WO2023131608A1 - Controlled release compositions - Google Patents

Abstract

The present disclosure related to a pharmaceutical formulation comprising particles comprising 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)acetic acid (selexipag metabolite), or a pharmaceutically acceptable salt, hydrate, solvate, or combinations thereof (active pharmaceutical ingredient), and poly(lactic-co-glycolic acid) (PLGA); as well as a process for preparing the pharmaceutical formation and uses thereof.

Description

CONTROLLED RELEASE COMPOSITIONS

TECHNICAL FIELD

[0001] The present invention is concerned with controlled release injectable formulations comprising 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)acetic acid (selexipag metabolite), or a pharmaceutically acceptable salt, hydrate, solvate or combinations thereof (active pharmaceutical ingredient), and poly(lactic-co-glycolic acid) (PLGA), as well as processes for preparing such controlled release formulations and uses thereof.

BACKGROUND

[0002] Selexipag and its metabolite have the following chemical structures:

(selexipag metabolite).

[0003] The preparation and the medicinal use of selexipag (2-{4-[N-(5,6- diphenylpyrazin-2-yl)-N-isopropylamino]butyloxy}-N-(methylsulfonyl)acetamide, NS-304, ACT-293987) and its active metabolite (2-(4-((5,6-diphenylpyrazin-2- yl)(isopropyl)amino)butoxy)acetic acid, MRE-269, ACT-333679, JNJ-68006861) are described in W02002/088084; W02009/157396; W02009/107736; W02009/154246; W02009/157397; WO2009/157398; W02010/150865; WO2011/024874; Nakamura et al., Bioorg Med Chem (2007), 15, 7720-7725; Kuwano et al., J Pharmacol Exp Ther (2007), 322(3), 1181-1188; Kuwano et al., J Pharmacol Exp Ther (2008), 326(3), 691-699; O. Sitbon et al., N Engl J Med (2015), 373, 2522-33; Asaki et al., Bioorg Med Chem (2007), 15, 6692-6704; Asaki et al., J. Med. Chem. (2015), 58, 7128-7137. Certain formulations are disclosed in WO2013/024051, WO2014/069401, WO2018/162527 and CN107811994. [0004] Selexipag was shown to be beneficial in the treatment of pulmonary arterial hypertension for adults. In a phase III clinical trial, among patients with pulmonary arterial hypertension, the risk of the primary composite end point of death or a complication related to pulmonary arterial hypertension was significantly lower among patients who received selexipag than among those who received placebo. Selexipag received market approval and is indicated for the treatment of pulmonary arterial hypertension (PAH, WHO Group I) to delay disease progression and reduce the risk of hospitalization for PAH.

[0005] So far, standard film-coated tablet formulations of selexipag intended for twice daily oral administration have been used, wherein excipients comprise D-mannitol, com starch, low substituted hydroxypropylcellulose, hydroxypropylcellulose, and magnesium stearate; the tablets are film coated with a coating material containing hypromellose, propylene glycol, titanium dioxide, and carnauba wax along with mixtures of iron oxides.

[0006] Moreover, a safety study of the switch from oral selexipag to intravenous selexipag in patients with PAH has been conducted (NCT03187678), whereby selexipag was administered twice daily as an infusion of approximately 87 minutes. The dose was individualized for each patient to correspond to his/her current oral dose of selexipag.

[0007] In certain instances, the use of an oral formulation of selexipag may be inappropriate or impossible, e.g. in urgent care, or in case a patient is for some reasons unable to swallow a tablet.

[0008] Moreover, in general, it is desirable to reduce the drug burden, particularly for treatment regimens that may last several months or more. The number and/or volume of dosage forms that need to be administered are commonly referred to as "drug burden". A high drug burden is undesirable for many reasons, such as the frequency of administration, often combined with the inconvenience of having to swallow large dosage forms, as well as the need to store and transport a large number or volume of pharmaceutical formulations. A high drug burden increases the risk of patients not taking their entire dose, thereby failing to comply with the prescribed dosage regimen.

[0009] Therefore, there is a need to develop a pharmaceutical composition or formulation, whose pharmaceutical effect is maintained, for example, for one week or longer, or one month or longer, whereby it only has to be administered at long time intervals such as one week or longer, or even one month or longer (a long-acting formulation). SUMMARY

[0010] The disclosure is directed to a pharmaceutical formulation comprising particles comprising 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)acetic acid (selexipag metabolite) or a pharmaceutically acceptable salt, hydrate, solvate, or combinations thereof (active pharmaceutical ingredient), and poly(lactic-co-glycolic acid) (PLGA). The PLGA has an inherent viscosity of from about 0.1 dL/g to about 1.0 dL/g, such as from about 0.1 dL/g to about 0.4 dL/g and about 0.15 dL/g to about 0.3 dL/g.

[0011] A process of preparing the pharmaceutical formulation is also disclosed, and involves dissolving an emulsion stabilizing surfactant in water to form a surfactant containing solution; dissolving the active pharmaceutical ingredient in a solvent to form a pharmaceutical solution; dissolving PLGA in the pharmaceutical solution to form a pharmaceutical-polymer solution; and adding the pharmaceutical-polymer solution into the surfactant containing solution under agitation to form and harden the particles.

[0012] The pharmaceutical formulation may be used in the prevention or treatment of ulcer, digital ulcer, diabetic gangrene, diabetic foot ulcer, pulmonary hypertension, pulmonary arterial hypertension, Fontan disease and pulmonary hypertension associated with Fontan disease, sarcoidosis and pulmonary hypertension associated with sarcoidosis, peripheral circulatory disturbance, connective tissue disease, chronic kidney diseases including glomerulonephritis and diabetic nephropathy at any stage, diseases in which fibrosis of organs or tissues is involved, or respiratory diseases.

[0013] A method for preventing and/or treating ulcer, digital ulcer, diabetic gangrene, diabetic foot ulcer, pulmonary hypertension, pulmonary arterial hypertension, Fontan disease and pulmonary hypertension associated with Fontan disease, sarcoidosis and pulmonary hypertension associated with sarcoidosis, peripheral circulatory disturbance, connective tissue disease, chronic kidney diseases including glomerulonephritis and diabetic nephropathy at any stage, diseases in which fibrosis of organs or tissues is involved, or respiratory diseases, comprising administering the pharmaceutical formulation to a human subject in need thereof is also disclosed.

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. l is a schematic of a process for manufacturing the pharmaceutical formulation. [0015] FIG. 2 is the particle size distribution of particles corresponding to an embodiment of the pharmaceutical formulation.

[0016] FIGs. 3-5 are the release profiles of certain embodiments of the pharmaceutical formulation. The cumulative release % is w/w. dl stands for drug loading.

[0017] FIG. 6 is a schematic of a process for manufacturing the pharmaceutical formulation.

[0018] FIGs. 7-26 are the in vitro release profiles of certain embodiments of the pharmaceutical formulation. The cumulative release % is w/w. dl stands for drug loading.

[0019] FIG. 27 is the in vivo release profile of certain embodiments of the pharmaceutical formulation in Male Wistar rats.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0020] The present disclosure relates to a pharmaceutical formulation comprising particles comprising 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)acetic acid (selexipag metabolite) or a pharmaceutically acceptable salt, hydrate, solvate, or combinations thereof (active pharmaceutical ingredient), and poly(lactic-co-glycolic acid) (PLGA). The PLGA has an inherent viscosity of from about 0.1 dL/g to about 1.0 dL/g, preferably from about 0.1 dL/g to about 0.4 dL/g, more preferably about 0.15 dL/g to about 0.3 dL/g. The term “active pharmaceutical ingredient” refers to selexipag metabolite, and pharmaceutically acceptable salts, hydrates, solvates, and combinations thereof.

[0021] In some embodiments, the active pharmaceutical ingredient is selexipag metabolite in its nonionized form. In other embodiments, pharmaceutically acceptable salts, hydrates, or solvates of the selexipag metabolite, or combinations thereof, are utilized. The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the selexipag metabolite, and exhibits minimal undesired toxicological effects. Such salts include inorganic or organic acid and/or base addition salts. For reference see for example “Handbook of Pharmaceutical Salts. Properties, Selection and Use.”, P. Heinrich Stahl, Camille G. Wermuth (Eds.), Wiley -VCH, 2008; and “Pharmaceutical Salts and Cocrystals”, Johan Wouters and Luc Quere (Eds.), RSC Publishing, 2012.

[0022] Examples of salts include, but are not limited to, calcium, ammonium, arginate, choline, 1,2-ethanedisulfonate, histidine, potassium, sodium, tromethamine, or combinations thereof; preferably the salt is calcium, ammonium, potassium, sodium, or combinations thereof. The pharmaceutically acceptable salt form may comprise two selexipag metabolite molecules and a single salt, such as calcium.

[0023] In addition, the pharmaceutical formulation may comprise a hydrate of the selexipag metabolite. In the hydrate form there are from about 0.01 to about 0.5 equivalents of water per selexipag metabolite, such as from 0.01 to 0.05, from 0.05 to 0.1, from 0.1 to 0.15, from 0.15 to 0.2, from 0.2 to 0.25, from 0.25 to 0.3, from 0.3 to 0.35, from 0.35 to 0.4, from 0.4 to 0.45, from 0.45 to 0.5. Depending upon the method of forming the active pharmaceutical ingredient, the amount of water present in the hydrate may vary, in addition, the storage conditions may influence the amount of water in the hydrate. For example, higher humidity will increase the amount of water in the hydrate. The hydrate is naturally formed in the presence of water. In some embodiments, the pharmaceutical formulation may comprise a solvate of the selexipag metabolite. Solvates are like hydrates except there is a solvent molecule instead of water, such as ethanol.

[0024] Poly(lactic-co-gly colic acid) (PLGA) is a copolymer of lactic acid and glycolic acid. The lactic acid monomer may be L-lactic acid, D-lactic acid, or a combination thereof. The PLGA may be a random or a block copolymer. Different ratios of lactic acid and glycolic acid may be used in the PLGA. In general, polymers comprising more lactic acid monomer tend to degrade more slowly. Example ratios of lactic acid monomer to glycolic acid monomer range from about 95:5 to about 25:75, such as from 90: 10 to 85: 15, from 85: 15 to 80:20, from 80:20 to 75:25, from 75:25 to 70:30, from 70:30 to 65:35, from 65:35 to 60:40, from 60:40 to 55:45, from 55:45 to 50:50, from 50:50 to 45:65, from 45:65 to 40:60, from 40:60 to 35:65, from 35:65 to 30:70, and from 30:70 to 25:75.

[0025] Uncapped PLGA polymers have a terminal carboxylic acid at one end and an alcohol at the other. Capped PLGA polymers have been esterified. It is believed that the terminal carboxylic acid of uncapped PLGA may autocatalyze the degradation of the PLGA, which would increase the rate of active pharmaceutical ingredient release. It is also believed that capped PLGA polymers have increased hydrophobicity which reduces the water accessibility, reducing polymer degradation rate and drug release. In some embodiments, the PLGA is capped with an ester, an ester alcohol, or polyethylene glycol (PEG). Examples of esters include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecanol esters formed from their respective alcohols and the terminal carboxylic acid. [0026] The inherent viscosity may be adjusted by using a higher molecular weight PLGA or by other means well known in the art. The PLGA has an inherent viscosity of from about 0.1 dL/g to about 1.0 dL/g, such as from 0.1 to 0.15, from 0.15 to 0.2, from 0.2 to 0.25, from 0.25 to 0.3, from 0.35 to 0.4 dL/g, from 0.4 dL/g to 0.45 dL/g, from 0.45 dL/g to 0.5 dL/g, from 0.5 dL/g to 0.55 dL/g, from0.55 dL/g to 0.6 dL/g, from 0.6 dL/g to 6.5 dL/g, from 0.65 dL/g to 0.7 dL/g, from 0.7 dL/g to 0.8 dL/g, from 0.8 dL/g to 0.9 dL/g, and from 0.9 dL/g to 1.0 dL/g. In some embodiments, the PLGA has an inherent viscosity from about 0.15 dL/g to about 0.3 dL/g. The inherent viscosity is measured at 25°C in CHCh by using a capillary viscometer (e.g. Ubbelohde tube).

[0027] The inherent viscosity is related to the molecular weight of the PLGA. The inherent viscosity is higher when the molecular weight is higher. Molecular weight may be measured with Size Exclusion Chromatography (SEC) at 35°C in CHCI3. The molecular weight is determined based on a comparison to polystyrene standards. Higher molecular weight PLGA degrades more slowly than lower molecular weight PLGA. In some embodiments, the molecular weight ranges from about 10,000 to about 50,000 g/mol, preferably about 10,000 to about 20,000 g/mol. Unless otherwise noted, the molecular weight is the weight average molecular weight.

[0028] The pharmaceutical formulation comprises particles of the active pharmaceutical ingredient and PLGA. The particles are microspheres which are mixtures of the active pharmaceutical ingredient and PLGA. The PLGA allows for a controlled release of the pharmaceutical ingredient. In some embodiments, the Dv50 of the particles is from about 70 pm to about 175 pm, such as from 70 pm to 75 pm, such as from 75 pm to 80 pm such as from 80 pm to 85 pm, from 85 pm to 90 pm, from 90 pm to 95 pm, from 95 pm to 100 pm, from 100 pm to 105 pm, from 105 pm to 110 pm, from 110 pm to 115 pm, from 115 pm to 120 pm, from 120 pm to 125 pm, from 125 pm to 130 pm, from 130 pm to 135 pm, from 135 pm to 140 pm, from 140 pm to 145 pm, from 145 pm to 150 pm, from 150 pm to 155 pm, from 155 pm to 160 pm, from 160 pm to 165 pm, from 165 pm to 170 pm or from 170 pm to 175 pm. In some embodiments the Dv50 is from about 80 pm to about 125 pm.

[0029] In some embodiments, the DvlO is from about 35 pm to about 110 pm, such as from 35 pm to 40 pm, 40 pm to 45 pm, from 45 pm to 50 pm, from 50 pm to 55 pm, from 55 pm to 60 pm, from 60 pm to 65 pm, from 65 pm to 70 pm, from 70 pm to 75 pm, from 75 pm to 80 pm, from 80 pm to 85 pm, from 85 pm to 90 pm, from 90 pm to 95 pm, from 95 pm to 100 pm, from 100 pm to 105 pm, or from 105 pm to 110 pm. In some embodiments, the Dv90 is from about 90 pm to about 220 pm, such as from 90 pm to 95 pm, from 95 pm to 100 pm, 100 pm to 110 pm, from 110 pm to 120 pm, from 120 pm to 130 pm, from 130 pm to 140 pm, from 140 pm to 150 pm, from 150 pm to 160 pm, from 160 pm to 170 pm, from 170 pm to 180 pm, from 180 pm to 190 pm, 190 pm to 200 pm, 200 pm to 210 pm ,or from 210 pm to 220 pm. In some embodiments, the DvlO is from about 40 pm to about 70 pm and the Dv90 is from about 100 pm about 230 pm. In some embodiments, the DvlO and the Dv90 may be any combination of the DvlO and Dv90 ranges described.

[0030] With respect to particle size distributions (PSD), Dv50 is known as the median diameter. Median values are defined as the value where half of the population resides above this point, and half resides below this point. The Dv50 is the size in microns (micrometer, pm) that splits the distribution with half above and half below this diameter. Similarly, the DvlO is the maximum diameter below which 10% of the sample volume exists, and Dv90 is the maximum diameter below which 90% of the sample volume exists. Herein, PSD is given in volume distribution. Particle size distributions can be measured by well- known methods in the art, for example, laser diffraction, sedimentation field flow fractionation, photon correlation spectroscopy, optical microscopy, or disk centrifugation. In the present disclosure, PSD is measured by optical microscopy. Diameter of 100 particles were measured to obtain a frequency distribution, which was then transformed into volume distribution by assuming a round sphere shape.

[0031] In some embodiments, the pharmaceutical formulation comprises particles comprising 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)acetic acid (selexipag metabolite) or a pharmaceutically acceptable salt, hydrate, solvate, or combinations thereof (active pharmaceutical ingredient), and poly(lactic-co-glycolic acid) (PLGA). The PLGA has an inherent viscosity of from about 0.1 dL/g to about 0.4 dL/g. The PLGA has a monomer ratio of lactic acid : glycolic acid of from 75:25 to 25:75, preferably 75:25 to 50:50. The particles have a particle size distribution Dv50 of from about 80 pm to about 125 pm. Optionally, the PLGA is further capped with polyethylene glycol (PEG), an alcohol, or an ester. [0032] In some embodiments, the pharmaceutical formulation comprises particles comprising 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)acetic acid (selexipag metabolite) or a pharmaceutically acceptable salt, hydrate, solvate, or combinations thereof (active pharmaceutical ingredient), and poly(lactic-co-glycolic acid) (PLGA). The PLGA has an inherent viscosity of from about 0.1 dL/g to about 1.0 dL/g. The PLGA has a monomer ratio of lactic acid : glycolic acid of from 75:25 to 25:75, preferably 75:25 to 50:50. The particles have a particle size distribution Dv50 of from about 80 pm to about 110 pm. Optionally, the PLGA is further capped with polyethylene glycol (PEG), an alcohol, or an ester. Optionally, the active pharmaceutical ingredient is from about 10% by wt. to about 40% by wt. based on the weight of the particles. Optionally, the particles release less than 10% of the active pharmaceutical ingredient after 24 hours and more than 90% after 3 months

[0033] The pharmaceutical formulation may be gamma-irradiated in order to sterilize the formulation. However, gamma-irradiation has the potential to degrade ingredients in the formulation. In some embodiments, after the formulation has been gammairradiated the content assay changes by less than 5%. The content assay is the amount of active pharmaceutical ingredient present in the formulation. In some embodiments, after the formulation has been gamma-irradiated the particles release less than 10% of the active pharmaceutical ingredient after 24 hours and more than 90% and less than 100% after 1 month, preferably after 2 months, more preferably after 3 months.

[0034] Suitable conditions for gamma irradiation are achieved by exposure to ionizing radiation in the form of gamma radiation from a suitable radioisotopic source such as ⁶⁰Co (cobalt 60) or of electrons energized by a suitable electron accelerator. Suitable conditions are radiation levels of 5 to 40 kGy, for instance 5 kGy, 25 kGy or 40 kGy, or in this case for purposes of evaluation, 27 kGy. Conditions relating to validation as prescribed in the Pharmacopeia, e.g. “US Pharmacopeia”, or “The International Pharmacopoeia, Ninth Edition 2019”, etc. should be taken into account.

[0035] In some embodiments, the loading of the active pharmaceutical ingredient is from about 10% by wt. to about 40% by wt. based on the weight of the particles. For example, the loading of the active pharmaceutical ingredient is from about 10% by wt. to about 40% by wt., such as from 10% by wt. to 12% by wt., from 12% by wt. to 15% by wt., from 15% by wt. to 20% by wt., from 20% by wt. to 25% by wt., from 25% by wt. to 30% by wt., from 30% by wt. to 35% by wt., or from 35% by wt. to 40% by wt. In some embodiments, the loading of the active pharmaceutical ingredient is about 20% by wt based on the weight of the particles. Unless otherwise noted, amounts of the active pharmaceutical ingredient are set forth on a free form basis. That is, the amounts indicate that amount of the free form of the selexipag metabolite administered, exclusive of, for example, solvent (such as in solvates), hydrates, or counterions (such as in pharmaceutically acceptable salts).

[0036] The pharmaceutical formulation is designed to be controlled release. The release rate is controlled by multiple factors, such as particle size, particle density, particle porosity, composition of the PLGA polymer such as the ratio of lactic acid : glycolic acid, the inherent viscosity of the PLGA polymer, whether the PLGA polymer is capped, and the amount (loading) of the active pharmaceutical ingredient inside of the particle. With respect to the release of the active ingredient, this can be characterized by diffusion and erosion. During diffusion the active pharmaceutical ingredient diffuses through the PLGA polymer. During erosion, the PLGA particle breaks down and additional active pharmaceutical ingredient is released. In some situations, there is an undesired initial rapid release of the active pharmaceutical ingredient. This may be reduced by, for example, using particles that have reduced amounts of active pharmaceutical ingredient on or close to the surface of the particle. The burst or initial release is the release of the active pharmaceutical ingredient over the first 24 hours. In some embodiments, the burst release is less than 10%, such as less than 8%, less than 5%, or less than 3%. The controlled release allows the active pharmaceutical ingredient to be released over time. The active pharmaceutical ingredient may be released over a period of 1 month, 2 months, or 3 months. In some embodiments, after 1 month the pharmaceutical formulation has released more than 90% and less than 100% of the active pharmaceutical ingredient. In some embodiments, after 2 months the pharmaceutical formulation has released more than 90% and less than 100% of the active pharmaceutical ingredient. In some embodiments, after 3 months the pharmaceutical formulation has released more than 90% and less than 100% of the active pharmaceutical ingredient. In some embodiments, after 1 month the pharmaceutical formulation has released more than 90% of the active pharmaceutical ingredient. In some embodiments, after 2 months the pharmaceutical formulation has released more than 90% of the active pharmaceutical ingredient. In some embodiments, after 3 months the pharmaceutical formulation has released more than 90% of the active pharmaceutical ingredient. [0037] The process for preparing the pharmaceutical formulation of the present disclosure comprises several steps. For example, an emulsion stabilizing surfactant is dissolved in water to form a surfactant containing solution. The active pharmaceutical ingredient is dissolved in a solvent to form a pharmaceutical solution. PLGA is dissolved in the pharmaceutical solution to form a pharmaceutical-polymer solution. The pharmaceutical- polymer solution is added into the surfactant containing solution under agitation to form microemulsion droplets. As the solvent is diffused and evaporated, the droplets are hardened to solidified particles. In some embodiments, the solvent is selected from methylene chloride, dimethyl sulfoxide (DMSO), or mixtures thereof. In some embodiments, the amount of the active pharmaceutical ingredient is from about 1% to about 35% by weight in the pharmaceutical-polymer solution (excluding the weight of the solvent), such as from 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 7%, 7% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%.

[0038] Examples of an emulsion stabilizing surfactant include, but are not limited to, polyvinyl alcohol, polyethylene glycol sorbitan monolaurate, sorbitan monooleate, sodium dodecyl sulfate, poloxamer 188, poloxamer 338, polysorbate 20, or polysorbate 80. The amount of emulsion stabilizing surfactant is from about 0.1% w/v to about 10% w/v in the aqueous phase, such as from 0.1% w/v to 0.5% w/v, 0.5% w/v to 0.7% w/v, 0.7% w/v to 1% w/v, 1% w/v to 1.5% w/v, 1.5% w/v to 2% w/v, 2% w/v to 3% w/v, 3% w/v to 4% w/v, 4% w/v to 5% w/v, 5% w/v to 6% w/v, 6% w/v to 7% w/v, 7% w/v to 8% w/v, 8% w/v to 9% w/v, or 9% w/v to 104% w/v. In some embodiments, the surfactant containing solution additionally comprises an acid. Examples of an acid include but are not limited to mineral acids, such as HC1. The acid may be used to adjust the pH to between 1.5 and 4.5, such as 1.5 to 2, 2 to 2.5, 2.5 to 3, 3 to 3.5, 3.5 to 4, 4 to 4.5. In some embodiments, the pH of the surfactant containing solution is about 3. The ratio of the water used in the surfactant containing solution to the solvent in the pharmaceutical-polymer solution can be varied from a ratio of 5: 1 to 100: 1, such as 5: 1 to 8: 1, 8: 1 to 10: 1, 10: 1 to 15: 1, 15: 1 to 20: 1, 20: 1 to 25: 1, 25: 1 to 30: 1, 30: 1 to 40: 1, 40: 1 to 50: 1, 50: 1 to 60: 1, 60: 1 to 70: 1, 70: 1 to 80: 1, 80: 1 to 90: 1, or 90: 1 to 100: 1

[0039] In some embodiments, after the particles are formed and hardened (solidified), they are washed with a solvent. Examples of the wash solvent include water or water with a buffer, such as a phosphate buffer. In some embodiments, the pH of the first wash is from about 2 to about 6 to remove the solvent. In some embodiments, a second wash is performed with a different pH. The purpose of the second wash(es) is to remove the active pharmaceutical ingredient from the surface of the particles. This reduces the initial release of the active pharmaceutical ingredient when the pharmaceutical formulation is administered. In some embodiments, the particles are washed during the hardening step by increasing the pH, such as to about a pH of 5- 9.

[0040] The pharmaceutical formulation can be part of a dosage form. For example, the pharmaceutical composition may be in the form of an aqueous suspension suitable for intramuscular and/or subcutaneous injection. In some embodiments, the pharmaceutical formulation is administered as subcutaneous injection (SC) or intramuscular injection (IM). Injectable suspensions may be prepared utilizing aqueous carriers along with appropriate additives. For SC or IM administration, the carrier will usually consist of sterile water and other ingredients which increase viscosity to prevent sedimentation between preparation and injection. Suitable dispersing or suspending agents for aqueous suspensions, include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, poly vinyl-pyrrolidone, gelatin, pol oxamer 338, PEG 4000, PEG 6000, PEG 8000, or PEG 10000. Isotonic preparations are employed when SC or IM administration is desired. In some embodiments, the carrier used in the parenteral formulations comprises sterile water. In some embodiments, the composition of the diluent is shown in the table below. It contains sodium CMC as resuspending agent, PS 20 as surfactant, phosphate buffer, sodium chloride as isotonic agent, sodium hydroxide as pH adjustment agent, and purified water as solvent.

Exemplary Composition of the Diluent

[0041] The term "therapeutically effective amount" refers to amounts, or concentrations, of the pharmaceutical formulation (or amounts/ concentrations of active ingredient within such formulation) that result in efficacious plasma levels for treating an indicated disease, condition, or disorder. For instance, a therapeutically effective amount may be 0.2 mg to 6.4 mg per day of the active pharmaceutical ingredient, preferably 0.4 mg to 3.2 mg per day, which may be found appropriate by the prescribing doctor. With "efficacious plasma levels" it is meant those plasma levels of the active pharmaceutical ingredient that provide effective treatment or effective prevention of the indicated disease, condition, or disorder.

[0042] As used herein, unless otherwise noted, the terms “treating”, “treatment” and the like, shall include the management and care of a patient for the purpose of combating a disease, condition, or disorder. The terms “treating” and “treatment” also include the administration of the pharmaceutical formulation as described herein to (a) alleviate one or more symptoms or complications of the disease, condition or disorder; (b) prevent the onset of one or more symptoms or complications of the disease, condition or disorder; and/or (c) eliminate one or more symptoms or complications of the disease, condition, or disorder.

[0043] In some embodiments, a dosage form comprises a therapeutically effective amount of the pharmaceutical formulation. The pharmaceutical formulation may be used in the prevention or treatment of ulcer, digital ulcer, diabetic gangrene, diabetic foot ulcer, pulmonary hypertension, pulmonary arterial hypertension, Fontan disease and pulmonary hypertension associated with Fontan disease, sarcoidosis and pulmonary hypertension associated with sarcoidosis, peripheral circulatory disturbance, connective tissue disease, chronic kidney diseases including glomerulonephritis and diabetic nephropathy at any stage, diseases in which fibrosis of organs or tissues is involved, or respiratory diseases. The pharmaceutical formulation may be used in the prevention or treatment of pulmonary arterial hypertension (PAH).

[0044] In some embodiments, a method for preventing and/or treating ulcer, digital ulcer, diabetic gangrene, diabetic foot ulcer, pulmonary hypertension, pulmonary arterial hypertension, Fontan disease and pulmonary hypertension associated with Fontan disease, sarcoidosis and pulmonary hypertension associated with sarcoidosis, peripheral circulatory disturbance, connective tissue disease, chronic kidney diseases including glomerulonephritis and diabetic nephropathy at any stage, diseases in which fibrosis of organs or tissues is involved, or respiratory diseases, comprising administering the pharmaceutical formulation to a human subject in need thereof.

[0045] [0001 ]In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a material” is a reference to at least one of such materials and equivalents thereof known to those skilled in the art, and so forth.

[0046] [0002]When a value is expressed as an approximation by use of the descriptor “about,” it will be understood that the particular value forms another embodiment. In general, use of the term “about” indicates approximations that can vary depending on the desired properties sought to be obtained by the disclosed subject matter and is to be interpreted in the specific context in which it is used, based on its function. The person skilled in the art will be able to interpret this as a matter of routine. In some cases, the gradations used in a series of values may be used to determine the intended range available to the term “about” for each value. Where present, all ranges are inclusive and combinable. That is, references to values stated in ranges include every value within that range. For example, a range defined as from 400 to 450 ppm includes 400 ppm and 450 ppm as independent embodiments.

[0047] [0003]It is to be appreciated that certain features of the invention which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. That is, unless obviously incompatible or specifically excluded, each individual embodiment is deemed to be combinable with any other embodiment(s) and such a combination is considered to be another embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. Finally, while an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment in itself, combinable with others. [0004]

[0048] [0005]When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C ”

EXAMPLES

[0049] [0006]The Examples set forth are provided to illustrate some of the concepts described within this disclosure. While each Example is considered to provide specific individual embodiments of composition, methods of preparation and use, these teachings should be considered representative of the more general disclosure; i.e., none of the Examples should be considered to limit the more general embodiments described herein.

[0050] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental error and deviation should be accounted for.

Example 1 : Microparticle Process

[0051] Figure 1 shows a schematic of a process for manufacturing the pharmaceutical formulation.

Step 1 - PVA solution preparation

[0052] The polyvinyl alcohol (PVA) solution is prepared by heating water to about 70°C, dispersing the polymer therein under agitation and allowing the surfactant containing solution to cool down to ambient temperature under continued agitation. Optionally, the pH of the solution is adjusted to pH 3 with hydrochloric acid.

Step 2 - Drug solution preparation

[0053] The drug solution is prepared by weighing the drug substance into a vial and adding methylene chloride (without or in combination with DMSO) and agitate until dissolved.

Step 3 - Drug-polymer solution preparation

[0054] PLGA is added to the clear drug solution and agitated until dissolved and then kept for at least 1 hour before use.

Step 4 - Emulsification / hardening

[0055] The drug-polymer solution is added to the PVA solution under agitation with an overhead stirrer. Step 5 - Separation

[0056] The microparticles are separated from the hardening bath using a stainless steel sieve with 12 pm pore size.

Step 6 - Washing

[0057] The microparticles collected were rinsed two or three times with water or other aqueous media.

Step 7 Dry ing

[0058] The washed microparticles were dried in a desiccator over silica gel, molecular sieve or under vacuum.

[0059] Batch manufacturing was performed at minimum lab scale (not more than 1 g) to establish the process and investigate effects of key parameters of the encapsulation process on particle size, drug encapsulation and in vitro (burst) release.

Example 2: Oil to Water Ratio Experiment

[0060] Microparticles were prepared as in Example 1 using process conditions in Table 2 and the PLGA grades in Table 1 (capped PLGA grades Resomer RG 502 and 752S (Resomer RG 502: 0.2 dL/g, MW 13,300 g/mol, end group: hydroxy-carboxylic acid ester, LA:GA ratio 50:50. Resomer 752S: 0.2 dL/g, MW 12,500 g/mol, end group: alcohol, LA:GA ratio 75:25.) In case of batches DF-19 and DF-21 a pre-emulsion of the complete external aqueous phase (400 g) and the oil phase (10 g) was made by adding the drug- polymer solution into the 1% PVA solution (adjusted to pH 3 with HC1) at 500 rpm (5 cm propeller stirrer). After removing the overhead stirrer and establishing magnetic stirring for hardening, polymer stuck to the magnetic stirrer bar and drug crystals grew in the external phase. The batches were discarded as no microencapsulated product was observable.

Table 1

cone, means concentration

Table 2

mag. means magnetic stirrer. MS means molecular sieves [0061] Batches DF-71 and DF-73 were prepared at a W:O (water to oil) ratio of

50: 1 without pre-emulsification step. Oil phase (8 g) was added to the aqueous 1% PVA solution (400 g, no pH adjustment) at 500 rpm (5 cm propeller stirrer).

[0062] Batches showed strong drug crystallization during hardening, which in case of batch DF-73 was so pronounced that it was not further processed after hardening. Unencapsulated drug of batch DF-71 could not be removed by washing as drug crystals were seen on the surface of the dried microparticles. [0063] Batches DF-22 and DF-23 were prepared at a W:O ratio of 80: 1. Oil phase (5 g) was added of the aqueous 1% PVA solution (400 g, adjusted to pH 3 with HC1) at 500 rpm (5 cm propeller stirrer). Minor drug crystallization was observed during hardening (start after about 20 min). A slight sticking was noted during drying over molecular sieve. However, individual particles were obtained for batch DF-23 after dry sieving. Unencapsulated drug could not be completely removed per washing step as drug crystals were seen in the dried product, especially for batch DF-22.

[0064] Batches DF-79 and DF-81 were prepared at a W:O ratio of 100: 1. Oil phase (4 g) was added to the aqueous 1% PVA solution (400 g) at 500 rpm (5 cm propeller stirrer). Again, minor drug crystallization was observed during hardening (start after about 15 min). Non-encapsulated drug was seen in the dried product.

[0065] The assay values were between 85 and 102% (table below).

[0066] Release studies indicated a controlled release of the active pharmaceutical ingredient over prolonged time intervals. The initial burst was lower for the higher W:O ratios, which was attributed to a better drug encapsulation. The release profile of the RG 502 batch was still incomplete after more than 2 months, see Figures 3 (RG 502) and 4 (RG 752S). Samples of batches DF-22 and DF-23 were extracted to test for remaining drug. The drug content was determined per UV assay and per HPLC method upon release of the residual microparticle mass at day 75 in acetonitrile:water (according to UV assay method). The HPLC method provided information about the chemical stability inside the microparticles as well.

[0067] There were no degradation peaks present in the chromatograms of the extracts indicating drug stability in the microparticles incubated in the aqueous release medium.

Example 3 : pH of the water phase

[0068] Microparticles were prepared as in Example 1 using process conditions in Table 4 and the PLGA grades in Table 3 (capped PLGA grades Resomer RG 752S). The drug concentration in the oil phase was set close to the solubility boundary of the drug in methylene chloride (3% w/w) and the polymer concentration adjusted to obtain a nominal drug loading of 20% in the microparticles (Table 3). About 5 g drug-polymer solutions were added in 72-73 seconds (“slow injection”) into 400 mL of external phase under agitation. After a hardening for 3 hours and separation, washing was performed with aqueous medium (lx pH 8 phosphate buffer, 2x water) and drying was conducted for 18.5 h over molecular sieve (3 A).

Table 3

Table 4

[0069] Drug crystals were seen in both dried (not sieved) products. The crystals appeared to be situated at or bound to the surface to a large extent. In case of batch DF-90, however, a considerable amount of free crystals were additionally present. [0070] The assays were high due to the mostly unremoved non-encapsulated drug (table below).

[0071] The advantage of the external phase acidification was substantiated by the release results, which highlighted that the increased amount of non-encapsulated drug obtained with 1% PVA in pure water as external phase leads to a higher initial drug burst (Figure 5), which is undesirable.

Example 5: Microparticle Process

[0072] Figure 6 shows a schematic of a process for manufacturing the pharmaceutical formulation.

Step 1 - PVA solution preparation

[0073] The polyvinyl alcohol (PVA) solution is prepared by heating water to about 70°C, dispersing the polymer therein under agitation and allowing the surfactant containing solution to cool down to ambient temperature under continued agitation. The pH of the solution is adjusted to pH 3 with hydrochloric acid.

Step 2 - Drug solution preparation

[0074] The drug solution is prepared by weighing the drug substance into a vial and adding methylene chloride under agitation until dissolved.

Step 3 - Drug-polymer solution preparation

[0075] PLGA is added to the clear drug solution and vortexed until dissolved.

Step 4 - Emulsification / hardening

[0076] The drug-polymer solution is added rapidly into the PVA solution under agitation at 500 rpm with an overhead stirrer and a 5 cm propeller. Hardening was continued 3 hours before separation.

Step 5 - Separation [0077] The microparticles are separated from the hardening batch under vacuum using a stainless steel sieve with 12 pm pore size.

Step 6 - Washing

[0078] The microparticles collected on the 12 pm stainless steel sieve were rinsed with water or pH 8 phosphate buffer.

Step 7 Dry ing

[0079] The washed microparticles were transferred onto glass dishes and dried in a desiccator over molecular sieve overnight.

[0080] This process leads to microparticles of a suitable size and with most of the API being encapsulated at a 20% drug loading. Capped polymer grades Resomer RG 502 and 752 S were used as PLGA and were suitable for a long acting API.

Example 6: DMSO as co-solvent

[0081] Batches were prepared with and without 20% DMSO for a high (20 % or 30%) and a low (10 %) drug loading taking advantage of the possibility to increase the drug and polymer concentration using the co-solvent (formulation table below). The process followed that of Example 5.

[0082] Drug crystals were seen in all dried (not sieved) products. Crystals were mostly situated at or bound to the surface, except for Batches DF-81 and DF-74 (batches prepared without DMSO and RG 752S as polymer), where lots of free crystals were present in the dried product. Microparticles prepared with DMSO (Batches DF-75 - DF-78) were larger than the particles from the methylene chloride process (Batches DF-79 - DF-81 and DF-74), which was attributed to the higher polymer concentration.

[0083] The release studies showed that DMSO incorporation had no effect on the initial release (1 d point) for Resomer RG 502 formulations independent of the drug loading (Figures 7 and 8). The release between 1 d and 17 d, which was attributed to a diffusional drug release before the polymer erosion started, was slower with DMSO-containing RG 502 formulations in line with their larger particle size.

[0084] DMSO incorporation affected the initial release (I d point) for Resomer RG 752S formulations (Figures 9 and 10). According to the microscopic appearance of the products, using DMSO as co-solvent resulted in a higher encapsulation of drug into the microparticles.

[0085] Batches based on Purasorb grade 5002 (Viscosity: 0.2 dL/g, MW 17,000 g/mol, LA/GA ratio 50:50, end-group: dodecanol) were prepared without and with 10% and 20% DMSO as co-solvent (formulation table below). The process followed that of Example 5 with a pH shift from pH 3 to pH 8 after 1 hour hardening. Polymer-drug solutions (5-6 g) were added into 400 ml of external phase under agitation. After a hardening for 3 hours and separation, washing was performed with water and drying was conducted for 1 (Batch DF- 93) or 3 days in a desiccator over molecular sieve (3 A).

[0086] Particles were mostly individual with minor sticking tendency and no free or surface bound drug crystals were noticed due to the application of a pH shift to slightly alkaline conditions during hardening, where the drug solubility is higher.

[0087] The particle size distribution was in the typical range (table below).

[0088] Drug release profiles were characterized by low initial bursts and an erosional release phase between about 2 and 5 weeks (see Figure 11). The release profiles appeared independent of the amount of co-solvent used. Example ?: API loading

[0089] The previously made batches in Example 6 used for evaluating the effect of the incorporation of DMSO, were also assessed in terms of the effect of the drug loading on the drug release. For this evaluation, the focus was put on the initial drug release.

[0090] An increase of the drug loading had no effect on the initial release (I d point) for Resomer RG 502 formulations independent of whether DMSO was used as co-solvent or not (Figures 7 and 8). The release between 1 d and 17 d was attributed to a diffusional drug release before the polymer erosion started.

[0091] The increase of the drug loading decreased the initial release (1 d point) for Resomer RG 752S formulations (Figures 9 and 10). This was attributed to a higher fraction of non-encapsulated drug resulting from the crystallization of drug in the external phase. [0092] The drug loading increase with RG 752S was accompanied by a noticeable change of the dry microparticles appearance from rather transparent to opaque. It was hypothesized, that a fraction of the drug could be molecularly dispersed and hence mobile in the PLGA matrix at a concentration close to 10% for RG 752S. As an increase of the drug loading would thus increase the crystallized, colloidally dispersed and hence immobile drug fraction, the cumulative release in its relative expression decreased.

Example 8: Capped PLGA Polymers

[0093] Formulations containing the Resomer grades RG 752S (end group: alcohol), RG 502 (end group: hydroxy-carboxylic acid ester, Evonik), and PDLG 5002 (end-group: dodecanol, Corbion-Purac) polymers were manufactured as shown in Example 1 and compared.

[0094] The encapsulation was efficient as free drug crystals were only seen with batch DF-82, prepared without acidification of the external phase.

[0095] In vitro release profiles of batches prepared with Resomer RG 502 and PDLG 5002 (see Figures 12) showed a similar initial release and a similar onset of the erosional release phase after about 14 days with its typical sigmoidal shape. There was no or only marginal release between 1 and 14 days, which would indicate diffusional drug release. The erosional release period lasted for about 3 weeks for PDLG 5002 and 4-5 weeks for RG 502, which was attributed to a broader molecular weight distribution of the Resomer batch.

[0096] The batch prepared with Resomer grade RG 752S (lactic-to-glycolic acid ratio 75:25) started to enter the sigmoidal release after 3 - 4 weeks, which then lasted for 5-6 weeks (see Figures 12). The later onset of about 1 - 2 weeks compared to the 50:50 PLGAs RG 502 and PDLG 5002 is believed to be due to increased hydrolytic stability of PLGA from the higher lactic acid content.

PLGA - PEG block co-polymer

[0097] PLGA-PEG block co-polymers are capped 50:50 PLGAs, which do not have an aliphatic end capping but are esterified with PEG. Expansorb 6P has nominally about 15% w/w PEG in the molecule and Expansorb 7P about 7%. Expansorb materials are PLGA endcapped with polyethylene glycol (PEG). 6P grade is 50:50 for LA:GA with an inherent viscosity range 0.45-0.65 dL/g and MW range 30,000-60,000 g/mol. 7P grade 50:50 for LA:GA with an inherent viscosity range 0.65-0.80 dL/g and a MW range 60,000-85,000 g/mol. The following formulations have been prepared for the evaluation as shown in Example 5.

[0098] All batches presented spherical microparticles with a no sticking tendency compared to Resomer (RG 502 and 752S) and Purasorb (PDLG 5002) grades, which was attributed to the higher molecular weight and hence the higher glass transition temperature. However, an effect of the PEG end cap could not be ruled out. Particles were larger when the co-solvent method was applied (batches DF-83 and DF-84) and all batches based on the 6P grade (batches DF-83, DF-85 and DF-87) appeared to contain some free crystals in the dried product.

[0099] Drug release revealed an instantaneous release for the Expansorb 6P batches (Figures 13 and 14). Expansorb 6P is the polymer with the higher amount of PEG mass fraction in the material. It was hypothesized that PEG could have offered enough porosity for an access of drug to the alkaline release medium.

[00100] Expansorb 7P, with the lower nominal PEG mass fraction, offered controlled release properties as a conventional capped polymer (Figure 15). The onset of the sigmoidal release phase was after about a month similar to Resomer RG 752S. The erosion phase lasted for about 4 weeks, which was also similar to the other capped polymers.

[00101] In contrast to the Resomer and Purasorb polymers, batches manufacture with DMSO as a co-solvent resulted in an increased initial release.

Blends of capped and uncapped PLGA [00102] Formulations with different drug polymer ratios of capped (e.g. 502) and uncapped PLGAs (e.g. 502H) polymer grades and drug loadings were tested. The following formulations were prepared as shown in Example 5.

[00103] There was a tendency for stickiness of the dry product, which caused particle aggregation during the process. Aggregation was less pronounced for batches DF-55 and DF-60. Free, non-encapsulated drug crystals were seen for all batches in similar degrees.

[00104] Drug release studies revealed that inclusion of uncapped PLGA changes the controlled release profile. Batch DF-53 prepared at a 1 : 1 ratio of Resomer grades 502 and 502H showed no controlled release anymore (Figure 16).

[00105] Batches prepared at the 3: 1 and 9: 1 ratios showed sustained release properties but the release was incomplete. There was also no impact on the onset of the drug release as expected comparing the profiles to batch DF-82, which was prepared without uncapped polymer.

[00106] The results indicated that the drug could interact with acidic residues leading to a pronounced effect on the erosional release phase. A similar, incomplete release was seen before with Resomer RG 502-based batch DF-22, where extraction of the drug from the material recovered after 75 days revealed proper mass balance and no signs of chemical degradation.

[00107] A higher initial release was obtained with the 25% compared to 15 and 20% drug loading (Figure 17). The batch prepared with the 3 : 1 blend of Purasorb grades 5002 and 5002A showed a much faster release compared to the 3: 1 blend of Resomer grades RG 502 and RG 502H (Figure 18). Also, the release was completed within one month and the onset of the release was shortened in the presence of the uncapped polymer compared to Purasorb 5002 alone (DF59, Figure 19).

[00108] It was noted that batches showing complete drug release on release testing (DF-59 and DF-60 made of Purasorb 5002 alone, and a 3: 1 mixture of Purasorb grades 5002 and 5002A, respectively; and DF-82 made of a 3: 1 blend of Resomer grades RG 502 and RG 502H) were characterized by predominantly individual particles whereas incomplete release of batches DF-22, DF-43 and DF-54 appeared to be linked to aggregated material (). Example 9: Washing pH

[00109] The washing step was tested at pH 3 and pH 9 (50 mM) phosphate buffer to see the effect on the release profile of batch DF-59 (Figure 20). Whereas pH 3 did not reduce the about 10% burst of DF-59 in the release medium, an almost complete removal (8.38% of 9.87%) was achieved at pH 9 within 1.5 hours. The rapid release of the API allowed integration of the washing step as a pH shift in the 3 -hour hardening step of the manufacturing process.

[00110] The following batches were prepared to develop the procedure for the pH shift (table below) as shown in Example 5.

[00111] Polarized microscopy of the emulsions showed that drug crystallization started in the external phase (1% PVA at pH 3) within 60 min. Without implementing the pH shift (batch DF-91) the drug crystals remained after the 3 hours of hardening, whereas no drug crystals were seen with batches prepared with the pH shift, independent of the time when the shift was conducted.

[00112] The longer the emulsions were kept at pH 8-9, the lower the initial drug release (Figure 21). However, the extent of drug diffusion or the start of the erosional release phase were not affected by the hardening time at pH 3 nor at pH 8-9.

Example 10: Gamma Irradiation

[00113] Microparticle batches were subjected to gamma irradiation (packaged in crimp glass vials) with a dose of 27 kGy. The content assay did not change considerably.

[00114] Release patterns of the batches were affected by gamma irradiation. The sigmoidal part of the profile of DF-119 shifted by 4-5 days (Figure 22). The sigmoidal part of the profile of the RG 502 batches DF-111, 112 and 117 shifted by about 1-3 days (Figures 23 and 24). The diffusion-controlled release was not affected for DF-111 and 112 but for DF- 117, where it increased. The sigmoidal part of the profile of the RG 752S batch DF-114 shifted by about one week (Figure 25).

Example 11 : Animal PK Study

[00115] The following batches were used in an in vivo study in Male Wistar rats with SC injection of the particles.

[00116] Release studies (Figure 26) were performed in parallel to the in vivo study (Figure 27) to verify the performance of the formulations. The suspension concentrations applied to the test systems (Male Wistar rats) are provided below.

Appearance / Particle size

[00117] Particle size distribution was measured by Malvern Mastersizer 3000 apparatus from Malvern Panalytical using the laser diffraction measurement method and the Mie theory. The results of the laser diffraction analysis are reported based on the particle size volume distribution as the cumulative undersize values dv50. The following settings were used:

[00118] Microparticles were added to a diluent to reach a concentration of 150 mg/mL. The suspension was dispersed for about 1 h with stirring. If agglomerates were visible, the mixture was sonicated for 30 minutes and stirred for an additional 30 minutes. Hereafter the suspension was added to the measuring media at a level with sufficient but not to high obscuration in the particle measuring equipment and the particle size determined Composition of the Diluent

Thermal characteristics per DSC

[00119] Ten (±5) milligrams of sample were filled into aluminum pans and closed with a lid. The temperature was raised from 25 to 180°C at 10 K/min (stage 1), cooled from 180 to 25°C at nominally lOOK/min (stage 2) and heated to 180°C again at 10 K/min (stage 3) using Perkin Elmer DSC 6000. Assay (content assay) and Purity

[00120] The assay and the impurities of selexipag metabolite were analyzed using reversed phase chromatography. Nominally 0.05 mg/ml selexipag metabolite from biodegradable selexipag metabolite microparticles in 40% (v/v) acetonitrile was analysed. This was obtained by dissolving about 25 mg of microparticles in a 100 ml volumetric flask with 40 mL acetonitrile with addition of water to the mark. The sample was centrifuged for 5 min at 13.000 rpm and the clear supernatant is analyzed by HPLC.

[00121] Analysis was performed using a Waters UPLC H-Class equipped with UV detector, column manager and auto-sampler. Separation was done on a Acquity UPLC BEH C18 (2.1 x 150mm, 1.7pm) analytical column using a column temperature of 45°C. UV detector was set on 230nm. A lOmM NH4Ac solution was used as mobile phase A. Acetonitrile was used as mobile phase B. A linear gradient program was applied starting in which the mobile phase B increases from 5% to 95% in 10 minutes, then held for 5 minutes. Hereafter the concentration of mobile phase B was brought back to 5% in 1 minute followed by an equilibration time of 5 minutes. The applied flow rate is 0.30 mL/min. An injection volume of 20 pL was used for the analysis. The assay value of the sample is calculated according following formula: [00122] % = [API peak responsesample x conc_ref x purity _ref x 100%]/[peak response_ref x concsampie x dose claim]

[00123] The concentration of an impurity is calculated according following formula:

[00124] % = [Impurity peak responsesample x conc_ref x purity _ref x 100%]/[peak responseref x concsampie x dose claim]

UV Assay

[00125] The UV assay was performed after dissolving microparticles in 40% acetonitrile. Selectivity in terms of other formulation ingredients and linearity were given at 339 pm.

In vitro Release Assay

[0001] In vitro drug release of 10-30 mg portion was performed under sink conditions in 100 ml of pH 7.4 phosphate buffer (PB) containing 0.02% NaNs as preservative. Samples were incubated in a horizontal shaker at 37°C and 80 rpm. Quantification of drug concentration in the buffer was performed per UV assay. Selectivity in terms of other formulation ingredients or compounds in the release medium as well as linearity were given at 299 pm. Samples were taken at different time points and analyzed for drug concentrations using the UV method. The total amount of drug released from microparticles to the release medium (pH 7.4 phosphate buffer) was calculated based on the drug concentration or concentration increase and total volume of buffer at different time points.

Claims

CLAIMS What is claimed:

1. A pharmaceutical formulation comprising particles comprising 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)acetic acid (selexipag metabolite) or a pharmaceutically acceptable salt, hydrate, solvate, or combinations thereof (active pharmaceutical ingredient), and poly(lactic-co-glycolic acid) (PLGA), wherein the PLGA has an inherent viscosity of from about 0.1 dL/g to about 1.0 dL/g, preferably from about 0.1 dL/g to about 0.4 dL/g, more preferably from about 0.15 dL/g to about 0.3 dL/g.

2. The pharmaceutical formulation of claim 1, wherein the particles have a particle size distribution Dv50 of from about 70 pm to about 175 pm, preferably from about 80 pm to about 125 pm.

3. The pharmaceutical formulation of claim 1 or 2, wherein the particles have a particle size distribution DvlO from about 35 pm to about 110 pm, and a particle size distribution

Dv90 from about 90 pm to about 220 pm.

4. The pharmaceutical formulation of any one of claims 1 to 3, wherein the PLGA is capped with ester, an ester alcohol, or polyethylene glycol (PEG).

5. The pharmaceutical formulation of any one of claims 1 to 4, wherein the PLGA has a monomer ratio of lactic acid : glycolic acid of from 75:25 to 25:75, preferably 75:25 to 50:50.

6. The pharmaceutical formulation of any one of claims 1 to 5, wherein after the formulation has been gamma-irradiated the content assay changes by less than 5%.

7. The pharmaceutical formulation of any one of claims 1 to 6, wherein after the particles have been gamma-irradiated the particles release less than 10% of the active

- 35 - pharmaceutical ingredient after 24 hours and more than 90% and less than 100% after 1 month, preferred 2 months, and more preferred 3 months.

8. The pharmaceutical formulation of any one of claims 1 to 6, wherein the particles comprise from about 10% by wt. to about 40% by wt. of the active pharmaceutical ingredient, preferably about 20% by wt.

9. The pharmaceutical formulation of any one of claims 1 to 8, wherein the particles are hardened.

10. The pharmaceutical formulation of any one of claims 1 to 9, wherein the particles release less than 10% of the active pharmaceutical ingredient after 24 hours and more than 90% and less than 100% after 3 months.

11. The pharmaceutical formulation of any one of claims 1 to 10, wherein the active pharmaceutical ingredient is selexipag metabolite.

12. The pharmaceutical formulation of any one of claims 1 to 11, wherein the PLGA has an inherent viscosity of from about 0.15 dL/g to about 0.3 dL/g, wherein the PLGA has a monomer ratio of lactic acid : glycolic acid of from 75:25 to 25:75, preferably 75:25 to 50:50, and wherein the particles have a particle size distribution Dv50 of from about 40 pm to about 80 pm.

13. The pharmaceutical formulation of claim 12, wherein the PLGA is capped with polyethylene glycol (PEG), an alcohol, or an ester.

14. A process of preparing the pharmaceutical formulation of any one of claims 1 to 13, comprising: dissolving an emulsion stabilizing surfactant in water to form a surfactant containing solution, dissolving the active pharmaceutical ingredient in a solvent to form a pharmaceutical solution, dissolving PLGA in the pharmaceutical solution to form a pharmaceutical -polymer

- 36 - solution, adding the pharmaceutical-polymer solution into the surfactant containing solution under agitation to form and harden the particles.

15. The process of claim 14, further comprising the step of washing the particles with a phosphate buffer.

16. The process of claim 14 or 15, wherein the solvent is methylene chloride, dimethyl sulfoxide (DMSO), or mixtures thereof.

17. The process of any one of claims 14 to 16, wherein the active pharmaceutical ingredient is from about 1% to about 35% by weight in the pharmaceutical-polymer solution (excluding the weight of the solvent).

18. The process of any one of claims 14 to 17, wherein the emulsion stabilizing surfactant comprises polyvinyl alcohol, and the surfactant containing solution additionally comprises an acid.

19. A dosage form comprising a therapeutically effective amount of the pharmaceutical formulation of any of claims 1 to 13.

20. The pharmaceutical formulation according to any one of claims 1 to 13 or 19, for use in the prevention or treatment of ulcer, digital ulcer, diabetic gangrene, diabetic foot ulcer, pulmonary hypertension, pulmonary arterial hypertension, Fontan disease and pulmonary hypertension associated with Fontan disease, sarcoidosis and pulmonary hypertension associated with sarcoidosis, peripheral circulatory disturbance, connective tissue disease, chronic kidney diseases including glomerulonephritis and diabetic nephropathy at any stage, diseases in which fibrosis of organs or tissues is involved, or respiratory diseases.

21. The pharmaceutical formulation according to claim 20 for the use in the prevention or treatment of pulmonary arterial hypertension (PAH).

22. A method for preventing and/or treating ulcer, digital ulcer, diabetic gangrene, diabetic foot ulcer, pulmonary hypertension, pulmonary arterial hypertension, Fontan disease and pulmonary hypertension associated with Fontan disease, sarcoidosis and pulmonary hypertension associated with sarcoidosis, peripheral circulatory disturbance, connective tissue disease, chronic kidney diseases including glomerulonephritis and diabetic nephropathy at any stage, diseases in which fibrosis of organs or tissues is involved, or respiratory diseases, comprising administering the pharmaceutical formulation according to any one of claims 1 to 13 or claim 19 to a human subject in need thereof.