Process for making a composition for pulmonary inhalation

Abstract

A process for manufacturing a Treprostinil-diketopiperazine dry powder for pharmaceutical use by spray drying a suspension without need to dissolve Treprostinil in an alcohol solution prior to processing. In some embodiments, the dry powder is for oral inhalation for pulmonary delivery using a dry powder inhaler.

Description

PCT Patent ApplicationDocket 1951300.00718 PCTPROCESS FOR MAKING A COMPOSITION FOR PULMONARY INHALATIONRELATED APPLICATION

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. provisional application Ser. No. 63 / 690,929, filed September 5, 2024. The subject matter of the provisional application is incorporated in its entirety by reference herein.TECHNICAL FIELD

[0002] An improved process for making an inhalable composition comprising a prostaglandin or prostaglandin analog, such as a prostacyclin, or a prostacyclin derivative and a diketopiperazine. The process is used in manufacturing bulk quantities of the composition which results in a more stable formulation for pharmaceutical use.BACKGROUND

[0003] Pulmonary arterial hypertension (PAH) is a complex, multifactorial, progressive syndrome characterized by persistent elevation of pulmonary arterial pressure and pulmonary vascular resistance (PVR) that leads to increase in right ventricular afterload and eventually culminates in right heart failure. Right ventricular failure limits cardiac output during exertion. The most common symptom at presentation is breathlessness, fatigue, angina, syncope, and abdominal distension, with impaired exercise capacity as a hallmark of the disease.

[0004] Pharmaceutical formulations for treating pulmonary hypertension can be made using various methods depending on the type of therapy use, including, e.g., injectables, tablets, capsules, or inhalers. There is always room for improving manufacturing processes for the various therapies to obtain better dispersion, solubility, stability, and delivery of the formulation for treating animals, in particularly, humans.

[0005] Inhalable pharmaceutical composition for treating lung conditions, including, pulmonary hypertension, idiopathic lung disease, such as pulmonary fibrosis and other lung diseases include the use of prostaglandins, such as prostacyclin, and derivatives and salts thereof, including Treprostinil. Prostaglandin-type drugs are difficult to solubilize and stabilize, such as when used for oral inhalation treatment of patients with PAH and other types of lung disease. Described in such patent application is a method for solubilizing prostaglandins and prostacyclin analogs, including Treprostinil, by placing the same in an alcohol, such as absolute ethanol, or a diluted ethanol in water solution. The alcoholic solution of the prostaglandin / prostacyclin or1508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT prostaglandin / prostacyclin analog, such as Treprostinil, to form a solution, is added to a water suspension containing diketopiperazine microparticles with the admixture spray dried to form a prostacy clin / Treprostinil and diketopiperazine dry powder which may be administered via a powder inhaler. More alcohol, such as ethanol, may cause the Treprostinil to remain in solution in the suspension making it more uniformly dispersed in the suspension.

[0006] It is a key principle of safety and green chemistry to reduce the amount of alcohol, such as ethanol, used in a pharmaceutical product, because ethanol has numerous drawbacks for manufacture of a pharmaceutical product. Manufacturing processes that use ethanol can lead to drug product impurities and, therefore, is not ideal for large scale processes. It can also be toxic, and any ethanol removed in the synthesis process can cause environmental and safety hazards due to its flammability. Reducing the amount of ethanol in pharmaceutical manufacturing can also help minimize the cost and environmental impact of the process.SUMMARY

[0007] Disclosed herein is a new process for making inhalable pharmaceutical formulations for treating lung disease with reduced drug impurities formation during processing. The method comprises admixing poorly water soluble prostaglandins, such as prostacyclin, and prostaglandin / prostacyclin analogs, such as Treprostinil, with a diketopiperazine in water suspension that does not require the use of an alcohol, or which requires substantially less alcohol, than as described previously, in terms of the chemical processing of associating the Treprostinil with the diketopiperazine particles to form a Treprostinil-diketopiperazine dry powder upon spray drying. This non-alcohol process results in a powder that is closer to the target Treprostinil content without the need to add excess or additional Treprostinil as is the case when high percentage alcohol content is used in the manufacturing process in making inhalation powders for treatment of diseases, including, pulmonary hypertension, interstitial lung disease, for example, idiopathic lung disease (ILD) such as idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, scleroderma, sarcoidosis and the like.

[0008] A non-alcohol-based process that facilitates the formulation of prostaglandin, such as prostacyclins, and prostaglandin / prostacyclin analogs, including, Treprostinil and derivatives thereof is provided, wherein dry powder composition for inhalation and deep lung delivery is disclosed. The process is a manufacturing process for making a dry powder composition which2508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT comprises, for example, Treprostinil, analog thereof, or a derivative thereof, or combinations thereof, which can be used for the treatment of disease, including, pulmonary arterial hypertension and / or idiopathic pulmonary fibrosis, progressive pulmonary fibrosis and scleroderma.

[0009] Prostacyclin and its analogs, such as Treprostinil, are typically hydrophobic compounds. Due to the same, such compounds have been found to associate well with crystalline particles of diketopiperazine, including (E)-3,6-bis[4-(jV-carbonyl-2-propenyl)amidobutyl]-2,5- diketopiperazine, suspended in water when prostacyclin / prostacyclin analogs are first dissolved in an alcoholic solution and then added to the diketopiperazine-water suspension. The admixture is then spray dried forming a Treprostinil - FDPK dry powder. The problem with such methodology is that it has been found that the amount of prostacyclin / prostacyclin analog, such Treprostinil, is utilized in excess of the amount required from the theoretical amount needed to obtain the percentage of Treprostinil in diketopiperazine spray-dried powder required for pharmaceutical use. Such excess can reach about 20% Treprostinil when Treprostinil is added to an ethanol containing solution to maximally dissolved the Treprostinil, and then adding the ethanolic solution to the diketopiperazine particles aqueous suspension, and the suspension / admixture is then spray dried to form the Treprostinil-diketopiperazine dry powder.

[0010] In one embodiment, it has now been found that some excess of prostacyclin / prostacyclin analog, such as Treprostinil, is needed to form the required prostacyclin / prostacyclin analog concentration in the prostacyclin-diketopiperazine, for example, from about 5% of the drug to obtain a target of 1% Treprosinil content in the dry powder. In one embodiment, this can be produced by dissolving the prostacyclin / prostacyclin analog in an alkaline solution, including a NaOH solution, or ammonium solution, and then add such alkaline solution to a diketopiperazine particles in water suspension, without the need for alcohol in the processing.

[0011] Methods are also disclosed for using the compositions using Treprostinil in diketopiperazine dry powder, such as (E)-3,6-bis[4-(jV-carbonyl-2-propenyl)amidobutyl]-2,5- diketopiperazine or fumaryl diketopiperazine (FDKP), in the treatment of pulmonary hypertension. In embodiments herewith, the method comprises providing a Treprostinil composition in a dry powder inhaler comprising a replaceable single dose cartridge comprising a dry powder for inhalation for delivery to the lungs for local, or systemic delivery into the pulmonary circulation. The dry powder inhaler may be a breath-powered inhaler which is compact, reusable or disposable, having various shapes and sizes, and comprising a system of3508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT airflow conduit pathways for the effective and rapid delivery of dry powder medicament to the lungs and the systemic circulation. In one embodiment, a dry powder composition for inhalation comprising Treprostinil or a salt thereof, is provided into the respiratory system in less than 10 seconds, or less than 5 seconds, or less than 3 seconds and the Treprostinil is detected at peak concentrations in the blood of the subject treated in less than 30 minutes, having a median Tmax of about 10 minutes or less.

[0012] In one embodiment, the process for making a dry powder composition comprising a hydrophobic prostacyclin / prostacyclin analog, such as Treprostinil, comprises in a step preparing a suspension of a dry powder composition comprising crystalline particles of a diketopiperazine, including (E)-3,6-bis[4-(A-carbonyl-2-propenyl)amidobutyl]-2,5-diketopiperazine or FDKP, by exposing the same to an acetic acid solution in a high shear mixer at a temperature ranging from 13°C to about 20°C while mixing the suspension; and washing the suspension in water for one or more times to remove the acetic acid. In one embodiment, the microcrystals of the diketopiperazine can be used after washing in water directly onto the next step of processing or can be dried and stored for future use. In one aspect of this embodiment, the process further comprises, pelletizing the suspension comprising the diketopiperazine in aqueous suspension in a cryogranulator, and drying the pellets formed from the suspension in a lyophilizer to collect the microcrystals of the diketopiperazine and collecting the dry powder and storing the dry powder.

[0013] In another embodiment, microcrystals of a diketopiperazine from storage are resuspended in deionized water to form a diketopiperazine suspension, or wet microcrystals of the diketopiperazine during processing, either are used for further processing in making the dry powder compositions. In an example embodiment, Treprostinil, and / or one or more other prostacyclin analog, is prepared separately in an alkaline solution, such as a sodium hydroxide aqueous solution. A preferred pH range for the resulting alkaline / Treprostinil solution is greater than pH 7.0, or from about pH 7.0 to pH 10, or from about pH 8.0 to about pH 9.5. In one embodiment, the starting pH of the Treprostinil solution is from about pH 8.0 to pH 9 so that when the solution is added to a second aqueous solution, the resultant pH is from about 3.0 to about 5.0 pH units, more preferably about 4.0 to about 4.5 pH units, and even more preferably about 4.1 to about 4.4 pH units. In one exemplary embodiment, the Treprostinil or other prostacyclin analog or combinations thereof, in the alkaline solution is then added to diketopiperazine microcrystals, such as FDKP in a water suspension, and the admixture then can4508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT be dried by either lyophilization, or spray dried to form a dry powder composition comprising Treprostinil (or other prostacyclin analog) and diketopiperazine for pharmaceutical use.

[0014] In another embodiment, there is disclosed a process for making a dry powder composition comprising a hydrophobic prostacyclin / prostacyclin analog, such as Treprostinil, The diketopiperazine, such as FDKP, is prepared by suspending the FDKP crystal in water after several deionized and / or distilled water washes. The diketopiperazine / FDKP suspension is pelletized in liquid nitrogen in a drop wise fashion using a cryogranulator to form pellets of substantially homogeneous size. The resulting diketopiperazine / FDKP pellets are collected from the liquid nitrogen using an automated conveyor belt system and they are transferred to a lyophilizer where they are dried by lyophilization. The resulting FDKP power is resuspended in water or an aqueous solution. Treprostinil, or other prostacyclin analog, is prepared in an alkaline solution, such as a sodium hydroxide aqueous solution. A preferred pH range for the resulting Treprostinil / alkaline solution is greater than pH 7.0. In one embodiment, the Treprostinil or other prostacyclin analog in the alkaline solution is then added to the diketopiperazine, such as FDKP, in water suspension, so that when the Treprostinil solution is combined with the suspension the resultant mixture has a pH ranging from about 3.0 to about 5.0 pH units, more preferably about 4.0 to about 4.5 pH units, and even more preferably about 4.1 to about 4.4 pH units; wherein the pH can be adjusted if necessary. In one embodiment, the admixture is spray-dried to form the dry powder comprising Treprostinil (or other prostacyclin analog) and a diketopiperazine dry powder.

[0015] By using such processes, Treprostinil or other prostacyclin analog need not be mixed with any alcohol, or substantially less alcohol, such as ethanol, before being added to the diketopiperazine in water suspension. In this manner, the methods of making the pharmaceutical dry powders for inhalation may utilize less alcohol and thus is starting materials and is more cost- effective, avoids use of flammable ingredient during manufacture, and may reduce impurities in the final pharmaceutical product.

[0016] In example embodiments, the instant process yields dry powders comprising Treprostinil and FDKP which bulk powders are devoid or have substantially reduced content of several impurities, including, a Treprostinil ethyl ester (0 wt / %), Treprostinil dimers 1 and 2 as compared to the Treprostinil-FDKP dry powder made by the process comprising ethanol. In some5508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT embodiments, Treprostinil dimers 1 and / or 2 are present at a concentration of less than about 0.3wt%; less than about 0.5%; less than about 0.7%; less thatn about 1.0%, less than about 1.5%; less than about 3%, or less than about 5%. In one embodiment, the pharmaceutical dry powders wherein the Treprostinil is processed in alkaline solution comprise less FDKP-Treprostinil esters 1, 2, 3 and 4, respectively, cyclopentyl / Trans FDKP, Side chain / Trans FDKP, cyclopentyl / Cis FDKP and Side chain / Cis FDKP, which are analytically determined by liquid chromatography, than samples made using alcohol solution to dissolved the Treprostinil.

[0017] In certain embodiments disclosed herein, a method for providing a prostacyclin analog in a diketopiperazine dry powder formulation to a patient in need thereof is disclosed. The method comprises, selecting a patient to be treated for PAH (pulmonary arterial hypertension), and administering to the patient a dry powder formulation comprising the prostacyclin analog, such as Treprostinil combined with a diketopiperazine, including, (E)-3,6-bis[4-( / V-carbonyl-2- propenyl)amidobutyl]-2,5-diketopiperazine or FDKP, by pulmonary inhalation. In this and other embodiments, the dry powder formulations can be provided more than once a day as needed by the patient and the composition is provided in a reconfigurable cartridge comprising from about 1 pg to about 200 pg of Treprostinil in the dry powder formulation per dose, or from 10 pg to about 100 pg, or from about 100 pg to about 150 pg, or from about 150 pg to about 300 pg of Treprostinil, a derivative thereof, an analog thereof or combinations thereof. In certain embodiments, the dry powder formulation can comprise from about 10 pg to about 300 pg of Treprostinil per treatment dose in a cartridge or capsule. In one embodiment, a cartridge for single use can comprise from about 10 pg to about 90 pg of Treprostinil for at least one inhalation. In some embodiments, the dry powder formulation is delivered using at least one inhalation per use or dose. In this and other embodiments, the dry powder formulation is delivered to a patient in less than 10 seconds, or less than 8 seconds or less than 6 seconds per inhalation or breath. In certain embodiments, one or more than one cartridge or capsules can be used per administration session for treating a patient depending on the patient’s need. In one embodiment, the pharmaceutical dry powder composition comprises microcrystalline particles of fumaryl diketopiperazine (FDKP), wherein the particles have a specific surface area ranging from about 59 m2 / g to about 63 m2 / g and have a pore size ranging from about 23 nm to about 30 nm.

[0018] In a particular embodiment, the formulation comprises a dry powder further comprising a sugar and an amino acid, wherein the sugar is mannitol or trehalose; and the amino acid is leucine6508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT or isoleucine and a cationic salt. In certain embodiments, the formulation can further comprise sodium chloride, potassium chloride, magnesium chloride or zinc chloride, sodium citrate, sodium tartrate, or combinations thereof.

[0019] In an exemplary embodiment, the Treprostinil - diketopiperazine dry powder dose is administered using a dry powder inhaler for oral inhalation using one or more inhalations from a dry powder inhaler. In this embodiment, a Treprostinil - diketopiperazine inhalation dry powder, is formed without the need for the employment of an alcohol, such as ethanol. Such is provided to a patient suffering with pulmonary arterial hypertension and in need of treatment. The dry powder inhaler may comprise a container including, a cartridge, and the container or cartridge comprises the dry powder comprising Treprostinil and (E)-3,6-bis[4-(A-carbonyl-2- propenyl)amidobutyl]-2,5-diketopiperazine and / or FDKP. Such composition may be administered in multiple daily doses for a period of about six months or as needed by the patient. The Treprostinil-diketopiperazine dry powder is administered by oral inhalation at an earlier time in the course of the disease to patients with Functional Class I, II or III as a first line monotherapy. In this and other embodiments, the Treprostinil composition comprises single dose capsules or cartridges comprising, 8 pg, 16 pg, 32 pg, 64 pg, 80 pg, 90 pg, 100 pg, or larger content depending on the patient’s need. Alternatively, a combination of cartridges thereof comprising Treprostinil or a salt thereof-diketopiperazine can be provided to the patient in a blister package for ease of use.

[0020] In one embodiment, a method for treating pulmonary arterial hypertension is provided comprising providing a patient in need of treatment a combination therapy using an inhalable dry powder comprising Treprostinil and fumaryl diketopiperazine (“FDKP”), and administering separately in combination with orally administered drugs selected from prostacyclin analogues, endothelin receptor antagonists (ERAs), including bosentran, ambrisentran and macitentan, soluble guanine cyclase agonists / stimulators such as riociguat, and PDE-5 inhibitors, including sildenafil, vardenafil and tadalafil. In some embodiments, the present compositions can be administered with one or more other active agents for treating lung disease, and not limited to a combination therapy comprising administering to a patient in need of treatment for lung disease, including, pulmonary arterial hypertension associated with idiopathic pulmonary fibrosis, a pharmaceutical composition comprising Treprostinil and a diketopiperazine, wherein the composition is substantially devoid of or does not contain a Treprostinil ethyl ester as an impurity.7508135580.1PCT Patent ApplicationDocket 1951300.00718 PCTIn one embodiment, the Treprostinil- diketopiperazine powders can be administered to a patient in need of treatment in combination with other standard lung therapies the patient is being treated at such time.

[0021] In yet another embodiment, the process for making dry powder pharmaceutical compositions comprising a prostacyclin, or a derivative, analog, or prodrug thereof, including, Treprostinil, an analog thereof, a pharmaceutical derivative thereof or an ester thereof, wherein the composition comprises sodium hydroxide as a base, and which composition is stable at room temperature for at least one month, at least three months, at least six months, at least 9 months, or at least one year.

[0022] In alternate embodiments, the dry powder for inhalation further comprises other carriers and / or excipients other than diketopiperazines, for example a sugar, including trehalose; buffers, including sodium citrate; salts, including, sodium chloride and zinc chloride, and one or more active agents, including, Treprostinil, vardenafd, and sildenafd.BRIEF DESCRIPTION OF DRAWINGS

[0023] Fig. l is a graph of the weight % of Treprostinil in a FDKP dry powder wherein the dry powder was formed by dissolving the Treprostinil in a sodium hydroxide solution of 1.25 eq NaOH, with suspension adjusted to pH 4.5 with additional NaOH solution, or a solution of 1.1 eq NaOH without adjustment of pH (resulting in a pH of about 4.4), at various overcharges (excess) of Treprostinil, before being added to FDKP microparticles in water and then spray drying.

[0024] Fig. 2 depicts a plot of the x50 (d50) and x84 (d84) of the particle distributions of a Treprostinil-FDKP dry powder formed after solution of the Treprostinil in a sodium hydroxide solution of 1.25 eq NaOH and with the suspension adjusted to pH 4.5 with additional NaOH solution, or in a solution of 1.1 eq NaOH without adjustment of pH (resulting in a pH of about 4.4) and addition of the same to FDKP microparticles in water, followed by spray drying.

[0025] Figs. 3a and 3b illustrate the particle distribution curves from which the x50 (d50) and (d84) of Fig. 2 are determined, density distribution versus particle size (pm).

[0026] Fig, 4 illustrates Treprostinil stability of a Treprostinil-diketopiperazine dry powder over a 1 month (4 weeks) period at 25°C and 60% relative humidity formed by treatment of Treprostinil with sodium hydroxide solution of 1.25 eq NaOH, with suspension adjusted to pH 4.5 with additional NaOH solution, or a solution of 1.1 eq NaOH without adjustment of pH (resulting in a8508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT pH of about 4.4) and then mixed into a FDKP microcrystal water suspension to form the Treprostinil-diketopiperazine dry powder upon spray drying.

[0027] Fig. 5 illustrates that Treprostinil wt% in treprostinil / FDKP dry powder versus the excess of Treprostinil (overage from theoretical) when the Treprostinil before addition to the FDKP powder suspended in water, is suspended in various concentrations of ethanol (0 -19.5%).

[0028] Fig. 6 is a plot of the x50 (d50) and x84 (d84) of the particle size distribution of a Treprostinil-FDKP dry powder for each overage (excess from theoretical) when the Treprostinil before addition to the FDKP powder suspended in water, is suspended in various concentrations of ethanol (0 -19.5%).

[0029] Fig. 7 depicts a graphic representation of Treprostinil content from resultant powders prepared (2% Treprostinil-FDKP dry powders) by the instant process using NaOH (green) and ammonium solutions (blue) from parallel experiments and compared to a dry powder made by a process using ethanol (black). Resultant dry powder made by each of the process yielded a 10- gram batch. Two powder batches each yielding 50 gram, from either NaOH solution process or an ammonium solution were also analyzed and shown as orange triangle, or an orange square, respectively.

[0030] Fig. 8 depicts a graphic representation of the particle size distribution of 2% Treprostinil- FDKP dry powders at X50 and X84 using a laser diffraction system (Sympatec) from Treprostinil-FDKP dry powders processed using NaOH (triangles) and ammonium solutions (solid and open squares) from parallel experiments and compared to a dry powder made by a process using ethanol (circles). Resultant dry powder made by each of the process yielded a 10 g batch. Two powder batches each yielding 50 g, from either NaOH solution process (orange triangle) or an ammonium solution (orange squares) were also analyzed.

[0031] Fig. 9 depicts a graph illustration of data acquired from stability studies of the Treprostinil- FDKP powders processed with NaOH (A-green) or ammonium solution (B-blue) for a 3 -month study.

[0032] Fig. 10 depicts a graphic illustration of data acquired from the stability samples study of the Treprostinil-FDKP dry powders showing the limit lines of pharmaceutically acceptable degradants (dotted lines) form during the incubation period. The data illustrates the content of each Treprostinil-FDKP ester (A, B, C, D) respectively for ester 1, 2, 3 and 4 respectively, in the9508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT dry powders processed with NaOH solution were more stable than the ones processed in an ammonium solution.

[0033] Fig. 11 depicts a graphic illustration of data acquired from samples prepared as in Example 1 and targeting 1% (wt%) Treprostinil-FDKP in the powders made with process yielding 5- 10 g of dry powders using from -5% to 20% (wt%) Treprostinil-FDKP powders processed with NaOH solution (solid triangles), or ammonium solution (solid squares) and compared to ethanol (solid circles).

[0034] Fig. 12 depicts a graphic representation of the particle size distribution of 1% Treprostinil- FDKP dry powders at X50 (solid triangles and X84 (open triangles) values using a laser diffraction system (Sympatec) wherein the Treprostinil-FDKP dry powders were processed using NaOH.DETAILED DESCRIPTION

[0035] As set forth in U.S. Patent Application Serial No. 17 / 975,403, Treprostinil, a hydrophobic compound, in an alcohol can be added to a suspension of diketopiperazine microparticles, such as FDKP, in water and the admixture spray dried to form a Treprostinil in diketopiperazine dry powder. However, it has been noted that when prostacyclin analogs, such as Treprostinil, are added to an alcohol, such as ethanol, that an excess of Treprostinil is needed to form the desired content of Treprostinil in the Treprostinil-FDKP dry powder that is formed when the Treprostinil alcoholic suspension is added to FDKP microcrystals in water and then spray dried to form the Treprostinil-FDKP dry powder. That is there is a need to overcharge, or add an excess of the prostacyclin analog, such as Treprostinil, to attain the theoretical yield of prostacyclin analog in the prostacyclin analog-diketopiperazine dry powder after spray drying.

[0036] Surprisingly, in one embodiment it has been discovered that the prostacyclin analog, such Treprostinil, can be added to an alkaline solution, such as one formed with NaOH, without the need for alcohol (such as ethanol), before addition to the water suspension comprising microcrystals of a diketopiperazine, and having the admixture spray dried, come much closer to the theoretical expected yield of the prostacyclin analog, such as Treprostinil, in the diketopiperazine dry powder that one would expect upon spray drying the admixture for pharmaceutical use. The process avoids wasting Treprostinil and FDKP materials, and the process proceeds in a safer, commercially feasible, and / or cost-saving manner.10508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT

[0037] The new method for manufacturing the prostacyclin analog, such as Treprostinil, in a diketopiperazine dry powder via spray drying, does not require the preparation of an alcohol- based solution of the prostacyclin analog before its addition to a diketopiperazine in water suspension. Such formulation technique has been found to have no impact on the particle size distribution in terms of measurements taken by laser diffraction at x50 or x84.

[0038] As would be understood by one of ordinary skill in the art, particle size analyzers differ on what parameter their analysis is based upon. Some particle size analyzers provide numberbased distributions (dynamic image analysis), others mass-based (sieve analysis) and others volume-based particle size distributions (laser diffraction). Particle size distributions are typically displayed as a histogram, where the width of bar corresponds to the lower or upper limit of the size class and the height of the bar corresponds to the quantity in that size class. In particle measurement technology it is common to generate a cumulative distribution for the classdependent values.

[0039] As used herein, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0040] As used herein, the term "microparticle" refers to a particle with a diameter of about 0.5 to about 1000 pm, irrespective of the precise exterior or interior structure. Microparticles having a diameter of between about 0.5 and about 10 microns can reach the lungs, successfully passing most of the natural barriers. A diameter of less than about 10 microns is required to navigate the turn of the throat and a diameter of about 0.5 pm or greater is required to avoid being exhaled. To reach the deep lung (or alveolar region) where most efficient absorption is believed to occur, it is preferred to maximize the proportion of particles contained in the "respirable fraction" (RF), generally accepted to be those particles with an aerodynamic diameter of about 0.5 to about 6 pm, though some references use somewhat different ranges, as measured using standard techniques, for example, with an Anderson Cascade Impactor. Other impactors can be used to measure aerodynamic particle size such as the NEXT GENERATION IMPACTOR™ (NGI™, MSP Corporation), for which the respirable fraction is defined by similar aerodynamic size, for example < 6.4 pm. In some embodiments, a laser diffraction apparatus is used to determine particle size, for example, the laser diffraction apparatus disclosed in U.S. Patents No. 8,508732, which disclosure is incorporated herein in its entirety for its relevant teachings related to laser diffraction, wherein the volumetric median geometric diameter (VMGD) of the particles is11508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT measured to assess performance of the inhalation system. For example, in various embodiments cartridge emptying of > 80%, 85%, or 90% and a VMGD of the emitted particles of <12.5 pm, < 7.0 pm, or < 4.8 pm can indicate progressively better aerodynamic performance.

[0041] As used herein, the term "dry powder" refers to a fine particulate composition that is not suspended or dissolved in a propellant, or other liquid. It is not meant to necessarily imply a complete absence of all water molecules.

[0042] In one embodiment, the present inventors have discovered that by admixing a prostacyclin analog, such as Treprostinil, with an alkaline base, such as a sodium hydroxide, or an ammonium solution rather than an alcohol, such as ethanol, before addition to diketopiperazine particles in a water suspension, that the need for the use of excess prostacyclin analog, such as Treprostinil, can be about 5% in reaching the theoretical amounts of prostacyclin analog, such as Treprostinil, in the spray dried Treprostinil-diketopiperazine admixture.

[0043] Using a method described in U.S. Patent Application Serial No. 17 / 975,403, in which Treprostinil is added to a 70% - 100% ethanol solution prior to addition into a FDKP microparticle water suspension before spray drying the Treprostinil-FDKP into a dry powder, it has been found that one needs to use an excess of Treprostinil of up to about 20% wt% to obtain 1-2 wt% Treprostinil in the Treprostinil-FDKP dry powder obtained after spray drying. To improve yield and reduce the waste of the Treprostinil, the inventors herein disclose that Treprostinil can be dissolved in an non-alcohol containing alkaline solution (such as a NaOH solution) at a pH greater than 7.0, and / or in solutions of equal to or less than 15% alcohol, more preferably in solutions of equal to or less than 10% alcohol, and yet more preferably in solutions of equal to or less than 5% alcohol, before adding the solution into an FDKP microcrystals in a water suspension, without any significant loss of Treprostinil from the theoretical amount needed to form a 1 wt% Treprostinil in Treprostinil-FDKP dry powder obtained after spray drying an admixture of Treprostinil in alkaline solution with FDKP microparticles in a water solution. Any excess of Treprostinil in the solution added to the FDKP microparticles in water is preferably equal to or less than 15%, or preferably equal to or less than 10%, and yet more preferably equal to or less than 5%.

[0044] In an embodiment, there is disclosed a process for making an inhalable dry powder composition of Treprostinil-diketopiperazine comprising:12508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT(a) washing microcrystals of a diketopiperazine, including, (E)-3,6-bis[4-(7V-carbonyl-2- propenyl)amidobutyl]-2,5-diketopiperazine or FDKP, in water washes;(b) forming a suspension of diketopiperazine in water;(c) mixing Treprostinil into an alkaline solution to form a Treprostinil alkaline aqueous solution in the pH range of about pH 8 to about pH 9, without the presence of an alcohol;(d) introducing the Treprostinil in alkaline solution of step (c) into the suspension of the microcrystals of the diketopiperazine in water to a resulting in a pH of about 4 - 4.5; and(e) spray drying the suspension of step (g) to form the inhalable dry powder composition of Treprostinil-diketopiperazine without a Treprostinil ethyl ester impurity.

[0045] In another embodiment, a process for making microcrystals of a diketopiperazine are described, including, (E)-3,6-bis[4-( V-carbonyl-2-propenyl)amidobutyl]-2,5-diketopiperazine or FDKP, entails mixing the diketopiperazine in a acetic acid solution in a high shear mixer at a temperature ranging from 13°C to about 20°C; or from about 15°C to about 20°C, while mixing the suspension; washing the suspension in water; pelletizing the suspension in a cryogranulator, and drying the suspension in a lyophilizer to collect the microcrystals of the diketopiperazine.

[0046] In another embodiment, a process for making the microcrystals of diketopiperazine includes adding diketopiperazine to an aqueous ammonia solution and combining an acetic acid solution in a high shear mixer at a temperature ranging from 13°C to about 20°C; or 15°C to about 25°C while mixing in a high shear mixer to form a suspension; washing the suspension in water; pelletizing the suspension in a cryogranulator using liquid nitrogen to form pellets of the suspension, collecting the pellets and drying the pellets to form dry microcrystals for later use.

[0047] Also disclosed in an embodiment is a process for forming an inhalable Treprostinil-FDKP dry powder comprising:(a) preparing microcrystals of a diketopiperazine and suspending the same in an aqueous solution;(b) preparing a Treprostinil solution in a non-alcoholic alkaline solution at a pH of about 8-9;.13508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT(c) introducing the non-alcoholic alkaline solution of Treprostinil into the aqueous suspension comprising the microcrystals of the diketopiperazine; and(d) spray drying the suspension of step (c) to form the inhalable Treprostinil-FDKP dry powder.

[0048] In some embodiments, the process for preparing the inhalable Treprostinil-FDKP dry powder optionally comprises, adjusting the pH of the solution to about pH 4.5 to pH 5.0 with additional basic or alkaline solution depending on the starting basic solution used to dissolve the treprostinil as in step b) above, for example, either sodium hydroxide or ammonium solution.

[0049] The diketopiperazine of such method may be (E)-3,6-bis[4-(A-carbonyl-2- propenyl)amidobutyl]-2,5-diketopiperazine, or may be FDKP.Example 1

[0050] Preparation of Treprostinil-FDKP dry powder without need for an alcohol

[0051] Treprostinil was added either to a sodium hydroxide (NaOH) solution containing 1.25 eq of NaOH based on the amount of Treprostinil present and added to microcrystals of FDKP in water suspension and adjusted to pH 4.5 with addition NaOH solution or added to a 1.1 eq NaOH solution with no further pH adjustment made once the solution was added to a microcrystalline FDKP in water suspension (resulting in a final pH of approximately 4.4). No ethanol was added to either suspension. The resultant admixture was sprayed dried using a Buchi B-290 using the following conditions: Inlet temperature: 220°C, aspiration pump: 90% and feed pump:55%. The resulting theoretical yield comprising the Treprostinil-FDKP dry powder was at 10 gram scale.

[0052] The Treprostinil-FDPK dry powder of Example 1 using a 20% overcharge (excess of Treprostinil versus theoretical or target need) was analyzed for Treprostinil concentration. The powder was found to contain 97.1% of the expected assay when compared to yield expected in respect to ethanol suspended Treprostinil as manufactured by a process set forth in U.S. Patent Application Serial No. 17 / 975,403 assigned to the present applicant.

[0053] Now turning to Fig. 1. Fig. 1 is a graph of the wt% of Treprostinil in a Treprostinil-FDKP dry powder wherein the Treprostinil was dissolved in a sodium hydroxide solution containing 1.25 eq NaOH, and added to a FDKP microcrystal aqueous suspension with suspension adjusted to pH 4.5 with additional NaOH solution, or a solution of 1.1 eq NaOH, which was added to a FDKP microcrystal aqueous suspension without adjustment of pH (resulting in a pH of about14508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT4.4), and then spray dried, using various overcharges (excess) of Treprostinil versus theoretical need. Comparison was made with the weight percent of Treprostinil that would theoretically be expected based on the amount of Treprostinil added to a diketopiperazine suspension in water, followed by spray drying. As shown by the dotted lines, the wt % of Treprostinil-diketopiperazine dry powder formed using the Treprostinil in alkaline solutions was found to be within specification for the target wt % of Treprostinil in a Treprostinil-diketopiperazine dry powder formed using the ethanol suspension method as described in U.S. Patent Application Serial No. 17 / 975,403 irrespective of the degree of excess of Treprostinil versus theoretical or target need.

[0054] Fig. 2 depicts a plot of the x50 (d50) and x84 (d84) of the particle size distributions by laser diffraction of Treprostinil in FDPK dry powder when the Treprostinil particles were added to a sodium hydroxide solution containing 1.25 eq NaOH, and added to a FDKP microcrystal aqueous suspension with suspension adjusted to pH 4.5 with additional NaOH solution, or a solution of 1.1 eq NaOH which was added to a FDKP microcrystal aqueous suspension without adjustment of pH (resulting in a pH of about 4.4) , followed by spray drying the admixture. No difference was seen in the x50 or x84 for no alcohol versus that of an excess of Treprostinil of 10 or 15% indicating that an overcharge was not necessary to obtain the desired the particle distribution of Treprostinil in the FDKP dry powder (ultimately formed after the alkaline solution of Treprostinil was added to a FDKP microparticle water solution and then the admixture spray dried to form a Treprostinil-FDKP dry powder).

[0055] Now turning to Figs. 3a and 3b illustrate the particle distribution curves from which the x50 (d50) and x84 (d84) of Fig. 2 are determined, density distribution versus particle size (um).Example 2

[0056] Stability testing of Treprostinil in FDKP dry powder formed by the method of Example 1 using a 5% overcharge (excess) of Treprostinil

[0057] The ph 4.5 and 1.1 eq. powders with a determined 5% overcharge of Treprostinil used in the manufacturing process were stored for 1 month in glass vials at a temperature of 25°C and 60% relative humidity. The percent of Treprostinil remaining after 1 month at the temperature of 25°C and 60% relative humidity was found to be no different than would be expected if the Treprostinil had been manufactured by the process described in U.S. Patent Application Serial No. 17 / 975,403 with exposure to ethanol, except with no Treprostinil-ethyl-ester impurity15508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT present. In addition, the bulk dry powder form made by the non-alcoholic process contained less other impurities. In certain embodiments, bulk powders developed impurities during incubation, comprising Treprostinil dimers were not seen to be any different in terms of percent of the dry powder over a 1 -month period of the stability studies. While ethyl ester content was seen to be slightly higher in the Treprostinil-FDKP powder processed with ethanol, none was found in the solution of 1.1 eq NaOH without adjustment of pH (resulting in a pH of about 4.4), than in the experiments wherein Treprostinil suspension was treated with sodium hydroxide solution of 1.25 eq NaOH, with suspension adjusted to pH 4.5 with additional NaOH solution.

[0058] Fig, 4 illustrates stability of the Treprostinil content in a Treprostinil-FDKP powder made by the method of Example 1, with a 5% overcharge of Treprostinil used in the manufacturing process, over a 1 -month (4 weeks) period at 25°C and 60% relative humidity. As seen the Treprostinil content did not change over 1 month at 25°C and 60% relative humidity. Studies of ethyl ester content and dimer 1 and dimer 2 content over the 1 -month period were not seen to change appreciably, with no impurity, or total impurities, approaching shelf-life specifications.

[0059] Fig. 5 illustrates that Treprostinil wt% in a Treprostinil / FDKP dry powder versus the excess of Treprostinil (overcharge from theoretical) when the Treprostinil is added to various concentrations of ethanol (0 -19.5%) before addition to FDKP microcrystals suspended in deionized and filtered water. As can be seen the data shows that as the ethanol content of the Treprostinil suspension is decreased from 19.5% ethanol to zero ethanol (Treprostinil dissolved in NaOH solution), the content of Treprostinil in the Treprostinil-FDKP dry powder formed by spray drying approaches that of the expected or theoretical content of the formulation, that is the slope of the Treprostinil content of the Treprostinil-FDKP dry powder line approaches that of the expected or theoretical content of the dry powder. Thus, the reduction or removal of ethanol in a Treprostinil suspension, which is subsequently added to the FDKP microcrystals suspended in deionized water, results in a reduction in the amount of Treprostinil excess needed to obtain the desired percentage of Treprostinil in a Treprostinil-FDKP dry powder obtained by spray drying the admixture. As can be seen, the non-alcoholic, ethanol-free solutions provide for a more effective and safe process for reducing danger when operating at large scale quantities in the commercial manufacturing setting.16508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT

[0060] Fig. 6 illustrates that the x50 (d50) and x84 (d84) of the particle size distribution of a Treprostinil-FDKP dry powders formed using various Treprostinil dissolved in no ethanol (0%) and in NaOH, 8.9% (EtOH) and 19.5% (EtOH) in ethanol as control. Treprostinil suspension is added to FDKP microparticles suspended in water, and the admixture spray dried to form a Treprostinil-FDPK dry powder. As can be seen the particle size data as adjudged by X50 and X84 did not vary with changing ethanol content for the treprositinil suspension added to the FDKP microparticle water suspension, in the resultant Treprostinil-FDKP dry powders.Example 3

[0061] Stability testing of Treprostinil in FDKP dry powder formed by the method of Example 1 using a range of up to 15% overcharge (excess) of Treprostinil using either NaOH or Ammonium.

[0062] In this study, Treprostinil-FDKP dry powders targeting a 2% (wt%) Treprostinil content were prepared using an alkaline solution comprising sodium hydroxide as described in Example 1 above for 1% (wt%) theoretical yield. The pH of these suspensions were, optionally adjusted to pH 4.5, as needed, with the addition 0.5% sodium hydroxide solution. In parallel experiments, another alkaline solution was tested comprising ammonium hydroxide, wherein Treprostinil was added to an ammonium hydroxide (NH30H) solution containing 1.25 eq of NH30H based on the amount of Treprostinil present and added to microcrystals of FDKP in water suspension. The pH of these suspensions were, optionally adjusted to pH 4.5 as needed with the addition 0.5% ammonia solution. In the experiments, powders prepared using excess Treprostinil charges ranging from -5% to 15% of target were used to determine various parameters of the dry powders, for example, percent Treprostinil content, particle size distribution and stability at room temperature. No ethanol was added to these suspensions. The resultant suspensions were sprayed dried using a Buchi B-290 using the following conditions: Inlet temperature: 220°C, aspiration pump: 90% and feed pump:55%. The resulting Treprostinil-FDKP dry powders targeted a theoretical yield of 5 or 10-grams. Two powders were prepared on 50-gram scale, one where Treprostinil was dissolved in a NaOH solution, as described above, using aa 5% excess of Treprostinil and another where the treprostinil was dissolved in a NH30H solution, as described above, without an excess of Treprostinil.17508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT

[0063] Dry powders comprising Treprostinil-FDKP made by an alcohol process wherein the Treprostinil is dissolved in ethanol prior to adding to an FDKP microcrystals in a water suspension were also made and evaluated in parallel to the dry powders made without ethanol. Samples of all the powders were tested at least in duplicates and the resultant data were analyzed.

[0064] FIG. 7 is a graph illustrating representative data resulting from the experiments herewith. As depicted in the graph, the Treprostinil content of the dry powders increases for all processes performed as the percentage of Treprostinil is increased in the suspension. The dry powders prepared with NaOH solution behave comparable to the dry powders prepared using ethanol. The data also show that the powders made using NH3OH had slightly higher Treprostinil content and that the 50 gram dry powder batches prepared without ethanol, match the data from the 5 and 10 gram preparation batches.

[0065] FIG. 8 depicts a graph of the particle size distribution of the dry powder herewith for the powders made using NaOH or NH3OH solutions to dissolve the Treprostinil. The data show that the powders made with the NaOH solution had similar or comparable particle size distribution as powders made using ethanol. Powders processed using NH3OH solution had larger particle sizes in particular at higher Treprostinil content in the powder at both 50% maximum distribution and at 84% of maximum particles distribution when compared to powders processed with alcohol based solutions, which served as control in this study. Samples of the 50-gram batches for both NaOH and NH3OH were comparable to the ethanol made powders.

[0066] The room temperature stability of dry powders was tested for several of the powders. In this study, dry powders from 2% NaOH made with 5% excess Treprostinil and dry powders made with NH3OH without excess Treprostinil were used. Samples were placed in glass vials and stored for three months at 25° C and 60% relative humidity. Samples were tested at different times during storage and analyzed for remaining Treprostinil content. FIG. 9 depicts data from the study and shows that the dry powders processed with the NaOH solution had no loss in Treprostinil content through the three months of incubation and it appears that the dry powders are very stable beyond the three months. Alternatively, the dry powders processed using NH3OH solution lost Treprostinil content beginning at one week into the study but retained > 90% Treprostinil content after eight weeks of incubation.18508135580.1PCT Patent ApplicationDocket 1951300.00718 PCT

[0067] FIG. 10 illustrate the degradation products / impurities that form after dry powder samples processed in alkaline solutions were stored at room temperature and 60% relative humidity for a period of three months. The data showed that degradation products, including FDKP-Treprostinil esters 1 (A), 2 (B), 3 (C) and 4 (D) formed and increased slightly with storage. Dry powders made using the sodium hydroxide solution were more stable, as the FDKP-Treprostinil esters were undetectable or less detectable after the three-month period of incubation. FDKP- Treprostinil esters 2 (B) and 4 (D) were undetectable for the entire three-month incubation. The data also indicate that the impurities appeared faster in the ammonium solution processed powders. The data also show that other degradants of FDKP-Treprostinil are Treprostinil dimers 1 (E) and 2 (F) and others impurities (G). The data also demonstrates that the dry powders processed in NaOH solution had less Treprostinil dimers after 12 weeks of incubation than the powders made using ammonium solution. Total other impurities for both sodium hydroxide or ammonium solution processed powders were similar up to about 4 weeks of incubation. Total other impurities increased for the sodium hydroxide processed powder and stabilized after 12 weeks and decreased for the ammonium solution processed powders. The data also showed that all impurities or degradation products resulting during the incubation period were at pharmaceutically acceptable levels as shown in the graphs A-G as a red dotted line.

[0068] FIG. 11 depicts a graphic illustrating data acquired from samples prepared as in Example 1 and targeting 1% (wt%) Treprostinil in the powders made targeting a yield of 5- 10 g of dry powders using from -5% to 20% (wt%) Treprostinil-FDKP powders processed with NaOH solution (solid triangles), or ammonium solution (solid squares) and compared to ethanol (solid circles). The data show that the Treprostinil-FDKP powders formed using the sodium hydroxide or ammonium solution contained slightly higher content of Treprostinil than powders processed with ethanol and the ammonium solution processed powders had the highest content of Treprostinil.

[0069] The particle size distribution of dry powders processed in alkaline solution of samples tested by laser diffraction range measured between 2 micron and 5.5 microns at X50 and X84 (See FIG. 12).19508135580.1

Claims

PCT Patent Application Docket 1951300.00718 PCTWe claim:

1. A process for making an inhalable dry powder composition comprising: spray drying a suspension including a non-alcoholic alkaline solution of Treprostinil and an aqueous suspension of microcrystals of diketopiperazine at a pH of about 4.0 - 4.5 to form the inhalable dry powder composition.

2. A process for making an inhalable dry powder composition comprising:(a) preparing microcrystals of a diketopiperazine and suspending the same in an aqueous suspension.(b) preparing a Treprostinil solution in a non-alcoholic alkaline solution at a pH of about 8.0 to about 9.0;(c) introducing the non-alcoholic alkaline solution of Treprostinil into the aqueous suspension of microcrystals of diketopiperazine to obtain an admixture having a pH of about 4.0 - 4.5; and(d) spray drying the suspension of step (c) to form the inhalable dry powder composition comprising Treprostinil and the diketopiperazine.

3. The method of claim 2, wherein the diketopiperazine is (E)-3,6-bis[4-(A-carbonyl-2- propenyl)amidobutyl]-2,5-diketopiperazine.

4. The method of claim 2, wherein the dry powder composition is devoid of a Treprostinil impurity.

5. The method of claim 4, wherein said impurity comprises Treprostinil ethyl ester, Treprostinil dimer 1, or Treprostinil dimer 2.

6. The method of claim 1, wherein the non-alcoholic alkaline solution is a sodium hydroxide solution.

7. The method of claim 1, wherein the non-alcoholic alkaline solution is ammonium hydroxide.

8. The method of claim 6, wherein the sodium hydroxide solution is 1.25 equivalent in the solution.

9. An inhalable dry powder composition made by the process of claim 1 or 2.20508135580.1PCT Patent Application Docket 1951300.00718 PCT10. The inhalable dry powder composition of claim 9, wherein said treprostinil content is between 1.8 and 2.4%.

11. The inhalable dry powder composition of claim 10, wherein said treprostinil content is 2%.21508135580.1

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