Dry powder composition of treprostinil prodrug and method of use thereof

Abstract

The present disclosure provides a dry powder composition of a treprostinil prodrug and a method for treating pulmonary hypertension (e.g., pulmonary arterial hypertension), portal hypertension, or pulmonary fibrosis in a patient requiring treatment. The dry powder composition comprises (a) about 0.1 wt% to about 3 wt% of a compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.01 wt% to about 3 wt% of DSPE-PEG2000, (c) about 10 wt% to about 50 wt% of leucine, and (d) the remainder being a sugar selected from the group consisting of trehalose and mannitol. The total of (a), (b), (c), and (d) is 100 wt%, and R1 is tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl. The present method comprises administering an effective amount of a dry powder composition into a patient's lungs by inhalation through a dry powder inhaler. In the specific compositions and methods provided herein, R1 is hexadecil, e.g., linear hexadecil.

Description

Technology Field Cross-reference literature for related applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 840,186 filed on April 29, 2019, the full text of which is incorporated herein by reference. Background Technology

[0002] Pulmonary hypertension (PH) is characterized by abnormally high blood pressure in the pulmonary vascular system. It is a progressive, fatal disease leading to heart failure and can occur in the pulmonary arteries, pulmonary veins, or pulmonary capillaries. Symptoms include shortness of breath, dizziness, syncope, and other symptoms, all of which are exacerbated by exercise. Various causes exist; the etiology may be unknown or idiopathic, and hypertension in other systems can induce portal hypertension in patients who have both portal hypertension and pulmonary hypertension.

[0003] Pulmonary hypertension has been classified into five groups by the World Health Organization (WHO). Group 1 is referred to as pulmonary arterial hypertension (PAH), which includes PAH of unknown cause (idiopathic), hereditary PAH (i.e., familial PAH or FPAH), PAH induced by drugs or toxins, and PAH caused by pathologies such as connective tissue diseases, HIV infection, liver disease, and congenital heart disease. Group 2 pulmonary hypertension is characterized as pulmonary hypertension associated with left heart disease. Group 3 pulmonary hypertension is characterized as PH associated with lung diseases such as chronic obstructive pulmonary disease and interstitial lung disease, as well as PH associated with sleep-related breathing disorders (e.g., sleep apnea). Group 4 PH is PH resulting from chronic thrombotic and / or embolic diseases, for example, PH caused by blood clots or blood coagulation disorders in the lungs. Group 5 includes pH caused by other disorders or conditions, e.g., 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) affects approximately 200,000 people worldwide, with about 30,000 to 40,000 such patients in the United States. Patients with PAH experience constriction of the pulmonary arteries, leading to high pulmonary artery pressure, which makes it difficult for the heart to pump blood to the lungs. Patients suffer from shortness of breath and fatigue, which often severely limits their ability to perform physical activities.

[0005] The New York Heart Association (NYHA) classified PAH patients into four functional classes to assess disease severity. Class I PAH patients classified by the NYHA do not restrict physical activity because routine physical activity does not cause excessive shortness of breath or fatigue, chest pain, or near-syncope. Class II PAH patients classified by the NYHA have slight limitations in physical activity. These patients feel comfortable at rest, but general physical activity causes excessive shortness of breath or fatigue, chest pain, or near-syncope. Class III PAH patients classified by the NYHA have significant limitations in physical activity. Although they feel comfortable at rest, Class III PAH patients experience excessive shortness of breath or fatigue, chest pain, or near-syncope as a result of less physical activity than usual. Class IV PAH patients classified by the NYHA are unable to perform any physical activity without symptoms. Class IV PAH patients experience shortness of breath and / or fatigue at rest, and discomfort increases with any physical activity. Right ventricular heart failure often occurs in patients with class IV PAH.

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

[0007] Portal hypertension (PPH) is defined by the coexistence of portal and pulmonary hypertension and is a serious complication of liver disease. The diagnosis of portal hypertension is based on hemodynamic criteria: (1) portal hypertension and / or liver disease (clinical diagnosis - ascites / varices / splenomegaly), (2) mean pulmonary artery pressure at rest > 25 mmHg, and (3) pulmonary vascular resistance > 240 dynes s / cm² 5 (4) Pulmonary artery occlusion pressure < 15 mmHg or transpulmonary gradient > 12 mmHg. PPH is a serious complication of liver disease and occurs in 0.25 to 4% of patients suffering from cirrhosis. Today, PPH is a concomitant in 4 to 6% of people referred for liver transplantation.

[0008] Pulmonary fibrosis is a respiratory disease characterized by the formation of scars in lung tissue, leading to severe breathing problems. Scar formation—that is, the excessive accumulation of fibrous connective tissue—thickens the lung walls and reduces the supply of oxygen to the blood. Consequently, patients with pulmonary fibrosis suffer from persistent shortness of breath. In some patients, a specific cause of the disease can be diagnosed, whereas in others, possible causes can be determined; this condition is referred to as idiopathic pulmonary fibrosis.

[0009] The present invention addresses the need for new treatment options for pulmonary hypertension (PH) (including pulmonary arterial hypertension (PAH)), portal hypertension (PPH), and pulmonary fibrosis by providing a dry powder composition of a treprostinil prodrug useful for pulmonary administration and a method for administering the same to a patient requiring treatment. (Patent Document 1) WO 2017 / 192993 A1

[0010] In one embodiment, the present disclosure relates to a dry powder composition. The dry powder composition comprises (a) about 0.1 wt% to about 3 wt% of a compound of the following formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt; (b) about 0.01 wt% to about 3 wt% of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); (c) about 10 wt% to about 50 wt% of leucine; and (d) the remainder being a sugar selected from the group consisting of trehalose and mannitol: [In the above formula, R 1 [It is tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl]. (a), (b), (c), and (d) total 100 wt%.

[0011] In one specific example, R 1 is tetradecyl. In another embodiment, R 1 It is a linear tetradecyl.

[0012] In one specific example, R 1 It is pentadecyl. In another embodiment, R 1 It is a linear pentadecyl.

[0013] In one specific example, R 1 is heptadecyl. In another embodiment, R 1 It is a linear heptadecyl.

[0014] In one specific example, R 1 is octadecyl. In another embodiment, R 1 It is a linear octadecyl.

[0015] In one specific example, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl.

[0016] In one embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 0.5% to about 2% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.05% to about 2% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.15 wt% to about 1.4 wt% of the total weight of the dry powder composition. In yet another embodiment, DSPE-PEG2000 is present in an amount of about 0.25 wt% to about 1 wt% of the total weight of the dry powder composition.

[0017] In one embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1% to about 2% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.1% to about 2% by weight of the total weight of the dry powder composition. In another embodiment, R1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.3% to about 1.4% by weight of the total weight of the dry powder composition. In yet another embodiment, DSPE-PEG2000 is present in an amount of about 0.5% to about 1% by weight of the total weight of the dry powder composition.

[0018] In one embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.2% to about 1.8% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.12% to about 1.8% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.36 wt% to about 1.26 wt% of the total weight of the dry powder composition. In yet another embodiment, DSPE-PEG2000 is present in an amount of about 0.6 wt% to about 0.9 wt% of the total weight of the dry powder composition.

[0019] In one embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1% to about 1.5% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.1% to about 1.5% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1is a linear hexadecyl. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.3% to about 1.05% by weight of the total weight of the dry powder composition. In yet another embodiment, DSPE-PEG2000 is present in an amount of about 0.5% to about 0.75% by weight of the total weight of the dry powder composition.

[0020] In one embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.4% to about 1.6% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.14% to about 1.6% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.42 wt% to about 1.12 wt% of the total weight of the dry powder composition. In yet another embodiment, DSPE-PEG2000 is present in an amount of about 0.7 wt% to about 0.8 wt% of the total weight of the dry powder composition.

[0021] In one embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.5% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.15% by weight to about 1.5% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.45 wt% to about 1.05 wt% of the total weight of the dry powder composition. In yet another embodiment, DSPE-PEG2000 is present in an amount of about 0.75 wt% of the total weight of the dry powder composition.

[0022] In one embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 0.1% by weight to about 3% by weight of the total weight of the dry powder composition, and the weight ratio of DSPE-PEG2000 to the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is in the range of about 0.1:1 (DSPE-PEG2000:compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt) to about 1:1 (DSPE-PEG2000:compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt). In another embodiment, R 1 is hexadecyl. In another embodiment, R 1is a linear hexadecyl. In other embodiments, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 0.5% to about 2% by weight of the total weight of the dry powder composition. In other embodiments, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1% to about 2% by weight of the total weight of the dry powder composition. In other embodiments, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.2% to about 1.8% by weight of the total weight of the dry powder composition. In another embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1% to about 1.5% by weight of the total weight of the dry powder composition. In another embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.4% to about 1.6% by weight of the total weight of the dry powder composition. In yet another embodiment, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.5% by weight of the total weight of the dry powder composition. In another embodiment, the weight ratio of DSPE-PEG2000 to the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is in the range of about 0.3:1 (DSPE-PEG2000:compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt) to about 0.7:1 (DSPE-PEG2000:compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt).In another embodiment, the weight ratio of DSPE-PEG2000 to the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is about 0.5:1 (DSPE-PEG2000: compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt).

[0023] In one embodiment of the dry powder composition provided herein, leucine is present in an amount of about 15% by weight to about 40% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, leucine is present in an amount of about 18% to about 33% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, leucine is present in an amount of about 20% to about 30% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, leucine is present in an amount of about 25% to about 30% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, leucine is present in an amount of about 27% by weight to about 30% by weight of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl. In another embodiment, leucine is present at about 30 weight percent of the total weight of the dry powder composition. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl.

[0024] In one embodiment, the sugar is mannitol. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl.

[0025] In one embodiment, the dry powder composition comprises (a) about 1.5 wt% of a compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.7 wt% of DSPE-PEG2000, (c) about 29.3 wt% of leucine, and (d) the remainder being mannitol. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl.

[0026] In one embodiment, the dry powder composition comprises (a) about 1.5 wt% of a compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.75 wt% of DSPE-PEG2000, (c) about 29.30 wt% of leucine, and (d) about 68.45 wt% of mannitol. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl.

[0027] In one embodiment, the dry powder composition is in the form of an aerosol having particles having a median mass aerodynamic diameter (MMAD) of about 1 μm to about 3 μm as measured by a next-generation impactor (NGI). In another embodiment, the dry powder composition is in the form of an aerosol having particles having an MMAD of about 1.3 μm to about 2.0 μm as measured by an NGI. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl.

[0028] In one embodiment, the sugar is mannitol, and the dry powder composition is in the form of an aerosol having particles having an MMAD of about 1 μm to about 3 μm as measured by NGI. In another embodiment, the sugar is mannitol, and the dry powder composition is in the form of an aerosol having particles having an MMAD of about 1.7 μm to about 2.7 μm as measured by NGI. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl.

[0029] In one embodiment, the dry powder composition is in the form of an aerosol having particles having a fine particle fraction (FPF) of about 30% to about 60% as measured by NGI. In another embodiment, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl.

[0030] In another aspect, the present disclosure relates to a method for treating pulmonary hypertension in a patient requiring treatment for pulmonary hypertension. The method comprises administering an effective amount of the dry powder composition disclosed herein into the patient's lungs by inhalation through a dry powder inhaler.

[0031] Pulmonary hypertension is, in one embodiment, pulmonary arterial hypertension (PAH). In one embodiment, the PAH is a Class I PAH as characterized by the New York Heart Association (NYHA). In another embodiment, the PAH is a Class II PAH as characterized by the NYHA. In another embodiment, the PAH is a Class III PAH as characterized by the NYHA. In another embodiment, the PAH is a Class IV PAH as characterized by the NYHA.

[0032] In one specific example, pulmonary hypertension is group 1 pulmonary hypertension, as characterized by the World Health Organization (WHO).

[0033] In another embodiment, pulmonary hypertension is group 2 pulmonary hypertension as characterized by the WHO.

[0034] In another embodiment, pulmonary hypertension is group 3 pulmonary hypertension as characterized by the WHO.

[0035] In another embodiment, pulmonary hypertension is group 4 pulmonary hypertension as characterized by the WHO.

[0036] In another embodiment, pulmonary hypertension is group 5 pulmonary hypertension as characterized by the WHO.

[0037] In another aspect, the present disclosure relates to a method for treating portal hypertension or pulmonary fibrosis in a patient requiring treatment for portal hypertension or pulmonary fibrosis. The method comprises administering an effective amount of the dry powder composition disclosed herein into the patient's lungs by inhalation through a dry powder inhaler.

[0038] In one embodiment of the treatment method described herein, administration is performed by administering medication once a day, twice a day, or three times a day.

[0039] In another embodiment of the treatment method described herein, administration comprises aerosolizing a dry powder composition and administering the aerosolized dry powder composition into the patient's lungs by inhalation. In one embodiment, the aerosolized dry powder composition comprises particles having an MMAD of about 1 μm to about 3 μm as measured by NGI. In another embodiment, the aerosolized dry powder composition comprises particles having an FPF of about 30% to about 60% as measured by NGI.

[0040] In another aspect, the present disclosure relates to a system for treating pulmonary hypertension, portal hypertension, or pulmonary fibrosis. The system comprises one of the dry powder compositions disclosed herein and a dry powder inhaler (DPI).

[0041] A DPI is, in one embodiment, a single-dose or multi-dose inhaler.

[0042] In other embodiments, DPI is pre-measured or device-measured. Brief explanation of the drawing

[0043] Figure 1 is a graph showing the effect of leucine content on spray drying recovery of mannitol-based C16TR (treprostinyl palmityl) dry powder.

[0044] Figures 2a to 2d are SEM images of dry mannitol-based C16TR (treprostinyl palmityl) powders containing different amounts of leucine as specified.

[0045] Figure 3 is a graph showing the effect of leucine content on particle size distribution measured by laser diffraction in mannitol-based C16TR (treprostinyl palmityl) dry powder.

[0046] Figure 4 is a graph showing the effect of C16TR (treprostinyl palmityl) content on MMAD of mannitol-based C16TR (treprostinyl palmityl) dry powder.

[0047] Figures 5a to 5c are SEM images of spray-dried mannitol-based C16TR (treprostinyl palmityl) powder at different inlet temperatures. The image in the upper panel was taken at high magnification, and the image in the lower panel was taken at low magnification.

[0048] Figures 6a and 6b are SEM images illustrating the morphology of mannitol-based C16TR (treprostinyl palmityl) dry powder spray-dried at an inlet temperature of 135°C with or without ammonium bicarbonate (ABC, 0.5 mg / mL). The image in the upper panel was taken at high magnification, and the image in the lower panel was taken at low magnification.

[0049] Figure 7a is a graph showing the DSC data of the mannitol-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-179.

[0050] Figure 7b is a graph showing X-ray diffraction data of mannitol-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-179.

[0051] Figures 8a and 8b are SEM images illustrating the effect of leucine content on the morphology of trehalose-based C16TR (treprostinyl palmityl) dry powder.

[0052] Figures 9a to 9c are SEM images illustrating the effect of the dry spray inlet temperature on the morphology of trehalose-based C16TR (treprostinyl palmityl) dry powder.

[0053] Figure 10a is a graph showing DSC data of trehalose-based C16TR (treprostinyl palmityl) dry powder.

[0054] Figure 10b is a graph showing X-ray diffraction data of trehalose-based C16TR (treprostinyl palmityl) dry powder.

[0055] Figure 11 is a DVS isotherm plot showing the moisture absorption of mannitol-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-167 (C16TR (treprostinyl palmityl) / DSPE-PEG2000 / Man / Leu, 1 / 0.5 / 80 / 20).

[0056] Figure 12 is a DVS isotherm plot showing the moisture absorption of trehalose-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-162 (C16TR (treprostinyl palmityl) / DSPE-PEG2000 / Treh / Leu, 1 / 0.5 / 80 / 20).

[0057] Figure 13 is a DVS isotherm plot showing the moisture absorption of trehalose-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-163 (C16TR (treprostinyl palmityl) / DSPE-PEG2000 / Treh / Leu, 1 / 0.5 / 70 / 30).

[0058] Figure 14 is a graph showing the change in MMAD in an accelerated stability study of mannitol-based C16TR (treprostinyl palmityl) dry powder.

[0059] Figures 15a and 15b are SEM images of mannitol-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-179 containing 1% C16TR (treprostinyl palmityl) at T0 and T3 (3 months), respectively.

[0060] Figures 16a and 16b are SEM images of mannitol-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-183 containing 1.5% C16TR (treprostinyl palmityl) at T0 and T3 (3 months), respectively.

[0061] Figures 17a and 17b are SEM images of a batch of mannitol-based C16TR (treprostinyl palmityl) dry powder SD-NNP-184 containing 2% C16TR (treprostinyl palmityl) at T0 and T3 (3 months), respectively.

[0062] Figures 18a and 18b are SEM images of mannitol-based C16TR (treprostinyl palmityl) dry powder batches containing 3% C16TR (treprostinyl palmityl) (SD-NNP-190) and 5% C16TR (treprostinyl palmityl) (SD-NNP-191), respectively, at T5 (5 months).

[0063] Figure 19 is a graph showing the change in MMAD in an accelerated stability study of trehalose-based C16TR (treprostinyl palmityl) dry powder.

[0064] Figure 20 is a graph showing the change in FPF in an accelerated stability study of trehalose-based C16TR (treprostinyl palmityl) dry powder.

[0065] Figures 21a and 21b are SEM images of the trehalose-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-162 containing 1% C16TR (treprostinyl palmityl) at T0 and T3.5 (3.5 months), respectively.

[0066] Figures 22a and 22b are SEM images of the trehalose-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-163 containing 1% C16TR (treprostinyl palmityl) at T0 and T3.5 (3.5 months), respectively.

[0067] Figures 23a and 23b are SEM images of a batch of trehalose-based C16TR (treprostinyl palmityl) dry powder SD-NNP-188 containing 1.5% C16TR (treprostinyl palmityl) at T0 and T3.5 (3.5 months), respectively.

[0068] Figures 24a and 24b are SEM images of the trehalose-based C16TR (treprostinyl palmityl) dry powder batch SD-NNP-189 containing 2% C16TR (treprostinyl palmityl) at T0 and T3.5 (3.5 months), respectively.

[0069] Figure 25 is a graph showing the pressure titrations of spray-dried treprostinyl palmityl dry powder formulations A, B, C, and D. For visibility, data points are offset sequentially (A, B, C, and D) within each pressure category (from left to right).

[0070] Figure 26 is a graph showing the particle size distribution of Treprostinyl Palmityl dry powder formulations A, B, C, and D.

[0071] Figure 27 is an SEM image of Treprostinyl Palmityl dry powder formulation A.

[0072] Figure 28 is an SEM image of Treprostinyl Palmityl dry powder formulation B.

[0073] Figure 29 is an SEM image of Treprostinyl Palmityl dry powder formulation C.

[0074] Figure 30 is an SEM image of Treprostinyl Palmityl dry powder formulation D.

[0075] FIG. 31 is a graph showing the fine particle dose (FPD) of Treprostinyl Palmityl dry powder formulation A after storage in capsules for 1 to 3 months at 25°C or 40°C as specified at T=0, or after storage as bulk for 3 months at 25°C or 40°C and then filled into capsules and administered on the same day.

[0076] FIG. 32 is a graph showing the fine particle dose (FPD) of Treprostinyl Palmityl dry powder formulation C after storage in capsules at T=0 and for 1 to 3 months at 25°C or 40°C as specified, or after storage as bulk at 25°C or 40°C for 3 months, filled into capsules, and administered on the same day.

[0077] FIG. 33 is a graph showing the fine particle dose (FPD) of Treprostinyl Palmityl dry powder formulation D after storage in capsules at T=0 and at 25°C or 40°C for 1 to 3 months as specified, or after storage as bulk at 25°C or 40°C for 3 months, filled into capsules, and administered on the same day.

[0078] Figure 34 is a dynamic vapor adsorption (DVS) isotherm plot of a trehalose-based C16TR (treprostinyl palmityl) dry powder formulation.

[0079] Figure 35 is a graph showing the aerosol particle size distribution of a trehalose-based C16TR (treprostinyl palmityl) dry powder formulation after storage at 40°C and uncontrolled ambient humidity for T=0 months and for 1.5 months, 2.5 months, and 3.5 months (n=1 per time point).

[0080] Figure 36 is a graph showing the concentration of C16TR (treprostinyl palmityl) equivalents (C16TR (treprostinyl palmityl) plus treprostinyl, ng / g) in the lungs after inhalation of trehalose-based C16TR (treprostinyl palmityl) dry powder formulation and nebulized INS1009.

[0081] Figure 37 is a graph showing the concentration of C16TR (treprostinyl palmityl) equivalent (C16TReq) in the lungs after inhalation of treprostinyl palmityl dry powder formulation D or formulation C.

[0082] Figure 38 is a graph showing the concentration of C16TR (treprostinyl palmityl) in the lungs after inhalation of treprostinyl palmityl dry powder formulation D or formulation C.

[0083] Figure 39 is a graph showing the concentration of TRE in the lungs after inhalation of Treprostinyl Palmityl dry powder formulation D or formulation C.

[0084] Figure 40 is a graph showing the concentration of TRE in plasma after inhalation of Treprostinyl Palmityl dry powder formulation D or formulation C.

[0085] Figure 41 is a graph showing the ΔRVPP response to a hypoxic challenge in rats exposed to Treprostinyl Palmityl dry powder formulation D or formulation C. Specific details for implementing the invention

[0086] Throughout this disclosure, the term “about” may be used with numerical values ​​and / or ranges. The term “about” is understood to mean such values ​​close to the quoted values. For example, “about 40 [units]” may mean within ± 25% of 40 (e.g., 30 to 50), ± 20%, ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, less than ± 1%, or any other value or range within or less than.

[0087] The term “pharmaceuticalally acceptable salt” refers to a salt prepared from a pharmaceutically acceptable, non-toxic base or acid comprising an inorganic or organic base and an inorganic or organic acid. The properties of the salt are not critical, provided they are pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts may be prepared from inorganic or organic acids. Exemplary pharmaceutical salts are disclosed in the literature [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. Non-limiting specific examples of inorganic acids include hydrochloric acid, hydrobromide, hydroiodide, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids are, without limitation, aliphatic, alicyclic, aromatic, aryliphatic, and heterocyclyl-containing carboxylic acids and sulfonic acids, e.g., 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, pyruvate, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, stearic acid, salicylic acid, p-hydroxybenzoic acid, phenylacetic acid, mandelic acid, emvonic acid (famosan), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pentothenic acid, toluenesulfonic acid, 2-hydroxyethanesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, algenic acid, 3-hydroxybutyric acid. It includes galactaric acid or 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 suitable alkali metal (Group Ia) salts, alkaline earth metal (Group IIa) salts, and other physiologically acceptable metals.These salts can be prepared from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Exemplary organic salts can be prepared from primary amines, secondary amines, tertiary amines, and quaternary ammonium salts. Examples include tromethamine, diethylamine, tetra-N-methylammonium, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine.

[0088] Throughout this specification, numerical ranges are provided for specific quantities. It should be understood that such ranges include all sub-ranges therein. Accordingly, the range “50 to 80” includes all possible ranges therein (e.g., 51 to 79, 52 to 78, 53 to 77, 54 to 76, 55 to 75, 60 to 70, etc.). Additionally, any value within the provided range may be an endpoint for the range included thereby (e.g., the range 50 to 80 includes ranges having endpoints such as 55 to 80, 50 to 75, etc.).

[0089] The term “treating” in one embodiment comprises: (1) preventing or delaying the onset of clinical symptoms of a condition, disorder, or pathology in a subject who has or may have had a condition, disorder, or pathology but has not yet experienced or may not exhibit clinical or subclinical symptoms of the condition, disorder, or pathology; (2) suppressing the condition, disorder, or pathology (e.g., preventing, reducing, or delaying the onset of a disease, or its recurrence in the case of maintenance therapy, or the onset of at least one clinical or subclinical symptom thereof); and / or (3) alleviating the pathology (e.g., causing a regression of at least one of the condition, disorder, or pathology or its clinical or subclinical symptoms thereof). In one embodiment, “treating” refers to suppressing the condition, disorder, or pathology (e.g., preventing, reducing, or recurring the onset of a disease, or its recurrence in the case of maintenance therapy, or the recurrence of at least one clinical or subclinical symptom thereof). In other embodiments, "treating" refers to alleviating a condition (e.g., by causing a regression of a condition, disorder, or pathology, or at least one of its clinical or subclinical symptoms). The benefit to the subject being treated is statistically significant compared to the condition or pathology of the same subject prior to treatment, or compared to the condition or pathology of an untreated control subject, or such benefit is at least recognizable to the subject or specialist.

[0090] "Effective amount" means an amount of the dry powder composition of the present disclosure sufficient to produce a desired therapeutic response.

[0091] In one aspect of the present invention, a dry powder composition of a treprostinil prodrug is provided. The dry powder composition is (a) a compound of the following formula (I), or a mirror image isomer, a diastereomer, or a pharmaceutically acceptable salt thereof, wherein [In the above formula, R 1 [is tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl], a compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, is present in an amount of about 0.1% to about 3% by weight of the total weight of the dry powder composition; (b) About 0.01 wt% to about 3 wt% of DSPE-PEG2000, (c) about 10 weight% to about 50 weight% of leucine, and (d) Contains a sugar selected from the group consisting of the remainder of trehalose and mannitol. (a), (b), (c), and (d) total 100 wt%.

[0092] In some embodiments, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 0.1 wt%, about 0.3 wt%, about 0.5 wt%, about 0.7 wt%, about 1 wt%, about 1.3 wt%, about 1.5 wt%, about 1.7 wt%, about 2.0 wt%, about 2.3 wt%, about 2.5 wt%, about 2.7 wt%, or about 3 wt% of the total weight of the dry powder composition. In other embodiments, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.5 wt% of the total weight of the dry powder composition. The compound of formula (I) and its pharmaceutically acceptable salt are treprostinil prodrugs disclosed in International Application Publication WO 2015 / 061720, the full text of which is incorporated herein by reference. In some embodiments, leucine is present in an amount of about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt% of the total weight of the dry powder composition.

[0093] PEG refers to polyethylene glycol, also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on its molecular weight. DSPE-PEG2000 may comprise branched or unbranched PEG molecules having an average PEG molecular weight of 2000 g / mol. In one embodiment, (b) is DSPE-PEG2000 present in an amount of about 0.03 wt% to about 2.1 wt% of the total weight of the dry powder composition. In another embodiment, (b) is DSPE-PEG2000 present in an amount of about 0.05 wt% to about 1.5 wt% of the total weight of the dry powder composition.

[0094] In one embodiment of a compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, R1 is tetradecyl. In another embodiment, R 1 It is a linear tetradecyl.

[0095] In other embodiments of the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, R 1 It is pentadecyl. In another embodiment, R 1 It is a linear pentadecyl.

[0096] In other embodiments of the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, R 1 is heptadecyl. In another embodiment, R 1 It is a linear heptadecyl.

[0097] In other embodiments of the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, R 1 is octadecyl. In another embodiment, R 1 It is a linear octadecyl.

[0098] In other embodiments of the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, R 1 is hexadecyl. In another embodiment, R 1 is a linear hexadecyl, that is, the compound of formula (I), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is the compound of formula (II) below, or its enantiomer, diastereomer, or pharmaceutically acceptable salt: In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is the compound of formula (II) or a pharmaceutically acceptable salt thereof. In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is the compound of formula (II). In another embodiment, the compound of formula (I) is the compound of formula (II). The compound of formula (II) is also referred to herein as C16TR or treprostinyl palmityl. In this application, C16TR and treprostinyl palmityl are used interchangeably and are referred to as the compound of formula (II).

[0099] In one embodiment, (a) is a compound of formula (I) or a pharmaceutically acceptable salt thereof. In another embodiment, (a) is a compound of formula (I). In another embodiment, (a) is a compound of formula (II) or a pharmaceutically acceptable salt thereof. In another embodiment, (a) is a compound of formula (II).

[0100] In one embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 0.5 wt% to about 2 wt% of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.05 wt% to about 2 wt% of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.15 wt% to about 1.4 wt% of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.25 wt% to about 1 wt% of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II), or its pharmaceutically acceptable salt is present in an amount of about 0.5 wt% to about 2 wt% of the total weight of the dry powder composition. In some embodiments, the compound of formula (I) or (II) is present in an amount of about 0.5% to about 2% by weight of the total weight of the dry powder composition.

[0101] In one embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1% to about 2% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.1% to about 2% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.3% to about 1.4% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.5% to about 1% by weight of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II), or its pharmaceutically acceptable salt is present in an amount of about 1% to about 2% by weight of the total weight of the dry powder composition. In some embodiments, the compound of formula (I) or (II) is present in an amount of about 1% to about 2% by weight of the total weight of the dry powder composition.

[0102] In one embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.2% to about 1.8% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.12% to about 1.8% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.36% to about 1.26% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.6% to about 0.9% by weight of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II), or its pharmaceutically acceptable salt is present in an amount of about 1.2% to about 1.8% by weight of the total weight of the dry powder composition. In some embodiments, the compound of formula (I) or (II) is present in an amount of about 1.2% to about 1.8% by weight of the total weight of the dry powder composition.

[0103] In one embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1% to about 1.5% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.1% to about 1.5% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.3% to about 1.05% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.5% to about 0.75% by weight of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II), or its pharmaceutically acceptable salt is present in an amount of about 1% to about 1.5% by weight of the total weight of the dry powder composition. In some embodiments, the compound of formula (I) or (II) is present in an amount of about 1% to about 1.5% by weight of the total weight of the dry powder composition.

[0104] In one embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.4% to about 1.6% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.14% to about 1.6% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.42% to about 1.12% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.7% to about 0.8% by weight of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II), or its pharmaceutically acceptable salt is present in an amount of about 1.4% to about 1.6% by weight of the total weight of the dry powder composition. In some embodiments, the compound of formula (I) or (II) is present in an amount of about 1.4% to about 1.6% by weight of the total weight of the dry powder composition.

[0105] In one embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.1% by weight to about 1% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.3% by weight to about 0.7% by weight of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.5% by weight of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II), or a pharmaceutically acceptable salt is present in an amount of about 1% by weight of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II) is present in an amount of about 1% by weight of the total weight of the dry powder composition.

[0106] In one embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.5 wt% of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.15 wt% to about 1.5 wt% of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.45 wt% to about 1.05 wt% of the total weight of the dry powder composition. In another embodiment, DSPE-PEG2000 is present in an amount of about 0.75 wt% of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II), or its pharmaceutically acceptable salt is present in an amount of about 1.5 wt% of the total weight of the dry powder composition. In some embodiments, the compound of formula (I) or (II) is present in an amount of about 1.5% by weight of the total weight of the dry powder composition.

[0107] In some embodiments, the compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 0.1 wt% to about 3 wt%, about 0.5 wt% to about 2 wt%, about 1 wt% to about 2 wt%, about 1.2 wt% to about 1.8 wt%, about 1 wt% to about 1.5 wt%, about 1.4 wt% to about 1.6 wt%, about 1 wt%, or about 1.5 wt% of the total weight of the dry powder composition, and the weight ratio of DSPE-PEG2000 to the compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is about 0.1:1 (DSPE-PEG2000:compound of formula (I) or (II)) to about 1:1 (DSPE-PEG2000:compound of formula (I) or (II), or in the range of about 0.3:1 (DSPE-PEG2000:compound of formula (I) or (II)) to about 0.7:1 (DSPE-PEG2000:compound of formula (I) or (II)), for example, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, or about 1:1. In one embodiment, the weight ratio of DSPE-PEG2000 to a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is in the range of about 0.1:1 (DSPE-PEG2000:compound of formula (I) or (II)) to about 1:1 (DSPE-PEG2000:compound of formula (I) or (II). In another embodiment, the weight ratio of DSPE-PEG2000 to a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is in the range of about 0.3:1 (DSPE-PEG2000:compound of formula (I) or (II)) to about 0.It is a range of 7:1 (DSPE-PEG2000: compounds of chemical formula (I) or (II).

[0108] In some embodiments, the weight ratio of DSPE-PEG2000 to the compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is about 0.5:1 (DSPE-PEG2000:compound of formula (I) or (II)). In this weight ratio, in one embodiment, the compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1% to about 2% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.5% to about 1% by weight of the total weight of the dry powder composition. In another embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.2% to about 1.8% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.6% to about 0.9% by weight of the total weight of the dry powder composition. In another embodiment, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.4% to about 1.6% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.7% to about 0.8% by weight of the total weight of the dry powder composition. In other embodiments, a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt is present in an amount of about 1.5% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of about 0.75% by weight of the total weight of the dry powder composition. In some embodiments, a compound of formula (I) or (II), or its pharmaceutically acceptable salt is present in each of the aforementioned weight percentages or weight percentages of the dry powder composition.In some embodiments, the compound of formula (I) or (II) is present in each of the aforementioned weight percentages or weight percentages of the dry powder composition.

[0109] In one embodiment, leucine is present in an amount of about 15% by weight to about 40% by weight of the total weight of the dry powder composition. In another embodiment, leucine is present in an amount of about 18% by weight to about 33% by weight of the total weight of the dry powder composition. In another embodiment, leucine is present in an amount of about 20% by weight to about 30% by weight of the total weight of the dry powder composition, for example, about 20% by weight, about 25% by weight, or about 30% by weight. In another embodiment, leucine is present in an amount of about 25% by weight to about 30% by weight of the total weight of the dry powder composition. In another embodiment, leucine is present in an amount of about 27% by weight to about 30% by weight of the total weight of the dry powder composition. In one embodiment, leucine is present in an amount of about 20% by weight of the total weight of the dry powder composition. In another embodiment, leucine is present in an amount of about 30% by weight of the total weight of the dry powder composition.

[0110] In some embodiments, the sugar in the dry powder composition is trehalose. In other embodiments, the sugar in the dry powder composition is mannitol.

[0111] In one embodiment, the dry powder composition comprises (a) about 1.5 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.7 wt% of DSPE-PEG2000, (c) about 29.3 wt% of leucine, and (d) the remainder being trehalose. In another embodiment, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In another embodiment, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I).1 is hexadecyl. In another embodiment, R in the compound of formula (I) 1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II).

[0112] In other embodiments, the dry powder composition comprises (a) about 1 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.5 wt% of DSPE-PEG2000, (c) about 29.6 wt% of leucine, and (d) the remainder being trehalose. In other embodiments, in the dry powder composition, (a) is about 1 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In other embodiments, in the dry powder composition, (a) is about 1 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I). 1 is hexadecyl. In another embodiment, R in the compound of formula (I) 1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II).

[0113] In other embodiments, the dry powder composition comprises (a) about 1 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.5 wt% of DSPE-PEG2000, (c) about 19.7 wt% of leucine, and (d) the remainder being trehalose. In other embodiments, in the dry powder composition, (a) comprises about 1 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In other embodiments, in the dry powder composition, (a) is about 1 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I). 1is hexadecyl. In another embodiment, R in the compound of formula (I) 1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II).

[0114] In other embodiments, the dry powder composition comprises (a) about 1.5 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.7 wt% of DSPE-PEG2000, (c) about 19.6 wt% of leucine, and (d) the remainder being trehalose. In other embodiments, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In other embodiments, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I). 1 is hexadecyl. In another embodiment, R in the compound of formula (I) 1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II).

[0115] In other embodiments, the dry powder composition comprises (a) about 1.5 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.7 wt% of DSPE-PEG2000, (c) about 29.3 wt% of leucine, and (d) the remainder being mannitol. In other embodiments, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In other embodiments, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I). 1 is hexadecyl. In another embodiment, R in the compound of formula (I)1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II).

[0116] In other embodiments, the dry powder composition comprises (a) about 1.5 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.75 wt% of DSPE-PEG2000, (c) about 29.30 wt% of leucine, and (d) about 68.45 wt% of mannitol. In other embodiments, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In other embodiments, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I). 1 is hexadecyl. In another embodiment, R in the compound of formula (I) 1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II). In one embodiment, the dry powder composition comprises (a) about 1.5 wt% of the compound of formula (II), (b) about 0.75 wt% of DSPE-PEG2000, (c) about 29.30 wt% of leucine, and (d) about 68.45 wt% of mannitol.

[0117] In other embodiments, the dry powder composition comprises (a) about 1 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.5 wt% of DSPE-PEG2000, (c) about 29.6 wt% of leucine, and (d) the remainder being mannitol. In other embodiments, in the dry powder composition, (a) is about 1 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In other embodiments, in the dry powder composition, (a) is about 1 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I). 1 is hexadecyl. In another embodiment, R in the compound of formula (I) 1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II).

[0118] In other embodiments, the dry powder composition comprises (a) about 1.5 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.7 wt% of DSPE-PEG2000, (c) about 19.6 wt% of leucine, and (d) the remainder being mannitol. In other embodiments, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In other embodiments, in the dry powder composition, (a) is about 1.5 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I). 1 is hexadecyl. In another embodiment, R in the compound of formula (I) 1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II).

[0119] In other embodiments, the dry powder composition comprises (a) about 1 wt% of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt, (b) about 0.5 wt% of DSPE-PEG2000, (c) about 19.7 wt% of leucine, and (d) the remainder being mannitol. In other embodiments, in the dry powder composition, (a) is about 1 wt% of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In other embodiments, in the dry powder composition, (a) is about 1 wt% of a compound of formula (I) or (II). In some embodiments, R in the compound of formula (I). 1 is hexadecyl. In another embodiment, R in the compound of formula (I) 1 is a linear hexadecyl, that is, the compound of formula (I) is the compound of formula (II).

[0120] The Mass Median Aerodynamic Diameter (MMAD) is the value of the aerodynamic diameter associated with particles smaller than the Medium Aerodynamic Diameter (MAD) for 50% of the mass of the provided aerosol and particles larger than the MAD for 50% of the mass. The MMAD can be determined by impactor measurements, for example, an Andersen Cascade Impactor (ACT) or a Next Generation Impactor (NGI). In some embodiments, the dry powder composition is in the form of an aerosol containing particles having an MMAD of about 1 µm to about 5 µm, about 1 µm to about 3 µm, about 1.3 µm to about 2.0 µm, or about 1.7 µm to about 2.7 µm when measured by an NGI. In one embodiment, the sugar in the dry powder composition is mannitol. In another embodiment, the sugar in the dry powder composition is trehalose.

[0121] In one embodiment, the sugar in the dry powder composition is mannitol, and the dry powder composition is in the form of an aerosol containing particles having an MMAD of about 1 μm to about 3 μm as measured by NGI. In another embodiment, the sugar in the dry powder composition is mannitol, and the dry powder composition is in the form of an aerosol containing particles having an MMAD of about 1.7 μm to about 2.7 μm as measured by NGI.

[0122] "Fine particle fraction" or "FPF" refers to the fraction of aerosols having a particle size of less than 5 μm in diameter when measured by cascade impact. FPF is usually expressed as a percentage. FPF indicates that it is correlated with the fraction of powder deposited in the patient's lungs. In some embodiments, the dry powder composition is in the form of an aerosol containing particles having an FPF of at least 20%, at least 30%, at least 40%, at least 50%, about 30% to about 60%, about 35% to about 55%, or about 40% to about 50% when measured by NGI. In one embodiment, the sugar in the dry powder composition is mannitol. In another embodiment, the sugar is trehalose.

[0123] The tap density of the powder is the ratio of the mass of the powder to the volume occupied by the powder after tapping for a specified period. The tap density of the powder represents its random dense packing. The tap density may be determined using the USP bulk density and tapped density method (United States Pharmacopeia convention, Rockville, Md., 10th Supplement, 4950-4951, 1999). In some embodiments, the dry powder composition comprises particles having a tap density of about 0.2 g / ml to about 0.8 g / ml, or about 0.3 g / ml to about 0.6 g / ml. In one embodiment, the sugar in the dry powder composition is mannitol. In another embodiment, the sugar in the dry powder composition is trehalose.

[0124] The dry powder composition of the present disclosure may be prepared from a liquid composition using freeze-drying or spray-drying techniques. When freeze-drying is used, the freeze-dried composition may be milled to obtain a finely divided dry powder containing particles within the desired size range described above. When spray-drying is used, the process is carried out under conditions that produce a finely divided dry powder containing particles within the desired size range described above. Exemplary methods for preparing a dry powder form of a pharmaceutical composition are disclosed in WO 96 / 32149, WO 97 / 41833, WO 98 / 29096, and U.S. Patents No. 5,976,574, 5,985,248, and 6,001,336, the full text of each of these disclosures is incorporated herein by reference. Exemplary spray drying methods are described in U.S. Patents No. 6,848,197 and 8,197,845, the full text of each of these disclosures is incorporated herein by reference.

[0125] In some embodiments, the dry powder composition of the present disclosure is prepared by the following process. A stock solution of a compound of formula (I) or (II), or its enantiomer, diastereomer, or pharmaceutically acceptable salt and DSPE-PEG2000 is prepared using an organic solvent, for example, an alcohol (e.g., 1-propanol). An aqueous stock solution of a sugar (e.g., mannitol or trehalose) and leucine is also prepared. Then, a desired amount of the stock solution is added to a mixture of water and an organic solvent to form a spray-drying feed solution. In the spray-drying feed solution, the volume ratio of water to organic solvent may be about 3:2 to about 1:1.

[0126] Spray drying is initiated by heating the drying gas by starting the drying gas flow and setting the desired inlet temperature, for example, to about 120°C to about 160°C, or to about 135°C to about 150°C. After the spray dryer outlet, the temperature reaches a suitable temperature, for example, about 55°C to about 65°C, and the liquid skid inlet is set so that the blank solvent can be atomized into the spray dryer with the help of nitrogen, and the system can be cooled and stabilized. Product filter pulsing is initiated, and the product filter purge flow is set, for example, to 10 to 20 scfh. After the system is stabilized, the liquid skid inlet is switched to the feed solution prepared above, and the process continues until the feed solution runs out. At the point when the feed solution runs out, the liquid skid inlet is switched back to the blank solvent, which can be atomized for about 5 to about 20 minutes. At this point, the powder is collected at the bottom of the product filter. After spraying the blank solvent for about 5 to about 20 minutes, the system is terminated by shutting off the liquid line, spray gas, drying gas heater, drying gas inlet, and finally the exhaust port.

[0127] In one embodiment, the dry powder composition of the present disclosure is delivered to the lungs of a subject by inhalation using a dry powder inhaler (DPI). In one embodiment, the dry powder inhaler is a single-dose dry powder inhaler. The propellant-free device, the DPI, delivers the dry powder to the lungs of the subject by utilizing the subject's inhalation. The unit dose of the dry powder composition used in the DPI device is often a dry powder blister disc in a hard capsule. Exemplary DPI devices suitable for delivering the dry powder composition of the present disclosure include the devices described in the following documents as well as the DPIs described in U.S. Patents No. 6,766,799, 7,278,425, and 8,496,002, each of which is incorporated herein by reference in its entirety.

[0128] The AIR® inhaler (Alkermes) comprises a small, breath-activated system that delivers porous powder from a capsule. The porous particles have an aerodynamic diameter of 1 to 5 µm [see International Patent Applications WO 99 / 66903 and WO 00 / 10541, the full text of which is incorporated herein by reference.

[0129] Aerolizer™ (Novartis) is a single-dose dry powder inhaler. In this device, the dry powder medication is stored in a capsule and released by piercing the capsule wall with a TEFLON-coated steel pin [see U.S. Patents No. 6,488,027 and 3,991,761, the full disclosure of each of these documents incorporated herein by reference].

[0130] Bang Olufsen provides a breath-actuated inhaler with blister strips having up to 60 doses. The dose is made available only during inhalation by a new trigger mechanism. The device is equipped with a dose counter and can be disposed of after all doses have been used [see EP 1522325, the full text of which is incorporated herein by reference].

[0131] Clickhaler® (Innovata PLC) is a large reservoir breath-activated multi-dose device [see U.S. Patent No. 5,437,270, the full text of which is incorporated herein by reference].

[0132] DirectHaler™ (Direct-Haler A / S) is a single-dose, pre-measured, pre-refillable, disposable DPI device made of polypropylene [see U.S. Patent No. 5,797,392, the full disclosure of which is incorporated herein by reference].

[0133] Diskus™ (GlaxoSmithKline) is a small, disposable DPI device containing up to 60 doses in a double foil blister strip to provide moisture protection. [Refer to GB2242134; the full text of the disclosure is incorporated herein by reference].

[0134] Eclipse™ (Aventis) is a reusable capsule device with a breath-actuated capacity to deliver up to 20 mg of a dry powder composition. The powder is drawn from the capsule into a vortex chamber where a rotating ball assists in the deagglomeration of the powder when the subject inhales [see U.S. Patent No. 6,230,707 and WO 9503846, the full text of which is incorporated herein by reference].

[0135] Flexhaler® is a plastic breath-activated dry powder inhaler and can be used with the dry powder composition provided herein.

[0136] FlowCaps® (Hovione) is a capsule-based, rechargeable, reusable, manual dry-powder inhaler that holds up to 14 capsules. The inhaler itself is moisture-proof [see U.S. Patent No. 5,673,686, the full disclosure of which is incorporated herein by reference].

[0137] Gyrohaler® (Vectura) is a manual, disposable DPI containing a blister strip [see GB2407042, the full text of which is incorporated herein by reference].

[0138] HandiHaler® (Boehringer Ingelheim GmbH) is a single-dose DPI device. It can deliver up to 30 mg of a dry powder composition in a capsule [see International Patent Application Publication WO 04 / 024156, the full text of which is incorporated herein by reference].

[0139] MicroDose DPI (Microdose Technologies) is a small electronic DPI device. It uses a piezoelectric vibrator (ultrasonic frequency) to deagglomerate drug powder in aluminum blisters (single or multiple doses) [see U.S. Patent No. 6,026,809, the full text of which is incorporated herein by reference].

[0140] The Nektar Dry Powder Inhaler® (Nektar) is a palm-sized, easy-to-use device. It provides convenient medication delivery from standard capsules and flow-independent lung immers.

[0141] The Nektar Pulmonary Inhaler® (Nektar) efficiently removes powder from its packaging, breaks down particles, and generates an aerosol cloud suitable for deep lung delivery. This enables the aerosolized particles to travel from the device to the deep lungs during the patient's respiration, thereby reducing losses in the throat and upper respiratory tract. Compressed gas is used to aerosolize the powder [see AU4090599 and U.S. Patent No. 5,740,794; the full disclosures of each document are incorporated herein by reference].

[0142] NEXT DPI™ is a device featuring multi-dose capability, moisture protection, and dose counting. The device may be used only when an appropriate suction flow is achieved, regardless of orientation (top to bottom) and volume [see EP 1196146, U.S. Patent No. 6,528,096, WO0178693, and WO0053158, the full text of each disclosure incorporated herein by reference].

[0143] Neohaler® is a capsule-based plastic breath-acting dry powder inhaler.

[0144] Oriel™ DPI is an active DPI that utilizes a piezoelectric membrane and nonlinear vibrations to aerosolize powder formulations [see International Patent Application Publication WO 0168169, the full text of which is incorporated herein by reference].

[0145] The RS01 single-dose dry powder inhaler developed by Plastiape of Italy features a compact size and a simple, effective perforation system, and is suitable for both gelatin and HMPC capsules.

[0146] Pressair™ is a plastic breath-actuated dry powder inhaler.

[0147] The Pulvinal® inhaler (Chiesi) is a breath-driven, multi-dose (100-dose) dry powder inhaler. The dry powder is stored in a transparent reservoir that is clearly marked to indicate when the 100th dose has been delivered [see U.S. Patent No. 5,351,683, the full disclosure of which is incorporated herein by reference].

[0148] Rotohaler® (GlaxoSmithKline) is a disposable device that uses a capsule [see U.S. Patents No. 5,673,686 and 5,881,721, the full text of which is incorporated herein by reference].

[0149] Rexam DPI (Rexam Pharma) is a single-dose, reusable device designed for use with capsules [see U.S. Patent No. 5,651,359 and EP 0707862, the full disclosure of each document incorporated herein by reference].

[0150] S2 (Innovata PLC) is a reusable or single-dose DPI for the delivery of high-concentration dry powder compositions. Its dispersion mechanism requires minimal patient effort to achieve excellent drug delivery to the patient's lungs. S2 is easy to use and features a passive engine, and thus does not require a battery or powder source [see AU3320101, the full text of which is incorporated herein by reference].

[0151] SkyeHaler® DPI (SkyePharma) is a multi-dose device containing up to 300 individual doses in a single-use or replaceable cartridge. The device is driven by breath and requires no adjustment between breath and operation [see U.S. Patent No. 6,182,655 and WO97 / 20589, the full disclosure of each of which is incorporated herein by reference].

[0152] Taifun® DPI (LAB International) is a multi-dose (up to 200) DPI device. It is breath-operated and flow-independent. The device includes a unique fluid-balanced drug reservoir combined with a volumetric dosage metering system for constant dosing [see U.S. Patent No. 6,132,394, the full disclosure of which is incorporated herein by reference].

[0153] The TurboHaler® (AstraZeneca) is described in U.S. Patent No. 5,983,893, the full text of which is incorporated herein by reference. This DPI device is an inspiratory flow-driven, multi-dose dry powder inhaler equipped with a multi-dose reservoir providing a dry powder composition for up to 200 doses and a dose range from several micrograms to 0.5 mg.

[0154] The Twisthaler® (Schering-Plough) is a multi-dose device with a dose-counting feature and can operate 14 to 200 times. The dry powder composition is packaged in a cartridge containing a desiccant [see U.S. Patent No. 5,829,434, the full disclosure of which is incorporated herein by reference].

[0155] Ultrahaler® (Aventis) combines accurate dose metering with good dispersion. It is an easy-to-use, detachable, pocket-sized device equipped with a numerical dose counter, a dose indicator, and a locking mechanism. The device can deliver up to 20 mg of a dry powder composition. Ultrahaler® is described in U.S. Patent No. 5,678,538 and WO2004026380, the full text of which is incorporated herein by reference.

[0156] Xcelovair™ (Meridica / Pfizer) has 60 pre-measured hermetic doses ranging from 5 to 20 mg. The device provides moisture protection under accelerated conditions of 40°C / 75% RH. The dispersion system maximizes the fine particle fraction, delivering up to 50% fine particle mass.

[0157] In another embodiment, a system is provided comprising (i) one of the dry powder compositions described herein, and (ii) a dry powder inhaler (DPI) for administering the dry powder composition. The DPI comprises (a) a reservoir containing the dry powder composition disclosed herein, and (b) means for introducing the dry powder composition to a patient by inhalation. In one embodiment, the reservoir contains the dry powder composition of the present invention in a capsule or blister pack. The material for the shell of the capsule may be gelatin, a cellulose derivative, starch, a starch derivative, chitosan, or a synthetic plastic. The DPI may be a single-dose or multi-dose inhaler. Additionally, the DPI may be pre-measured or device-measured. In one embodiment, the dry powder inhaler is a single-dose dry powder inhaler.

[0158] In one embodiment, the system is used to treat pulmonary hypertension, portal hypertension, or pulmonary fibrosis. The system comprises the dry powder composition disclosed herein, namely, a dry powder composition comprising a compound of formula (I) or (II), or a mirror image isomer, a diastereomer, or a pharmaceutically acceptable salt thereof, and a DPI. In one embodiment, the dry powder composition comprises a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In another embodiment, the dry powder composition comprises a compound of formula (I) or (II). The dry powder inhaler may be as described above and / or may be a single-dose or multi-dose inhaler and / or may be pre-measured or device-measured. In one embodiment, the dry powder inhaler is a single-dose dry powder inhaler.

[0159] In another aspect of the present invention, a method for treating pulmonary hypertension (PH) in a patient requiring treatment for pulmonary hypertension (PH) is provided. The method comprises administering an effective amount of the dry powder composition disclosed herein, namely, a dry powder composition comprising a compound of formula (I) or (II), or a mirror image isomer, a diastereomer, or a pharmaceutically acceptable salt thereof, to the patient's lungs by inhalation through a dry powder inhaler. In one embodiment, the dry powder composition comprises a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In another embodiment, the dry powder composition comprises a compound of formula (I) or (II). In one embodiment, administration comprises aerosolizing the dry powder composition through a DPI to provide an aerosolized dry powder composition, and administering the aerosolized dry powder composition to the patient's lungs by inhalation through the DPI. In some embodiments, the aerosolized dry powder composition comprises particles having an MMAD of about 1 μm to about 10 μm, about 1 μm to about 7 μm, about 1 μm to about 5 μm, or about 1 μm to about 3 μm as measured by NGI. In one embodiment, the aerosolized dry powder composition comprises particles having an FPF of about 40% to about 70%, about 30% to about 60%, or about 50% to about 60% as measured by NGI.

[0160] The World Health Organization (WHO) has classified pulmonary hypertension (PH) into five groups. Group 1 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, Group 1 PH includes 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. Group 2 PH includes pulmonary hypertension associated with left ventricular disease, e.g., atrial or ventricular disease, or valvular disease (e.g., or mitral stenosis). WHO Group 3 pulmonary hypertension is characterized as pulmonary hypertension associated with lung disease, e.g., chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD), and / or hypoxemia. Group 4 pulmonary hypertension is pulmonary hypertension caused by chronic thrombotic and / or embolic disease. Group 4 PH is also referred to as chronic thromboembolic pulmonary hypertension. Patients with Group 4 PH experience blocked or narrowed blood vessels due to blood clots. Group 5 PH is the "miscellaneous" category and includes blood disorders (e.g., polycythemia vera, essential thrombocytopenia), systemic disorders (e.g., sarcoidosis, vasculitis), and / or metabolic disorders (e.g., thyroid disease, glycogen storage disease).

[0161] The method provided herein may be used to treat patients with Group 1 (i.e., pulmonary arterial hypertension or PAH), Group 2, Group 3, Group 4, or Group 5 PH as characterized by the WHO. In one embodiment of the method, the pulmonary hypertension being treated is chronic thromboembolic pulmonary hypertension.

[0162] In other embodiments of the method, the pulmonary hypertension being treated is pulmonary arterial hypertension (PAH). In some embodiments, the PAH being treated is Class I PAH, Class II PAH, Class III PAH, or Class IV PAH, as defined by the New York Heart Association (NYHA).

[0163] In one embodiment, the PAH is a Class I PAH, as characterized by NYHA.

[0164] In another embodiment, the PAH is a Class II PAH, as characterized by NYHA.

[0165] In another embodiment, the PAH is a Class III PAH, as characterized by NYHA.

[0166] In another embodiment, the PAH is a Class IV PAH, as characterized by NYHA.

[0167] In another aspect, the present disclosure provides a method for treating portal hypertension (PPH) in a patient requiring treatment for portal hypertension (PPH). The method comprises administering an effective amount of the dry powder composition disclosed herein, namely, a dry powder composition comprising a compound of formula (I) or (II), or a mirror image isomer, a diastereomer, or a pharmaceutically acceptable salt thereof, to the patient's lungs by inhalation through a dry powder inhaler. In one embodiment, the dry powder composition comprises a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In another embodiment, the dry powder composition comprises a compound of formula (I) or (II). In one embodiment, administration comprises aerosolizing the dry powder composition with a dry powder inhaler (DPI) to provide an aerosolized dry powder composition, and administering the aerosolized dry powder composition to the patient's lungs through the DPI. In some embodiments, the aerosolized dry powder composition comprises particles having an MMAD of about 1 μm to about 10 μm, about 1 μm to about 7 μm, about 1 μm to about 5 μm, or about 1 μm to about 3 μm as measured by NGI. In one embodiment, the aerosolized dry powder composition comprises particles having an FPF of about 40% to about 70%, about 30% to about 60%, or about 50% to about 60% as measured by NGI.

[0168] In some embodiments, a patient with PH, PAH, or PPH treated by the disclosed method exhibits one or more of the following treatment responses: (1) a decrease in the pulmonary vascular resistance index (PVRI) from the pre-treatment value, (2) a decrease in mean pulmonary artery pressure from the pre-treatment value, (3) an increase in the hypoxemia score from the pre-treatment value, (4) a decrease in the oxygenation index from the pre-treatment value, (5) an improvement in right ventricular function compared to the pre-treatment value, and (6) an improvement in exercise capacity compared to the pre-treatment value (e.g., as measured by a 6-minute walk test).

[0169] In one embodiment of the disclosed method, a dry powder composition is administered once a day to a patient with PH, PAH, or PPH. In another embodiment of the disclosed method, a dry powder composition is administered twice a day to a patient with PH, PAH, or PPH. In yet another embodiment of the disclosed method, a dry powder composition is administered three or more times a day to a patient with PH, PAH, or PPH. In one embodiment, administration is made with food. In one embodiment, each administration comprises 1 to 5 doses (puffs) from a 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 compact and portable by the patient. In one embodiment, the dry powder inhaler is a single-dose dry powder inhaler.

[0170] In another aspect, the present disclosure provides a method for treating pulmonary fibrosis in a patient requiring treatment for pulmonary fibrosis. The method comprises administering an effective amount of the dry powder composition disclosed herein, namely, a dry powder composition comprising a compound of formula (I) or (II), or a mirror image isomer, a diastereomer, or a pharmaceutically acceptable salt thereof, into the patient's lungs by inhalation through a dry powder inhaler. In one embodiment, the dry powder composition comprises a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In another embodiment, the dry powder composition comprises a compound of formula (I) or (II). In one embodiment, administration comprises aerosolizing the dry powder composition with a DPI to form an aerosolized dry powder composition, and administering the aerosolized dry powder composition into the patient's lungs through the DPI. In some embodiments, the aerosolized dry powder composition comprises particles having an MMAD of about 1 µm to about 10 µm, about 1 µm to about 7 µm, about 1 µm to about 5 µm, or about 1 µm to about 3 µm as measured by NGI. In one embodiment, the aerosolized dry powder composition comprises particles having an FPF of about 40% to about 70%, about 30% to about 60%, or about 50% to about 60% as measured by NGI. In one embodiment, the dry powder composition is administered to a patient once a day, twice a day, or three or more times a day. In one embodiment, administration is made with food. In one embodiment, each dose 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 compact and portable by the patient. In one embodiment, the dry powder inhaler is a single-dose dry powder inhaler. Examples

[0171] The present invention is further illustrated with reference to the following examples. However, it should be noted that, as with the specific examples described above, these examples are illustrative and should not be interpreted as limiting the scope of the invention in any way. Example 1 - Preparation and Characterization of an Inhalable Dry Powder Formulation Containing the Compound of Formula (II) (Treprostinyl Palmityl)

[0172] The present example describes a mannitol and trehalose-based dry powder formulation containing treprostinyl palmityl represented by chemical formula (II), the preparation thereof by spray drying using a Buchi B-290 spray dryer equipped with an inert loop condenser B-295 and a dehumidifier B-296, and the characterization and stability testing of the formulation.

[0173] A mannitol-based treprostinyl palmityl dry powder formulation was successfully prepared with the ingredients treprostinyl palmityl / DSPE-PEG2000 / mannitol / leucine (1 / 0.5 / 80 / 20, 1.5 / 0.75 / 80 / 20, 2 / 1 / 80 / 20, w / w). A feed stock was prepared by dissolving all ingredients in a 1-propanol / H2O co-solvent system (50 / 50, v / v) without the addition of ammonium bicarbonate. The spray drying yield for the mannitol-based dry powder exceeded 90%. The collected dry powder has spherical particles, a crystalline XRD profile, and low moisture content.

[0174] A trehalose-based treprostinyl palmityl dry powder formulation having the components of treprostinyl palmityl / DSPE-PEG2000 / trehalose / leucine (1 / 0.5 / 80 / 20, 1 / 0.5 / 70 / 30, 1.5 / 0.75 / 80 / 20, 2 / 1 / 80 / 20, w / w) was produced by spray-drying a feed stock containing all components dissolved in a 1-propanol / H2O co-solvent system (50 / 50, v / v) without the addition of ammonium bicarbonate. The trehalose-based dry powder contains disintegrated particles, exhibiting crystalline leucine and amorphous trehalose. The trehalose-based dry powder exhibited good physical stability over 3 months. Materials and Methods 1. Matter

[0175] Phosphate buffered saline: PBS, pH 7.4, Cat. No. 10010 (Life Technologies), or equivalent

[0176] Sodium chloride: ACS Reagent (JT Baker, Cat. No. 3628-05), or equivalent

[0177] Treprostinyl palmityl, the above chemical formula II

[0178] DSPE-PEG2000: N-(methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt, SUNBRIGHT®DSPE-020CN (NOF, Tokyo, Japan), or equivalent

[0179] D-Lactose, monohydrate (Sigma)

[0180] L-Leucine (Sigma)

[0181] Ammonium bicarbonate (Sigma)

[0182] Anhydrous ethanol (Fisher Sci)

[0183] 1-Propanol (Fisher Sci) 2. Equipment

[0184] Buchi B-290 spray dryer (Buchi) equipped with an inert loop condenser B-295, a dehumidifier B-296, a 2-fluid nozzle ID 0.7 mm, and a high-performance cyclone separator.

[0185] SEM: Zeiss-Sigma FE-SEM (Germany)

[0186] XRD: (PANalytical, Netherlands)

[0187] DSC 250, TA Instruments, New Castle, DE, USA.

[0188] Tapping Density Tester, JV 1000 (Copley Scientific, UK)

[0189] NGI: Next Generation Shocker (MSP Corpo n, MN, USA)

[0190] PSD: RODOS / M (Sympatec, Germany)

[0191] Karl Fischer titrators: Aquastar, AQV33, EMD.

[0192] DLS: Mobiuζ ® , Atlas (Wyatt Technology, Santa Barbara, CA)

[0193] High-performance liquid chromatograph: Waters Alliance Model 2695. HPLC software: Waters Empower™ 3

[0194] magnetic stirring plate 3. Preparation of a dry powder formulation comprising treprostinyl palmityl, DSPE-PEG2000, trehalose, and leucine in a weight ratio of 1:0.5:80:20 Preparation of stock solution:

[0195] Treprostinyl palmityl: 10 mg / mL in 1-propanol

[0196] DSPE-PEG2000: 10 mg / mL in 1-propanol

[0197] Trehalose: 150 mg / mL in DI water

[0198] Leucine stock: 20 mg / mL in DI water Preparation of spray-drying supply solution:

[0199] According to Table 1, a spray-dried feed solution was prepared in a weight ratio of treprostinyl palmityl to total trehalose and leucine of 1:100. The final feed solution contains 50% 1-propanol and 20.3 mg / mL of solid (Table 2).

[0200] A trehalose and leucine stock solution was first added to the water phase, followed by the addition of 1-propanol and sonication in a water bath. Subsequently, treprostinyl palmityl (Formula (II)) and DSPE-PEG2000 were added individually. Stirring was applied throughout the entire process. Spray drying process of trehalose-based dry powder containing treprostinyl palmityl / DSPE-PEG2000 / Treh / Leu(1 / 0.5 / 80 / 20):

[0201] Spray drying was performed using a Buchi B-290 spray dryer under the following parameters: inlet temperature of 150°C, outlet temperature of 64°C, spray airflow of 414 L / h (36 mm, height from rotameter), and suction velocity of 35% m 3 / h, and a feed rate of 7.5 mL / min (22%). Table 3 summarizes the process parameters. 4. Preparation of a dry powder formulation comprising treprostinyl palmityl, DSPE-PEG2000, mannitol, and leucine in a weight ratio of 1:0.5:80:20 Preparation of stock solution:

[0202] Treprostinyl palmityl: 10 mg / mL in 1-propanol

[0203] DSPE-PEG2000: 10 mg / mL in 1-propanol

[0204] Mannitol: 150 mg / mL in DI water

[0205] Leucine stock: 20 mg / mL in DI water Preparation of spray-drying supply solution:

[0206] According to Table 4, a spray-dried feed solution was prepared in a weight ratio of treprostinyl palmityl to total mannitol and leucine of 1:100. The final feed solution contains 50% 1-propanol and 20.3 mg / mL of solid (Table 5).

[0207] Mannitol and leucine stock solutions were first added to the aqueous phase, followed by the addition of propanol and sonication in a water bath. Subsequently, treprostinyl palmityl and DSPE-PEG2000 were added separately. Stirring was applied throughout the entire process. Spray drying process of mannitol-based dry powder containing treprostinyl palmityl / DSPE-PEG2000 / mannitol / Leu(1 / 0.5 / 80 / 20):

[0208] Spray drying was performed using a Buchi B-290 spray dryer under the following parameters: inlet temperature 135°C, outlet temperature 60°C, spray airflow 414 L / h (36 mm, height from rotameter), and suction velocity 35% m 3 / h, and a feed rate of 7.5 mL / min (22%). Table 6 summarizes the process parameters. 5. Characteristics analysis of dry powder Surface electron microscope (SEM)

[0209] The dry powder sample (as received) was poured onto carbon tape and subsequently coated with 20 nm of gold (Au) using an Electron Microscopy Sciences (EMS150T ES) sputter coater. Particle morphology was observed using a Zeiss-Sigma FE-SEM (Germany) with an operating voltage of 5 keV. Relatively high resolution was obtained by maintaining the working distance at 8 to 10 mm. X-ray diffraction test (XRD)

[0210] A dry powder sample (as received) was packed into a zero-background sample holder, and subsequently, X-ray diffraction (XRD) was used to evaluate structural features using a PANalytical (Netherlands) X'Pert diffractometer equipped with Cu Kα (λ= 1.540598Å) at 45 kV and 40 mA at a scanning speed of 0.04 rad degrees per minute. The scanning range was 4° to 40° (2θ), the time per step was 97.92 seconds, and the step size was 0.0131°. Differential Scanning Calorimetry (DSC)

[0211] Approximately 5 to 10 mg of dry powder was weighed into a DSC sample pan and then airtightened. The test was performed as follows: equilibrium was maintained at 20°C, and the temperature was controlled at 0.32°C for 60 seconds, isothermally at 1.0 min, and ramp from 5°C / min to 180.0°C. Particle size distribution (PSD) by laser diffraction

[0212] Approximately 15 to 20 mg of dry powder was placed in the desired glass tube. The Sympatech-HOLOS-REDOS mode was used. The test was performed as follows:0 Moisture content test (Karl Fischer)

[0213] The moisture content of the dry powder was analyzed using a Karl Fischer. Approximately 30 mg of the sample was weighed and transferred to a titration vessel. The equipment, materials, and operating parameters were used as described below: Material / Equipment: (1) Titrator: Aquastar AQV33 Karl Fischer titrator with 5 mL burette and associated balance (2) Balance: An analytical balance capable of weighing up to 4 decimal places with an interface that can be connected to an Aquastar titrator. (3) Water standard 1% NIST (4) 100% desiccant or molecular sieve, type 4A, 1 / 16 pellet (5) Lint-free fabric, Kimwipe (6) Weighing boat (7) 3 mL syringe solution: (1) Titration: Aquastar CombiTitrant 2 (2) Solvent: 60 / 40 methanol / formamide Device Parameters and Conditions: (1) Drift: <50 µg / min (2) Stirring speed: 40% (3) Maximum time: 300 seconds (4) End: Rival Drift Bulk and tap density

[0214] Density tests were performed using a tapped density tester, JV 1000 (Coply Scientific, UK). The following procedure was performed: the glass tube was washed; dried with compressed air; the glass tube was weighed and recorded as W1; the dried powder was transferred to the glass tube, the height was marked as A, and the weight was recorded as W2; the top was sealed with Parafilm; the glass tube was placed in a 5 mL graduated cylinder and tapped for 10 minutes; the height after tapping was marked as B; the powder was removed from the tube, washed, and dried with compressed air; the glass tube was weighed and recorded as W3; water was added to level B and the weight was recorded as W5; water was added to level A and the weight was recorded as W4 (assuming the density of water is 1 g / mL). Aerodynamic Particle Size Distribution (APSD) using NGI

[0215] Approximately 20 mg of powder loaded into size 3 Vcaps HPMC capsules was dispersed into a next-generation cascade shocker (NGI) (Copley Scientific, UK) operated for 4 seconds at a volumetric flow rate of 60 L / min via a commercial inhaler (Plastiape RS01). The drug content collected at each stage from the NGI device was evaluated by HPLC-MS. The fine particle fraction (FPF) was defined as the mass of drug (<5 µm) deposited in the NGI divided by the released dose. HPLC assay

[0216] The concentrations of treprostinyl (TRE) and treprostinyl palmityl were determined using a Waters Alliance Model 2695 equipped with a PDA detector (Waters 2996) and a corona-charged aerosol detector (Thermo Fisher Scientific). Column: ACE 3 C8 HPLC Column 4.6x50 (Mac-Mod Analytical, Cat. No. ACE1120546) Column temperature: 25℃ Mobile Phase A: Acetonitrile 25%, Methanol 25%, Water 50%, Formic Acid 0.1%, Triethylamine 0.01% Mobile Phase B: Acetonitrile 50%, Methanol 50%, Formic Acid 0.1%, Triethylamine 0.01% Flow rate: 1 mL / min Gradient for measuring TRE: Injection volume: 50 µl UV wavelength: 270 ± 2.4 nm Samples and standards were dissolved in 33% acetonitrile, 33% methanol, and 33% water. The test was fitted using the power function Log(Area) = A + B*Log(Conc). Duration of stay: TRE approx. 1.8 minutes, C16TR approx. 7.6 minutes Total record time 9 minutes result 1. Batch of Mannitol-Based Treprostinyl Palmityl Dry Powder

[0217] Different batches of mannitol-based treprostinyl palmityl dry powder were prepared by spray drying. In such batches, the amount of treprostinyl palmityl in the dry powder varied from 1% to 5% (weight ratio, w / w), while the ratio of treprostinyl palmityl to DSPE-PEG2000 was maintained constant at 2 to 1. The leucine content ranged from 0 to 30% (w / w) of the dry powder. The effect of ammonium bicarbonate was also investigated. During the spray drying process, the inlet temperature varied from 120°C to 150°C. Table 7a shows the composition and inlet temperature for different batches of mannitol-based treprostinyl palmityl dry powder. For each batch, the amounts of treprostinyl palmityl, DSPE-PEG2000, mannitol, and leucine are expressed as weight ratios. The amounts of treprostinyl palmityl and leucine are also indicated by their approximate weight percentages, expressed as their proportions in weight ratios. Table 7b shows the targeted weight percentages of treprostinyl palmityl, DSPE-PEG2000, mannitol, and leucine in each batch when calculated based on weight ratios. 1.1. Effect of Leucine Content on Spray Drying Recovery Rate

[0218] The effect of leucine on the properties of mannitol-based treprostinyl palmityl dry powder was evaluated. Four leucine loads, namely 0%, 10%, 20%, and 30%, were evaluated. An increase in mannitol load was applied to offset the decrease in leucine content.

[0219] The spray drying recovery rate (%) was low from the batch containing no leucine. The recovery rate increased significantly when the leucine level was increased to 10% and 20%. Subsequently, the recovery rate decreased slightly when the leucine content was further increased to 30% (Fig. 1). The batch with 20% leucine has the highest value for powder density (Table 8). 1.2. Effects of Leucine Content (30%, 20%, 10%, and 0%) on Powder Morphology

[0220] SEM was performed to test the effect of leucine content on the powder surface (Figs. 2a to 2d). Changes in leucine content occurred with different weight ratios of mannitol to leucine, namely 70 / 30, 80 / 20, 90 / 10, and 100 / 0.

[0221] Dry powder samples of treprostinyl palmityl were produced with different weight ratios of mannitol to leucine (70 / 30, 80 / 20, 90 / 10, and 100 / 0). SEM data indicated that increasing the leucine content from 20% to 30% resulted in the formation of powders with a wrinkled surface (Figs. 2a and 2b). Dry powder without leucine was destroyed with a low recovery rate after spray drying (Fig. 2d). For further studies, 20% leucine was used. 1.3. Effect of leucine content on PSD (tested by laser diffraction)

[0222] Three batches of mannitol-based dry powder containing 10, 20, and 30% leucine (w / w) were investigated by laser diffraction. Batch information is shown in Table 9.

[0223] As shown in FIG. 3, the formulation having 20% ​​leucine has the smallest particle size (D50). The formulation having 20% ​​leucine will be used in further studies in this embodiment. 1.4. Effects of various amounts of treprostinyl palmityl on MMAD

[0224] To investigate the effect on dry powder characteristics, different amounts of treprostinyl palmityl were introduced into mannitol-based dry powder formulations. As shown in Table 10, five dry powder batches were prepared with treprostinyl palmityl in the range of 1% to 5% and a weight ratio of treprostinyl palmityl / DSPE-PEG2000 of 2:1.

[0225] The value of MMAD was constant when 1 to 2% of treprostinyl palmityl was present in the dry powder, and it increased significantly when treprostinyl palmityl was increased from 3% to 5% (Fig. 4). 1.5. Effect of Spray Drying Inlet Temperatures at 150°C, 135°C, and 120°C on Powder Morphology

[0226] Mannitol-based treprostinyl palmityl dry powders with the same component ratio were produced at different inlet temperatures, namely 150°C, 135°C, and 120°C (Table 11). The effect of inlet temperature on the dry powder morphology was investigated first. SEM indicated that less surface destruction was present in the spray-dried dry powder samples at lower inlet temperatures of 135°C and 120°C (Figs. 5a to 5c). Since no significant difference was observed between 135°C and 120°C (Figs. 5b and 5c), an inlet temperature of 135°C was used for further investigation. 1.6. Effects of Ammonium Bicarbonate (ABC) in Feed Stock on Mannitol-Based Treprostinyl Palmityl Dry Powder Morphology

[0227] The effect of ABC on the morphology of mannitol-based treprostinyl palmityl dry powder was tested by adding or not adding ABC to the feed stock when preparing the dry powder (Table 12). No changes in the powder surface were observed with the addition of ABC (Figs. 6a and 6b). Accordingly, ABC will not be applicable to mannitol-based treprostinyl palmityl dry powder. 1.7. Physico-chemical properties of mannitol-based treprostinyl palmityl dry powder

[0228] Mannitol-based treprostinyl palmityl dry powder was produced by spray-drying a solution containing all components. It was expected that the mannitol-based dry powder would exhibit slightly amorphous characteristics, such as Tg in DSC tests and broad peaks in powder X-ray diffraction (XRD) tests. Figures 7a and 7b show the DSC and XRD data for batch SD-NNP-179 containing 1% treprostinyl palmityl and 20% leucine, respectively. Tg was not detected, and sharp peaks were observed in the powder XRD for this batch. These two characteristics were also confirmed in other batches, regardless of differences in composition and spray-drying conditions. 2. Batch of Trehalose-Based Treprostinyl Palmityl Dry Powder

[0229] Different batches of trehalose-based treprostinyl palmityl dry powder were prepared using spray drying. Table 13A shows the inlet temperatures used for the spray drying process and the composition of the batches. For each batch, the amounts of treprostinyl palmityl, DSPE-PEG2000, trehalose, and leucine are indicated by weight ratio. Table 13B shows the targeted weight percentages of treprostinyl palmityl, DSPE-PEG2000, trehalose, and leucine in each batch when calculated on a weight basis. The batches varied in treprostinyl palmityl content from 1% to 2% (weight ratio, w / w), while the ratio of treprostinyl palmityl to DSPE-PEG2000 remained constant at 2 to 1. The leucine content in the batches was 20% or 30% (w / w). During the spray drying process, the inlet temperature varied from 110℃ to 155℃. 2.1. Effect of Leucine Content on Powder Morphology

[0230] Trehalose-based treprostinyl palmityl dry powders with two levels of leucine (20% and 30%) were prepared (Table 14). SEM data indicated that increasing the leucine content from 20% to 30% resulted in a wrinkled powder surface (Figs. 8a and 8b). 2.2. Effect of Leucine Content on Powder Aerosol Performance

[0231] The inventors introduced two levels of leucine, namely 20% and 30%, into trehalose-based treprostinyl palmityl dry powder and compared its particle size (D50, laser diffraction) and MMAD (Table 14).

[0232] The data in Table 14 indicate that the dry powder with 30% leucine has a larger geometric particle size (D50) and lower deposition of the powder on the throat and pre-separator compared to the powder with 20% leucine. Due to the low solubility of leucine, leucine will precipitate first. The higher the amount of leucine, the faster it will trigger precipitation and produce larger particles. However, the difference in MMAD was not significant. 2.3. Effect of Treprostinyl Palmityl Content on Dry Powder Aerosol Performance

[0233] To investigate the effect on the dry powder characteristics, different amounts of treprostinyl palmityl were introduced into trehalose-based treprostinyl palmityl dry powder. As shown in Table 15, four batches of dry powder were prepared with treprostinyl palmityl content ranging from 1% to 2% and a weight ratio of treprostinyl palmityl / DSPE-PEG2000 fixed at 2:1.

[0234] In the case of having more treprostinyl palmityl in the dry powder, deposition on MMAD and the throat and pre-separator increased, while the fine particle fraction (FPF) decreased (Table 15). 2.4. Effect of Spray Drying Inlet Temperature on Powder Morphology

[0235] In the spray drying process, the inlet temperature was expected to affect the characteristics of the dried powder, such as moisture content, particle size, and powder morphology. Two inlet temperatures of 130°C and 150°C were investigated with batches of trehalose-based vehicle dried powder shown in Table 16.

[0236] SEM showed that the powder was destroyed at a high inlet temperature of 150°C (Figs. 9a to 9c). However, to prevent high moisture in the final dried powder, 150°C will be used for the trehalose-based dried powder containing treprostinyl palmityl. 2.5. Physico-chemical properties of trehalose-based dry powder

[0237] Similar to the mannitol-based treprostinyl palmityl dry powder, trehalose-based treprostinyl palmityl dry powder was prepared by spray-drying a solution containing all components. The dry powder was expected to exhibit slightly amorphous characteristics, such as Tg in DSC and broad peaks in powder X-ray diffraction (XRD).

[0238] The batches of trehalose-based treprostinyl palmityl dry powders shown in Table 17 were treated with DSC and XRD. In the DSC test, Tg ranging from 64°C to 80°C was observed for all batches. Tg can increase when the powder is treated with secondary drying via overnight freeze-drying, as observed in batches SD-NNP-162 and SD-NNP-163, due to the reduction of moisture in the dry powder (Fig. 10a). Compared to the XRD from the mannitol-based dry powder, the XRD from the trehalose-based dry powder showed several sharp peaks (Fig. 10b), which is attributed to the amorphous state of trehalose. All batches exhibited similar XRD data regardless of differences in the weight ratios of the components and the drying conditions. Table 17 presents additional characteristics of the dry powder batches, including MMAD, FPF, and throat + pre-separator deposition. 3. Dynamic Vapor Adsorption (DVS) Profiles of Mannitol and Trehalose-Based Dry Powders

[0239] Moisture can be introduced into dry powder formulations during spray drying, packaging, and storage, potentially causing product instability and packaging issues. During spray drying, moisture in the dry powder can be reduced through secondary drying. However, during packaging, the powder can absorb moisture when exposed to the environment, even under moisture-controlled conditions. The moisture absorption of mannitol and trehalose-based treprostinyl palmityl dry powders was tested.

[0240] As shown in Fig. 11, the mannitol-based treprostinyl palmityl dry powder can absorb up to 0.3% moisture when the RH% is increased from 0 to 40%. Compared to the lactose-based treprostinyl palmityl dry powder, the mannitol-based dry powder absorbed much less moisture, which is possible because the mannitol-based dry powder contains crystalline mannitol and leucine, and the stable form for both is the non-hydrated form.

[0241] The moisture absorption profiles for the trehalose-based dry powders are shown in Fig. 12 (powder with 20% leucine) and Fig. 13 (powder with 30% leucine). The weight change for the trehalose-based dry powders reached a peak at 50% RH%. Subsequently, moisture absorption decreased due to the physical form of trehalose changing from amorphous to crystalline. Powder formulations with 20% and 30% leucine exhibit moisture absorption data, with the latter formulation showing less than 1% absorption. However, a significant difference was observed during the desorption process. The powder with 30% leucine showed a higher moisture remainder at 0% RH%. 4. Stability test for mannitol-based treprostinyl palmityl dry powder

[0242] Stability tests for mannitol-based treprostinyl palmityl dry powder were conducted over 3 months at 40°C without humidity control. Five batches of mannitol-based treprostinyl palmityl dry powder containing 1, 1.5, 2, 3, and 5% treprostinyl palmityl were examined (Table 18). The NGI test generally indicated that the MMAD of the dry powder increased sharply at the 2-month mark and subsequently dropped back to a level similar to that at the 1-month mark (Fig. 14). SEM data indicated the growth of dense small fibers on the surface of the powder (Figs. 15a, 15b, 16a, 16b, 17a, 17b, 18a, and 18b).

[0243] Table 19 shows the details of the stability data for the batches of mannitol-based treprostinyl palmityl dry powder. In all batches of mannitol-based dry powder containing 20% ​​leucine, significant changes in MMAD and FPF were observed. Additionally, the initial MMAD was lower in the dry powders containing 1, 1.5, and 2% treprostinyl palmityl compared to the dry powders containing 3 and 5% treprostinyl palmityl. 5. Stability test for trehalose-based dry powder

[0244] Stability studies for trehalose-based dry powder formulations containing 20% ​​or 30% leucine and 1% to 2% treprostinyl palmityl were also performed under the same conditions as the mannitol-based dry powder formulations. Over a study period of 3.5 months, no significant changes were observed (Table 20, and Figures 21a, 21b, 22c, 22b, 23a, 23b, 24a, and 24b).

[0245] Table 21 shows details of the stability data for the batches of trihalose-based treprostinyl palmityl dry powder. For batches SD-NNP-162 and SD-NNP-188, MMAD was significantly increased (Table 21 and Fig. 19). For batch SD-NNP-162, the FPF value decreased the most (Fig. 20). Batch SD-NNP-163 (treprostinyl palmityl 1%, leucine 30%) showed the lowest and most stable MMAD and the highest stable FPF. Summary of Findings on Mannitol-Based Treprostinyl Palmityl Dry Powder Formulations

[0246] In mannitol-based treprostinyl palmityl dry powder, the addition of leucine resulted in a high spray drying recovery rate. Mannitol-based dry powder with 20% leucine exhibited spherical particle shape and a low geometric diameter (D50 = 2.75 μm).

[0247] In the spray drying process, the inlet temperature varying from 120°C to 150°C did not affect the morphology of the mannitol-based treprostinyl palmityl dry powder. Since the moisture content of the dry powder produced at 135°C is approximately 1%, the inlet temperature was set at 135°C.

[0248] No glass transition was detected in the mannitol-based treprostinyl palmityl dry powder in the DSC test, which supports the X-ray diffraction (XRD) results indicating a crystalline material. Additionally, the spray-dried mannitol-based treprostinyl palmityl dry powder is less hygroscopic and absorbs less than 1% moisture at 90% RH%.

[0249] In a stability test of mannitol-based treprostinyl palmityl dry powder, MMAD increased sharply at 2 months for formulations containing 1 to 2% treprostinyl palmityl, and at 1 month for formulations with higher treprostinyl palmityl content. At subsequent times, MMAD decreased. All formulations exhibited fibers on the powder surface after storage. Summary of Findings on Trehalose-Based Treprostinyl Palmityl Dry Powder Formulations

[0250] Trehalose-based treprostinyl palmityl dry powder containing 30% leucine has a wrinkled surface and less powder is deposited in the throat and pre-separator compared to that containing 20% ​​leucine. However, there is no significant difference in MMAD between the two.

[0251] In the main drying process, the inlet temperature was selected at 150°C to achieve a low moisture content in the final dried powder.

[0252] A glass transition temperature (Tg) in the range of 64 to 80°C was observed, which indicates the amorphous state of trehalose in the dry powder. The trehalose-based treprostinyl palmityl dry powder exhibited higher moisture absorption compared to the mannitol-based treprostinyl palmityl dry powder.

[0253] In stability tests, most trehalose-based treprostinyl palmityl dry powder formulations tested did not show a significant change in FPF. All formulations showed hair-like crystals on the powder surface after storage.

[0254] In summary, the data of this example indicate that a treprostinyl palmityl content in the range of up to 2 wt% does not affect the physical properties of the treprostinyl palmityl dry powder. At 3 and 5 wt% treprostinyl palmityl, an increase in the initial MMAD of the mannitol-based powder was observed. Leucine content was found to be important for the dry powder aerosol properties. Example 2 - Preparation, Encapsulation, and Characterization of Inhalable Mannitol and Trehalose-Based Treprostinyl Palmityl Dry Powder Formulations

[0255] The present example describes the preparation and encapsulation by spray drying of four dry powder formulations of treprostinyl palmityl, namely, formulations A, B, C, and D. Formulations A and D are mannitol-based, and their compositions in both weight ratios and targeted weight percentages calculated based on weight ratios are shown in Table 22. Formulations B and C are trehalose-based, and their compositions in both weight ratios and targeted weight percentages calculated based on weight ratios are shown in Table 23. The present example also describes the characterization of formulations A through D regarding particle size, morphology, moisture content, solvent content, physical state, vapor adsorption profile, thermal properties, and weight loss at different temperatures. 1. Spray-drying preparation of formulations A, B, C, and D

[0256] Treprostinyl palmityl dry powder formulations A, B, C, and D were prepared using a BLD-200 spray dryer with approximately 55 grams of incoming solid, respectively. Between each condition, a blank solvent solution was sprayed to remove the previous formulation from the solution line. No additional cleaning of the spray dryer was performed between conditions.

[0257] Each of the four formulations was prepared as an independent solution. The solutions were prepared at room temperature without light protection. For the preparation of each solution, the following steps were performed: 1. Dissolve leucine in deionized water. 2. Dissolve a sugar (mannitol or trehalose) in deionized water. 3. Filter the aqueous solution through a 0.2 µm PVDF membrane. 4. Dissolve DSPE-PEG2000 in 1-propanol. 5. Dissolve treprostinyl palmityl in 1-propanol. 6. Add the organic solution to a stirred aqueous solution.

[0258] The spray-drying formulations and process conditions are listed in Table 24. The manufacturing yield ranges from 54 to 80 mass%. The bulk-dried powder of each formulation was packaged in a drying glove box. 2. Powder encapsulation

[0259] The dry powder formulations were encapsulated using Xcelodose 600S, and 50 to 51 capsules were packed per formulation. The relative humidity of the suite was less than 30%. A summary of the encapsulation is shown in Table 25. For example, in each capsule Capsules were prepared from a conventional batch of Formulation D (containing 1.50 wt% treprostinyl palmityl, 0.75 wt% DSPE-PEG2000, 68.45 wt% mannitol, and 29.30 wt% leucine) by filling each capsule with 112.5 µg of treprostinyl palmityl, 56.2 µg of DSPE-PEG2000, 5133.8 µg of mannitol, and 2197.5 µg of leucine. It was observed that other batches of Formulation D having different weight percentages for each component independently at the conventional weight percentage values ​​specified above or within ±5% thereof had equivalent characteristics and performance. The capsules were collected in glass vials and heat-sealed in foil bags containing 0.5 g of molecular sieve desiccant. 3. Analytical Feature Analysis

[0260] Each of the four formulations was evaluated for particle size distribution, particle morphology, moisture content, residual solvent, physical state, water absorption, and thermal properties. A summary of the results is shown in Table 26. 3.1. Particle Size Distribution

[0261] Since these formulations were targeted for respiratory delivery, the target particle size was less than 5 µm. The particle size distribution was measured by laser diffraction on a Malvern Mastersizer 2000 equipped with a Scirocco 2000 dry powder dispersion unit. An initial pressure titration screening was performed on all samples for method development (n=1), and it was observed that the results were nearly identical across the three dispersion air pressures used (2.5, 3.0, and 3.5 bar) (Fig. 25). Based on this initial screening, two additional repetitions (n=2) were measured at a dispersion air pressure of 3.0 bar. The results were averaged into the dispersions shown in Fig. 26 and Table 27. For these measurements, the Fraunhofer approximation model was used. Small sample trays were used at a feed rate of 65%, a background measurement time of 10 seconds, and a sample measurement time of 30 seconds. Blind filtering was used to capture between 1% and 6% of the data. 3.2. Particle Morphology

[0262] Each of the four formulations was imaged at magnifications of 500, 1500, and 5000 using a Hitachi SU3500 scanning electron microscope. Images taken at magnification of 5000 are shown in Fig. 27 (Formulation A), Fig. 28 (Formulation B), Fig. 29 (Formulation C), and Fig. 30 (Formulation D). All formulations contained whole and collapsed spherical particles with a diameter of approximately 3 μm or less, consistent with laser diffraction results. Formulation C appeared to be the most wrinkled, while Formulation A appeared to be the "smoothest." Surface roughness can improve aerosol performance. For all formulations, no significant crystalline surface formation or particle fusion was observed. 3.3. Moisture Content

[0263] Since the formulations were spray-dried from a solvent mixture containing water, the samples were analyzed for moisture content using a Metrohm 874 oven sample processor. Three blanks and three moisture standards were tested first to determine system suitability before testing the samples. 20 milligram samples (n=3 replicates) were heated for each formulation from a starting temperature of 50°C to 140°C at a heating rate of 2.5°C / min. The moisture content for all formulations was less than 3% by weight, as shown in Table 28. Formulations containing trehalose (B and C) exhibited higher moisture content than those formulated with mannitol (A and D). Formulations containing a higher leucine content (C and D) also exhibited higher moisture content. 3.4. Residual Solvent

[0264] Since the other component in the spray solvent is 1-propanol, the residual amount of 1-propanol was determined using the headspace method on an Agilent 7890 gas chromatography system. All samples have less than 0.5 wt% of 1-propanol (Table 29), and formulations with more leucine (C and D) have less residual 1-propanol as well as formulations containing mannitol (A and D). 3.5. Powder X-ray Diffraction

[0265] Sample crystallinity was evaluated using a Rigaku MiniFlex 600 Powder X-ray diffractometer. Samples were prepared on a 0.2 mm Zero Background Holder (ZBH) disc and tested on the instrument at 3 to 40 2θ. Formulations A and D exhibited crystalline mannitol (a nearly polymorphic mixture) and leucine, whereas formulations B and C exhibited crystalline leucine with amorphous trehalose.

[0266] The components may exhibit different diffraction intensities as pure substances compared to the spray-dried formulation. The formulation containing mannitol appeared to have a similar diffraction pattern. The formulation containing trehalose also appeared to have a similar amorphous diffraction pattern. 3.6. Differential Scanning Calorimetry (DSC)

[0267] Thermal transitions can be used to predict the stability of the formulation. Four formulations were scanned on a TA Instruments Q2000 differential scanning calorimeter. Samples were hermetically sealed and held in equilibrium overnight in a dry environment (< 5 % RH) prior to being performed on the instrument. The control was set to ± 1.5°C / min with a heating ramp rate of 2.5°C / min from 0 to 180°C. A thermal event at 64°C was observed for all samples, and this event may correspond to the melting of treprostinyl palmityl or DSPE-PEG2000.

[0268] No crystallization events, which serve as a positive indicator of thermal stability, were observed for the samples. No glass transition was detected for the mannitol-based formulations (A and D), which supports the powder X-ray diffraction (PXRD) results for the crystalline materials. Formulations A and D exhibited a melt at 164°C, which is consistent with the melting temperature of mannitol. Formulations B and C exhibited a glass transition at 83°C, likely due to the amorphous trehalose. Formulation B also exhibits thermal events at 133°C and 158°C, which are not observed in the other samples. 3.7. Thermogravimetric Analysis (TGA)

[0269] Thermal decomposition data for the formulation was measured using a TA Instruments Discovery Thermogravimetric Analyzer. The sample was passed from 0°C to 300°C at a rate of 2.5°C / min. The formulation began to decompose rapidly after 180°C. Weight change was observed at approximately 100°C, which corresponds to the moisture content. 3.8. Dynamic Vapor Adsorption

[0270] Moisture adsorption and desorption profiles were measured on the DVS Advantage 1 surface measurement system. Samples were processed from 0 to 90% RH at 25°C with a phase transition of 109% RH. All four formulations appeared to have a weight change event starting at approximately 50% or 60% RH, depending on the formulation. A second cycle was performed on the samples to evaluate the water absorption rate after crystallization. No change was observed in the samples during the second cycle, and most samples did not retain water during the final desorption phase.

[0271] The adsorption results from Formulation D show a change at higher humidity (approximately 70% RH) compared to the results from Formulation A. A slight loss of mass of Formulation D was observed at approximately 50% RH compared to Formulation A, which appears to result in continued mass loss even when absorbing added moisture at higher humidity.

[0272] The uptake rate of the trehalose formulation indicates that it takes approximately 50 to 90 minutes (depending on the level of leucine content) to reach moisture equilibrium at 40% relative humidity. The lower the leucine content, the faster the moisture was absorbed compared to those with a higher leucine content. 3.9. Aerosol performance of capsules of formulations A, C, and D evaluated by aerodynamic particle size distribution (APSD) by NGI

[0273] Capsules of Treprostinyl Palmityl dry powder formulations A, C, and D were stored at 25°C or 40°C for 1 to 3 months. The appearance of the encapsulated dry powder did not change, and, for example, no browning or apparent growth or change in hardness was observed. The particle size distribution (PSD) of the dry powder formulations, as measured by laser diffraction, was not affected after 3 months of storage at 25°C or 40°C.

[0274] Treprostinyl palmityl dry powder formulations A, C, and D were encapsulated, and the capsules were stored at 40°C for 1, 2, or 3 months, or at 25°C for 3 months. The initial (T=0) FPD of the formulations, as well as the microparticle dose (FPD) of the formulations from the stored capsules, was measured by NGI. Treprostinyl palmityl dry powder formulations A, C, and D were also stored as bulk at 40°C or 25°C for 3 months, subsequently filled into capsules, and administered on the same day for FPD determination. The FPD results for formulations A, C, and D are shown in Figures 31, 32, and 33, respectively. These data indicate that formulation D exhibited the minimum change in FPD (-3.7%) after being stored in capsules at 25°C for 3 months. Additionally, Formulation D exhibited a change of -5.2% in FPD when stored as bulk at 25°C for 3 months, filled into capsules, and administered on the same day. Furthermore, for Formulations A, C, and D, storage at 40°C did not yield the predicted values ​​for aerosol performance as measured by FPD for long-term storage at 25°C. Based on these data, conditioning or pretreatment of the powder or capsules is not required to alter aerosol performance.

[0275] The released doses and total recovery rates of formulations A, C, and D from the capsules described above were also determined, and the results are shown in Table 30.

[0276] The above data indicates that Formulation D represents the minimum change in released dose after being stored in capsules at 25°C for 3 months, and after being stored in bulk at 25°C for 3 months, filled into capsules, and administered on the same day. Taken together, based on changes in aerosol performance, Formulation D was found to be stable for at least 3 months at 25°C. Additionally, the latest stability data supports a shelf life of 6 months when stored at 2 to 8°C. Example 3 - Determination of Aerosol Performance of Trahalose-Based Treprostinyl Palmityl Dry Powder Formulation and Pulmonary Pharmacokinetic Profile in Rats After Inhalation of Dry Powder Formulation under Accelerated Storage Conditions

[0277] A trihalose-based treprostinyl palmityl dry powder containing treprostinyl palmityl, DSPE-PEG2000, trehalose (Treh), and leucine (Leu) in a weight ratio of 1:0.5:70:30 was prepared by spray drying using a Buchi B-290 system as described in Example 1. The dry powder was stored in sealed glass vials at 40°C and uncontrolled ambient humidity for accelerated stability studies. The aerosol performance of the powder was measured after storage for 1.5, 2.5, and 3.5 months. Method and material 1. Dynamic Vapor Adsorption (DVS) Study

[0278] Moisture absorption curves were obtained using a dynamic vapor adsorption (DVS) automated gravimetric adsorption system (DVS Intrinsic1 Plus, Surface Measurement System, PA, USA). Approximately 20 mg of powder was loaded, and the adsorption / desorption cycle was performed at 25°C using an adsorption / desorption isotherm (from 0% relative humidity (RH) to 90% RH, then to 0% RH, and increasing by 10% RH). The change (%) in powder mass according to RH was determined and plotted. 2. DPI Device and Aerodynamic Particle Size Distribution (APSD) Test

[0279] The RS01 Mod.7 DPI device (High Resistance, code 239700002AA, Plastiape, Italy) was used in this study. The median mass aerodynamic diameter (MMAD) of the trehalose-based treprostinyl palmityl dry powder was measured at 60 L / min using a next-generation impactor (NGI).

[0280] Approximately 15 mg of dry treprostinyl palmityl powder was loaded into Size # 3 HPMC capsules (Qualicaps, Inc.). These capsules were loaded into a DPI device and operated to characterize the aerosol particle size distribution (APSD). The amount of drug deposited on each impactor stage and filter was analyzed by high-performance liquid chromatography (HPLC). 3. Nose-dedicated suction

[0281] Treprostinyl palmityl dry powder was delivered through a 12-port nose-only inhalation chamber using a dry powder dispenser (Vilnius Aerosol Generator (VAG), CH Technologies, USA). Approximately 1 g of dry powder was loaded into the VAG. The VAG had a flow rate of 8 L / min at 1.0 V for a total of 20 minutes. The dry powder delivery system is described in more detail in the literature [see "Inhaled INS1009 Demonstrates Localized Pulmonary Vasodilation," European Respiratory Society (ERS) International Congress, 3-7 September 2016, London, United Kingdom, Abstract No: 853952 (poster PA2845); the full text of this literature is incorporated herein by reference]. 4. PK Sample Collection and Analysis

[0282] Rat lung tissues were harvested immediately after delivery (approx. 0.5 hours), and at 6, 12, and 24 hours after administration. Lung tissue samples were analyzed by LC-MS / MS for treprostinyl palmityl and treprostinyl (TRE). The results are reported as "treprostinyl palmityl equivalents" to explain the post-mortem hydrolysis of treprostinyl palmityl. Treprostinyl palmityl equivalent, ng / g = [Treprostinyl palmityl, ng / g] + [TRE, ng / g] * (MM Treprostinyl palmityl / MM TRE) MM: Molar mass result 1. Dynamic Vapor Adsorption (DVS)

[0283] Dynamic vapor adsorption (DVS) of the treprostinyl palmityl dry powder formulation is shown in Fig. 34. When the dry powder was exposed to an increase in RH up to 50%, the absorbed moisture increased from 0% to over 8%, was not completely reversibly desorbed, and remained at over 5.5%. To maintain the moisture content at approximately 4% or less, the RH exposure to this powder can be controlled to <30% during manufacturing. 2. Aerodynamic Particle Size Distribution (APSD)

[0284] APSD data of dry treprostinyl palmityl powder stored for 1.5 months (T=1.5M), 2.5 months (T=2.5M), and 3.5 months (T=3.5M), as well as the initial APSD (T=0) obtained by RS01 Mod. 7 DPI (high resistance) at 60 L / min, are shown in Fig. 35 and Table 31. The distributions from all four time points were similar.

[0285] The fine particle fraction (FPF), MMAD, and geometric standard deviation (GSD) values ​​of the aerosolized dry powder after storage for up to 3.5 months at 40°C and uncontrolled ambient humidity are summarized in Table 32. The FPF values ​​ranged from 54.6% to 58.7%. The MMAD values ​​ranged from 1.25 µm to 1.44 µm. The GSD values ​​ranged from 3.5 to 4.1. 3. Rat lung PK results

[0286] The concentrations of treprostinyl palmityl equivalents (treprostinyl palmityl plus treprostinyl) in the lungs after inhalation of nebulized INS1009 or aerosolized treprostinyl palmityl dry powder are summarized in Table 33 and Figure 36. Nebulized INS1009 contained treprostinyl palmityl in a molar ratio of 45:45:10 suspended in PBS, along with the excipients squalene and DSPE-PEG2000 [see Corboz et al., "Preclinical Pharmacology and Pharmacokinetics of Inhaled Hexadecyl-Treprostinil (C16TR), a Pulmonary Vasodilator Prodrug," J Pharmacol Exp Ther. 363:348-357 (2017); the full text of this literature is incorporated herein by reference]. Both nebulized INS1009 and C16TR (treprostinyl palmityl) dry powders have similar lung PK profiles after inhalation in rats. A statistical comparison of these two profiles is summarized in Table 34, which shows similar slopes for both profiles.

[0287] In summary, this study indicates that the aerosol particle size distribution of the trehalose-based treprostinyl palmityl dry powder formulation is reproducible during storage for up to 3.5 months at 40°C in sealed vials and uncontrolled RH, and that the pulmonary PK profile of the treprostinyl palmityl dry powder formulation is similar to that of nebulized INS1009. Example 4 - Pharmacokinetic evaluation of mannitol and trehalose-based treprostinyl palmityl dry powder formulations in rats

[0288] In this study, the pulmonary and plasma pharmacokinetics (PK) of two different treprostinyl palmityl dry powder formulations having mannitol (i.e., Formulation D as described in Example 2) or trehalose (i.e., Formulation C as described in Example 2) as the major excipient were evaluated. The compositions of Formulations D and C expressed in weight ratios, the targeted weight percentages calculated based on weight ratios, and the actual weight percentages of the components from a typical batch of each formulation are summarized in Tables 35A and 35B, respectively. method

[0289] Male Sprague Dooley rats (300 to 400 g) were exposed to the aerosol of dry powder formulation D or formulation C using a 12-port nose-only inhalation chamber and a Vilinius aerosol generator (VAG). The rats were placed in a restraint tube attached to the nose-only port in the chamber. In two separate studies with formulation D and formulation C, nine rats were used in each study, and one port was used for collecting the aerosol drug on the filter. An abbreviated study was also performed with formulation D, where six rats were used, and one port was used for collecting the amount of drug deposited on the filter.

[0290] For drug exposure, 1 gram of the substance was placed in the VAG. The output from the VAG was established at 1 volt (V), and the drug was dispersed and delivered to the nose-only chamber with a bias airflow of 8 L / min. Air entered the bottom of the nose-only chamber and was expelled from the top. The exposure period was set to 20 minutes. A vacuum source (0.5 L / min) was attached to the filter, and the drug sampling time was set to 5 minutes. The amount of treprostinyl palmityl deposited on the filter was measured by HPLC and a rechargeable aerosol detector (CAD). The delivered drug dose was calculated from the concentration of the inhaled drug (from the filter data), the exposure period, the respiratory volume per minute, and the weight with a deposition fraction of 1.0 used for the drug delivered to the nose and a deposition fraction of 0.1 used for the drug delivered to the lungs. It was expressed as a dose per kg of body weight.

[0291] In two primary studies with formulations D and C, blood samples were collected at 0.5, 2, 4, 6, 12, and 24 hours after drug exposure. Blood samples were centrifuged to extract plasma. In these studies, respiratory tissues from the larynx, trachea, keel + bronchus, and lungs were collected at 0.5, 6, 12, and 24 hours after drug exposure. In a simplified study with formulation D, blood samples were obtained at 0.5, 2, 4, 12, and 24 hours, and respiratory tissues were harvested at 0.5 and 24 hours after drug exposure. The concentrations of treprostinil (TRE) in plasma and treprostinil palmityl and TRE in respiratory tissues were measured by LC-MS / MS. For all respiratory tissues, the concentration of treprostinyl palmityl (C16TR) equivalent (C16TReq) was derived from the concentrations of treprostinyl palmityl and TRE (C16TReq = treprostinyl palmityl + [TRE x 615 / 390.5 ng / g]), where 615 and 390.5 are the molecular weights of treprostinyl palmityl and TRE, respectively. Lung treprostinyl palmityl equivalent and plasma TRE data were used to derive the following PK parameters using the Microsoft Excel PK Solver program: lambda z (terminal clearance constant), T½, Tmax, Cmax, and AUC0-inf. result

[0292] Exposure of rats to Formulation D and Formulation C resulted in a slightly higher total delivery dose of 78 µg / kg body weight for Formulation D compared to a total delivery dose of 58 µg / kg body weight for Formulation C. The concentration of treprostinyl palmityl equivalents in the lungs, measured at 0.5 hours after exposure, was also slightly higher for Formulation D, averaging 3072 ng / g compared to 1711 ng / g for Formulation C. 1. Lungs and upper airways treprostinyl palmityl, TRE and treprostinyl palmityl equivalents

[0293] For both Formulation D and Formulation C, the peak concentrations of treprostinyl palmityl, TRE, and treprostinyl palmityl equivalent in the lungs occurred at 0.5 hours after exposure. Over 24 hours, treprostinyl palmityl and TRE decreased slowly in a single-exponential manner, and the concentrations of both treprostinyl palmityl and TRE were consistently higher for Formulation D at all time points. These results are illustrated in Fig. 37 for treprostinyl palmityl equivalent (C16TReq), which showed a slow decrease in the uniform concentration of treprostinyl palmityl in the lungs over 24 hours, and the concentration of treprostinyl palmityl equivalent was consistently higher for Formulation D compared to Formulation C. Fig. 38 shows the concentration of treprostinyl palmityl (C16TR) in the lungs after inhaling Formulation D or Formulation C. Figure 39 shows the concentration of TRE in the lungs after inhaling formulation D or formulation C.

[0294] Comparison of induced PK parameters for lung treprostinil palmityl equivalents did not show significant differences between formulation D and formulation C for lambda z, T½, and Tmax, but showed a 79% higher lung treprostinil palmityl equivalent Cmax and a 130% higher AUC0-24 h for formulation D (Table 36).

[0295] Regarding deposition in the larynx, trachea, keel + bronchus, and lungs, most of the treprostinil palmityl (> 97%) was deposited in the lungs. This was observed in both formulations D and C. Nasal tissues were not collected in this study. 2. Plasma TRE

[0296] The peak concentration of TRE in plasma was observed at 0.5 hours, and for formulation D, it decreased slowly over 24 hours, while for formulation C, it decreased more rapidly. The plasma TRE concentration for formulation C remained below detectable levels until 24 hours (Fig. 40).

[0297] Comparison of induced PK parameters for plasma TRE revealed that Formulation D had a 34% lower lambda z and a 51% higher T½ compared to Formulation C (Table 37). Plasma TRE Cmax was 10% higher and AUC0-t was 51% higher for Formulation D compared to Formulation C (Table 37).

[0298] In summary, the inhalation of treprostinil palmityl dry powder formulations D and C resulted in lower plasma Cmax and sustained levels of treprostinil in plasma and lungs compared to the inhalation of treprostinil. A comparison of PK profiles in rats between formulations D and C delivered under similar conditions of a VAG output of 1 V for 20 minutes resulted in a 34% higher delivery dose for formulation D (78 µg / kg) compared to formulation C (58 µg / kg). Concentrations of treprostinil palmityl, TRE, and treprostinil palmityl equivalents in the lungs were consistently higher with formulation D, likely due to the higher delivery dose, and both formulations exhibited a slow, single-exponential decline over 24 hours. A similar trend was observed with higher plasma TRE concentrations in formulation D and a slow decline over 24 hours for both formulations. Most TRE and treprostinil palmityl exposure occurred in the lungs, and less than 3% was deposited in the upper respiratory tract of the larynx, trachea, keel, and bronchi.

[0299] These results for formulations D and C show a PK profile similar to that previously observed in nebulized INS1009, which exhibits peak concentrations of treprostinyl palmityl equivalent in the lungs and TRE in plasma by 30 minutes, with a slow, single-exponential decline over 24 hours. However, there appears to be a significant difference in the lung treprostinyl palmityl equivalent:plasma TRE Cmax ratio between the treprostinyl palmityl dry powder formulation on one hand and nebulized INS1009 on the other. The typical pulmonary treprostinyl palmityl equivalent:plasma TRE Cmax ratio for nebulized INS1009 is about 800, and the ratio for treprostinyl palmityl dry powder formulation is in the range of about 1,600 to 13,000 [see Corboz et al., J Pharmacol Exp Ther 363: 1-10 (2017), the full text of which is incorporated herein by reference]. This is because pulmonary vasodilation is more associated with the local activity of TRE in the lungs and less associated with the level of TRE in plasma [Chapman RW et al., Pulm. Pharmacol. Ther. [See 49:104-111 (2018), the contents of which are incorporated herein by reference in their entirety], the unexpected difference in the pulmonary treprostinyl palmityl equivalent:plasma TRE Cmax ratio indicates that administration of the treprostinyl palmityl dry powder formulation results in low systemic exposure to TRE and, consequently, minimizes potential systemic side effects such as a decrease in systemic blood pressure. Example 5 - Evaluation of the efficacy of mannitol and trehalose-based treprostinyl palmityl dry powder formulations in hypoxic inoculated remotely controlled rats

[0300] This example describes the evaluation of the in vivo efficacy of two different treprostinyl palmityl dry powder formulations described in Examples 2 and 4: mannitol-based formulation D and trehalose-based formulation C (Tables 35A and 35B). Efficacy was determined in rats prepared with a telemetry probe implanted in the right ventricle to measure the inhibition of the increase in right ventricular pulse pressure (RVPP) induced by exposure to an inhaled hypoxic gas mixture. method

[0301] Experiments were performed in male Sprague Dooley rats implanted with telemetry props in the right ventricle and descending aorta to measure RVPP and mean systemic arterial blood pressure (mSAP). These cardiovascular parameters were measured during the transition from breathing normal oxygen air (21% O2 / residual O2) to breathing normal oxygen air after 10 minutes of exposure to hypoxic air (10% O2 / residual O2), and then returning to breathing normal oxygen air. In each experiment, the increase in RVPP due to hypoxia inoculation (Δ RVPP attributable to hypoxia) was measured before drug exposure and at 1, 6, 12, and 24 hours after exposure to inhaled treprostinyl palmityl dry powder formulation D or formulation C. The drug formulations were supplied to a Vilinius aerosol generator (VAG) at an output of 1 volt (V) and delivered to a 12-port nose-only inhalation chamber for 20 minutes. The aerosol was dispersed from the VAG at a bias flow of 8 L / min and delivered to the bottom of the nose-only inhalation chamber. A filter was connected to one of the nose-only ports and attached to a vacuum flow of 0.5 L / min for 5 minutes. The amount of treprostinyl palmityl deposited on the filter was measured by LC-MS / MS. Plasma and respiratory tissue samples from rats were analyzed for the concentration(s) of treprostinyl palmityl and / or TRE. result

[0302] Exposure of rats to inhaled hypoxia increased the RVPP by 10 to 13 mmHg above normal oxygen values. At the end of hypoxia inoculation, the RVPP decreased immediately within minutes and returned to the pre-hypoxia value within 10 minutes. The drug effect was determined by comparing the hypoxia-attributed △RVPP at various times up to 24 hours after drug exposure to the sum of 2 to 3 measurements obtained before drug exposure.

[0303] The experimental results are illustrated in Fig. 41. In Fig. 41, values ​​are mean ± SEM (n = 6 to 8 rats for Formulation D and n = 4 rats for Formulation C). Day -1 represents the baseline value before drug exposure, and Day 0 represents the value after drug exposure. ΔRVPP indicates an increase in right ventricular pulse pressure. "*" indicates P < 0.05 compared to the combined value before drug exposure (at 1, 6, and 12 hours) at Day -1. The results indicate that exposure to Formulation D reduces ΔRVPP due to hypoxia, with a statistically significant (P < 0.05) inhibition observed at 1, 6, 12, and 24 hours. Similar results were obtained for Formulation C, with a statistically significant (P < 0.05) inhibition observed at 1, 6, 12, and 24 hours. Up to 24 hours after treatment with formulations D and C, RVPP tended to return to baseline values ​​observed before drug exposure. As a control, exposure to an inhaled mannitol vehicle (containing 69.48 wt% targeted weight percent mannitol, 29.78 wt% targeted weight percent leucine, and 0.74 wt% targeted weight percent DSPE-PEG2000) suppressed the ΔRVPP response to hypoxia at 1, 6, 12, and 24 hours after administration (data not shown). Based on the concentration of the inhaled drug, duration of exposure, body weight, respiratory volume per minute, and a deposition fraction of 1.0, the delivery dose of Formulation D was 78 µg / kg body weight (equivalent concentration of pulmonary treprostinil palmityl 3072 ng / g) and the delivery dose of Formulation C was 58 µg / kg body weight (equivalent concentration of pulmonary treprostinil palmityl 1711 ng / g) [see Alexander DJ et al., Inhal. Toxicol. 20:1179-1189 (2008); the full text of this literature is incorporated herein by reference].

[0304] In summary, efficacy evaluations in hypoxic-inoculated telemetric rats indicate that inhaled treprostinil palmityl dry powder formulations C and D inhibited the increase in RVPP up to 24 hours after drug exposure. Up to 24 hours, the RVPP response to hypoxia tended to return to baseline values ​​observed prior to drug exposure. In comparison, nebulized INS1009 at an achieved delivery dose of 76 µg / kg inhibited the hypoxia-induced increase in RVPP up to 12 hours while returning to baseline values ​​up to 24 hours. Inhaled treprostinil at the highest dose used (215 µg / kg) inhibited the hypoxia-induced increase in RVPP for 2 hours, and at lower inhaled doses (15, 53, or 110 µg / kg) for 1 hour, respectively. These results demonstrated the prolonged inhibition of hypoxia-induced increase in RVPP by the treprostinyl palmityl dry powder formulation in telemetric rats. Example 6 - Evaluation of Mannitol and Trehalose-Based Treprostinyl Palmityl Dry Powder Formulations for Cough and Ventilation in Guinea Pigs

[0305] This example describes a study on the effects of mannitol-based treprostinyl palmityl dry powder formulation D and trehalose-based treprostinyl palmityl dry powder formulation C (described in Examples 2 and 4; see formulation composition in Tables 35A and 35B) to show changes in cough, ventilation, and Penh, and a dimensionless index of the altered breathing pattern typically seen during bronchoconstriction in self-aware male guinea pigs. method

[0306] Experiments were performed on male Hartley guinea pigs. After a 3-day acclimatization period, guinea pigs were placed on a whole-body plethysmograph using established techniques to measure ventilation (tidal volume, respiratory rate, and volume per minute), Penh, and cough [see Corboz et al., J Pharmacol Exp Ther 363: 1-10 (2017); Chong BTY et al., J. Pharmacol. Toxicol. Methods 39, 163-168 (1998); Lomask, Exp. and Toxicol. Pathol. 57,13-20 (2006); the full text of each of these references is incorporated herein by reference]. Cough was measured from plethysmograph recordings indicating a large expiration following a large inhalation, and was verified by manual observation, video recording, and cough audible. Ventilation, Penh, and cough data were measured during a 15-minute baseline period prior to exposure to the dry powder aerosol. Test items, including Formulation D, Formulation C and their individual vehicles, namely the mannitol vehicle (containing 69.48 wt% targeted weight percent mannitol, 29.78 wt% targeted weight percent leucine, and 0.74 wt% targeted weight percent DSPE-PEG2000), and the trehalose vehicle (containing 69.48 wt% targeted weight percent trehalose, 29.78 wt% targeted weight percent leucine, and 0.74 wt% targeted weight percent DSPE-PEG2000), were subsequently delivered as dry powder for 15 minutes, and then observed for a period of 120 minutes after the aerosol compound was administered. Air for aerosol delivery for all experimental steps was supplied by an air compressor. The typical humidity of the supplied air was approximately 30% when measured. Ventilation, Penh, and cough were measured before, during, and after exposure to the test product. The aerosolized test product was formed using a Vilnius aerosol generator (VAG).The air flow rate through the VAG was set to 4.5 L / min to disperse the aerosol, and combined with air with 1 L / min of moisture (30% humidification) to facilitate aerosol delivery to the blood volume meter and minimize electrostatic adhesion problems. Accordingly, the total air inflow was 5.5 L / min. The generator output from the VAG was 1, 0.75, and 0.5 volts, and each VAG output was provided for 15 minutes to deliver three different doses of the drug, with higher doses delivered at higher voltages. A vacuum flow of 8 L / min was formed at the bottom of the blood volume meter so that air and aerosol entered from the top and were discharged from the bottom of the system. A separate vacuum source of 0.5 L / min was connected to a filter attached to the port of the blood volume meter to sample the concentration of the drug (treprostinyl palmityl). Filter sampling was maintained for the entire duration of the study, i.e., 135 minutes, and a 15-minute exposure time was used to calculate the aerosol concentrations in the nose-only chamber and the total inhaled drug dose. Filter samples were analyzed for treprostinyl palmityl concentrations. At the end of the study, guinea pigs were euthanized, and blood (plasma), lungs, trachea, larynx, and keel + trachea were collected to measure treprostinyl palmityl and TRE concentrations in these samples. result

[0307] Exposure to formulation D, formulation C, or mannitol and trehalose vehicles was well tolerated and did not result in any death.

[0308] Aerosolized formulation D (total inhaled dose = 35.8 µg / kg body weight) administered at 1 V for 15 minutes induced coughing in 4 out of 6 guinea pigs. The average number of coughs for this exposure was 24 ± 12. At a setting of 0.75 volts and administration for 15 minutes (total inhaled dose = 12.8 µg / kg body weight), coughing was observed in 3 out of 4 guinea pigs, with an average cough count of 19 ± 7. At a setting of 0.5 volts and administration for 15 minutes (total inhaled dose = 2.3 µg / kg body weight), no coughing was observed in 4 out of 4 guinea pigs. Mannitol vehicle aerosols produced at a 1 V setting administered for 15 minutes did not cause coughing in 4 out of 4 guinea pigs (Table 38). There were no consistent changes in ventilation with formulation D or mannitol vehicle, and the slight increase in PanH observed with these drugs did not reach statistical significance compared to mannitol vehicle.

[0309] Aerosolized formulation C (total inhaled delivery dose = 10.2 µg / kg body weight) administered at 1 V for 15 minutes induced coughing in 3 out of 6 guinea pigs, with an average cough count of 10 ± 5 coughs. In settings administered at 0.75 V (total inhaled delivery dose = 4.7 µg / kg body weight) and 0.5 V (total inhaled delivery dose = 1.5 µg / kg body weight) for 15 minutes of exposure, formulation C did not induce coughing in 4 out of 4 guinea pigs in each group. Trehalose vehicle administered at a voltage of 1 V for 15 minutes did not induce coughing in 4 out of 4 guinea pigs (Table 38). There were no consistent changes in ventilation with formulation C or trehalose vehicle.

[0310] Lung treprostinil palmityl homologous concentrations increased with the inhaled drug dose for both Formulation D and Formulation C, with Formulation D having approximately three times higher levels of lung treprostinil palmityl homologous concentration compared to Formulation C (Table 38). Since most of the inhaled drug was deposited in the lungs (data not shown), there was no difference between these two formulations in the percentage of drug deposited in the upper airway tissues of the larynx, trachea, and keel + bronchi. Nasal tissues were not collected in this study.

[0311] The results from these studies showed that coughing occurred with both formulations at critical inhalation doses of 12.8 µg / kg for Formulation D and 10.2 µg / kg for Formulation C. These individual doses are 10 and 8 times higher than the critical dose of inhaled TRE that induces coughing in guinea pigs, which is 1.23 µg / kg. After exposure to Formulation D or Formulation C, the first cough occurred between 17 and 35 minutes, which is later than the timing of coughing with neubulidized TRE occurring within the first 10 minutes of exposure.

[0312] The uniform concentration of treprostinil palmityl in the lungs was approximately three times higher than that of formulation D (Table 38), and because most of the drug was deposited in the lungs (data not shown), there was no difference between these two formulations in the percentage of drug deposited in the upper airways of the larynx, trachea, and keel plus bronchi. The concentration of TRE in plasma was two to three times higher in formulation D compared to formulation C (Table 38). Example 7 - Evaluation of the effect of treprostinyl palmityl dry powder formulation in the treatment of an 8-week Sugen-hypoxia (SuHx)-induced pulmonary arterial hypertension rat model

[0313] The Sugen-hypoxia (SuHx)-induced PAH model in rats is a well-documented model. This model replicates many pathologies seen in clinical diseases. In this example, the effects of inhaled treprostinil (TRE), intravenous treprostinil (TRE), and oral celexipeg on pulmonary arterial pressure (PAP) and other cardiovascular parameters, right ventricular hypertrophy, pulmonary and cardiac histopathology, and biomarkers associated with PAH were evaluated on an 8-week SuHx-induced pulmonary arterial hypertension (PAH) model in rats, as well as treprostinil palmityl dry powder formulation D (described in Examples 2 and 4; see formulation composition in Table 35A). Research group, test item and vehicle and administration thereof

[0314] Male Sprague Dooley rats weighing 200 to 250 g were randomized into study groups based on body weight to ensure that the weight range was evenly distributed across the groups. Table 39 summarizes the study groups and the treatments received by each study group. 1. Group 1 - Old Tree Food Control Group

[0315] The control group (Group 1) received a single subcutaneous injection of 100% DMSO at 2 mL / kg (vehicle for Sugen) and was not treated. 2. Group 2 - Treatment group of vehicle for Treprostinyl Palmityl Dry Powder Formulation D

[0316] The vehicle for Treprostinyl Palmityl dry powder formulation D has a targeted composition of 70 wt% mannitol and 30 wt% leucine. Rats in this vehicle treatment group (Group 2) were weighed, placed in a cup of a Vilnius aerosol generator (VAG) (loaded with 170 mg of vehicle), and administered in a nose-cone chamber at 1 volt once a day until the substance was aerosolized. The aerosolization period was measured. In parallel with Groups 3 and 4, vehicle treatment for these groups was performed for 35 days. 3. Groups 3 and 4 - Treatment group with Treprostinyl Palmityl Dry Powder Formulation D

[0317] For groups 3 and 4, Treprostinyl palmityl dry powder formulation D was weighed and placed in a VAG cup (90 mg used for a targeted dose of 57 µg / kg and generated at 0.5 volts (V), and 170 mg used for a targeted dose of 138 µg / kg and generated at 1 volt (V)). The dry powder formulation was administered once daily for 35 days, and for each administration, the VAG was left until all material was aerosolized. The aerosolization period was measured. The dry powder formulation was stored at 4 ± 2°C. 4. Group 5 - Treatment group with nebulized vehicle for inhaled treprostinil (TRE)

[0318] The nebulized vehicle for inhaled treprostinil (TRE) is phosphate-buffered saline (PBS). In parallel with Group 6, rats in this nebulized vehicle treatment group (Group 5) received nebulized PBS in a nose cone chamber four times a day for 12 hours over a period of 35 days. 5. Group 6 - Treatment group with nebulized treprostinil (TRE)

[0319] The nebulized treprostinil (TRE) solution contained 0.5 mM TRE in PBS at a pH of 7.4. This solution was stored at 4 ± 2°C, and the expiration date was set to 7 days after preparation. Using this solution, a targeted solution of 110 µg / kg TRE was delivered and administered to rats in Group 6 by four inhalations over a 12-hour period daily for 35 days. 6. Group 7 - Treatment group with vehicle for intravenous treprostinil via continuous infusion

[0320] The vehicle for intravenous treprostinil was an aqueous solution containing 3.0 mg / mL m-cresol, 5.3 mg / mL NaCl, and 6.3 mg / mL sodium citrate at a pH of 6.0 to 7.2. Rats in this group (Group 7) were each implanted with an osmotic pump filled with the vehicle and administered continuous infusion at the infusion rate specified for Group 8 below. In parallel with Group 8, continuous vehicle infusion for Group 7 was continued for 35 days. 7. Group 8 - Treatment group with intravenous treprostinil (TRE) via continuous infusion

[0321] Two solutions were prepared for the intravenous administration of TRE to Group 8. These solutions differed only in the concentration of TRE. Specifically, the first solution was an aqueous solution containing 8.75 mg / mL TRE, 3.0 mg / mL m-cresol, 5.3 mg / mL NaCl, and 6.3 mg / mL sodium citrate, with a pH of 6.0 to 7.2. The second solution was an aqueous solution containing 10.7 mg / mL TRE, 3.0 mg / mL m-cresol, 5.3 mg / mL NaCl, and 6.3 mg / mL sodium citrate, with a pH of 6.0 to 7.2. Each rat in this group (Group 8) received an intravenous infusion of TRE using an implanted osmotic pump (ALZET pump). Each ALZET pump was first filled with 2 mL of Solution 1 containing 8.75 mg / mL TRE, which was sufficient to achieve continuous infusion over a 28-day period (based on an infusion rate of 2.5 µL / h) for 450 g rats and to achieve a targeted dose of 810 ng / kg / min TRE. The ALZET pump was replaced on day 19 of infusion (day 40 of the total study) and filled with 2 mL of Solution 2 containing 10.7 mg / mL TRE, based on the rat body weight increasing to approximately 550 g. The induction of TRE concentrations in Solution 1 and Solution 2 is shown below. Continuous IV TRE infusion continued for 35 days.

[0322] Derivation of TRE concentration (8.75 mg / mL) in the first solution: o Assume rat body weight is 450 g o Infused TRE = 810 ng / kg / min; = 364.5 ng / min; = 21.87 µg / h; = 524.88 µg / day; = 18.37 mg / 35 days o AlZET infusion rate = 2.5 µl / hr; = 60 µl / day; = 2100 µl / 35 days; = 2.1 mL / 35 days o TRE concentration = 18.37 mg / 2.1 mL = 8.75 mg / mL

[0323] Induction of TRE concentration (10.7 mg / mL) in the second solution o Assume rat body weight is 550 g o Infused TRE = 810 ng / kg / min; = 445.5 ng / min; = 26.73 µg / h; = 641.52 µg / day; = 22.45 mg / 35 days o ALZET infusion rate = 2.5 µl / hr; = 60 µl / day; = 2100 µl / 35 days; = 2.1 mL / 35 days o TRE concentration = 22.45 mg / 2.1 mL = 10.7 mg / mL 8. Group 9 - Treatment group of celexipeg with vehicle via oral administration

[0324] The vehicle for celexipeg is an aqueous solution containing 0.5% (w / v) methylcellulose with a pH of 7.5 to 8.0. In parallel with Group 10, the vehicle was administered to rats of this group by oral feeding twice a day for 35 days. 9. Group 10 - Treatment group with celexipeg via oral administration

[0325] A celexipeg solution containing 3.0 mg / mL celexipeg in 0.5% (w / v) methylcellulose at pH 7.5 was prepared. The solution was stored at room temperature, and the expiration date after preparation was set to 7 days. The solution was administered to this rat group (Group 10) by oral feeding twice daily at a targeted dose of 30 mg / kg with a volume of 10 mL / kg at each administration. Selexipeg treatment was continued for 35 days. Research Design

[0326] Table 39 describes an overview of the study design, the details of which are as follows. 1. Introduction of PAH

[0327] On the 21st, rats were randomized into treatment groups based on their body weight as described above.

[0328] On day 0, a solution of 10 mg / mL of sugen in DMSO was prepared, and rats from groups 2 through 10 (see Table 39) received a single subcutaneous injection of the sugen solution (20 mg / kg at a volume of 2 mL / kg) and were returned to their rat cages. Additionally, on day 0, rats from group 1 received a single subcutaneous injection of 100% DMSO (sugen vehicle) at 2 mL / kg and were returned to their individual rat cages.

[0329] Rats from Groups 2 through 10 were placed in air-controlled cages equipped with a mixture of nitrogen and ambient air controlled by a ventilated cage system to accommodate an FiO2 of 0.10 (10%). The rats were maintained under these hypoxic conditions for 21 days. Under hypoxic conditions, the cages were cleaned and replaced once a week to expose the rats to ambient oxygen levels for less than 10 minutes. The rats were exposed to ambient oxygen levels from days 22 to 56. Group 1 rats were placed in cages exposed to ambient oxygen (normal oxygen) levels for 56 days. The rats were observed daily for any changes in their behavior and general health status.

[0330] Treatment with the test product or vehicle was administered from day 22 to day 56. Food and water were provided freely. Daily observations of the rats' behavior and general health status were performed. Weekly body weight was measured. 2. Echocardiogram

[0331] Echocardiographic monitoring of disease progression was performed on all rats on day 0, day 21 (before treatment), and the day of surgery (day 56). 3. Blood and lung PK sampling

[0332] Venous blood (0.5 ml, anticoagulated with EDTA) was sampled from all rats (including normal oxygen and vehicle groups) on day 23 (immediately before the second dose), day 38 (immediately before the next dose), and day 57 (24 hours after the last dose). Blood was sampled from the saphenous vein for rats and from the jugular vein for all other rats using an ALZET pump. Whole blood was centrifuged to obtain plasma, which was frozen and stored at -80°C for analysis. 4. Dry powder inhalation (Groups 2 to 4)

[0333] During the treatment period, each rat was placed in a nose-cone restraint chamber, which was connected to a 12-port nose-only inhalation chamber (CH Technologies). Treprostinyl palmityl dry powder formulation D or its vehicle was delivered using a VAG. Airflow was introduced into the VAG at a flow rate of 7 L / min and connected to the nose-only inhalation chamber. For Group 3 treated with a low dose of treprostinyl palmityl dry powder formulation D, 90 mg of treprostinyl palmityl dry powder formulation D was metered and loaded into the VAG deagglomerator. The VAG was set to a voltage of 0.5 V, which corresponds to a powder concentration of 0.5 mg / L (approx. 7 µg / L treprostinyl palmityl). For Group 4 treated with a high dose of treprostinyl palmityl dry powder formulation D, 170 mg of treprostinyl palmityl dry powder formulation D was weighed, loaded into a VAG deagglutinator, and delivered at a voltage of 1 V, which corresponds to a powder concentration of 1.0 mg / L (approx. 14.7 µg / L treprostinyl palmityl). The powder aerosol output concentration was continuously monitored using a portable aerosol monitor (Casella MicroDust Pro). The exact delivery time was recorded. The dry powder remaining inside the VAG deagglutinator was weighed to calculate the actual amount of aerosolized test dry powder. A glass fiber filter connected to a vacuum source at a vacuum flow of 0.5 L / min was placed on one of the exposure ports 5 minutes after the start of aerosolization, and the aerosol was collected from the chamber onto the filter over a 5-minute period. All filter samples were maintained at 4°C until analysis.

[0334] Rats from group 2 received a vehicle for 170 mg of treprostinil palmityl dry powder formulation D and were administered with a 1.0 V setup.

[0335] In this study, two different suction towers and VAG and laser sets were used, one for dry powder vehicle and the other for treprostinyl palmityl dry powder formulation D.

[0336] After removing the remaining powder from the VAG deagglomerator, all parts of the VAG were blown with dry air. The tower was blown with dry air between the administration of Group 3 (low dose) and Group 4 (high dose), and after the administration of Group 4, it was washed with an aqueous solution of 0.5% sodium dodecyl sulfate (SDS), tap water, and distilled water. 5. Nebulization inhalation (Groups 5 and 6)

[0337] Treprostinil and its vehicle PBS were administered using a nebulizer and controller from Aerogen (Aeroneb Pro), which is designed to deliver a mass average aerosol diameter (MMAD) of 2.5 to 4 µm and a flow rate in the range of 0.2 to 0.4 mL / min. During the treatment period, each rat was placed in a nose-cone resistance chamber and connected to a 12-port nose-dedicated inhalation chamber (CH Technologies). The volume of the solution to be nebulized was 6 mL, the airflow was 6 L / min, and the concentration of treprostinil was 0.5 mM. A glass fiber filter was placed on one of the exposure ports and connected to a vacuum source with a vacuum flow of 0.5 L / min for a 5-minute period, starting at 5 minutes after the onset of nebulization and ending at 10 minutes.

[0338] Two intake towers and two separate nose-cone sets were used. One was for group 5 receiving PBS, and one was for group 6 receiving TRE.

[0339] The nebulizer was cleaned by sequentially administering aqueous solutions of 0.5% SDS, tap water, and distilled water through the nebulizer, and by nebulizing PBS between each use to wash away any residual drug from the drug cup and through the perforated plate. The nebulized tower tube and other materials used during the nebulization process were also washed once a day with the preparation described above. Additionally, the aerosolized tower tube and other materials used during the aerosolization process were washed with the preparation described above after each chamber administration. 6. Continuous IV Infusion (Groups 7 and 8)

[0340] Rats from Group 8 were anesthetized with 2% isoflurane and medical-grade air. The back of each rat was incised to place an ALZET pump filled with a first solution containing 8.75 mg / mL TRE. A catheter was implanted into the jugular vein and connected to the ALZET pump. For continuous infusion over a 35-day period, on day 19 of infusion, the catheter was temporarily clamped, and the ALZET pump was replaced with a new pump filled with a second solution containing 10.7 mg / mL TRE. A vehicle-filled ALZET pump was implanted in each rat from Group 7. 7. Oral administration (Groups 9 and 10)

[0341] Rats from Group 10 received the reference compound, celexipeg, by oral feeding twice daily from day 22 to day 56 (35 days). With continuous stirring to maintain a homogeneous suspension of celexipeg, the dose was withdrawn into a gastric syringe, one syringe was filled at a time, and that dose was carefully administered before filling the next syringe. The dose was provided by oral feeding at a rate of 10 mL / kg body weight at each administration. Rats from Group 9 were administered the vehicle by oral feeding twice daily from day 22 to day 56 (35 days). Surgical instruments and measurement of hemodynamic and functional parameters in efficacy study rats

[0342] 1. On the selected surgery day, 24 hours after the last administration, rats were anesthetized with a mixture of 2 to 2.5% isoflurane USP (Abbot Laboratories) in oxygen and placed on a heating pad to maintain body temperature.

[0343] 2. The rat was tracheostomyed and immediately ventilated using a positive pressure rodent respirator set to approximately 10 ml / kg body weight at a frequency of 90 strokes / minute.

[0344] 3. A cannula connected to a pressure transducer was inserted into the left femoral artery to measure systemic arterial blood pressure (SAP).

[0345] 4. The heart was exposed through a sternotomy, Insyte 20GA 1.16 was introduced into the right ventricle, and a saline-filled PE-50 catheter connected to a transducer was rapidly connected.

[0346] 5. After recording right ventricular pressure for a few seconds, the Insyte was advanced further into the pulmonary artery to allow for an additional 60 seconds of PAP recording.

[0347] 6. Hemodynamic parameters were recorded continuously during the duration of the procedure or until the loss of PAP signal.

[0348] 7. After hemodynamic monitoring, blood was obtained by cardiac puncture for biomarker analysis (described below).

[0349] 8. After collecting blood samples, the thoracic cavity was further opened to expose the lungs. The muscle above the trachea was incised to remove the lungs and heart. The harvested tissue was washed with PBS to remove any excess blood before weighing.

[0350] 9. The right lung was ligated and the four lobes were separated to be immediately collected for drug concentration and biomarker analysis, weighed, frozen at liquid concentration, and stored at -80℃.

[0351] 10. For the histology and casting of the heart, the procedure was as follows: 1) 5 out of 11 rats from each of groups 2 to 10 were retained to evaluate the histological and biochemical parameters of the heart and were treated accordingly as described in point 11 below; 2) The other 6 rats from each of groups 2 to 10 were used to determine the Fulton index and were treated as described in point 12. After the collection of data for the Fulton index, the heart tissues were stored at -80°C for biomarker analysis.

[0352] 11. For histological examination, the left lung was flushed with 0.9% NaCl. The left lung was inflated using a 10 mL syringe filled with 10% neutral buffered formalin (NBF) and a fixative attached to a blunt-tip needle (23 g). The tip of the needle was inserted into the trachea and secured in place with a suture, while the other syringe was ligated to the pulmonary artery. The lung was gently inflated at physiological pressure until it was fully, uniformly, and consistently expanded (to prevent the fixative from seeping through the lung surface). This provides optimal vasodilation and airway expansion without causing excessive tissue destruction. Subsequently, the needle was removed, the trachea was sutured, and the tissue was impregnated in 10% NBF at a tissue-to-fixative ratio of 1:20. The heart was irrigated in PBS and subsequently impregnated in 10% NBF at a tissue-to-fixative ratio of 1:20. The tissues were maintained in formalin for 24 to 48 hours. The left lung and heart were subsequently resected and transferred in 70% ethanol.

[0353] All fixed tissues were embedded, sliced, and stained. Lung sections were stained with hematoxylin and eosin (H&E) for morphological determination or with von Willebrand factor (VWF) for endothelial cell staining. Heart sections were stained with H&E, and with Sirius Red or Trichrome staining for collagen fiber visualization and quantification.

[0354] 12. As part of the Fulton index, the heart was dissected to separate the left ventricle with a septum from the right ventricle, and subsequently quantified separately. After collecting data for the Fulton index, the heart tissue was stored at -80°C for biomarker analysis. Acquisition and analysis of experimental data

[0355] Experiment tracking was analyzed using Clampfit software from Axon Instruments.

[0356] PAP recorded continuously for at least 1 minute or until signal loss was used to extract average, diastolic, and systolic lung pressures.

[0357] The recorded whole-body arterial pressure (SAP) is used to extract the average, diastolic, and systolic arterial pressures.

[0358] At the end of the recording of hemodynamic parameters, the right and left ventricles including the septum and the lung lobes were resected to determine the wet weight.

[0359] The following hemodynamic and cardiac function parameters are quantified through appropriate statistical analysis. ● Mean arterial systemic pressure ● Mean arterial pulmonary pressure ● Diastolic pulmonary pressure ● Systolic pulmonary pressure ● Systolic right ventricular pressure ● Saturation (SO2) ● Weight gain ● Lung weight ● Fulton Index ● Heart rate ● Pulse pressure

[0360] In addition, molecules exhibiting cardiac biochemistry were tested, including oxidative stress, collagen (Sircol assay) and hydroxyproline content, uric acid, sodium dilution peptide: BNP, NT-proBNP (biomarker of myocardial stress), endothelin-1 (heart failure), angiopoetin (neovascularization), von Willebrand factor (endothelial cells), interleukin-6 (biomarker of heart attack and stroke), Toll receptor C (biomarker of heart disease), plasma cytokines, atrial natriuretic peptide ANP (biomarker of stroke, coronary artery disease, myocardial infarction, and heart failure), toponin T / I (biomarker of cardiac ischemia), and CPK-MB (cardiac biomarker for myocardial infarction).

[0361] In addition, genes linked to PSH in this embodiment, e.g., bone morphogenesis type 2 (BMPR1), BMP-9, ABCC8, TBX4, ACVRL, SMAD 4 / 9, KCNA5, and TET2, are investigated. Additionally, in the heart and lungs, genes such as collagen type 1 alpha 1 (COL1A1), collagen type 1 alpha 2 (COL1A2), and collagen type 3 alpha 1 (COL3A1) are associated with the formation and secretion of collagen. P4HA1 is an important gene in collagen biosynthesis. ACTG2 is a gene associated with myofibroblast differentiation. Changes in the expression of such genes are also investigated.

[0362] Treprostinil palmityl dry powder formulation D is expected to improve pathophysiology and histopathology in the pulmonary blood vessels and heart of rats inoculated with Su / Hx. * * * * * * * * *

[0363] Although the described invention 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 substituted without departing from the actual spirit and scope of the invention. Furthermore, many variations may be made to adapt specific situations, materials, compositions of materials, processes, process steps, or steps to the purpose and scope of the described invention. All such variations are intended to fall within the scope of the claims appended herein.

[0364] Patents, patent applications, patent application publications, journal articles, and protocols referenced herein are included in their entirety for all purposes.

Claims

Claim 1 A dry powder composition comprising the following: (a) 0.1% to 3% by weight of a compound of the following formula (I) or a pharmaceutically acceptable salt: ,in the above formula, R 1 (a) is linear hexadecyl, (b) DSPE-PEG2000 comprising 0.01 wt% to 1.5 wt% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000, (c) 20 wt% to 30 wt% of leucine, and (d) the remainder being a sugar selected from the group consisting of trehalose and mannitol, wherein the total of (a), (b), (c), and (d) is 100 wt%. Claim 2 A dry powder composition according to claim 1, wherein the sugar is mannitol. Claim 3 A dry powder composition according to claim 1, wherein DSPE-PEG2000 is present in an amount of 0.05% to 1.5% by weight of the total weight of the dry powder composition. Claim 4 A dry powder composition according to claim 1, wherein a compound of formula (I) or a pharmaceutically acceptable salt is present in an amount of 1% to 2% by weight of the total weight of the dry powder composition, and DSPE-PEG2000 is present in an amount of 0.5% to 1% by weight of the total weight of the dry powder composition. Claim 5 A dry powder composition according to claim 1, comprising (a) 1.5 wt% of a compound of formula (I) or a pharmaceutically acceptable salt, (b) 0.75 wt% of DSPE-PEG2000, (c) 29.30 wt% of leucine, and (d) 68.45 wt% of mannitol. Claim 6 A dry powder composition according to claim 1, comprising (a) 1.5 wt% of a compound of formula (I), (b) 0.75 wt% of DSPE-PEG2000, (c) 29.30 wt% of leucine, and (d) 68.45 wt% of mannitol. Claim 7 A dry powder composition according to any one of claims 1 to 6, wherein the dry powder composition is administered to the patient's lungs by inhalation through a dry powder inhaler. Claim 8 A dry powder composition in which, in claim 7, pulmonary hypertension is pulmonary arterial hypertension. Claim 9 A dry powder composition according to claim 8, wherein pulmonary arterial hypertension is Class I, Class II, Class III, or Class IV pulmonary arterial hypertension as characterized by the New York Heart Association (NYHA). Claim 10 A dry powder composition according to claim 7, wherein the pulmonary hypertension is Group 1, Group 2, Group 3, Group 4, or Group 5 pulmonary hypertension as characterized by the World Health Organization (WHO). Claim 11 A dry powder composition according to claim 7, wherein the pulmonary hypertension is chronic thromboembolic pulmonary hypertension, idiopathic pulmonary arterial hypertension, familial pulmonary arterial hypertension, or pulmonary hypertension associated with interstitial lung disease. Claim 12 A dry powder composition according to claim 7, wherein the pulmonary hypertension is pulmonary hypertension associated with interstitial lung disease. Claim 13 A dry powder composition according to any one of claims 1 to 6, wherein the dry powder composition is administered to the patient's lungs by inhalation through a dry powder inhaler, for treating portal hypertension or pulmonary fibrosis in a patient requiring treatment. Claim 14 A dry powder composition according to claim 7, wherein the administration of the dry powder composition comprises aerosolizing the dry powder composition and administering the aerosolized dry powder composition by inhaling it into the patient's lungs. Claim 15 A dry powder composition according to claim 8, wherein the administration of the dry powder composition comprises aerosolizing the dry powder composition and administering the aerosolized dry powder composition into the patient's lungs by inhalation. Claim 16 A dry powder composition according to claim 11, wherein the administration of the dry powder composition comprises aerosolizing the dry powder composition and administering the aerosolized dry powder composition by inhaling it into the patient's lungs. Claim 17 A dry powder composition according to claim 13, wherein the administration of the dry powder composition comprises aerosolizing the dry powder composition and administering the aerosolized dry powder composition by inhaling it into the patient's lungs. Claim 18 A dry powder composition according to claim 14, wherein the aerosolized dry powder composition comprises particles having an MMAD of 1 μm to 3 μm when measured by a Next Generation Impactor (NGI). Claim 19 A dry powder composition according to claim 15, wherein the aerosolized dry powder composition comprises particles having an MMAD of 1 μm to 3 μm when measured by a Next Generation Impactor (NGI). Claim 20 In claim 16, the aerosolized dry powder composition comprises particles having an MMAD of 1 μm to 3 μm when measured by a Next Generation Impactor (NGI). Claim 21 In claim 17, the aerosolized dry powder composition comprises particles having an MMAD of 1 μm to 3 μm when measured by a Next Generation Impactor (NGI). Claim 22 A dry powder composition according to claim 14, wherein the aerosolized dry powder composition comprises particles having a fine particle fraction of 30% to 60% when measured by a Next Generation Impactor (NGI). Claim 23 A dry powder composition according to claim 15, wherein the aerosolized dry powder composition comprises particles having a fine particle fraction of 30% to 60% when measured by a Next Generation Impactor (NGI). Claim 24 In claim 16, the dry powder composition is composed of particles having a fine particle fraction of 30% to 60% when the aerosolized dry powder composition is measured by a Next Generation Impactor (NGI). Claim 25 A dry powder composition according to claim 17, wherein the aerosolized dry powder composition comprises particles having a fine particle fraction of 30% to 60% when measured by a Next Generation Impactor (NGI). Claim 26 A system for treating pulmonary hypertension, portal hypertension, or pulmonary fibrosis, comprising a dry powder composition of any one of claims 1 to 6 and a dry powder inhaler (DPI). 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