Sterilization of ensifentrine by gamma irradiation

Abstract

The present invention relates to the technical field of sterilizing pharmaceutically active compounds. More specifically, the present invention relates to the technical field of ensifentrine sterilization by means of gamma irradiation.

Description

[0001] STERILIZATION OF ENSIFENTRINE BY GAMMA IRRADIATION FIELD OF THE INVENTION

[0002] The present invention relates to the technical field of sterilizing pharmaceutically active compounds. More specifically, the present invention relates to the technical field of ensifentrine sterilization by means of gamma irradiation.

[0003] BACKGROUND OF THE INVENTION

[0004] Ensifentrine, also known as 9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one, or RPL-554 among others, is a dual inhibitor of phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4). PDE3 inhibitors act as bronchodilators whilst PDE4 inhibitors have anti-inflammatory properties. There is also evidence which suggests that combined inhibition of PDE3 and PDE4 can have additive or synergistic antiinflammatory and bronchodilator.

[0005] The structure of ensifentrine is shown below:

[0006]

[0007] Ensifentrine was firstly described in WO 2000 / 58308 A1, which further describes a process for its preparation, compositions comprising it as well as its use in the treatment of asthma or chronic obstructive pulmonary disease (COPD). Ensifentrine was approved for medical use by the FDA in June 2024 and is sold under the commercial name of OHTUVAYRE, in the form of inhalation suspension.

[0008] As most of the pharmaceutical products, ensifentrine must meet stringent sterility requirements before being administered to a patient in need thereof. In this regard, patent document WO 2023 / 156794 A1 describes a process for producing a sterilised ensifentrine suspension, by heating ensifentrine particles to obtain sterile ensifentrine particles and mixing said ensifentrine particles with a sterile liquid vehicle. In this document, there is a comparison among different sterilization techniques, such as terminal sterilization or dry gamma-ray sterilization. However, the inventors concludedthat terminal heat treatment, terminal gamma irradiation and dry gamma irradiation caused significant degradation of ensifentrine.

[0009] In view of its recent approval by the FDA, ensifentrine is therefore seen as an interesting product by the pharmaceutical industry. Thus, the provision of ensifentrine sterilization processes that meet the stringent requirements are highly desired.

[0010] SUMMARY OF THE INVENTION

[0011] Contrary to what is disclosed in WO 2023 / 156794 A1, inventors have surprisingly found that it is possible to use gamma irradiation for sterilizing ensifentrine in particle form, particularly micronized ensifentrine, by using gamma irradiation to obtain a final sterilized product which remains chemically stable and no significant chemical degradation was observed with respect to the non-irradiated product (i.e. contains less than 3.1% of total impurities, preferably less than 2% of total impurities, more preferably less than 1% of total impurities, even more preferably less than 0.5% of total impurities), when the sample is in a sealed container and is irradiated under an inert gas (such as nitrogen or argon) or has been subjected to vacuum.

[0012] Therefore, in a first aspect the invention relates to a process for the preparation of a sterile ensifentrine in particle form, comprising subjecting the ensifentrine in particle form to gamma irradiation at a dose of from 10 to 50 KGys, wherein the irradiation is applied to ensifentrine packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum.

[0013] In a particular embodiment, sterile ensifentrine in particle form obtained according to the process of the invention is in micronized form and does not experience any change neither in its crystalline characteristics, as demonstrated by DSC (Differential Scanning Calorimetry), TGA (Thermal Gravimetric Analysis), XPRD (X-ray powder diffractometry), IR (infrared spectrum), nor in its particle size as proved by Malvern analysis. Also the corresponding pharmaceutical compositions containing it turned out to be physically and chemically stable after long-term and accelerated storage conditions.

[0014] The process of the invention also allows to solve the technical problem of preparing micronized ensifentrine sterile suspensions to be used for nebulization. Sterilization methods of the prior art carried out directly on the final formulation are indeed not suitable; aseptic filtration cannot be utilized due to non-filterability of suspended particles for ensifentrine, particularly in the polymorphic Form I, while wet steam (autoclaving) involves a degree of heat which can be only tolerated by thermostable compounds. Thus, in a second aspect, the invention relates to a process for preparing a sterile liquid pharmaceutical composition comprising a) providing ensifentrine in particle form prepared by the process according to the first aspect, and b) combining the sterileensifentrine in particle form obtained in step (a) with a sterile liquid vehicle to produce the sterile liquid pharmaceutical composition. The sterile liquid pharmaceutical composition is suitable for administration by inhalation, particularly by nebulization (i.e. administered by oral inhalation using a standard jet nebulizer with a mouthpiece).

[0015] In a third aspect, the invention refers to a process for preparing an ampule comprising a sterile liquid pharmaceutical composition comprising (i) producing a sterile liquid pharmaceutical composition suitable comprising ensifentrine in particle form by a process according to the second aspect of the invention; and (ii) filling an ampule with the sterile liquid pharmaceutical composition. The sterile liquid pharmaceutical composition is suitable for administration by inhalation, particularly by nebulization (i.e. administered by oral inhalation using a standard jet nebulizer with a mouthpiece).

[0016] It also forms part of the invention a pharmaceutical composition comprising the sterile therapeutically acceptable ensifentrine in particle form obtainable by the process of the invention with one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the pharmaceutical composition with the sterile therapeutically acceptable ensifentrine in particle form obtainable by the process of the invention comprises one or more active ingredients selected from the group of p2-adrenergic receptor agonist (such as salbutamol, albuterol, formoterol, and salmeterol) or a muscarinic receptor antagonist (such as glycopyrrolate).

[0017] In a further aspect the invention relates to an ampule with the pharmaceutical composition comprising the sterile therapeutically acceptable ensifentrine in particle form obtainable by the process of the invention with one or more pharmaceutically acceptable carriers, diluents or excipients, optionally wherein the ampule is sealed in a foil pouch.

[0018] DESCRIPTION OF THE FIGURES

[0019] Figure 1. Provides a representative X-ray Powder Diffraction (XRPD) pattern of an ensifentrine sample obtained as disclosed herein.

[0020] DEFINITIONS

[0021] In the context of the present invention, the term “container” refers to a package used to contain a determined quantity of a pharmaceutical product, in the case of the present invention, ensifentrine in particle form.

[0022] In a preferred embodiment the ratio between the amount of ensifentrine in particle form and the volume of the container is equal or less than 7:1 w / v, for example equal or less than 6:1 w / v, equal or less than 5:1 w / v, equal or less than 4:1 w / v, equal or less than 3:1 w / v, equal or less than 2:1 w / v or equal or less than 1:1 w / v.

[0023] In the context of the present invention, the term “ensifentrine in particle form” is used to designate particles comprising at least 95 wt% of ensifentrine, preferably, 96 wt% ofensifentrine, more preferably 97 wt% of ensifentrine, more preferably 98 wt% of ensifentrine, more preferably 99 wt% of ensifentrine, still more preferably 99.5 wt% of ensifentrine wherein the individual particles do not have a volume exceeding 10 mm3. In the context of the present invention, the term “sachet” is used a synonym of the term “pouch”, “bag” or “vessel” to designate a sealed container typically made of paper, plastic, or foil.

[0024] In the context of the present invention, the term “transparent” means that the material permits the light to pass through, meaning that the material has a visible light transmission greater than 0%, up to 100%.

[0025] In the context of the present invention, the term “sterile” means substantially free from microorganism, meaning that the product meets the pharmaceutical standards. In particular, a material is considered to be sterile when a) upon validation according to the International Standard Organization Procedure ISO 11137 it ensures a Sterility Assurance Level (SAL) of at least 10'6(preferably 10'7) and / or when it yields a sterile material according to the criteria of the European Pharmacopoeia (Ph. Eur. 5.1.1). The containers used in the present invention are made of materials, which are stable and retain their properties after exposure to gamma irradiation. Examples of said materials are aluminum and plastic materials.

[0026] Examples of plastic materials that may be used for the containers of the invention include the following:

[0027] • Thermoplastics: Acrylonitrile / Butadiene / Styrene copolymers.

[0028] • Aromatic polyesters such as PET, PETG, cellulose derivatives such as cellulose ester and cellulose ethers, paper, carboard.

[0029] • Fluoropolymers such as polychlorotrifluoroethylene (ECTFE; ethylenechlorotrifluoroethylene) copolymer), polyvinyl fluoride, polyvinylidene fluoride (PVDF), ethylene-tetrafluoroethylene (ETFE) and fluorinated Ethylene Propylene (FEP).

[0030] • Polyacrylics such as polymethylmethacrylate, polyacrylonitrile, polyacrylate and polycyanoacrylate.

[0031] • Aliphatic and aromatic polyamides and polyimides.

[0032] • Polycarbonates

[0033] • Polyethylenes such as LDPE, LLDPE, HDPE, UHMPE, UHMWPE).

[0034] Polypropilenes.

[0035] • Polystyrenes.

[0036] • Polysulfones.

[0037] • Polyurethanes.

[0038] • Polyvinylbutyral.

[0039] • Polyvinylchloride (PVC) and Polyvinylidene Chloride (PVDC)• Styrene / Acrylonitrile copolymers (SAN)

[0040] • Thermosets such as allyl diglycol Carbonate Polyester, epoxies, phenolics, polyesters and polyurethanes.

[0041] • Elastomers such as ethylene-propylene diene monomer (EPDM), fluoro elastomers, natural rubber (isoprene), nitrile, polyacrylics, chloroprene (neoprene), silicones (peroxide and platinum catalyst systems), styrenebutadiene copolymers, urethanes.

[0042] In the context of the present invention, the term “exerting vaccum” is used to indicate a reduction of the pressure to less than 103Pa, or less than 1 atm, or less than 1.01325 bar.

[0043] In the context of the present invention, the term “micronization” as used herein refers to the process of reducing the average diameter of particles of a solid material. Usually, the term micronization is used when the particles that are produced are only a few micrometers (typically less than 10 pm) in diameter. Traditional micronization techniques are based on the use of friction to reduce particle size. Such methods include milling and grinding. Reduction in particle size may also take place as a result of collision and impact of the particles to each other. A product is micronized when it has been subjected to micronization as above defined.

[0044] In the context of the present invention, the term “Dvx” as used herein means that x% of the particles in a composition (based on volume) have a diameter of equal to or below a specified Dv value. Thus, a Dvgo of 10 pm means that 90% of the particles, by volume, have a diameter of equal to or below 10 pm. Both Dvgo and Dvso are commonly used to characterize particle size distributions, being Dvgo used more often. A Dvso of 5 pm means that 50% of the particle population, by volume, have a diameter of equal to or below 5 pm. The particle size distribution (PSD) may be (and has been) determined by dynamic light scattering using a HELOS / BR apparatus offered by SYMPATEC which uses a parallel beam laser diffraction set-up using a dispersion pressure of 2.0 bar.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] As previously mentioned, the invention relates to a process for the preparation of a sterile therapeutically acceptable ensifentrine in particle form, said process comprising subjecting ensifentrine in particle form to gamma irradiation at a dose of from 10 to 50 KGys wherein the irradiation is applied to ensifentrine packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum.

[0047] In an embodiment of the process, the ensifentrine in particle form is subjected to gamma irradiation at a dose of from 10 to 50 KGys, preferably of from 10 to 40 KGys, even more preferably of from 10 to 30 KGys, even more preferably of from 25 to 30 KGys.In an embodiment the product to be sterilized is subjected to gamma irradiation in more than one irradiation cycle. In said embodiment the accumulated amount of irradiation of the various cycles is as defined in the preceding sentence.

[0048] In another embodiment of the process, the container comprises a first sachet made of a material selected from the group consisting of plastic material made of polyethylene. In a preferred embodiment, the material of the first sachet is selected from LDPE, HDPE and combinations thereof.

[0049] In another embodiment of the process, the container consists of a first sachet and a second sachet. In a preferred embodiment, the container consists of a first sachet and a second sachet, made of a material as disclosed above.

[0050] In an embodiment of the process, the first sachet is a transparent sachet.

[0051] In another embodiment of the process, the container further comprises a second sachet surrounding the first sachet. In a preferred embodiment, the second sachet surrounding the first sachet is made of aluminum or made of a plastic material or a combination of both.

[0052] In an embodiment of the process, vacuum is exerted at least in the first sachet prior to applying gamma irradiation. In another embodiment, vacuum is exerted in the first sachet and in the second sachet prior to applying gamma irradiation.

[0053] In another embodiment of the process, vacuum is exerted in the aluminum sachet prior to gamma irradiation.

[0054] In an embodiment of the process, vacuum is exerted to reduce the pressure to 700 mbar, preferably 100 mbar, even more preferably to 1 mbar.

[0055] In an embodiment of the process, the ensifentrine in particle form is irradiated during the sufficient time to achieve the desired irradiation dose.

[0056] In an embodiment of the process, the process is carried out at a temperature comprised between 20 °C and 50 °C, preferably between 20 °C and 40 °C.

[0057] In an embodiment of the process the ensifentrine in particle form is characterized by a particle size distribution having a Dvso (median particle size by volume) value comprised between 10 and 200 pm, preferably between 10 and 100 pm, more preferably between 10 and 60 pm, as measured by dynamic light scattering (DLS).

[0058] In another embodiment of the process, the ensifentrine in particle form subjected to gamma irradiation is micronized. In another embodiment, the micronized ensifentrine in particle form is characterized by a particle size distribution having a Dvso (median particle size by volume) value of from 0.2 pm to 5 pm, preferably of from 0.8 to 3 pm, as measured by dynamic light scattering.In another embodiment of the process, the ensifentrine in particle form is crystalline. In another embodiment of the process, the ensifentrine in particle form is in the crystalline polymorphic Form I as disclosed in WO 2012 / 020016 A1. In another embodiment of the process, at least 95% of the ensifentrine in particle form is in the polymorphic Form I, preferably at least 97% of the ensifentrine in particle form is in the polymorphic Form I, more preferably at least 99% of the ensifentrine in particle form is in the polymorphic Form I.

[0059] In certain embodiments of the first aspect of the invention, the process for producing a sterile ensifentrine in particle form comprises subjecting ensifentrine in particle form to gamma irradiation at a dose of from 10 to 30 KGys, wherein the irradiation is applied to ensifentrine in particle form packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum.

[0060] In certain embodiments of the first aspect of the invention, the process for producing a sterile ensifentrine in particle form comprises subjecting micronized ensifentrine in particle form to gamma irradiation at a dose of from 10 to 30 KGys, wherein the irradiation is applied to micronized ensifentrine packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum.

[0061] In certain embodiments of the first aspect of the invention the process for producing a sterile ensifentrine in particle form comprises subjecting micronized ensifentrine in particle form to gamma irradiation at a dose of from 10 to 30 KGys, wherein the irradiation is applied to micronized ensifentrine packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum, wherein the micronized ensifentrine in particle form is characterized by a particle size distribution having a Dvso (median particle size by volume) value of from 0.2 pm to 5 pm, preferably of from 0.8 pm to 3 pm, as measured by dynamic light scattering.

[0062] In certain embodiments of the first aspect of the invention, the process for producing a sterile therapeutically acceptable ensifentrine in particle form comprises subjecting micronized ensifentrine in particle form to gamma irradiation at a dose of from 10 to 30 KGys, wherein the irradiation is applied to micronized ensifentrine packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum, wherein the micronized ensifentrine in particle form is characterized by a particle size distribution having a Dvso (median particle size by volume) value of from 0.2 pm to 5 pm, preferably of from 0.8 pm to 3 pm, as measured by dynamic light scattering, and wherein micronized ensifentrine in particle form is at least 95% in the crystalline polymorphic Form I.

[0063] In certain embodiments of the first aspect of the invention, the process for producing a sterile therapeutically acceptable ensifentrine in particle form comprises subjectingmicronized ensifentrine in particle form to gamma irradiation at a dose of from 10 to 30 KGys, wherein the irradiation is applied to micronized ensifentrine packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum, wherein the micronized ensifentrine in particle form is characterized by a particle size distribution having a Dvso (median particle size by volume) value of from 0.2 pm to 5 pm, preferably of from 0.8 pm to 3 pm, as measured by dynamic light scattering, wherein micronized ensifentrine in particle form is at least 95% in the crystalline polymorphic Form I and wherein the gamma irradiation produces less than 0.5% w / w increase in total impurities.

[0064] In certain embodiments of the first aspect of the invention, the process for producing a sterile therapeutically acceptable ensifentrine in particle form comprises subjecting micronized ensifentrine in particle form to gamma irradiation at a dose of from 10 to 30 KGys and at a temperature between 20 °C and 50 °C, wherein the irradiation is applied to micronized ensifentrine packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum, wherein the micronized ensifentrine in particle form is characterized by a particle size distribution having a Dvso (median particle size by volume) value of from 0.2 pm to 5 pm, preferably of from 0.8 pm to 3 pm, as measured by dynamic light scattering, wherein micronized ensifentrine in particle form is at least 95% in the crystalline polymorphic Form I and wherein the gamma irradiation produces less than 0.5% w / w increase in total impurities.

[0065] The following examples represent specific embodiments of the present invention. They do not intend to limit in any way the scope of the invention defined in the present description.

[0066] EXAMPLES

[0067] Synthesis of ensifentrine

[0068] A mixture of (E)-2-(mesitylimino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimidino[6,1-a]isoquinolin-4-one (398 mg 1.0 mmol), / \ / -(2-chloroethyl)urea (623 mg, 5.1 mmol, 5.0 equiv.), lithium carbonate (225 mg 3.1 mmol), and potassium iodide (84 mg, 0.5 mmol) were stirred in acetonitrile (2 mL) at reflux temperature for 4 days. The resulting reaction mixture was cooled down to 20-25 °C. After the cooling, dichloromethane (10 mL) and aqueous saturated ammonium chloride (10 mL) were added. The organic and the aqueous phases were separated, and the aqueous phase was extracted with dichloromethane (5 mL). The organic phases were joined and washed with aqueous saturated ammonium chloride (5 mL). The solvent of the organic phase was distilled off at reduced pressure and the obtained residue was purified by column chromatography. A fraction of 200 mg of ensifentrine was obtained (yield: 40%). The ensifentrine obtained has a purity of 97.40%, as measured by HPLC. The average particle size of the productwas in the range of 20 to 40 microns. Purity was measured by HPLC (Agilent, UV detection, 360 nm) using an octadecyl silane silica gel for chromatography (4.6 mm x 25 cm, 3.5 pm), with a flux of 0.8 mL / min. Column Temperature: 40 °C. The mobile phase was a mixture of mobile phase A and mobile phase B.

[0069] Mobile phase A: 0.63 g of ammonium formate is solved in 900 mL of water HPLC grade. The pH of the solution is adjusted to pH=5.0 ± 0.1 with formic acid, raise up to 1000 mL and filter (0.45pm).

[0070] Mobile phase B: CH3CN.

[0071] Injection Volume: 5 pL (use needle wash with mobile phase A:B in 25:75) Gradient elution:

[0072] >

[0073]

[0074] System equilibration before injection: 5 minEnsifentrine XPRD characterization Sample preparation: Approximately 20 mg of ensifentrine were prepared in standard sample holders using two foils of polyacetate.

[0075] Data acquisition: Powder diffraction pattern was acquired on a Bruker D8 Advance Series 2Theta / Theta powder diffraction system using CuKcd -radiation in transmission geometry. The system is equipped with a VANTEC- 1 single photon counting PSD, a Germanium monochromator, a ninety positions auto changer sample stage, fixed divergence slits and a radial soller. Programs used: Data collection with DIFFRAC plus XRD Commander V.2.5.1, and evaluation with HighScore Plus 4.9 (Malvern Panalytical). Measurement conditions: The samples were measured at room temperature in a range from 4° to 40° in 20 in 0.5 hours measurement using an angular step of 0.05° and a time per step of 2198 s.

[0076] Ensifentrine PSD characterization (dynamic light scattering)

[0077] Equipment: SYMPATEC model HELOS BR. Dry disperser: RODOS / M. Feeder:

[0078] ASPIROS. Lent: R1 (< 35pm).

[0079] Sample preparation: 30 mg in an Aspires vial well closed.Measuring range: Density: 1.000 g / cm3; Heywood shape factor: 1.000; Caicuiation Mode: FREE; Forced stability: 0; REMO: Not used; Limit curves: Not used.

[0080] Trigger Conditions:

[0081] Reference measurement: Duration 10 s Single / Time base 50.0 ms

[0082] Normal measurement start / stop trigger:

[0083] Start: 0.000 s after opt. concentr. > 1 %

[0084] Valid: always

[0085] Stop after: 2.000 s opt. concentr. < 1 % or after 10.000 s real time

[0086] Trigger timeout: 60 s

[0087] Repetition and series

[0088] Repeat: 0 times

[0089] Disperser: RO DOS / Type RODOS / M

[0090] Injector: 4mm

[0091] With 0 cascade elements

[0092] Primary pressure: 2.0 bar

[0093] Feeder: Type Aspires / Aspires speed 50 mm / s

[0094] Vacuum: Nilfisk / Delay 2 s

[0095] Irradiation process

[0096] Samples were irradiated in a cobalt-60 irradiator, Marsh type, which has 95 containers (320 x 320 x 320 cm, each).

[0097] Irradiation was carried out by ionizing cobalt-60 radiation. Cobalt-60 is a radioactive isotope that emits gamma photons of 1.17 and 1.33 MeV, which is packaged in doubleencapsulated stainless-steel cylinders.

[0098] The bombardment of gamma photons on the products causes the displacement of electrons, these reactions generate free radicals and / or other reactions that can break the chemical bonds of the molecules. Likewise, this breaking of links in the microbial DNA means that any microorganism that survives the treatment is non-viable or incapable of reproducing and consequently its death.

[0099] The effect of the sterilizing agent is sterilization, decontamination, or physicochemical improvements depending on the absorbed dose, initial contamination, etc.

[0100] Routine process control was applied to ensure that products are treated in accordance with the requirements specified in ISO 11137, Good Manufacturing Standards and other applicable regulations. (DT-PR-007 “Process Control Procedure”).

[0101] Irradiator operation

[0102] The installation was designed to work in continuous mode. The type of irradiation was through stainless steel transport containers and their movement is of the “shuffle-dwell”type, start-stop. The product enters through a conveyor belt at the top of the irradiation cell, where irradiation took place while the source is always exposed (“continuous operation”) and slides along this belt to the output belt once the process is finished. The product was transported around the sources (source frame) by a series of discrete movements (pushers) separated by a controlled period of time (time selector) during which the load is stationary.

[0103] The load (the set of containers inside the irradiation cell) was transported around the sources, configured in such a way that said load exceeds above and below the set of sources (“source overlap”), moving along six levels integrating doses throughout the journey. The tempo is the control parameter and proportional to the absorbed dose, the longer the time, the higher the dose.

[0104] The operation was controlled through a control panel in which a programmable controller executes the indicated movements, and the process parameters are set. All data was dumped into a computer from which process records can be obtained. By design, the position of the source frame was automatically set within the working conditions, as well as the irradiation time.

[0105] The security system associated with this panel determined that in the event of any alarm, both from a radiological protection type and from an industrial point of view (lack of container, extension of the actuation time of a pusher, time selector failure, etc.) it causes the installation to stop, the source frame descends to the safety position and until it has reached its position, access to the irradiation cell was not possible.

[0106] Results

[0107] Non-micronized ensifentrine irradiation

[0108] Non-micronized ensifentrine was sterilized with gamma radiation at 10 KGys, at 15 KGys, at 20 KGys and at 25 KGys.

[0109] The sterilization process is carried out on ensifentrine in particle form packed in containers made of polyethylene, after having been subjected to vacuum. The containers are, in turn, sealed in bags made of LDPE.

[0110] Particularly, the samples were introduced in double sachets, meaning that the first sachet with the sample is placed into the second sachet. In some cases, both sachets are transparent sachets and in other cases, the first sachet with the sample is a plastic sachet, preferably a transparent sachet which is placed into a second aluminum sachet. In the cases wherein vacuum is used, vacuum was made in both sachets to eliminate the air.A set of gamma irradiation tests were performed with non-micronized ensifentrine (ENS-127-01) to determine if degradation is produced by analyzing the percentage amount of main impurities as measured by HPLC.

[0111]

[0112] Composition of the non-micronized ensifentrine before the irradiation:

[0113]

[0114] n.d.: Not detectec

[0115] Sterilization of packed ensifentrine with gamma radiation at 10 KGys:

[0116]

[0117] Sterilization of packed ensifentrine with gamma radiation at 20 KGys:

[0118]

[0119] I I | |

[0120]

[0121] | Sterilization of packed ensifentrine with gamma radiation at 25 KGys:

[0122]

[0123] All of the experiments performed led to practically any degradation. An increase in the percentage of total impurities between 0.07% and 0.46% was produced, which was less than 0.5%. It was also found that when the sample was placed in a first transparent sachet which was placed into a second aluminum sachet, and the packed sample was subjected to vacuum, the degradation observed was less than when the sample was placed into a double transparent sachet, in which the sample was under air atmosphere. Determination of impurity increase respect to the sample before irradiating:

[0124]

[0125] Further, when the irradiation was carried out using a transparent sachet and an aluminum sachet the degradation was less than when a double transparent sachet was used. Noteworthy, sterile ensifentrine particles showed no change neither in its crystalline characteristics, as demonstrated by XPRD, and infrared spectrum, nor in its particle size as proved by dynamic light scattering analysis using a Sympatec HELOS / BR apparatus and a dispersion pressure of 2.0 bar.

[0126] Micronized ensifentrine preparation

[0127] Ensifentrine (53 g) was micronized in a Micromazinazinone brand micronizer, model GB50D, under the following conditions: pressure 4.5 bars, Teflon chamber, feed speed 2-2.5 rpm and with nitrogen as gas. Micronized ensifentrine was obtained with a particle distribution having Dv of 0.5 pm, Dvso of 1.60 pm and Dvgo of 3.50 pm, measured by dynamic light scattering using a HELOS / BR apparatus offered by SYMPATEC. Chemical purity of 99.76%, as measured by HPLC.

[0128] Micronized ensifentrine irradiation

[0129] Micronized ensifentrine was sterilized as described for non-micronized ensifentrine with gamma radiation at 25 KGys.

[0130] Composition of micronized ensifentrine before the irradiation.

[0131]

[0132] Sterilization of micronized ensifentrine with gamma radiation at 25 KGys:

[0133]

[0134] Determination of impurity increase respect to the sample before irradiating:

[0135]

[0136] The micronized ensifentrine in particle form also withstood irradiation, with totals remaining below 0.5% and the vacuum-packed sample seemed to degrade less than the non-micronized product. Noteworthy, sterile micronized ensifentrine showed no change either in its crystalline characteristics, as demonstrated by DSC, TGA, XPRD, and infrared spectrum, or in its particle size as proved by dynamic light scattering analysis using a Sympatec HELOS / BR apparatus and a dispersion pressure of 2.0 bar.

[0137] A double irradiation of a micronized sample of ensifentrine (PB-M(1)) under vacuum was performed, i.e. each sample was irradiated twice at 25 KGy under vacuum as shown below.

[0138] Composition of micronized ensifentrine before irradiation:

[0139] I

[0140]

[0141] Sterilization with gamma radiation, radiating twice at 25 KGys:

[0142]

[0143] mpurity increase respect to the sample irradiating twice at 25 KGys:

[0144]

[0145] These experiments confirm that when the micronized sample was under vacuum, the degradation observed was below 0.3%, whereas when the sample was under air atmosphere, the degradation observed was above 0.5%. Noteworthy, sterile micronized ensifentrine showed no change either in its crystalline characteristics, as demonstrated by DSC, TGA, XPRD, and infrared spectrum, or in its particle size as proved by dynamic light scattering analysis using a Sympatec HELOS / BR apparatus and a dispersion pressure of 2.0 bar.Also, sterile micronized ensifentrine packed in a container made of a material capable of withstanding gamma irradiation, wherein the container is sealed and contained an inert gas or was subject to vacuum turned out to be physically and chemically stable after accelerated storage conditions (i.e. 40°C i 75%HR) for at least two months.

Claims

CLAIMS1. Process for the preparation of sterile ensifentrine in particle form, said process comprising subjecting the ensifentrine in particle form to gamma irradiation at a dose of from 10 to 50 KGys, wherein the irradiation is applied to ensifentrine in particle form packed in a container made of a material capable of withstanding gamma irradiation and wherein the container is sealed and contains an inert gas or is subject to vacuum.

2. Process according to claim 1 , wherein gamma irradiation is at a dose of from 10 to 30 KGys.

3. Process according to any one of claims 1 to 2, wherein ensifentrine particles have a particle size distribution having a Dvgo between 10 and 200 pm, preferably between 10 and 100 pm, more preferably between 10 and 60 pm as measured by dynamic light scattering.

4. Process according to any one of claims 1 to 3, wherein the particles of ensifentrine are micronized.

5. Process according to any one of claims 1 to 4, wherein the particles have a particle size distribution characterized by a Dvso value of from 0.2 pm to 5 pm, preferably a Dvso value of from 0.8 pm to 3 pm.

6. Process according to any one of claims 1 to 5, wherein the ensifentrine particles comprise at least 95% of ensifentrine in polymorphic Form I, preferably at least 97% in polymorphic Form I, more preferably 99% in polymorphic Form I.

7. Process according to any one of the preceding claims, wherein the process is carried out at a temperature comprised between 20 °C and 50 °C, preferably between 20 °C and 40 °C.

8. Process according to any one of the preceding claims, wherein the ratio between the volume of the container and the weight of ensifentrine in particle form is equal or less than 7:1 w / v.

9. Process according to any one of the preceding claims, wherein the container comprises a first sachet made of plastic material.

10. Process according to any one of the preceding claims, wherein the container further comprises a second sachet surrounding the first sachet which second sachet can be made of aluminum or made of plastic material or a combination of both.

11. Process according to any of the preceding claims, wherein vacuum is exerted at least in the first sachet prior to applying gamma irradiation.

12. Process according to any of the preceding claims, wherein vacuum is exerted also in the aluminum sachet prior to applying gamma irradiation.

13. Process according to any of the preceding claims, wherein the gamma irradiation produces less than 0.5% w / w increase in total impurities.

14. A process for preparing a sterile liquid pharmaceutical composition comprising ensifentrine particles, the process comprising:(a) preparing sterile ensifentrine in particle form by a process according to claims 1 to 13, and(b) combining the sterile ensifentrine in particle form obtained in step (a) with a sterile liquid vehicle to produce the sterile liquid pharmaceutical composition.

15. A process for producing an ampule comprising a sterile liquid pharmaceutical composition, the process comprising:(i) producing a sterile liquid pharmaceutical composition comprising ensifentrine in particle form by a process as defined in any one of claims 1 to 14; and(ii) filling an ampule with the sterile liquid pharmaceutical composition.

16. A sterile liquid pharmaceutical composition comprising sterile ensifentrine particles suitable for administration by inhalation, wherein the composition is produced by a method comprising:(i) producing a sterile liquid pharmaceutical composition comprising ensifentrine in particle form by a process as defined in any one of claims 1 to 14; wherein the gamma irradiation produces less than 0.5% w / w increase in total impurities and (ii) combining the sterile ensifentrine in particle form obtained in step (a) with a sterile liquid vehicle to produce the sterile liquid pharmaceutical composition.

17. An ampule with the sterile liquid pharmaceutical composition according to claim 16, optionally sealed in a foil pouch.

18. The sterile liquid pharmaceutical composition according to claim 16 and the ampule according to claim 17 for use in the treatment of COPD.

Images