Methods for treating systemic sclerosis and frost bite by inhalation therapy

Abstract

The present invention relates to methods for treating systemic sclerosis and frost bite, by administering a pharmaceutical composition by inhalation. The pharmaceutical composition comprises a plurality of liposomes, encapsulating prostacyclin or a prostacyclin analogue. The treatment method of the present invention is convenient for home care and can be delivered for over 5 days to effectively treat systemic sclerosis and frost bite.

Description

METHODS FOR TREATING SYSTEMIC SCLEROSIS AND FROST BITE BYINHALATION THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application No. 63 / 640,175 filed on 29 April 2024, the entire disclosure of which is incorporated herein by reference.FIELD

[0002] Disclosed herein are methods for treating systemic sclerosis and frost bite, by administering a pharmaceutical composition by inhalation. The pharmaceutical composition comprises a plurality of liposomes, encapsulating prostacyclin or a prostacyclin analogue.BACKGROUND

[0003] Systemic sclerosis (SSc, also known as scleroderma) is a rare autoimmune disease of connective tissue that causes fibrosis and vascular damage. The most prominent feature of SSC is progressive fibrosis, resulting from the excessive deposition of extracellular matrix components in skin, blood vessels, heart, lungs, kidneys, gastrointestinal tract, and the musculoskeletal system.

[0004] About 95% of all SSc patients are affected with Raynaud’s phenomenon (RP), which can result in digital ulcer (DU). DUs are associated with substantial morbidity (reduced quality of life, pain, disability, and disfigurement) that can escalate to gangrene and amputation of affected digits. In addition, SSc causes joint dysfunction, esophageal and gastrointestinal dysfunction, pulmonary dysfunction, cardiac and vascular dysfunction and renal dysfunction.

[0005] Intravenous (IV) iloprost infusion is marketed in Europe for multiple indications, including the treatment of SSc, including SSc-related RP and DU and frost bite. For example, Ilomedin® is administered by IV infusion pump at 0.5 - 2.0 ng / kg / min dosing rate for 6 hours at hospitals for consecutive 5-6 days to achieve a therapeutic plasma concentration of 80 pg / mL for 2.0 ng / kg / min dosing. The lengthy intravenous infusions is due to a combination of narrow therapeutic window, poor oral route bioavailability and tolerability of iloprost. Themetabolic clearance of iloprost from plasma is high (about 20 ± 5 mL / kg / min) and the half-life of the terminal disposition phase from plasma is 0.5 hours. As a result, plasma iloprost level falls to less than 10% of the equilibrium concentration just two hours after the end of infusion (haps: / / w ¥w.medsafe.govt.nz / profs / datasheet / i / noniedininf.pdf). The intravenous infusion imposes logistical challenges that impact patients’ quality of life and substantially increase healthcare costs.

[0006] Therefore, there is a need for the treatment of SSc and frost bite that can be selfadministered in a non-hospital setting for over 5 days.BRIEF SUMMARY OF THE INVENTION

[0007] The present invention provides methods for treating systemic sclerosis (SSc) or frost bite in a subject, comprising the step of administering a pharmaceutical composition to the subject in need thereof by inhalation. The pharmaceutical composition comprises a plurality of liposomes, each liposome comprises:(a) a lipid bilayer, comprising a vesicle-forming lipid that is a mixture of a first phospholipid, a second phospholipid and optionally a sterol or a mixture of a first phospholipid, a charged lipid and optionally a sterol; and(b) an internal aqueous medium inside the lipid bilayer, comprising (i) a weak acid salt and (ii) prostacyclin or a prostacyclin analogue, wherein the liposomes are suspended in an external medium and the pH of the external medium is above the pKaof prostacyclin or the prostacyclin analogue.

[0008] The present invention also provides the use of a pharmaceutical composition for the manufacture of a medicament for the treatment of SSc or frost bite, wherein the pharmaceutical composition comprises a plurality of liposomes, each liposome comprises:(a) a lipid bilayer, comprising a vesicle-forming lipid that is a mixture of a first phospholipid, a second phospholipid and optionally a sterol or a mixture of a first phospholipid, a charged lipid and optionally a sterol; and(b) an internal aqueous medium inside the lipid bilayer, comprising (i) a weak acid salt and (ii) prostacyclin or a prostacyclin analogue, wherein the liposomes are suspended in an external medium and the pH of the external medium is above the pKaof prostacyclin or the prostacyclin analogue.

[0009] The invention will become more apparent when read with the accompanying figures and detailed description which follow.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Illustrative embodiments of the present invention are described in detail below with reference to the following figures.

[0011] FIG.l is a line graph illustrating the iloprost concentrations of IV administered free iloprost, intra-tracheal administered free iloprost and intra-tracheal administered liposomal iloprost of the present invention in rat plasma.

[0012] FIG. 2 A to FIG. 2D are line graphs of simulated plasma iloprost concentration in human after intra-tracheal administration of liposomal iloprost at different doses [FIG. 2A dosage is 20 pg / dose, 3 times per day (once every 2 hrs); FIG. 2B dosage is 30 pg / dose, 3 times per day (once every 2 hrs); FIG. 2C dosage is 20 pg / dose, 2 times per day (once every 3 hrs); and FIG. 2D dosage is 30 pg / dose, 2 times per day (once every 3 hrs)].

[0013] FIG. 3 is a bar graph showing the simulated human maximum serum concentration (Cmax) of the liposomal iloprost of the present invention, comprising 20 pg, 30 pg and 40 pg of iloprost, after 3 inhalations within a 6-hour interval. The Cmax of 2 ng / kg / min of IV iloprost administration (80 pg / mL) is the dotted line.

[0014] FIG. 4A is a line graph showing the simulated human plasma iloprost concentration on Days 1 , 3, 7 and 28, after inhaling 20 pg of the liposomal iloprost of the present invention, 3 times daily within a 6 -hour interval over 28 days (solid line). The dotted line shows the plasma iloprost concentration of IV iloprost infusion at 2 ng / kg / min per day over 6 hours.

[0015] FIG. 4B is a line graph showing the simulated human plasma iloprost concentration onDays 1 , 3, 7 and 28, after inhaling 30 pg of the liposomal iloprost of the present invention, 3 times daily within a 6-hour interval over 28 days (solid line). The dotted line is the plasma iloprost concentration of IV iloprost infusion at 2 ng / kg / min per day over 6 hours.

[0016] FIG. 4C is a line graph showing the simulated human plasma iloprost concentration on Days 1, 3, 7 and 28, after inhaling 40 pg of the liposomal iloprost of the present invention, 3 times daily within a 6-hour interval over 28 days (solid line). The dotted line is the plasma iloprost concentration of IV iloprost infusion at 2 ng / kg / min per day over 6 hours.

[0017] FIG. 5 A and FIG. 5B are line graphs illustrating simulated human plasma iloprost concentration, after inhaling 30 pg of the liposomal iloprost of the present invention, 4 times daily 4 hours apart over 7 days (FIG. 5A) and after inhaling 30 pg of the liposomal iloprost of the present invention, 3 times daily 4 hours apart over 7 days (FIG. 5B).

[0018] FIG. 6 is a schematic diagram showing the clinical trial design in the prophetic example.DETAIL DESCRIPTION OF INVENTION

[0019] As used herein, the articles “a” and “an” refer to one or more than one (z.e. , at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0020] All numbers are modified by the term “about”. As used herein, the term “about” refers to a range of ±10% of a specified value.

[0021] The term “comprise” or “comprising” is generally used in the sense of include / including which means permitting the presence of one or more features, ingredients or components.

[0022] The term “subject” can refer to a vertebrate having systemic sclerosis (SSc) or frost bite or a vertebrate deemed to be in need of treatment for systemic sclerosis or frost bite. Subjects include warm-blooded animals, such as mammals, such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includesdomesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

[0023] The term "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with SSc or frost bite or those in which SSc or frost bite is to be prevented. The administration of the pharmaceutical composition described herein aims to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect SSc or frost bite, by reducing the symptoms or signs of SSc or frost bite (such as painful digital ulcers), the disabilities induced by SSc or frost bite, or the number or severity of attack of RP. For DU, healing is defined as complete re- epithelialization of the ulcer.

[0024] As used herein, the terms “encapsulation”, “loaded” and “entrapped” can be used interchangeably, and refer to the incorporation or association of a biologically active agent (e.g., iloprost) in the internal aqueous medium of a liposome.

[0025] The encapsulation efficiency of prostacyclin or its analogue in the liposomes (the percentage of the intemal / encapsulated amount of the drug relative to the total amount of the drug for loading) is above about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0026] As used herein, “substantially free” means that pharmaceutical composition contains less than 5 %, 4%, 3%, 2% or 1% of a specific substance. In some embodiments, pharmaceutical composition does not contain the specific substance.

[0027] The present invention provides methods for treating systemic sclerosis (SSc) or frost bite in a subject, comprising the step of administering a pharmaceutical composition to the subject in need thereof by inhalation. The pharmaceutical composition comprises a plurality of liposomes, each liposome comprises:(a) a lipid bilayer, comprising a vesicle-forming lipid that is a mixture of a first phospholipid, a second phospholipid and optionally a sterol or a mixture of a first phospholipid, a chargedlipid and optionally a sterol; and(b) an internal aqueous medium inside the lipid bilayer, comprising (i) a weak acid salt and (ii) prostacyclin or a prostacyclin analogue, wherein the liposomes are suspended in an external medium and the pH of the external medium is above the pKaof prostacyclin or the prostacyclin analogue.

[0028] The inhalation of the pharmaceutical composition described herein has the following unexpected characteristics:

[0029] The therapeutically effect of the pharmaceutical composition of the present invention for treating SSc or frost bite, delivered by inhalation twice a day, three times a day, four times a day, five times a day or 6 times a day, is comparable to or better than that of daily 6-hour IV infusion of prostacyclin or the analogue thereof. In one embodiment, the severity or the number of attacks of SSc, SSc-related RP, SSc-related DU or frost bite is reduced after administering the pharmaceutical composition by inhalation at least twice a day. In another embodiment, symptoms and signs of SSc, including RP and DU are improved after administering the pharmaceutical composition by inhalation at least twice a day. For DU, improvement is defined as reduction in ulcer depth or visible signs of re-epithelialization.

[0030] The pK profile of the pharmaceutical composition of the present invention, delivered by inhalation, is comparable to or better than that of daily 6-hour IV infusion of prostacyclin or the analogue thereof.

[0031] In one embodiment, the pharmaceutical composition of the present invention, comprises a plurality of liposomes encapsulating iloprost (hereafter “liposomal iloprost”), delivered by inhalation as a single dose or multiple doses (e.g., two to three times a day, two to four times a day, two to five times a day or two to six times a day) achieves the plasma level that is comparable to that of IV iloprost infusion.

[0032] In one embodiment, the inhalation of the pharmaceutical composition provided herein, as a single dose or multiple doses at least twice a day, provides maximum serum concentration(Cmax) of the prostacyclin or the prostacyclin analogue (e.g., iloprost) that is in the range of about 15 to 200 pg / mL or 20 to 160 pg / mL. In another embodiment, the inhalation of the pharmaceutical composition provided herein, at least twice a day, provides maximum serum concentration (Cmax) of the prostacyclin or the prostacyclin analogue (e.g., iloprost) that is in the range of about 70 to 200 pg / mL or 80 to 180 pg / mL.

[0033] In some embodiments, the liposomal iloprost with 20 pg of iloprost, administered by inhalation 3 times a day, provides the Cmax in the range of about 80 to about 110 pg / mL on Day 1, Day 3, Day 7 and Day 28 of administration. In other embodiments, the Cmax of the liposomal iloprost with 30 pg of iloprost, administered by inhalation 3 times a day, is in the range of about 110 pg / mL to about 140 pg / mL on Day 1 , Day 3, Day 7 and Day 29 of administration. In yet another embodiment, the Cma of the liposomal iloprost with 40 pg of iloprost, administered by inhalation 3 times a day, is in the range of about 140 pg / mL to about 170 pg / mL on Day 1, Day 3, Day 7 and Day 28 of administration. In yet another embodiment, the Cmax of the liposomal iloprsot with 30 pg of iloprost, administered by inhalation 4 times a day, is in the range of about 110 pg / mL to about 125 pg / mL on Day 1 and about 140 pg / mL to about 155 pg / mL on Day 3 of administration.

[0034] In one embodiment, the inhalation of the pharmaceutical composition provided herein, at least twice a day, provides an area under the concentration-time curve from 0-24 hours (AUCo-24) of the prostacyclin or the prostacyclin analogue (e.g., iloprost) that is in the range of about 300 pg*h / mL to about 950 pg*h / mL.

[0035] In some embodiments, the AUCo-24 of the liposomal iloprost comprising 20 pg of iloprost, administered by inhalation 3 times a day, is in the range of about 300 pg*h / mL to about 450 pg*h / mL on Day 1 and Day 28 of administration. In another embodiment, the AUCO-24 of the liposomal iloprost comprising 30 pg of iloprost, administered by inhalation 3 times a day, is in the range of about 500 pg*h / mL to about 600 pg*h / mL on Day 1 , about 600 to 700 pg*h / mL on Day 3 of administration, about 500 to 600 pg*h / mL on Day 28 ofadministration. In other embodiments, the AUC0-24 of the liposomal iloprost comprising 40 pg of iloprost, administered by inhalation 3 times a day, is in the range of about 700 pg*h / mL to about 800 pg*h / mL on Day 1 and Day 28 of administration. In yet another embodiment, the AUCO-24 of the pharmaceutical composition comprising 30 pg of iloprost, administered by inhalation 4 times a day, is in the range of about 750 pg*h / mL to about 900 pg*h / mL on Day 1 and 850 pg*h / mL to about 950 pg*h / mL Day 3 of administration.

[0036] In one embodiment, the inhalation of the pharmaceutical composition provided herein, at least twice a day, provides minimum serum concentration (Cmin) of the prostacyclin or the prostacyclin analogue (e.g., iloprost) that is in the range of about 5 pg / mL to about 20 pg / mL.

[0037] The inhalation of the pharmaceutical composition provided herein, at least twice a day, provides a plasma concentration of the prostacyclin or the prostacyclin analogue (e.g., iloprost) over 80 pg / mL for about 10 minutes to about 50, 60, 70, 80, 90, 100, 110 minutes after the inhalation, or for about 15 minutes to about 60, 70, 80, 90, 100, 110 minutes after the inhalation. In some embodiments, the inhalation of the liposomal iloprost comprising 20 pg of iloprost, administered by inhalation 3 times a day, provides a plasma iloprost concentration over 80 pg / mL for about 5 minutes to about 30, 40 or 50 minutes after the inhalation or about 10 minutes to about 30, 40 or 50 minutes after the inhalation. In some embodiments, the inhalation of the liposomal iloprost comprising 30 pg of iloprost, administered by inhalation 3 times a day, provides a plasma iloprost concentration over 80 pg / mL for about 15 minutes to about 35, 40, 50 or 60 minutes after the inhalation or about 20 minutes to about 40, 50 or 60 minutes after the inhalation. In some embodiments, the inhalation of the liposomal iloprost comprising 40 pg of iloprost, administered by inhalation 3 times a day, provides a plasma iloprost concentration over 80 pg / mL for about 30 minutes to about 50, 60 or 70 minutes after the inhalation or for about 35 minutes to about 55, 65 or 75 minutes after the inhalation.

[0038] The pharmaceutical composition described herein is administered by inhalation, including but not limited to oral inhalation.

[0039] In some embodiments, inhalation is administered via a metered dose inhaler, a dry powder inhaler, a soft mist inhaler or dispersing via aerosol (including intranasal and pulmonary administration).

[0040] In some embodiments, the pharmaceutical composition may be administrated twice, three times, four times, five times or six times a day. In other embodiment, the frequency of dosing of the pharmaceutical composition can be about every 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours or some mixture thereof. In another embodiment, the pharmaceutical composition is administered for over 5 days or for about 28 days.

[0041] In some embodiments, the pharmaceutical composition is administered with another therapeutic agent for treating SSc or frost bite. Non limiting examples of the therapeutic agent for treating SSc are calcium channel blocker and phosphodiesterase-5 (PDE5) inhibitor. Non limiting examples of the therapeutic agent for treating frost bite are a thrombolytic and a nonsteroidal anti-inflammatory drug.Liposomal components

[0042] The term “liposome” as used herein refers to microscopic vesicles or particles made up of one or more lipid bilayers enclosing an internal aqueous medium. To form liposomes, the presence of at least one “vesicle-forming lipid” is needed, which is an amphipathic lipid capable of either forming or being incorporated into a lipid bilayer. Any suitable vesicleforming lipid may be used to form the lipid bilayer constituting the liposomes. Vesicleforming lipids include, but not limited to, phospholipids such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylethanolamine (PE) or phosphatidylserine (PS), and charged lipids, such as a positively charged lipid or a negatively charged lipid.

[0043] In some embodiments, the vesicle-forming lipid is a mixture of a fist phospholipid and a second phospholipid and optionally a sterol. In certain embodiments, the first phospholipidis phosphatidylcholine (PC), which is selected from the group consisting of hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoyl phosphatidylcholine (DPPC), distearyloyl phosphatidylcholine (DSPC), diarachidoyl phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC), oleoyl palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), dipetroselinoyl phosphatidylcholine, palmitoylelaidoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine, dilauroyl phosphatidylcholine (DLPC), diundecanoyl phosphatidylcholine (PC(11:0 / 11 :0)), didecanoyl phosphatidylcholine (PC(10:0 / 10:0)), dinonanoyl phosphatidylcholine (PC(9:0 / 9:0) and any combination thereof. In certain embodiments, the second phospholipid is PE modified by a polyethylene glycol, for example, a polyethylene glycol having a molecular weight of about 500 to about 10,000 daltons, such as l,2-distearoly-.v77-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (DSPE-PEG2000) or a negatively charged phospholipid, such as phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylserine (PS) or any combination thereof. In certain embodiments, PG is distearyloyl phosphatidylglycerol (DSPG), Dipalmitoyl phosphatidylglycerol (DPPG) or dimyristoyl phosphatidylglycerol (DMPG) or dioleoyl phosphatidylglycerol (DOPG). In certain embodiments, PA is 1,2- distearoyl-sn-glycero-3-phosphate (DSPA), l,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), l,2-dimyristoyl-sn-glycero-3-phosphate (DMPA) or l,2-dioleoyl-sn-glycero-3-phosphate (DOPA).

[0044] In other embodiments, the vesicle-forming lipids are a mixture of a first phospholipid, a charged lipid and optionally a sterol. The charged lipid includes stearylamine, 1 ,2-dioleoyl- 3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol (DC-Cholesterol), N4-Cholesteryl -Spermine (GL67), dimethyldioctadecylammonium (DDAB), 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), ethylphosphocholine (ethyl PC) or any combination thereof.

[0045] The sterol is cholesterol, cholesteryl hemisuccinate, ergosterol, lanosterol, or any combination thereof. In one embodiment, the sterol is cholesterol. In some embodiments, the lipid bilayer comprises 20-45 mole%, less than 15 mole %, 0-14.99 mole % or 0-13 mole % of sterol. In other embodiments, the sterol in the external lipid bilayer is less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 mole %. In another exemplary embodiment, the external lipid bilayer is substantially free of sterol.

[0046] In an exemplary embodiment, the mole percent of the first phospholipid:sterol:the second phospholipid is 75-99:0-14.9: 0.1-25.

[0047] In an exemplary embodiment, the mole percent of the first phospholipid:sterol:the second phospholipid is 50-70:20-45:0.1-10, 50-70:20-45:0.5-8 or 55-65:25-40:1-6.

[0048] In another exemplary embodiment, the mole percent of the first phospholipid: sterol: charged lipid is 75-99:0-14.9: 0.1-25.

[0049] In another exemplary embodiment, the mole percent of the first phospholipid: sterol: the charged lipid is 50-70: 20-45: 0.1-10, 50-70: 20-45: 0.5-8 or 55-65:25-40: 1-6.

[0050] In an embodiment, the mole % of HSPC:cholesterol:DSPG or DSPE-PEG2000 in the lipid bilayer is 75-99:0-14.9: 0.1-25.

[0051] In an embodiment, the mole % of HSPC:cholesterol:DSPG or DSPE-PEG2000 in the lipid bilayer is 50-70: 20-45: 0.1-10, 50-70: 20-45: 0.1-5 or 55-65:25-40:0.5-8.

[0052] In another embodiment, the mole % of DPPC:Cholesterol:DSPE-PEG2000 in the lipid bilayer is 50-70: 20-45: 0.5-8, 50-70: 20-45: 0.1-5 or 55-65:25-40:1-6.

[0053] In one embodiment, the external lipid bilayer of the liposomes further comprises a surfactant, which can be a non-ionic surfactant, a cationic surfactant or a zwitterionic surfactant. A non-ionic surfactant has no formally charged groups in its head. A cationic surfactant carries a net positive charge in its head. A zwitterion surfactant is electrically neutral but carries formal positive and negative charges on different atoms.

[0054] Non-limiting examples of non-ionic surfactant include non-ionic water soluble mono-, di-, and tri-glycerides; non-ionic water soluble mono- and di-fatty acid esters of polyethyleneglycol; non-ionic water soluble sorbitan fatty acid esters (e.g. sorbitan monooleates such as TWEEN 20 (polyoxyethylene 20 sorbitan monooleate), SPAN 80); non-ionic water soluble triblock copolymers (e.g., poly(ethyleneoxide) / poly-(propyleneoxide) / poly(ethyleneoxide) triblock copolymers such as POLOXAMER 406 (PLURONIC F- 127), or derivatives thereof.

[0055] Non-limiting examples of cationic surfactant include dimethyldialkylammonium bromide or dodecyltrimethylammonium bromide.

[0056] Non-limiting examples of zwitterionic surfactant include 3-(N,N-dimethyl palmity lammonio) -propanes ulfonate,

[0057] The liposome in the suspension is subjected to size reduction. A liposome's size is typically referred to its diameter. Liposome size reduction can be accomplished by a number of methods, such as extrusion, sonication, homogenization techniques or milling techniques, which are well known and can be performed by a person skilled in this art. Extrusion includes passing liposomes, under pressure, one or more times through filters having defined pore sizes. The filters are generally made of polycarbonate, but can also be made of any durable material which does not interact with the liposomes and which is sufficiently strong to allow extrusion under sufficient pressure. The size of the liposomes can be reduced by sonication, which employs sonic energy to disrupt or shear liposomes that will spontaneously reform into smaller liposomes. For example, sonication can be conducted by immersing a glass tube containing the liposome suspension into the sonic epicenter produced in a bath-type sonicator, or a probe type sonicator may be used in which the sonic energy is generated by vibration of a titanium probe in direct contact with the liposome suspension. In the present invention, the liposomes generally have a diameter of about 50 nm to 500 nm, such as about 500 nm or less, about 400 nm or less, about 300 nm or less, about 200 nm or less or about 100 nm or less.

[0058] After sizing, the concentration of the weak acid salt in the external medium is adjusted to provide a pH gradient between the internal aqueous medium and the external medium, which can be carried out by a number of ways, for example, by exchanging the external medium witha suitable buffer lacking the weak acid salts such as citric acid buffer (H3C6H5O) and phosphoric acid buffer (H3PO4), by methods such as diafiltration, dialysis, ultrafiltration, or tangential flow filtration. The internal aqueous medium of the liposome further comprises a cyclodextrin. Non-limiting examples of cyclodextrin include a-CD, 0-CD, y-CD, 2- Hydroxypropyl 0-CD (HP-P-CD), sulfobutylether -CD (SBE-P-CD), randomly methylated P- CD (RM-P-CD) or a combination thereof. Preferably, the cyclodextrin is HP-P-CD, RM-P- CD or a combination thereof.

[0059] The weak acid salt provides a lower outside and a higher inside pH gradient between the external medium and the internal aqueous medium of the liposomes. In one embodiment, the pH of the external medium pH is at least 0.1 unit, 0.5 unit or 1 unit higher than the pKa of prostacyclin or prostacyclin analogue. In yet another embodiment, the pH of the internal aqueous medium is about 7, 8, 9 or 10 and the pH of the external medium is less than 7, less than 6, less than 5, less than 4, less than 3, about 3-7, about 3.5-6.5, or about 4-6.

[0060] Non-limiting examples of weak acid salt include carboxylic acid salt and bicarbonate salt.

[0061] “Bicarbonate salt” as used herein refers to a pharmaceutically acceptable salt compound including a bicarbonate anion and a cationic component. In one embodiment, the cationic component of the salt compound is a metal. Non-limiting examples of the metal include a Group IA or IIA metal, such as potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), cesium (Cs), and lithium (Li) or a metal other than Group IA or IIA metal, such as ferrous iron (Fe) and nickel (Ni). Examples of bicarbonate salt include, but not limited to, potassium bicarbonate, sodium bicarbonate, calcium bicarbonate, magnesium bicarbonate, cesium bicarbonate, lithium bicarbonate, nickel bicarbonate, ferrous iron bicarbonate or any combination thereof.

[0062] “Carboxylic acid salt” as used herein includes, but not limited to, formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate or a combination thereof. In oneexemplary embodiment, the acetate is sodium acetate, calcium acetate, or a combination thereof

[0063] The concentration of the bicarbonate salt or carboxylic acid salt is 50 mM or above, 100 mM or above, 150 mM or above, 200 mM or above, 250 mM or above, 300 mM or above, 350 mM or above, 400 mM or above, 450 mM or above, 500 mM or above, 600 mM or above, 700 M or above, 800 mM or above 900 mM, less than 1000 mM, from 50 mM to less than 1000 mM, from 50 mM to 800 mM, from 200 mM to less than 1000 mM, from 200 mM to 800 mM, or from 200 mM to 600 mM, from 250 mM to less than 1000 mM, from 250 mM to 800 mM, or from 250 mM to 600 mM, from 300 mM to 600 mM.

[0064] The prepared liposome can be stored for substantial periods of time prior to weak acid drug loading and administration to a subject. For example, liposomes can be stored at refrigerated conditions for substantial periods of time prior to weak acid drug loading. Alternatively, liposomes can be dehydrated, stored, and subsequently rehydrated and loaded with a weak acid drug prior to administration. Liposomes may also be dehydrated after being loaded with the weak acid drug. Dehydration can be performed by a number of methods available and known in the art. In some embodiments, liposomes are dehydrated using standard freeze-drying apparatus i.e. dehydration under low pressure conditions. Also, liposomes can be frozen e.g. using liquid nitrogen. Saccharides can be added to the liposomal environment, e.g., to the buffer containing the liposomes, prior to dehydration, to ensure stability and integrity of the liposome during dehydration. Examples of saccharides include but are not limited to maltose, lactose, sucrose, trehalose, dextrose, sorbitol, mannitol, xylitol, or a combination thereof.

[0065] Prostacyclin or prostacyclin analogue is added to the external medium of the liposome and the resultant suspension is incubated under a condition to effect drug loading into the internal aqueous medium of the liposome, allowing diffusion of the drug into the internal aqueous medium of the liposome and until a desired loading concentration and encapsulationefficiency is achieved.

[0066] Table 1 shows the non-limiting examples of prostacyclin and the analogue thereof for loading.

[0067] Table !.

[0068] The term "receptor agonist" is considered an analogue, as the receptor agonist is a synthetic molecule designed to mimic the action of a naturally occurring ligand / molecule (such as prostacyclin) by binding to the same receptor and activating it, essentially acting as an "analogue" of the naturally occurring ligand / molecule.

[0069] In one embodiment, the prostacyclin analogue is iloprost. In another embodiment, the dosage of iloprost is between 5 pg to 1000 pg, 5 pg to 500 pg, 5 pg to 400 pg, 5 pg to 300 pg, 5 pg to 200 pg, 5 pg to 150 pg, 5 pg to 100 pg, 5 pg to 90 pg, 5 pg to 80 pg, 5 pg to 70 pg, 5 pg to 60 pg, 5 pg to 50 pg, 5 pg to 40 pg, 5 pg to 30 pg, 5 pg to 20 pg, 10 pg to 1000 pg, 10 pg to 500 pg, 10 pg to 400 pg, 10 pg to 300 pg, 10 pg to 200 pg, 10 pg to 150 pg, 10 pg to 100 pg, 10 pg to 90 pg, 10 pg to 80 pg, 10 pg to 70 pg, 10 pg to 60 pg, 10 pg to 50 pg, 10 pg to 40 pg, 10 pg to 30 pg, 10 pg to 20 pg, 15 pg to 1000 pg, 15 pg to 500 pg, 15 pg to 400 pg, 15 pg to 300 pg, 15 pg to 200 pg, 15 pg to 150 pg, 15 pg to 100 pg, 15 pg to 90 pg, 15 pg to 80 pg, 15 pg to 70 pg, 15 pg to 60 pg, 15 pg to 50 pg, 15 pg to 40 pg, 15 pg to 30 pg, 15 pgto 20 pg, 20 |ig to 1000 |ig, 20 g to 500 pg, 20 pg to 400 pg, 20 pg to 300 pg, 20 pg to 200 pg, 20 pg to 150 pg, 20 pg to 100 pg, 20 pg to 90 pg, 20 pg to 80 pg, 20 pg to 70 pg, 20 pg to 60 pg, 20 pg to 50 pg, 20 pg to 40 pg, 20 pg to 30 pg, 25 pg to 1000 pg, 25 pg to 500 pg, 25 pg to 400 pg, 25 pg to 300 pg, 25 pg to 200 pg, 25 pg to 150 pg, 25 pg to 100 pg, 25 pg to 90 pg, 25 pg to 80 pg, 25 pg to 70 pg, 25 pg to 60 pg, 25 pg to 50 pg, 25 pg to 40 pg, 25 pg to 30 pg, 30 pg to 1000 pg, 30 pg to 500 pg, 30 pg to 400 pg, 30 pg to 300 pg, 30 pg to 200 pg, 30 pg to 150 pg, 30 pg to 100 pg, 30 pg to 90 pg, 30 pg to 80 pg, 30 pg to 70 pg, 30 pg to 60 pg, 30 pg to 50 pg, 30 pg to 40 pg per dose / inhalation.

[0070] The present disclosure will be further described in the following examples. However, it should be understood that the following examples are solely intended for the purpose of illustration and should not be construed as limiting the present disclosure in practice.EXAMPLES

[0071] Example 1 : Liposomal Iloprost Preparation

[0072] A liposomal colloidal suspension comprising iloprost was prepared using ethanol injection technique as follows: a first phospholipid (HSPC) and a second phospholipid (i.e., DSPE-PEG2000) at a molar ratio of 98:2 were dissolved in 2.86 mL of ethanol solution at approximately 60°C. The resultant lipid solution was injected into 17.14 mL of sodium bicarbonate solution (100-400 mM; pH 8.5) that contains 45-120 mM hydroxypropyl-0- cyclodextrin (CD) and mixed under vigorous stirring at 60°C for liposome hydration. Then, the obtained mixture was extruded 6 to 10 times through polycarbonate membranes with specific pore size (200 and / or 100 nm, respectively), so as to obtain a suspension of liposomes having a mean particle size within a range of around 100 nm to 140 nm. Subsequently, the suspension of liposomes was dialyzed with a tangential flow filtration system against 10 mM of a weakly acidic buffer (with pH 5.0-7.0) to form a transmembrane pH gradient between the internal aqueous core of the liposome and the external medium (i.e., a higher inside and loweroutside pH gradient). The suspension of liposomes having such pH gradient was then stored at 4°C before drug loading process.

[0073] Iloprost (commercially available from Cayman Chemical, USA) was dissolved in 50 mM of a buffer solution, then added into the suspension of liposomes at a drug-to-phospho lipid ratio of 0.25 mg / mL-to-10 mM and incubated at 40°C for 30 min. The resultant product was adjusted with a weak acid buffer (pH 5.0-7.0) to obtain a liposomal iloprost composition having a pH of 5.0-5.7 and a phospholipid concentration of 8.2-8.6 mg / mL.

[0074] Analytical Method of Iloprost

[0075] Iloprost assay was analyzed using Waters HPLC system with PDA Detector. An aliquot of 30 pL of iloprost sample solution was directly injected into the HPLC system with a mixture of acetonitrile, methanol, phosphate buffer (pH 2.5) at volume ratio of 36:17:47 as the mobile phase at flow rate of 1.0 mL / min. Separation was performed in 3.9 mm x 15.0 cm, 5.0 pm, at 25 °C and the peak was detected at 205 nm.

[0076] Example 2: Animal Pharmacokinetic (PK) Evaluation

[0077] For intra-tracheal administration of the liposomal iloprost or free iloprost solution, each rat (Sprague Dawley rats, >250 g) was anesthetized with isofluran and positioned securely at a 45 to 50° angle by the upper teeth. The microspray aerosol tip (MicroSprayer, PennCentury, Philadelphia, PA) was attached to a high-pressure syringe (PennCentury) and inserted to the bifurcation. About 0.5 mL / kg of liposomal iloprost or free iloprost solution was administered.

[0078] At a predetermined time point, a blood sample of each rat was collected into a heparin- containing tube and maintained on wet ice. The blood sample was then centrifuged at approximately 2500 x g for 15 min and at 4 ± 2°C within 1 hour of collection. Approximately 0.1 mL of the rat plasma sample was added into a new storage tube and stored at a temperature of -70 ± 2°C.

[0079] To quantify the iloprost concentration, 30 pL of the rat plasma sample and 3 mL of methyl tert-butyl ether were added into a test tube and vortex-mixed for 1 minute. 1 mL offormic acid solution (2%) was added to the tube and the resultant mixture was vortexed for 2 minute to disrupt the binding of plasma proteins to iloprost, followed by centrifuge at 3000 rpm for 5 minutes. The supernatant (150 pL) was transferred to another clean tube and evaporated under N2 Stream for 15 min at 35°C, then mixed with 200pL of H2O for LC-MS / MS analysis (Table 2).

[0080] Table 2: Rat plasma analytical method information

[0081] FIG. 1 shows the rat plasma iloprost concentration following IV administration of free iloprost, intra-tracheal administration of liposomal iloprost and free iloprost solutions.

[0082] Example 3: Simulated Human PK Profile Based on Rat PK Data

[0083] The recommended dosage of Ilomedin® is between 0.5 to 2 ng / kg / min for an infusion of 6h / day, depending on the patient’s tolerance. FIG. 2A to FIG. 2D illustrate the simulated plasma profile in human based on the rat data Example 1. FIG. 2A represents the simulated human plasma iloprost concentration of the 20 pg / dose, 3 times per day (once every 2 hrs) dosage; FIG. 2B represents simulated human plasma iloprost concentration of the 30 pg / dose, 3 times per day (once every 2 hrs) dosage; FIG. 2C represents the simulated human plasma iloprost concentration of the 20 pg / dose, 2 times per day (once every 3 hrs) dosage; and FIG. 2D represents the simulated human plasma iloprost concentration, derived from the 30 pg / dose, 2 times per day (once every 3 hrs) dosage. The dotted line in FIG. 2A to FIG. 2D representsthe plasma concentration of IV iloprost infusion at 2 ng / kg / min.

[0084] Example 4: Human PK Profile of Single Inhalation of Liposomal Iloprost

[0085] A randomized controlled trial was conducted to evaluate the pK profile of a single inhaled liposomal iloprost of the present invention.

[0086] Subjects were randomized to (a) inhale a single dose of the liposomal iloprost of Example 1, comprising 5 pg to 40 pg of iloprost (in 5 pg increment) within 20 minutes or (b) placebo. Eight subjects were included in each dosage group (i.e., 8 subjects in the 5 pg iloprost dose group, 8 subjects in the 10 pg iloprost dose group, 8 subjects in the 15 pg iloprost dose group ... until 8 subjects in the 40 pg iloprost dose group).

[0087] Plasma was collected at the following post-dose time, for pK profiling:Day 1: Predose, 0.033, 0.083, 0.166, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 18 hours.Day 2 : 24 hours post-dose.

[0088] The plasma concentration of iloprost was quantified using the LC-MS / MS method.

[0089] The pK profile of the singled inhaled liposomal iloprost is summarized in Table 3 below.

[0090] Table 3.Area under the drug concentration-time curve, from time zero to the last measurable concentration.

[0091] Example 5 : Simulated Human PK Profile of Multiple Inhalations of Liposomal Iloprost

[0092] The human pK data from Example 4, derived from a single inhaled liposomal iloprost, comprising 5-40 pg of iloprost, was used to develop and validate a population pharmacokinetic (PK) model.

[0093] A two-compartment model (2CBM) with dual absorption kinetics (slow and fast) provided the best fit for the data. Model performance was confirmed using visual predictive checks (VPCs), which demonstrated that the model adequately predicted plasma iloprost concentrations across all dosages. Validation was further performed based on the single dose human data from Example 4, where most observed data points fell within the 95% confidence interval of the model’s median predicted concentrations, even though these had sampling durations of 10 hours or less.

[0094] Subsequent stochastic simulations were conducted using Phoenix version 8.3 (Certara, Princeton, NJ) to predict the pK parameters (e.g., Cmax and AUC0-24) of a multiple administration schedule (three inhalations over a 6-hour period each day for 28 days).

[0095] Simulations were conducted for up to 28 days (n = 100 replicates per scenario) to estimate Cmax, Cmin, and AUC of the first dose on Day 1 and at steady state (Days 3, 7, and 28). Table 4 summarizes these pK parameters for each dose. FIG. 3 illustrates the simulated Cmax for liposomal iloprost with 20 pg to 40 pg of iloprost remain within the target exposure range relative to IV iloprost infusion at 2 ng / kg / min (the dotted line). FIG. 4A to FIG. 4C are concentration-time graphs of the liposomal iloprost of the present invention, comprising 20 pg, 30 pg and 40 pg of iloprost.

[0096] Table 4. Predicted human pK data of the liposomal iloprost of the present invention, comprising 20 to 40 pg of iloprost, inhaled three times per day during a 6-hour interval over 28 Days (N=100).

[0097] Table d

[0098] Example 6: Simulated Human PK Profile of Multiple Inhalations of Liposomal Iloprost

[0099] The human pK data from Example 4 and the validation and simulation method as described in Example 5, were used to develop and validate a population pharmacokinetic (PK) model of the liposomal iloprost of the present invention comprising 30 pg of iloprost, with the following administration schedules:Schedule A: inhalation four times a day, each dose is 4 hours apart; andSchedule B: inhalation three times a day, each dose is 4 hours apart.

[0100] FIG. 5 A shows the simulated human plasma concentration of iloprost for Schedule A and FIG. 5B shows the simulated human plasma concentration of illoprost for Schedule B.

[0101] For both administration schedules, the liposomal iloprost of the present invention provides a simulated human plasma concentration of iloprost over the therapeutic plasma iloprost concentration for IV iloprost infusion (80 pg / mL) for at least 33 minutes after each inhalation.

[0102] The stimulation also shows Cmax after the 1stinhalation was about 117 pg / mL for both Schedule A and Schedule B, while AUC0-24 after 1stinhalation was 830 and 640 pg*h / mL for Schedule A and Schedule B, respectively. Cmax and AUC0-24 on Day 3 (steady state) were 149 pg / mL and 885 pg*h / mL for Schedule A and 145 pg / mL and 664 pg*h / mL for Schedule B respectively.

[0103] Table 5 summarizes the simulated pK parameters (Cmax and AUC0-24) of Schedule A and Schedule B.

[0104] Table 5a(SS) - estimate @ steady statebAUC (24) Steady state estimate; exposure over 24 hours

[0105] Prophetic Example: Clinical Trial Evaluating the Effectiveness of Inhaled Liposomal Iloprost in Treating SSc-DU

[0106] A multicenter, randomized and double-blind clinical trial will be conducted to evaluate the efficacy of the liposomal iloprost of the present invention in treating patients with SSc associated DU. FIG. 6 illustrates the study design of the clinical trial.

[0107] Approximately 50 eligible participants will be randomized to receive the liposomal of the present invention, comprising 30 pg of iloprost, or placebo (liposomes without iloprost). Eligible participants are aged 18 years and older with at least one SSc associated digital ulcer.

[0108] The visible, painful digital ulcer (DU) digital ulcer is defined by the American College of Rheumatology (ACR) / European Alliance of Associations for Rheumatology (EULAR) 2013 criteria, with the following characteristics(a) An open wound without eschar or scab(b) Onset within 3 months prior to Screening(c) Diameter > 4 mm at screening, with visible depth and dermal loss, located exclusively on the volar surface of the finger or at least involving the volar surface at or distal to the proximal interphalangeal joint, as confirmed by central reviewer

[0109] The trial is designed to evaluate the efficacy of the liposomal iloprost of the present invention in promoting the healing of DU in patients with systemic sclerosis.

[0110] Outcome measurements, including DU assessments, hand pain related to DU, numberof RP attack, hand disability in systemic sclerosis-digital ulcers (HDISS-DU), patient-reported global assessment (PtGA), physician-reported global assessment (PhGA), and safety assessments will be conducted at specified timepoints.

[0111] Participants will inhale the liposomal ilopsort solution or the placebo solution three times daily via oral inhalation over 28 consecutive days. Each of the three inhalations will be separated by an interval of approximately 6 hours (± 30 minutes on Day 1 and ± 1 hour for Day 2 to Day 28). The initial dose will be 20 pg. If the initial dose is tolerated, the second dose may be increased to 30 pg. Conversely, if a patient does not tolerate the 30 pg dose, 20 pg will be administered for subsequent inhalations.

[0112] The inventors predict the liposomal iloprost of the present invention, comprising 20-30 pg of iloprost for oral inhalation three times a day, is effective in treating and / or promote healing of SSc associated DU and reduce the number and severity of RP attacks.

Claims

AMENDED CLAIMS received by the International Bureau on 20 October 2025 (20.10.2025)1. A method of treating systemic sclerosis (SSc) in a subject, comprising the step of administering a pharmaceutical composition to the subject in need thereof by inhalation, said pharmaceutical composition comprises a plurality of liposomes, each liposome comprises:(a) a lipid bilayer, comprising a vesicle-forming lipid that is a mixture of a first phospholipid, a second phospholipid and optionally a sterol or a mixture of a first phospholipid, a charged lipid and optionally a sterol; and(b) an internal aqueous medium inside the lipid bilayer, comprising (i) a weak acid salt and (ii) prostacyclin or a prostacyclin analogue, wherein the liposomes are suspended in an external medium and the pH of the external medium is above the pKaof prostacyclin or the prostacyclin analogue.

2. The method of claim 1, wherein the weak acid salt is a carboxylic acid salt or bicarbonate salt.

3. The method of claim 2, wherein the carboxylic acid salt is selected from the group consisting of formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, benzoate and a combination thereof.

4. The method of claim 2, wherein the concentration of the bicarbonate salt is about 50 mM to less than about 1000 mM.

5. The method of claim 1, wherein the first phospholipid is phosphatidylcholine (PC), , the second phospholipid is phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS) or any combination thereof, and the charged lipid is a positively charged lipid or a negatively charged lipid.

6. The method of claim 1, wherein the first phospholipid is hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), diarachidoyl phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC), oleoyl palmitoyl phosphatidylcholine, dioleoylphosphatidylcholine (DOPC), dipetroselinoyl phosphatidylcholine, palmitoylelaidoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine, dilauroyl phosphatidylcholine (DLPC), diundecanoyl phosphatidylcholine (PC(11 :0 / 11 :0)), didecanoyl phosphatidylcholine (PC(10:0 / 10:0)), dinonanoyl phosphatidylcholine (PC(9: 0 / 9:0)) or any combination thereof, the second phospholipid is l ,2-distearoly-.s / / -glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] (DSPE-PEG2000), distearoyl phosphatidylglycerol (DSPG), Dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), l,2-dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG) or any combination thereof, and the charged lipid is stearylamine, l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol (DC-Cholesterol), N4-Cholesteryl-Spermine (GL67), dimethyldioctadecylammonium (DDAB), 1 ,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), ethylphosphocholine (ethyl PC) or any combination thereof.

7. The method of claim 1, wherein the sterol is cholesterol, cholesteryl hemi succinate, ergosterol, lanosterol, or any combination thereof.

8. The method of claim 7, wherein the first phospholipid is hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), diarachidoyl phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC), oleoyl palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), dipetroselinoyl phosphatidylcholine, palmitoylelaidoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine, dilauroyl phosphatidylcholine (DLPC), diundecanoyl phosphatidylcholine PC(11 :0 / 11 :0), didecanoyl phosphatidylcholine PC(10:0 / 10:0), dinonanoyl phosphatidylcholine PC(9: 0 / 9:0) or any combination thereof, the second phospholipid is l,2-distearoly-sn-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] (DSPE-PEG2000), distearoyl phosphatidylglycerol (DSPG), Dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), l,2-dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG) or any combination thereof, the charged lipid is stearylamine, l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol (DC-Cholesterol), N4-Cholesteryl-Spermine (GL67), dimethyldioctadecylammonium (DDAB), 1 ,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), ethylphosphocholine (ethyl PC) or any combination thereof and the sterol is cholesterol.

9. The method of claim 1, wherein the mole percent of the first phospholipid: sterol: the second phospholipid or the charged lipid is 75-99:0-14.9:0.1-10.

10. The method of claim 1, further comprising the step of administering a therapeutic agent for treating SSc that is a calcium channel blocker or a phosphodiesterase-5 (PDE5) inhibitor or a therapeutic agent for treating frost bite that is a thrombolytic and a nonsteroidal anti- inflammatory drug.

11. The method of claim 1, wherein the prostacyclin analogue is iloprost, epoprostenol, treprostinil or cisaprost.

12. The method of claim 11, wherein the prostacyclin analogue is iloprost,.

13. The method of claim 12, wherein pharmaceutical composition provides a maximum serum concentration (Cmax) of iloprost at least 80 pg / mL.

14. The method of claim 12, wherein the pharmaceutical composition provides an area under the plasma concentration-time curve over the last 24-h dosing interval (AUC0-24) of iloprost at least 350 pg*h / mL.

15. The method of claim 12, wherein the pharmaceutical composition provides an area under the plasma concentration-time curve from time zero to the end of the dosing interval (AUCtau) of iloprost at least 90 pg*h / mL.

16. The method of claim 12, wherein the pharmaceutical composition provides an area under the plasma concentration-time curve from time of dosing to the last time point with measurable concentration serum concentration (AUCo-iast) of iloprost at least 400 pg*h / mL after the inhalation.

17. The method of claim 12, wherein the pharmaceutical provides a plasma concentration over 80 pg / mL for at least 15 minutes after the inhalation.

18. The method of claim 12, wherein the dose of iloprost is at least 5 μg per inhalation.

19. The method of claim 1, wherein the pharmaceutical composition is administered at least twice a day.

20. A method of treating frost bite in a subject, comprising the step of administering a pharmaceutical composition to the subject in need thereof by inhalation, said pharmaceutical composition comprises a plurality of liposomes, each liposome comprises:(a) a lipid bilayer, comprising a vesicle-forming lipid that is a mixture of a first phospholipid, a second phospholipid and optionally a sterol or a mixture of a first phospholipid, a charged lipid and optionally a sterol; and(b) an internal aqueous medium inside the lipid bilayer, comprising (i) a weak acid salt and (ii) prostacyclin or a prostacyclin analogue, wherein the liposomes are suspended in an external medium and the pH of the external medium is above the pKaof prostacyclin or the prostacyclin analogue.

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