ka]

[0219] Scheme 1:

[0220] Treprostinil esterification was catalyzed by the strong acid resin Amberlyst® 15 (Rohm and Haas). Treprostinil acid was dissolved in anhydrous dioxane / alcohol (approximately 4 mL) at a concentration of 10 mg / mL. The added alcohol (R2-OH) was appropriate to create the corresponding chain length at the R2 group. As an example, for the C2 (ethyl ester) compound, the alcohol was ethanol. The molar amount of alcohol in the solvent was 10 times the molar amount of treprostinil.

[0221] Treprostinil in dioxane / alcohol solution was added to the Amberlyst resin, which was then washed and dried. For each 40 mg of treprostinil, 1 g of resin was added to a glass vial. The mixture was placed on a shaker and incubated overnight at 40°C. The liquid portion was then removed from the vial and washed twice with 3 mL of dioxane. The recovered solvent was then collected. The solvent was dried under a stream of nitrogen until evaporation ceased. The remaining treprostinil alkyl ester and nonvolatile alcohol (if a long-chain alcohol was used) were dissolved in 2 mL of hexane / ethyl acetate (1:1) and purified by liquid-liquid extraction against an equal volume of phosphate buffer, followed by water. The organic layer was then separated, dried under a stream of nitrogen, and further dried under vacuum. When a long-chain alcohol was used, an additional purification step was required to separate the alcohol by liquid chromatography. An ACE CN, 5 μm, Ultra-Inert HPLC column, 100×21.2 mm, was used with a mobile phase of hexane / propanol 98:2%.

[0222] Scheme 2:

[0223] To a solution of (1R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-hexahydro-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-1H-benz[f]inden-5-yl]oxy]acetic acid (treprostinil) (78.1 mg, 200 μmol) dissolved in 1,4-dioxane (2.0 mL) was added Amberlyst® 15 resin (2.0 g) and the alcohol R2-OH (2.0 mmol, 10 equiv.). The reaction mixture was heated to 40 °C and shaken at approximately 100 rpm for 18-196 h. The solvent was removed, and the resin was washed with acetonitrile (MeCN) (3 × 3 mL). The 1,4-dioxane and MeCN extracts were combined and dried using a gentle stream of warm N2 gas and gentle heating to yield a thick, waxy solid. The crude material was purified by 20% HCl. n The purified material was dissolved in PrOH / hexane and subjected to preparative HPLC purification. The solvent was removed from the purified material using a gentle stream of warm N2 gas and gentle heating to give an off-white waxy solid. For storage, the pure material was suspended in ethyl lactate and subjected to analytical HPLC for concentration determination.

[0224] As an example, the compound of formula (A) below was synthesized by the method of Scheme 2. [Table 1A]

[0225] A general diagram for the synthesis of ethyl esters of treprostinil is shown below in Scheme 1. The alcohol is substituted with an alkyl ester of the desired chain length (e.g., linear or branched C5-C6 with even or odd chain lengths). 18 It can be modified based on alkyl esters. [ka] Example 2 Rates of spontaneous esterase-mediated hydrolysis of treprostinil alkyl esters

[0226] Spontaneous and / or esterase-mediated hydrolysis rates were measured for the prostacyclin alkyl ester compositions shown in Table 2. Cx indicates the alkyl chain length at the R2 position of the compound of formula (A) shown above. [Table 2]

[0227] Furthermore, the rate of spontaneous hydrolysis can be determined by determining whether the carboxylic acid group is C3, C4, C5, C6, C8 or C 10 Alkyl-derivatized treprostinil compounds at 200 μM were measured in 20% ethanol at 40° C. at six time points (0 h, 1 h, 2 h, 4 h, 6 h, 24 h).

[0228] Each sample was prepared as a 200 μM solution in 20% ethanol. At each time point, an aliquot was removed for HPLC analysis, and the remaining reaction (C3, C4, C5, C6, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26 10 ) or their degradation products (treprostinil). For each sample, the hydrolysis rate was calculated from the measured peak areas of the reactants and products: % hydrolysis = (product peak area / (reactant peak area + product peak area) * 100).

[0229] The results of a time course experiment are shown in Figure 1A. The results indicate that the rate of hydrolysis correlates with the length of the alkyl ester moiety.

[0230] The rate of esterase-mediated hydrolysis of treprostinil compounds and compositions is evaluated by determining the C2, C4, C6, C8, and C9 positions in the carboxylic acid group. 10 The compounds derivatized with alkyl groups and compositions containing them were measured. The experiment was carried out at 37°C, and the hydrolysis rate was measured 15 minutes, 30 minutes, and 1 hour after adding esterase to the compound solution. The reaction mixture for each sample was prepared in a final volume of 500 μL containing 200 μM treprostinil compound, 0.05 U esterase, 20% ethanol, and PBS. The hydrolysis rate was measured as described above.

[0231] The results of this experiment are shown in Figure 1B. The results show that the rate of degradation of the compound decreases with increasing alkyl ester chain length.

[0232] The conversion of treprostinil alkyl esters to treprostinil was also measured in the presence of rat, dog, and monkey lung tissue homogenates at 37°C. Data were calculated based on an exponential growth fit to a maximum value (experiments were performed in duplicate). The results of this study are shown in Table 2A below and in Figure 37. Specifically, the left side of Figure 37 shows that the conversion rate to treprostinil depends on the alkyl chain length. In this experiment, treprostinil alkyl esters were prepared in water and incubated for 4 hours at a final concentration of 200 nM in 1 mL of tissue homogenate normalized to 10 mg / mL protein.

[0233] The right side of Figure 37 shows the effect of C in the presence of lung tissue homogenates from rats, dogs, or monkeys. 12 The conversion rate (percentage) of -TR to treprostinil is shown. 12 -TR was prepared in water and incubated for 4 hours at a final concentration of 200 nM in 1 mL of tissue homogenate normalized to 10 mg / mL protein. Both experiments in Figure 37 (left and right graphs) were performed in duplicate. The lines represent nonlinear exponential regression assuming one-phase decay. [Table 2A] Example 3 Particle size characterization of treprostinil compositions

[0234] The compositions in Table 3 were subjected to particle size characterization, where Cx represents the alkyl chain length at the R2 position of formula (A) shown above. [Table 3]

[0235] All particle size measurements were performed using a Wyatt Technology Mobius™ Zeta Potential / Particle Sizing Instrument in quasi-elastic light scattering (QELS) mode. An aliquot of the composition was diluted 10-fold with pre-filtered (0.02 μm pore filter) ultrapure deionized H2O. Light scattering data was collected and converted to particle size and size distribution using Dynamics® v.7.2.4 instrument software. The reported average particle size is based on a cumulative model, which mathematically fits the particle diffusion constant (determined by the raw scattering intensity of particles in suspension) to obtain the average particle size and the distribution of particle sizes around the average diameter.

[0236] The particle size (mean particle size) of the treprostinil composition increases in size in compositions containing C2-C5 alkyl ester derivatized treprostinil, and decreases in size in compositions containing C6-C 12 A decrease in size was observed in compositions containing alkyl ester-derivatized treprostinil. These results are shown in Figure 2. The largest mean particle size (316 nm) was found for compositions containing treprostinil pentyl ester (i.e., treprostinil derivatized with a C5 alkyl ester). The composition containing treprostinil ethyl ester had a mean particle size of 41 nm. It should be recognized that by manipulating processing parameters, the same composition can be produced with different mean diameters and size distributions. Manipulation of the composition in combination with manipulation of processing parameters can also be used to produce particles of various sizes.

[0237] It was also found that under the conditions used herein, treprostinil compounds derivatized with longer chains formed more uniform particles than compounds with shorter alkyl ester chains. Particle uniformity was determined using software-calculated polydispersity (PD%). Polydispersity is defined as the standard deviation of the particle size distribution from the mean particle size value. PD% normalizes polydispersity to the mean diameter by dividing by the mean size and multiplying by 100. These parameters indicate whether a particle suspension has one or more particle size populations (unimodal vs. multimodal). This also provides insight into the width of the particle size distribution around the mean (or the degree of particle uniformity) for each particle population.

[0238] When PD%≦15, the Dynamics® polydispersity parameter indicates a monodisperse population of particles. A calculated PD%≧57% indicates a polydisperse population of particles. For example, the PD% data plotted in FIG. 2 provides information about the uniformity of the particle size population from the treprostinil compounds tested. C8-TR (TR=treprostinil), C 10 -TR, C 12 -TR and C 14 The C2-TR, C6-TR, C6-TR alkyl esters yielded near monodisperse particles with PD% of 15 or approximately 15. 16 -TR and C 18 The C8-TR alkyl esters yielded particles with a PD% slightly above 15, suggesting that there was a single population of particles. However, these particles were not significantly different from the C8-TR, C 10 -TR, C 12 -TR and C 14 Compared with C1-TR, C2-TR, C3-TR, C4-TR, and C5-TR had a wider particle size distribution around the mean particle size. C3-TR, C4-TR, and C5-TR showed values ​​much greater than 15 PD%, with some showing values ​​≥ 57. These values ​​indicate the presence of multiple populations of particles with a wide particle size distribution. Example 4 Measurement of cyclic adenosine monophosphate (cAMP) levels in CHO-K1 cells in response to treprostinil compositions

[0239] A cell-based Chinese hamster ovary-K1 (CHO-K1) assay based on the GloSensor™ cAMP assay (Promega) was used to characterize the effects of treprostinil alkyl ester compounds on cAMP levels.

[0240] cAMP is a second messenger involved in signal transduction of G-protein-coupled receptors (GPCRs) that act through Gα-s and Gα-i proteins. Because the treprostinil receptor is a GPCR, this assay provides an indication of whether the respective prostacyclin compound (or its metabolite) binds to the receptor and activates the GPCR cell signaling cascade.

[0241] The GloSensor™ assay utilizes a genetically engineered form of firefly luciferase into which a cAMP-binding protein moiety has been inserted. Upon binding of cAMP, a conformational change is induced, resulting in an increase in light output.

[0242] The EP2 prostanoid receptor was cotransfected into CHO-K1 cells with the GloSensor™ plasmid (Promega) as follows. CHO-K1 cells were harvested when the monolayer was 50–90% confluent. First, the cells were washed with 5 mL of PBS. 2 mL of prewarmed (37°C) 0.05% trypsin-EDTA (Life Technologies, catalog number: 25300054) was added, and the cells were dislodged by gently tapping the side of the flask. Next, 10 mL of antibiotic-free growth medium (Life Tech, catalog number: 31765092) containing 10% fetal bovine serum (FBS; Hyclone, catalog number: SH30071.03) was added, and the cells were centrifuged at 250 × g for 5 minutes at room temperature. The medium was aspirated, and the cell pellet was resuspended in 10 mL of growth medium. Cell counts were determined using a hemocytometer. In each well of a culture-treated 96-well flat-bottom plate (Costar, Cat. No. 3917), 1 × 10 cells were added per 100 μL of antibiotic-free growth medium. 4 The cells were incubated overnight in a water-jacketed incubator at 37°C and 5% CO2.

[0243] For small-scale transfections of up to 20 wells, a ratio of pGLoSensor-22F cAMP plasmid (Promega, catalog number: E2301) (2 μg):(EP2) (10 ng) (Origene, catalog number: SC126558):pGEM-3Zf(+) (10 ng) (Promega, catalog number: P2271) was diluted to a final concentration of 12.6 ng / μL (total plasmid) in Opti-MEM I Reduced Serum Medium (Life Technologies, catalog number: 1985062). Next, 6 μL of FuGENE HD Transfection Reagent (Promega, catalog number: E2311) was added to 160 μL of diluted plasmid and mixed carefully by gentle pipetting. The complex was incubated at room temperature for 0-10 minutes, then 8 μL of complex was added per well of a 96-well white assay plate (Costar, Cat. No. 3917) and gently mixed without disturbing the cell monolayer. The plate was incubated for 20-24 hours at 37°C and 5% CO2 in a water-jacketed incubator. Following incubation, the cells were processed and analyzed.

[0244] For larger-scale transfections, the above steps were scaled up accordingly, and after the final incubation, the cells were frozen. To prepare frozen transfected CHO-K1 cells, the medium was aspirated from the culture flask and the cells were rinsed with 5 mL of PBS. As described above, 2 mL of pre-warmed (37°C) 0.05% trypsin-EDTA (Life Technologies, Catalog No. 25300054) was added, and the cells were dislodged by gently tapping the side of the flask. Next, 10 mL of antibiotic-free growth medium (Life Technologies, Catalog No. 31765092) containing 10% FBS (Hyclone, Catalog No. SH30071.03) was added, and the cells were centrifuged at 250 × g for 5 minutes at room temperature. Cell number was determined using a hemocytometer. The medium was aspirated, and the cell pellet was collected at 2.5 × 10 6Cells / vial in freezing media (Millipore, Cat. No. S-002-5F ) transfected cells were incubated overnight at -80°C and then transferred to liquid nitrogen for long-term storage. The frozen stock was then thawed one day before use in the assay, and cells were plated at 2.5 x 10 per well in 100 µL of antibiotic-free complete medium (F12 (Life Technologies, Cat. No. 31765092) + 10% FBS (Hyclone, Cat. No. SH30071.03)). 4 After overnight incubation at 37°C and 5% CO2 in a water-jacketed incubator, the cells were ready for the cAMP response assay.

[0245] In preparation for cAMP measurement, cells were equilibrated with GloSensor cAMP Reagent before treatment. For equilibration, the medium was carefully removed from each well. Next, 100 μL of equilibration medium (6% v / v Glosensor Reagent Stock Solution (Promega, Cat. No.: E291), 10% FBS (Hyclone, Cat. No.: SH30071.03), and 88% CO2-independent medium (Life Technologies, Cat. No.: 18045088)) was added per well of a 96-well plate to the side of each well. The plate was then incubated at room temperature for 2 hours. The first pre-read measurement was performed using a microplate reader (MicroLumat Plus). The plate was incubated for an additional 10 minutes at room temperature, followed by a second pre-read measurement.

[0246] Working solutions of free treprostinil and treprostinil alkyl ester compounds were prepared in 10x concentrated solutions to give a final concentration of 1x when added to cells. Following treatment, each plate was read every 5 minutes for the duration of the assay using a microplate reader (MicroLumat Plus). To determine the fold change in cAMP relative to the control, transfection efficiency was first determined by dividing the second pre-read measurement by the mean value of the corresponding pre-read measurement. The normalized relative light units (RLU) of the samples were then calculated by multiplying the plate read measurements by the transfection efficiency. The fold change in cAMP relative to the control was then determined by dividing the normalized RLU of the sample by the normalized RLU of the control. Validation of the cAMP assay using free treprostinil

[0247] The cAMP assay was validated using free treprostinil. Treprostinil (10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0.0001 μM, 0.00001 μM, and 0.000001 μM) was added to equilibrated CHO-K1 cells, and the cells were then incubated for 30 minutes. Luminescence was then measured at room temperature. Alkyl ester treprostinil compositions

[0248] CHO-K1 cells co-transfected with EP2 receptor and GloSensor™ plasmid were treated with free treprostinil (10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0.0001 μM, 0.00001 μM, 0.000001 μM) and treprostinil alkyl ester compounds, i.e., compounds of formula (A) shown above with C6, C8 or C at the R2 position. 10 The cells were challenged with compounds having linear alkyl groups.

[0249] The following concentrations of compound were measured: 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0.0001 μM, 0.00001 μM, and 0.000001 μM. cAMP levels were then measured every 5 minutes over an 8-hour time course. Results from the three highest concentrations are shown in Figure 3A (10 μM), Figure 3B (1 μM), and Figure 3C (0.1 μM). The components of the treprostinil compositions shown in Figures 3A, 3B, and 3C are listed in Table 4 below.

[0250] Treprostinil decyl ester (C 10 cAMP levels in response to treprostinil (-TR) (10 μM) were comparable to free treprostinil and persisted for at least 6 hours. Sustained cAMP levels were not observed in response to free treprostinil.

[0251] CHO-K1 cells co-transfected with the EP2 receptor and GloSensor™ plasmid were treated with free treprostinil (5 μM) and the compounds C2, C6, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, 10 or C 12 The animals were challenged with treprostinil compositions containing either treprostinil derivatized with a linear group (5 μM), or treprostinil derivatized with a linear group (5 μM). The components of the treprostinil compositions are shown in Table 5 below. cAMP levels were then measured every 5 minutes over an 8-hour time course.

[0252] The results of these experiments using a dose of 5 μM are shown in Figures 4 and 5. 10 The cAMP response to treprostinil alkyl esters (5 μM) was greater than or comparable to the response induced by free treprostinil (Figure 4). 10 cAMP levels in response to treprostinil alkyl ester compounds were significantly increased by free treprostinil and C6, C8, and C 12 It lasted significantly longer than the treprostinil derivative. [Table 4] [Table 5] Treprostinil Compounds

[0253] A cell-based (CHO-K1) cAMP assay was also used to characterize the effect of unformulated treprostinil compounds (i.e., compounds without hydrophobic additives and / or amphiphiles, e.g., PEGylated lipids) on cAMP levels.

[0254] CHO-K1 cells co-transfected with the EP2 receptor and GloSensor™ plasmid were treated with free treprostinil (5 μM) and C2, C3, C4, C5, C6, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C 10 or C 12 After challenge with a treprostinil derivative (5 μM) bearing a linear alkyl ester moiety, cAMP levels were then measured every 5 minutes over an 8-hour time course.

[0255] The results of these experiments are shown in Figure 5. C2 and C 10 Treprostinil alkyl esters induced cAMP response levels comparable to those of free treprostinil. 12 The derivatized treprostinil compounds were found to elicit minimal cAMP responses. Nebulized Treprostinil Ester Compositions

[0256] The cell-based (CHO-K1) cAMP assay described above was also used to characterize the effect of nebulization of various treprostinil compositions on cAMP levels.

[0257] CHO-K1 cells co-transfected with the EP2 receptor and GloSensor™ plasmid were treated with 10 μM free treprostinil (control or nebulized) and the compounds of formula (A) shown above with C2, C8, C at the R2 position. 10 or C 12The mice were challenged with 10 μM treprostinil compositions (control or nebulized) containing compounds derivatized with either a linear alkyl group.

[0258] The compositions tested in this experiment are shown in Table 6 below (with results in Figure 6). cAMP levels were then measured every 5 minutes over an 8 hour time course.

[0259] The nebulizer Aeroneb Pro (Aerogen) was used to nebulize the treprostinil derivative composition. The desired volume of formulation (usually 3 mL) was loaded onto the mesh head of the nebulizer. This head was directly connected to a glass impinger with an airtight seal. Nebulization was carried out using the factory settings until the entire sample was nebulized. After nebulization was complete, the head was removed; the impinger was capped and centrifuged at 600 × g for 5 minutes to settle the aerosol inside the impinger. This procedure ensured nearly 100% collection of the nebulized sample.

[0260] As shown in Figure 6, nebulization of the derivatized treprostinil composition did not adversely affect the cAMP response level or duration of the response. [Table 6] Comparison of Treprostinil Compounds and Compositions Containing Them

[0261] The median effective concentration (EC) of various treprostinil compounds 50 ) was determined using the results from the cAMP assay. Table 7 (below) shows the EC 20 values ​​for the cAMP response in CHO-K1 cells for the following compositions and compounds: 50 Summarize the data: T554 (C2-TR 40mol%, squalane 40mol%, Chol-PEG2k 10mol%, DOPC 10mol%), T612 (C2-TR 10mol%, DMPE-P1K 90mol%), T501 (C5-TR 40mol%, squalane 40mol%, Chol-PEG2k 20 mol%), T601 (C6-TR 40mol%, squalane 40mol%, Chol-PEG2k 10mol%, DOPC 10mol%), T555 (C8-TR 40mol%, Squalane 40mol%, Chol-PEG2k 10mol%, DOPC 10mol%), T556(C 10 -TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%), T568(C 12 -TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%), T621(C 12 -TR 10mol%, DPPE-P2K 90mol%), T623(C 16 -TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%), T622(C 16 -TR 10mol%, DPPE-P2K 90mol%), C2-TR (100 mol%), C8-TR (100 mol%), C 12 -TR (100 mol%) and Free treprostinil. A subset of dose-response curves for selected treprostinil compounds and compositions are shown in Figures 7-14. For free treprostinil, efficacy decreases with increasing incubation time (supporting an immediate response), while the various treprostinil compositions all show increasing efficacy with incubation time (suggesting a delayed-release profile). [Table 7] Example 5 Determining the Effects of Treprostinil Compounds on Cell Proliferation

[0262] To determine the effect of treprostinil compounds on cell proliferation, cell-based assays using CHO-K1 cells and rat alveolar cells (NR8383 cells) were performed. CHO-K1 cells

[0263] CHO-K1 cells were harvested when the cell monolayer was 50-90% confluent (using passage numbers 4-11). The medium was aspirated from the flask, and the cells were rinsed with 2 mL of F12 medium. Next, 1 mL of pre-warmed (37°C) 0.25% trypsin-EDTA (Life Technologies, Inc.) was added. Technologies, catalog number: 25300054) was added, and the cells were dislodged from the flask by gently tapping the side of the flask. Complete growth medium (F12 (Life Technologies, catalog number: 31765092) + 10% FBS (Hyclone, catalog number: SH30071.03) + 1X Pen-Strep (Life Technologies, catalog number 15140-122) was then added to a volume of 10 mL. The cells were centrifuged at 250 × g for 5 minutes at room temperature, and the medium was aspirated. The cell pellet was resuspended in 10 mL of complete growth medium. Cell number was determined using a hemocytometer. Cells were then seeded at 2000 cells per well of a 96-well plate in 100 μL of complete growth medium. Plates were incubated overnight at 37°C and 5% CO2 in a water-jacketed incubator.

[0264] The next day, 80 μL of fresh complete medium was added to each well, and CHO-K1 cells were challenged with the treprostinil compound and composition treatment. A working solution was prepared at 10× concentration, and after two-fold serial dilutions, 20 μL aliquots were added per well to a final 1× concentration. Following 48 hours of incubation at 37°C and 5% CO2 in a water-jacketed incubator, the inhibitory effect on cell proliferation was determined. The plates were analyzed using 20 μL of Presto Blue reagent (Life Technologies, Catalog No. A13262) per well. The reagents were mixed, and the plates were incubated for 1 hour at 37°C and 5% CO2 in a water-jacketed incubator. The plates were read using a CytoFluor Series 4000 (PerSeptive BioSystems) or a Synergy Neo microplate reader (BioTek) at emission λ: 590 nm and excitation λ: 560 nm. Percent inhibition was determined using the following formula: % inhibition = 100% - (treated sample / control x 100%). NR8383 cells

[0265] Rat alveolar NR8383 cells were harvested when the monolayer was 50–90% confluent (using passage numbers 5–11). Because NR8383 cells contain both adherent and non-adherent cells, the medium was transferred to a 50 mL Falcon tube. To obtain the remaining cells in the flask, 2 mL of plain medium was added, and the remaining cells were transferred to a 75 cm 2The cells were removed from the flask with a cell scraper and added to a 50 mL tube. The cells were centrifuged at 200 × g for 5 minutes at room temperature, and the medium was aspirated. The cell pellet was resuspended in 10 mL of complete growth medium (F12 (Life Technologies, Cat. No.: 31765092) + 15% FBS-heat inactivated (Hyclone, Cat. No.: SH30071.03) + 1X Pen-Strep (Life Technologies, Cat. No.: 15410-122)). Cell numbers were determined using a hemocytometer. The cells were then seeded at 4000 cells per well of a 96-well plate in 100 μL of complete growth medium. The plate was incubated overnight at 37°C and 5% CO2 in a water-jacketed incubator.

[0266] The next day, 80 μL of fresh complete medium was added to each well, and the NR8383 cells were challenged with the treprostinil compound treatment. Following 72 hours of incubation at 37°C and 5% CO2 in a water-jacketed incubator, the inhibitory effect on cell proliferation was determined. Measurements and calculations were performed as described above for CHO-K1 cells. Effect of treprostinil alkyl ester compositions on CHO-K1 cell proliferation

[0267] CHO-K1 cells were treated with treprostinil alkyl ester derivatives: T554 (C2-TR 40mol%, squalane 40mol%, Chol-PEG2k 10mol%, DOPC 10mol%), T543 (C6-TR 40mol%, Toco Acet 40mol%, Chol-PEG2k 10mol%), T555 (C8-TR 40mol%, Squalane 40mol%, Chol-PEG2k 10mol%, DOPC 10mol%), T556(C 10 -TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%), T568(C12 -TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%), T623(C 16 The cells were challenged with a composition containing 40 mol% treprostinil-TR, 40 mol% squalane, 10 mol% Chol-PEG2k, and 10 mol% DOPC. Following a 48-hour incubation period, the inhibitory effect of the treprostinil derivative composition on cell proliferation was determined.

[0268] Table 8 below summarizes the effects of the above treprostinil compositions on CHO-K1 cell proliferation. At a maximum concentration of 100 μM, T543 (C6-TR) and T623 (C 16 -TR) showed a significant inhibitory effect on cell proliferation. [Table 8] Effect of Treprostinil Compositions on NR8383 Cell Proliferation

[0269] Rat alveolar NR8383 cells were challenged with the same treprostinil derivative compositions:

[0270] At the same concentration as the CHO-K1 cells (0.55 μM to 125 μM), T543 (C6-TR 40mol%, Toco Ace 40mol%, Chol-PEG2k 20mol%), T554 (C2-TR 40mol%, squalane 40mol%, Chol-PEG2k 10mol%, DOPC 10mol%), T555 (C8-TR 40mol%, Squalane 40mol%, Chol-PEG2k 10mol%, DOPC 10mol%), T556(C 10 -TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%), T568(C 12-TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%) and T623(C 16 -TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%). Following a 72 hour incubation period, the inhibitory effect of the treprostinil derivative composition on cell proliferation was determined.

[0271] Table 8 summarizes the effects of the treprostinil compositions on NR8383 cell proliferation. At the maximum dose of 100 μM, all of the treprostinil derivative compositions showed some inhibition of cell proliferation, with T543 (C6-TR) showing the highest inhibitory effect. Effects of treprostinil alkyl ester compounds on cell proliferation

[0272] To determine the effect of treprostinil derivative compounds (unformulated) on cell proliferation, the cell-based assays described above were performed using CHO-K1 cells and rat alveolar cells (NR8383 cells). CHO-K1 cell proliferation assay

[0273] CHO-K1 cells were treated with treprostinil alkyl esters, i.e., the following R2 groups: C2, C3, C4, C5, C6, C8, C 10 or C 12 The cells were challenged with TR compounds of formula (A) having a linear alkyl chain at dosages ranging from 0.098 μM to 25 μM. Following a 48-hour incubation period, the inhibitory effect on cell proliferation was determined.

[0274] The effects of the above treprostinil alkyl esters on CHO-K1 and NR8383 cell proliferation are summarized below in Table 9. At the highest concentration, only the treprostinil octyl ester compound showed inhibition of cell proliferation. [Table 9] NR8383 cell proliferation assay

[0275] Rat alveolar NR8383 cells were cultured with C2, C3, C4, C5, C6, C8, and C at the R2 position of formula (A) at concentrations ranging from 0.195 μM to 25 μM. 10 or C 12 The cells were challenged with a treprostinil compound derivatized with a linear alkyl moiety, and following a 72 hour incubation period, the inhibitory effect on cell proliferation was determined.

[0276] The effects of the treprostinil alkyl esters on NR8383 cell proliferation are summarized in Table 11. Similar to the CHO-K1 cell assay, only the treprostinil octyl ester showed some inhibition of cell proliferation at the highest concentration. Treprostinil Derivative Compositions - Effects on Cell Proliferation

[0277] To determine the effect of the treprostinil derivative compositions on cell proliferation, cell-based assays were performed using CHO-K1 cells and rat alveolar cells (NR8383 cells). Effect of Treprostinil Compositions on CHO Cell Proliferation

[0278] CHO-K1 cells were cultured in a treprostinil derivative composition: T596(C2-TR 45mol%, DSG-P2K 55mol%), T597(C6-TR 45mol%, DSG-P2K 55mol%), T598(C8-TR 45mol%, DSG-P2K 55mol%), T599(C 10 -TR 45mol%, DSG-P2K 55mol%) and T600(C 12 -TR 45mol%, DSG-P2K 55mol%), T612(C2-TR 10mol%, DMPE-P1K Cells were challenged with T613 (C8-TR 10 mol%, DMPE-P1K 90 mol%) at concentrations ranging from 0.23 μM to 29 μM. Following a 72-hour incubation period, the inhibitory effect on cell proliferation was determined. Following a 48-hour incubation period, the inhibitory effect on cell proliferation was determined.

[0279] The effects of treprostinil compositions on CHO-K1 cell proliferation are summarized below in Table 10. None of the compositions tested showed a significant inhibitory effect on CHO-K1 cell proliferation. [Table 10]

[0280] Similarly, CHO-K1 cells were challenged with treprostinil compositions T612 (R2=C2) and T613 (R2=C8) at concentrations ranging from 1.41 μM to 180 μM. After 48 hours, the inhibitory effect on cell proliferation was determined, and all four treprostinil compositions showed 100% inhibition of cell proliferation at higher concentrations. Effect of Treprostinil Compositions on NR8383 Cell Proliferation

[0281] Rat alveolar NR8383 cells were cultured at the same concentrations (0.23 μM to 29 μM) as those used for CHO-K1 cells, with the same treprostinil compositions (as described above) as well as T596 (C2-TR 45 mol%, DSG-P2K 55 mol%), T612 (C2-TR 10 mol%, DMPE-P1K 90 mol%), T597 (C6-TR 45 mol%, DSG-P2K 55 mol%), T598 (C8-TR 45 mol%, DSG-P2K 55 mol%), T613 (C8-TR 10 mol%, DMPE-P1K 90 mol%), T599 (C 10 -TR 45mol%, DSG-P2K 55mol%), T600(C 12 The cells were challenged with 45 mol% DSG-TR and 55 mol% DSG-P2K. Following a 72-hour incubation period, the inhibitory effect on cell proliferation was determined.

[0282] Table 10 above summarizes the effect of treprostinil compositions on NR8383 cell proliferation. All of the treprostinil compositions showed some (≦10%) inhibition of NR8383 cell proliferation. Example 6 Treprostinil compounds in vivo

[0283] The in vivo effects of treprostinil derivative compounds were determined using a rat model. Young male Sprague-Dawley rats (Charles River) were used for the study. Rats were anesthetized with ketamine / xylazine, placed on a heating pad, and their tracheas were surgically isolated and catheterized, and then mechanically ventilated throughout the study.

[0284] A catheter was placed in the femoral artery to measure systolic (sys) and diastolic (dias) blood pressure. A thoracotomy was performed, and a catheter was inserted into the right ventricle and placed in the pulmonary artery to measure pulmonary artery systolic and diastolic blood pressure. Oxygen saturation (SaO2) was measured with a pulse oximeter placed on the foot.

[0285] Cardiovascular measurements were performed under these normoxic conditions in rats ventilated with room air (FiO2 = 0.21). To induce hypoxia, FiO2 was reduced over 30 min until SaO2 fell to a value of 50-60%, and baseline hypoxic values ​​for each of the parameters were determined.

[0286] Groups of four rats were treated with PBS, free treprostinil (1.7 μg / kg and 10 μg / kg), or C2-TR (T554); C8-TR (T555: C8-TR 40 mol%, squalane 40 mol%, Chol-PEG2k 10 mol%, DOPC 10 mol%) (38.6 μg / kg), C 10 -TR(T556:C 10 -TR 40mol%, squalane 40mol%, Chol-PEG2k 10mol%, DOPC 10mol%)(40.8μg / kg)), C12 -TR(T568) was administered.

[0287] As shown in Table 11 below, due to differences in molecular weight of the treprostinil derivative compositions, the target doses were varied slightly by weight. The actual lung doses achieved were approximately 5x lower than those shown in Table 11 (e.g., a dose of 10 μg / kg resulted in approximately 2 μg / kg in the lungs). The various treatments were delivered (by inhalation of nebulized drug into the lungs of rats). The pulmonary artery pressure (PAP), systemic arterial pressure (SAP), and heart rate of the rats were continuously measured for 180 minutes. The PAP signal was collected at 200 points per second. [Table 11]

[0288] Normalized variations in mean PAP (mPAP) are shown in Figure 15 as a percentage from the hypoxic baseline value at (T=0). The hypoxic baseline PAP value was 100% and changes in pressure were measured relative to the hypoxic baseline. Normalized variations in mean SAP (mSAP) are shown in Figure 16 as a percentage from the hypoxic baseline value. Heart rate is shown in Figure 17 as a percentage of the hypoxic baseline value over time. Example 7 Measurement of cyclic adenosine monophosphate (cAMP) levels in CHO-K1 cells in response to 5-nonanyl-TR

[0289] A cell-based Chinese hamster ovary-K1 (CHO-K1) assay based on the GloSensor™ cAMP assay (Promega) was used as described above in Example 4 to characterize the effects of the following compounds on cAMP levels: 5-nonanyl-TR, i.e., R2 = 5-nonanyl [ka] a compound of formula (A) C12 -TR, i.e., R2=C 12 Alkyl [ka] a compound of formula (A) C 14 -TR, i.e., R2=C 14 Alkyl [ka] a compound of formula (A) C 16 -TR, i.e., R2=C 16 Alkyl [ka] A compound of formula (A)

[0290] CHO-K1 cells co-transfected with the EP2 receptor and GloSensor™ plasmid were treated with 5-nonanyl-treprostinil (branched chain, 5C9-TR), or the compound with C at the R2 position. 12 , C 14 or C 16 The rats were challenged with treprostinil alkyl ester compounds with either a linear alkyl group or a treprostinil alkyl ester. cAMP levels were then measured every 5 minutes over an 8-hour time course. 14 -TR and C 16 Dose-response curves for -TR at incubation times of 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 hours are shown in Figures 18, 19 and 20, respectively. 14 -TR and C 16 Similar to treprostinil-TR, the potency of 5-nonanyl-TR increased with incubation time, indicating a delayed release profile. 50) was determined using the results from the cAMP assay. 14 -TR and C 16 -EC about TR 50 are shown in Figures 18, 19 and 20, respectively.

[0291] C at 10 μM (top panel) and 5 μM (bottom panel) concentrations 12 -TR, C 14 -TR, C 16 The kinetic profile results from 5-nonanyl-TR or 5-nonanyl-TR are shown in Figure 21. C at both concentrations 12 -TR, C 14 cAMP levels in response to treprostinil-TR and 5-nonanyl-TR increased over the first 1 to 1.5 hours and were sustained for at least 8 hours. 12 -TR>C 14 -TR>5-nonanyl-TR>C 16 -TR.

[0292] The results of this study show that C 12 , C 14 or C 16 Similar to treprostinil alkyl ester compounds with linear alkyl ester groups, 5-nonanyl-TR is functional and has been shown to exhibit sustained cAMP activity. Thus, unlike free treprostinil (see Example 4), 5-nonanyl-TR has a delayed release profile. Example 8 C 14 Comparison of cyclic adenosine monophosphate (cAMP) activation in CHO-K1 cells in response to -TR formulations

[0293] A cell-based Chinese hamster ovary-K1 (CHO-K1) assay based on the GloSensor™ cAMP assay (Promega) was used as described above in Example 4 to assess the effect of different C α-glucan-1 (CHO-K1) on cAMP levels. 14 The effects of the C-TR formulation were characterized. 14-TR formulations are shown below in Table 12. Composition T679 does not contain DOPC; composition T647 does not contain DOPC or squalane. C 14 The structure of -TR is: [ka] [Table 12]

[0294] CHO-K1 cells co-transfected with the EP2 receptor and GloSensor™ plasmid were transfected with C at the carboxylic acid position. 14 The animals were challenged with treprostinil alkyl ester formulations having a linear alkyl ester group and components as shown in Table 12. cAMP levels were then measured every 5 minutes over an 8 hour time course.

[0295] Dose response curves for compound T679 at incubation times of 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours are shown in Figure 22. The potency of T679 increased over the incubation time, indicating a delayed release profile. Results from the cAMP assay were used to determine the half maximal effective concentration (EC 50 ) was determined, which is also shown in Figure 22.

[0296] A comparison of the kinetic profiles for free treprostinil, T631, and T679 at 10 μM (top panel) and 5 μM (bottom panel) is shown in Figure 23. Both T631 and T679 were less potent than free treprostinil. However, unlike free treprostinil, cAMP activation increased over time in response to both T631 and T679 and was sustained for at least 8 hours. The results of the study showed that the DOPC-free C 14 -TR T679 formulation is functional and C 14-TR T631, showing a delayed release profile similar to that of T631.

[0297] Dose-response curves for compound T647 at incubation times of 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours are shown in Figure 24. Similar to T679, the potency of T647 increased over the incubation time, indicating a delayed release profile. Results from the cAMP assay were used to determine the half maximal effective concentration (EC 50 ) was determined, which is also shown in Figure 24.

[0298] A comparison of the kinetic profiles for free treprostinil, T631, and T647 at 10 μM (top panel) and 5 μM (bottom panel) is shown in Figure 25. Both T631 and T647 were less potent than free treprostinil. However, unlike free treprostinil, cAMP activation increased over time in response to both T631 and T647 and was sustained for at least 8 hours. The results of the study demonstrated that C647, which does not contain DOPC or squalane, significantly increased cAMP activity. 14 -TR T647 formulation is functional and C 14 -TR T631 was shown to exhibit a delayed release profile similar to that of T631. Example 9 Functional cAMP studies on treprostinil alkyl ester nanoparticle formulations

[0299] A cell-based Chinese hamster ovary-K1 (CHO-K1) assay based on the GloSensor™ cAMP assay (Promega) was used as described above in Example 4 to characterize the effect of treprostinil compositions on cAMP levels. The cAMP profiles of the following treprostinil compositions were tested in this study (see also Table 13): T555:C8-TR (i.e., R2 = [ka] a compound of formula (A) T556:C 10 -TR (i.e., R2 = [ka] a compound of formula (A) T568:C 12 -TR (i.e., R2 = [ka] a compound of formula (A) T631:C 14 -TR (i.e., R2 = [ka] a compound of formula (A) T623:C 16 -TR (i.e., R2 = [ka] a compound of formula (A) T637:C 18 -TR (i.e., R2 = [ka] a compound of formula (A) [Table 13]

[0300] CHO-K1 cells co-transfected with the EP2 receptor and GloSensor™ plasmids were challenged with the treprostinil alkyl ester compositions listed above, and cAMP levels were then measured every 5 minutes over an 8-hour time course.

[0301] Composition T637(C 18 Dose-response curves for T679 (-TR) at 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hour incubation times are shown in Figure 26. The potency of T679 increased over the initial incubation period and then maintained at sustained levels for at least 8 hours, indicating a delayed release profile. Results from the cAMP assay were used to determine the half maximal effective concentration (EC 50 ) was determined, which is also shown in Figure 26.

[0302] Figure 27 shows a comparison of the kinetic profiles for free treprostinil, T555, T556, T568, T631, T623, and T637 at 10 μM (upper panel) and 5 μM (lower panel). Each of the treprostinil alkyl ester compounds was less potent than free treprostinil. However, unlike free treprostinil, cAMP activation increased and then maintained at sustained levels for at least 8 hours in response to each of the treprostinil alkyl ester compounds. This indicates that each of these compounds is functional and exhibits a delayed-release profile. The activity ranking for these compounds was T555 / T556>T568>T631>T623>T637. Example 10 Kinetics of enzymatic conversion of branched treprostinil compounds.

[0303] A series of studies was conducted to determine the conversion kinetics of linear versus various branched treprostinil compounds to treprostinil. 0.4 mM linear C8-TR or the branched treprostinil compounds 2-dimethyl-1-propanyl-TR, 3,3-dimethyl-1-butanyl-TR, 2-ethyl-1-butanyl-TR, 5-nonanyl-TR, or 3-pentanyl-TR (see below for structures) was incubated with 0.2 U esterase at 37°C for 1 hour, and the conversion rate (% of total) was calculated for incubation times of 0.25, 0.5, 0.75, or 1 hour. C8-TR (i.e., R2 = [ka] a compound of formula (A) 2-dimethyl-1-propanyl-TR (i.e., R2 = [ka] a compound of formula (A) 3,3-dimethyl-1-butanyl-TR (i.e., R2 = [ka] a compound of formula (A) 2-Ethyl-1-butanyl-TR (i.e., R2 = [ka] a compound of formula (A) 5-nonanyl-TR (i.e., R2 = [ka] a compound of formula (A) 3-pentanyl-TR (i.e., R2 = [ka] a compound of formula (A)

[0304] FIG. 28 shows that 5-nonanyl-TR exhibited a slower conversion rate than linear C8-TR.

[0305] FIG. 29 shows the esterase-mediated conversion rates to treprostinil of the following treprostinil compounds: C8-TR, C9-TR, C 10 -TR, C 12 -TR, C 14 -TR and C 16 -TR, i.e., R2, is as follows: The conversion rate is for the C8-TR compound and was measured after 1 hour of esterase incubation. C8-TR (i.e., R2 = [ka] a compound of formula (A) C9-TR (i.e., R2 = [ka] a compound of formula (A) C 10 -TR (i.e., R2 = [ka] a compound of formula (A) C 12 -TR (i.e., R2 = [ka] a compound of formula (A) C14 -TR (i.e., R2 = [ka] a compound of formula (A) C 16 -TR (i.e., R2 = [ka] a compound of formula (A)

[0306] 30 shows the esterase-mediated conversion of branched treprostinil compounds (below) to treprostinil relative to the esterase-mediated conversion of C8-TR to treprostinil. Conversion rates were measured after 1 hour of esterase incubation. 4C7-TR (i.e., R2 = [ka] a compound of formula (A) 4C8-TR (i.e., R2 = [ka] a compound of formula (A) 3C8-TR (i.e., R2 = [ka] a compound of formula (A) 2C8-TR (i.e., R2 = [ka] a compound of formula (A) 5C9-TR (i.e., R2 = [ka] a compound of formula (A) 2C9-TR (i.e., R2 = [ka] a compound of formula (A)

[0307] Each of the branched compounds 4C7-TR, 4C8-TR, and 5C9-TR showed a slower conversion rate than the linear compound C8-TR. The asymmetric branched compound (4C8-TR) showed a slower conversion rate than the symmetric compounds (4C7-TR and 5C9-TR). Furthermore, there was no significant difference between the conversion rates of the R and S isomers of 2C8-TR ((R)-2C8-TR vs. (S)-2C8-TR). Example 11 Determination of treprostinil pharmacokinetics in rats

[0308] Table 14 shows the treprostinil alkyl ester formulations used in this study. The first three compositions (T568, T631, and T623) are believed to form lipid nanoparticles, while the last three compositions (T630, T635, and T636) are believed to form micelles. [Table 14]

[0309] T568 and T630:C 12 -TR (i.e., R2 = [ka] a compound of formula (A)

[0310] T631 and T635:C 14 -TR (i.e., R2 = [ka] a compound of formula (A)

[0311] T623 and T636:C 16 -TR (i.e., R2 = [ka] a compound of formula (A)

[0312] Nebulized treprostinil (TRE) solution and treprostinil alkyl ester formulations were administered (15 nmol / kg or 6 mg / kg) by nose-only inhalation (24-hour study) to anesthetized, ventilated rats (6-hour study) or conscious rats. Blood and lung samples were collected at specific time points. The concentrations of TRE and treprostinil alkyl esters in plasma and lung tissue were measured by HPLC / MS / MS analysis. Anesthetized and ventilated rats

[0313] Male Sprague-Dawley rats were anesthetized and prepared with an endotracheal tube for ventilation. The right femoral vein was cannulated to facilitate blood collection. Terminal lung samples were collected for analysis no later than 6 hours after dosing. An Aeroneb® nebulizer and control unit (Aerogen, Galway, Ireland) was used to generate an aerosol with a median aerodynamic diameter (MMAD) of 2.5 μm to 4 μm at a rate of 0.1 mL / min, providing an estimated lung dose of 6 μg / kg. A SAR-830 / AP small animal ventilator (CWE Inc., Ardmore, PA) set at a ventilator tidal volume (VT) of 8 mL / kg and a rate of 90 breaths / min was used to deliver a 250 μL volume of nebulized test article. Systemic blood pressure (mSAP), heart rate (HR) and arterial oxygen saturation (SaO2). Physiological parameters were measured during normoxia (fraction of inspired oxygen [FrO2] = 0.21, SaO2 ≈ 90%) and during hypoxia (FrO2 = 0.10, SaO2 ≈ 50%) for 2–3 h. Conscious nose-only inhalation

[0314] Male Sprague-Dawley rats were placed in a restraining tube and J Nebulized drug exposure was achieved using an Aeger-NYU nose-only, directed-flow inhalation exposure system (CH Technologies, Westwood, NJ) (FIG. 31). Test articles (6 mL at a specified concentration) were nebulized using an Aeroneb nebulizer to deliver a predetermined estimated lung dose. Blood and lung tissue samples were collected at selected times after drug nebulization over a 24-hour period.

[0315] Ventilated rats treated with nebulized TRE solution showed a maximum plasma concentration (C max ) (3.5 ng / mL), which occurred immediately after administration (Figure 32, left side). Measurable levels of TRE were not seen in plasma for more than 4 hours and in lungs for up to 6 hours. In contrast, ventilated rats treated with nebulized TPD-LNP had lower plasma TRE C levels, ranging from 0.2 ng / mL to 0.6 ng / mL. max values ​​(Table 15, Figure 32, left side). At 6 hours, treprostinil alkyl esters remained in the lungs at levels ranging from 100 ng / g to 400 ng / g tissue TRE equivalent (Figure 33). The treprostinil detected in the lungs was presumably generated by treprostinil alkyl ester hydrolysis during sample preparation. When administered with micellar TPD, plasma levels of TRE were higher than with TPD lipid nanoparticle formulations, indicating that the nanoparticles play an additional role in the delayed-release effect (Figure 32, compare left and right graphs). In a 24-hour study in rats administered the TPD-lipid nanoparticle formulation (nose-only inhalation), TRE Cmax was higher than in ventilated animals, indicating a near first-order exponential decline. 14 - and C 16 Plasma concentrations of TRE in rats treated with the treprostinil alkyl ester lipid nanoparticle formulation were maintained above 0.1 ng / mL for up to 24 hours (a level comparable to activity in acute hypoxia studies) (Figure 34, top). Lung levels of total TRE+TPD were approximately 10 times higher than plasma TRE. 3 The inhaled C was 2-fold higher and also showed an exponential decline in rats administered the treprostinil alkyl ester lipid nanoparticle formulation (Figure 34, bottom). Table 16 further shows the pharmacokinetics of treprostinil in rats after administration via a nose-only system with a nebulized treprostinil alkyl ester lipid nanoparticle formulation at an estimated lung dose of 6 μg / kg. Figure 35 shows the inhaled C 16 This further demonstrates that the 24-hour release kinetics of treprostinil from the -TR formulation is independent of dose (nose-only administration). The times in Figure 35 correspond to the time after the start of nebulization of 6 mL of suspension (nebulization duration was 30 to 60 min). Figure 38 shows that animals treated with T568 and T623 had a survival benefit (survival for over 200 minutes) compared with animals treated with free treprostinil or PBS. Specifically, Figure 38 shows pulmonary artery pressure in animals treated with various lipid nanoparticle formulations, PBS, and treprostinil. Furthermore, treatment with T568 and T623 was shown to have little effect on systemic hemodynamics (Figure 39). Finally, the treprostinil alkyl ester nanoparticle formulation was shown to gradually convert to treprostinil, resulting in consistent plasma levels with low peak values ​​(Figure 40). [Table 15] [Table 16]

[0316] Inhaled TPD has a long-term presence in the lungs, associated with a slow, sustained release of TRE into the blood, and this duration of activity is increased with TPD formulated in lipid nanoparticles. Example 12 C in dogs 16 Pharmacokinetic profile of -TR alkyl ester lipid nanoparticle formulation

[0317] Twelve beagle dogs of either sex were treated with different inhaled doses of treprostinil in PBS, both given via nebulizer, or the compound of formula (A) formulated in a lipid nanoparticle formulation suspended in PBS (where R = [ka] )(C 16 Patients were randomly assigned to receive either the 2- or 3-mg dose of acetaminophen (-TR) (T623) (see Table 14). The formulation was nebulized using an Aeroneb nebulizer (MMAD: 2.5-4 μm) and delivered to a 500 ml expansion chamber. The formulation was nebulized for 2 min with ventilator settings of 90 ml / breath and 15 breaths / min (delivered volume = 2.7 L) and collected on a filter. The amount of drug (μg) on ​​the filter was measured by HPLC to calculate the concentration of drug (μg / L) delivered through the ventilator circuit.

[0318] Dosimetry was performed in dogs anesthetized with propofol and nebulized The drug was introduced into a mixing chamber inserted onto the inspiratory rim of the canine respirator. Before each experiment, a technical trial was performed to measure the concentration of drug (μg / L) delivered for each breath. The inhaled drug dose (μg / kg) was calculated using the formula: inhaled drug dose (μg / kg) = drug concentration (μg / L) × minute ventilation (L / min) × Calculations were made using time (min) / body weight (kg). After drug delivery, the dogs were removed from the respirator and blood samples were collected over 72 h to measure treprostinil plasma concentrations by HPLC / MS / MS. Clinical signs were monitored over this 72 h period.

[0319] The use of an anesthetized, intubated, and ventilated approach provided reproducibility between dogs and demonstrated the efficacy of treprostinil (5±1 and 16±2 μg / kg) and C 16 The target inhaled dose was achieved for both -TR (7±1, 22±1, 46±1, and 95±1 μg / kg). Treprostinil plasma C for dogs receiving treprostinil (2.7 and 5.9 ng / ml, respectively) at inhaled doses of 5 and 16 μg / kg. max The values ​​are C values ​​for similar inhaled doses (7 and 22 μg / kg) in the T623 formulation (0.2 and 0.3 ng / mL, respectively). 16 The treprostinil levels were 15-20 times higher than those achieved with inhaled T623 (Figure 36). Furthermore, plasma levels of treprostinil were sustained over 48 hours with inhaled T623 but disappeared within hours after inhalation of treprostinil (Figure 36). Coughing and rapid shallow breathing were absent during delivery of treprostinil to anesthetized, ventilated dogs, but were present during the recovery period. Dogs receiving T623 showed no signs of respiratory irritation at inhaled doses as high as 46 μg / kg. C 16 Alkyl ester lipid nanoparticle treprostinil formulations and C 12 and C 14 Comparison of alkyl ester lipid nanoparticulate treprostinil formulations

[0320] Twelve beagle dogs were treated with inhaled treprostinil and three treprostinil alkyl ester lipid nanoparticle formulations: T568 (dodecyl-treprostinil, C 12 -TR), T631 (tetradecyl-treprostinil, C 14 -TR) and T623 (hexadecyl-treprostinil, C 16-TR). The components of each formulation are shown in Table 14 above.

[0321] Posology measurements were performed in propofol-anesthetized, artificially ventilated dogs; nebulized drug was introduced into a mixing chamber inserted above the inspiratory rim of the respirator. A technical trial was performed before each experiment, measuring the drug concentration per breath (μg / L), minute ventilation, and the time required to achieve the target lung dose. After recovery from anesthesia, blood samples were collected over 72 h, and plasma levels of TRE were measured by HPLC / MS / MS. Clinical signs (coughing, rapid shallow breathing, vomiting, and pale gums) were also monitored.

[0322] At a target lung dose of 18 μg / kg, plasma levels of treprostinil were significantly higher than free treprostinil (C max =5.9±0.6ng / ml), but C 12 -TR, C 14 -TR and C 16 -TR max The values ​​were 5-, 13-, and 20-fold lower. Plasma treprostinil was below the level of quantification for up to 4 h after inhalation of free treprostinil, but persisted for 48 to 72 h after inhalation of the treprostinil alkyl ester formulation.

[0323] C max and AUC dose-dependent increases were observed after inhalation C 16 A prolonged presence of treprostinil in plasma was observed with TR (6-90 μg / kg), up to 72 h at higher doses. Free treprostinil and C at the target dose of 18 μg / kg 12 - Adverse clinical signs were observed in TR but C 14 -TR and C 16 -TR was not observed. 16 In a dose-response study with -TR, adverse clinical signs were seen in only one dog at a target lung dose of 90 μg / kg.

[0324] Based on PK studies in dogs, the inhaled C 16 -TR results in a sustained presence of treprostinil in the plasma and a lower potential for side effects than inhaled free treprostinil at comparable doses. Example 13 Lipid Nanoparticle C 16 Characterization of alkyl ester treprostinil formulations

[0325] T748, lipid nanoparticles C with the following ingredients: 16 Alkyl ester treprostinil formulations were characterized. [Table 16A]

[0326] Evaluation of the tolerability and pharmacokinetics (PK) of treprostinil in rats administered T748 lipid nanoparticle formulation

[0327] Inhalation C 16 To assess whether repeated dosing with -TR is well tolerated and alters PK, rats were treated with C 16 -TR.

[0328] Five groups of Sprague-Dawley rats (n=4 per group) were treated with either inhaled phosphate-buffered saline (PBS) or four doses of C given by nebulization in a nose-only inhalation chamber. 16 Cohorts of rats were exposed to 100 mg / kg of 10 ... 16 After daily inhalation administration of treprostinil-TR for 1, 7, and 14 days, blood samples were taken at 1, 3, 6, and 24 hours, and lung samples were collected 24 hours after the last administration of the drug. 16 The concentration of -TR was measured by HPLC / MS / MS. Body weight was recorded daily, and organ weights (lung, heart, liver) were measured 24 hours after the last drug administration.

[0329] 14 consecutive days C 16There were no tolerability issues or significant changes in body and organ weights (vs. PBS) after inhalation of C-TR. 16 Increasing the inhaled dose of -TR (0.6-18 μg / kg) increased plasma C max The C and AUC increased, but this was not consistently affected with repeated dosing. There was some variability in AUC between days 1 and 14 among the different dose groups, with two of the four doses (1.8 and 18 μg / kg) showing no difference, and the other two doses (0.6 and 6 μg / kg) showing a 3- to 4-fold increase in AUC by day 14. 16 The presence of -TR was not detected at any dose. However, relatively high concentrations of C 16 -TR (approximately 1,000-fold higher than plasma treprostinil) was found in the lungs. 16 -TR is the C 16 -induced a dose-dependent increase in the concentration of TR, which was unchanged by repeated administration for 14 consecutive days.

[0330] Inhalation C 16 -TR (0.6-18 μg / kg) was well tolerated, with no evidence of changes in body or organ weights after 14 consecutive days of administration. C on the cough reflex in guinea pigs 16 Efficacy of alkyl ester lipid nanoparticle treprostinil formulations

[0331] In this study, inhaled treprostinil and the alkyl ester hexadecyl-treprostinil (C 16 The antitussive effect of a lipid nanoparticle formulation of acetaminophen (-TR) was studied in guinea pigs.

[0332] Three groups of male Dunkin Hartley guinea pigs were placed in a whole-body The subjects were then subjected to a thysmograph and administered aerosolized phosphate-buffered saline (PBS), TRE (1–300 μg / ml), and C 16-TR lipid nanoparticle formulation T748 (30 μg / ml). T623 has the following components:

[0333] The aerosol was mixed with inspired air delivered at a rate of 2 L / min. Coughs were generated using a Pro nebulizer (nebulizer output 0.36 mL / min). PBS or drug was delivered for 10 min, and the number of coughs was recorded during and 20 min after delivery. Coughs were detected by visual observation, plethysmographic recording, and cough sounds.

[0334] Exposure to aerosolized PBS did not induce coughing. TRE exposure did not consistently induce coughing in the animals tested until exposure concentrations equaled or exceeded 30 μg / mL. The cough response was characterized by frequent coughing bouts with a lower coughing sound compared to the typical coughing sound induced by citric acid or capsaicin. TRE at a 30 μg / mL nebulized concentration produced consistent coughing in seven of seven guinea pigs, with one to four coughing bouts and a total cough count averaging 36 ± 9 coughs. In contrast, inhaled CRE at a 30 μg / mL nebulized concentration produced consistent coughing in seven of seven guinea pigs, with one to four coughing bouts and a mean total cough count of 36 ± 9 coughs. 16 The -TR lipid nanoparticle formulation did not induce coughing in 6 of 6 guinea pigs, with no events.

[0335] The results of this study demonstrate that inhaled TRE induces cough in guinea pigs, the profile of which is somewhat similar to that previously described with inhaled prostaglandins in guinea pigs (type II cough) (Maher and Belvisi, 2010). 16 The -TR lipid nanoparticle formulation did not induce coughing, suggesting that this formulation may eliminate some of the local adverse side effects, such as coughing, seen with inhaled TRE therapy in humans. Example 14 Acylation of Treprostinil Derivatives

[0336] Treprostinil or a treprostinil ester derivative (eg, derivatized with an alkyl or alkenyl group in the carboxylic acid moiety as prepared in Example 1) is acylated as follows.

[0337] The compound of Example 1 (0.05 mol) or treprostinil is dissolved in 10 mL of dichloromethane at 0° C. Dimethylaminopyridine is added (20 mol%), and then a solution of acyl chloride R(CO)Cl (R is R5 or R6 as described herein) (2.1 equivalents) at 0° C. is added to the compound of Example 1 or treprostinil. The solution is stirred and warmed to 23° C. for 1 hour. The reaction is monitored by thin-layer chromatography. If no further change is observed, the reaction is quenched with NaHCO3(sat), and the quenched mixture is extracted with dichloromethane (3×10 mL). The combined organic extracts are dried over anhydrous sodium sulfate, and the solvent is removed under vacuum to obtain the crude product. Purification is carried out by column chromatography on silica gel using 2% methanol in dichloromethane.

[0338] A general scheme for the synthesis of acylated treprostinil derivatives is shown below (R2 is depicted herein as, for example, H or a straight-chain or branched alkyl group): [ka]

[0339] Other acylation techniques known in the art can be used, including selective acylation of each secondary alcohol. Furthermore, R2 can be selected so that after acylation of the secondary hydroxyl function, the R2 group is selectively removed from the compound of Example 11. Such protecting group strategies are well known to those skilled in the art and are described, for example, in Peter GM Wutes and Theodora W. Greene, Greene's Protective Groups in Organic Synthesis, 4th Edition, Wiley (2006), which is incorporated herein by reference in its entirety for all purposes. An exemplary scheme of such a process is shown below: [ka]

[0340] C 16 Synthesis of TR-OAc: [ka]

[0341] To a solution of (1R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-hexahydro-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-1H-benz[f]inden-5-yl]oxy]acetic acid (treprostinil) (78.1 mg, 200 μmol) dissolved in 1,4-dioxane (2.0 mL) was added triethylamine (TEA) (98 μL, 700 μmol, 3.5 equiv.), acetic anhydride (166 μL, 1,760 μmol, 8.8 equiv.), and a catalytic amount of dimethylaminopyridine (DMAP). The reaction mixture was shaken at 40°C for 72 h. The solvent was removed under reduced pressure to give a thick, colorless oil. This crude material was dissolved in hexane and washed with a solution of saturated NaHCO3 (3 × 5 mL). The organic layers were combined and the solvent was removed using a gentle stream of warm N2 gas and gentle heating to give a thick colorless oil. nThe product was dissolved in PrOH / hexane, passed through a 0.45 μm syringe filter, and subjected to preparative HPLC purification. The solvent was removed from the purified material using a gentle stream of warm N2 gas and gentle heating to give a thick, colorless oil. The pure material was suspended in ethyl lactate for storage and subjected to analytical HPLC for concentration determination.

[0342] C 16 -TR-OAc: 73% overall yield. This compound was also characterized by NMR spectroscopy:

[0343] 1 H NMR (500 MHz, CDCl3) δ 0.89 (t, J = 7.0 Hz, 6H), 1.17-1.32 (m, 33H), 1.43-1.46 (m, 2H), 1.49-1.66 (m, 8H), 1.89-1.93 (m, 1H), 1.99 (s, 3H), 2.06 (s, 3H), 2.30-2.35 (m, 2h), 2.47 (d, J = 14.5 Hz, J = 6.0 Hz, 1H), 2.55 (d, J = 15.0 Hz, J = 6.0 Hz, 1H), 2.76 (d, of d, J = 14.5 Hz, J = 6.0 Hz, 1H), 2.90 (d of d, J = 15.0 Hz, J = 6.0 Hz, 1H), 4.19 (t, J = 7.0 Hz, 2H), 4.62 (s, 2H), 4.70-4.74 (m, 1H), 4.87 (p, J = 6.0 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H) ppm; 13C NMR (125 MHz, CDCl3) δ 14.2, 14.3, 21.5 (2), 22.7, 22.9, 25.1, 26.0 (2), 28.3, 28.8, 29.4, 29.6, 29.7, 29.8, 29.9, 31.9, 32.1, 33.6, 33.7, 34.3, 37.8, 40.7, 49.0, 65.6, 66.2, 74.6, 79.0, 109.8, 121.8, 126.4, 127.6, 140.7, 155.1, 169.6, 171.0, 171.1 ppm. Example 15 Synthesis of Treprostinilamide Derivatives

[0344] Treprostinil is commercially available and can be synthesized by the methods disclosed in, for example, U.S. Patent No. 6,765,117 and U.S. Patent No. 8,497,393. The synthesis of prostaglandin derivatives is described in U.S. Patent No. 4,668,814. The disclosures of U.S. Patent No. 6,765,117, U.S. Patent No. 8,497,393 and U.S. Patent No. 4,668,814 are each incorporated by reference in their entirety for all purposes.

[0345] To a solution of (1R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-hexahydro-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-1H-benz[f]inden-5-yl]oxy]acetic acid (i.e., treprostinil) (78.1 mg, 200 μmol) dissolved in 1,4-dioxane (2.0 mL) was added triethylamine (TEA) (98 μL, 700 μmol, 3.5 equiv.), alkylamine R1-NH2 (240 μmol, 1.2 equiv.), and a solution of PyBOP (364 mg, 700 μmol, 3.5 equiv.) dissolved in 2.0 mL MeCN (acetonitrile).

[0346] The reaction mixture was heated to 40° C. and shaken at approximately 100 rpm overnight. The solvent was removed under reduced pressure to give the crude product as a thick yellow oil. 20% nThe product was extracted from the oil by repeated washing with PrOH / hexane (3 x 3 mL) (1-1 extraction). The solvent was removed from the organic extract using a gentle stream of warm N2 gas and gentle heating to give a thick pale yellow oil. The crude material was purified by 20% distillation. n The product was dissolved in PrOH / hexane, passed through a 0.45 μm syringe filter, and subjected to preparative HPLC purification. The solvent was removed from the purified material using a gentle stream of warm N2 gas and gentle heating to give a thick, colorless oil. The pure material was suspended in ethyl lactate for storage and subjected to analytical HPLC for concentration determination.

[0347] The following treprostinil amide derivatives of formula B were prepared according to the synthetic scheme shown above (Table 17), with the % yields shown in parentheses. [ka] [Table 17]

[0348] C6-TR-A and C 12 -TR-A was characterized by NMR spectroscopy. NMR characterization of C6-TR-A 1 H NMR (500 MHz, CDCl3) δ 0.90 (q, J = 7.0 Hz, 6 H), 1.17 (q, J = 12.0 Hz, 1H), 1.30-1.70 (m, 18 H), 1.81-1.83 (m, 1H), 1.80-1.93 (m, 1H), 2.20 (p, J = 6.0 Hz, 1H), 2.22-2.23 (m, 1H), 2.47-2.54 (m, 2H), 2.75-2.82 (m, 2H), 3.16 (sextet, J = 4.0 Hz, 1H), 3.35 (q, J = 7.0 Hz, 2H), 3.63 (s, 1H), 3.70-3.80 (m, 1H), 4.48 (s, 2H), 6.55 (s, 1H), 6.70 (d, J = 7.5 Hz, 1H), 6.85 (d, J = 7.5 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H) ppm; 13 C NMR (125 MHz, CDCl3) δ 14.2, 14.3, 22.8, 22.9, 25.6, 26.4, 26.7(2), 28.8, 29.7, 31.6, 32.1, 33.0, HRMS (ESI, 2:2:1 MeCN, MeOH, H2O): m / z = 474.35717 ([M+H] + ). NMR characterization of C12-TR-A HRMS (ESI, 2:2:1 MeCN, MeOH, H2O): m / z=558.45099([M+H] + ). Example 16 Solubility of treprostinilamide derivatives in hydrofluoroalkane propellants.

[0349] Selected treprostinil derivatives were evaluated for use in metered dose inhalers (MDIs). Four ester derivatives, dodecyl-treprostinil (C 12 -TR), tetradecyl-treprostinil (C 14 -TR), hexadecyl-treprostinil (C 16 -TR) and branched-chain nonanyl-treprostinil (5C9-TR) and two amide derivatives, C 16 -TR-A and C 12-TR-A (see Table 17) was tested for solubility in the hydrofluoroalkane propellants HFA-134a and HFA-227 spiked with ethanol.

[0350] 5mg of each treprostinil compound was added to a glass bottle. A specific amount of ethanol was added by weight. An MDI valve was crimped onto each bottle, and HFA propellant was added through the valve to a total volume of 5mL. The compound was allowed to dissolve at room temperature for 24 hours. The formulation was visually evaluated for solubility. The objective was to estimate the minimum ethanol concentration required to solubilize each compound in the propellant.

[0351] Soluble samples existed as clear, colorless solutions. Less soluble samples had thin liquid-vapor rings of varying densities visible on the surface of the bottle at the liquid-vapor interface. Insoluble samples had the formation of a white precipitate or crystals. Ethanol was added as a solubilizing agent. As can be seen from the solubility tables below (Tables 18 and 19), compounds that were insoluble in 3% added ethanol became soluble in 10 or 13% added ethanol. [Table 18] [Table 19] Example 17 In ventilated rats, C 12 Plasma treprostinil pharmacokinetics after inhalation of an amide-linked treprostinil nanoparticle formulation

[0352] Male Sprague-Dawley rats (N=3) were anesthetized and provided with an endotracheal tube for ventilation. A cannula was inserted into the right femoral vein to facilitate blood collection. The rats were administered the following components: 12 -T763, a lipid nanoparticle formulation with 45 mol% TR-A, 45 mol% squalane, and 10 mol% DSPE-PEG2000, was administered.

[0353] An Aeroneb® nebulizer and control unit (Aerogen, Dangan, Galway, Ireland) was used to generate aerosols with mass median aerodynamic diameters (MMAD) of 2.5 μm to 4 μm at a rate of 0.1 mL / min.

[0354] A 300 μL volume of nebulized test article was delivered using a SAR-830 / AP small animal ventilator (CWE Inc., Ardmore, PA) set at a ventilator tidal volume (VT) of 8 mL / kg and a rate of 90 breaths / min. The target dose was 6 μg / kg treprostinil equivalent.

[0355] C 12 Nanoparticle formulation T568(C) containing -TR alkyl esters 12 The plasma levels of treprostinil were significantly lower than those obtained with the same dose of -TR 40 mol%, squalane 40 mol%, chol-PEG2k 10 mol%, and DOPC 10 mol%, suggesting that the conversion rate of the amide prodrug is much slower than that for the ester prodrug of treprostinil.

[0356] While the invention as described has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the intended spirit and scope of the described invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0357] All patents, patent applications, patent application publications, journal articles, and protocols referenced herein are incorporated by reference in their entirety for all purposes.

Claims

[Claim 1] Improved adverse event profile.

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