Method of treating COPD patients resistant to standard therapy

Abstract

The present invention relates to a compound for use in a method of improving the CAT score in a subject suffering from chronic obstructive pulmonary disease (COPD) who is resistant to prior treatment for COPD, wherein the compound is ensifentrine or a pharmaceutically acceptable salt thereof.

Description

METHOD OF TREATING COPD PATIENTS RESISTANT TO STANDARD THERAPYFIELD OF THE INVENTION

[0001] The present invention relates to increasing CAT scores i subjects suffering from chronic obstructive pulmonary disease (COPD).BACKGROUND OF THE INVENTION

[0002] Ensifentrine (7V-(2-{(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6,7-dihydro-2J / -pyrimido[6,l-a]isoquinolin-3(4J7)-yl}ethyl)urea; also known as RPL554) is a dual PDE3 / PDE4 inhibitor and is described in WO 00 / 58308 Al.

[0003] As a combined PDE3 / PDE4 inhibitor, ensifentrine has both bronchodilatory and antiinflammatory activity and is useful in the treatment of respiratory disorders including chronic obstructive pulmonary disease (COPD). The chemical structure of ensifentrine is shown below.

[0004] COPD is a progressive, long-term condition affecting a large number of people worldwide. Ongoing symptoms include breathlessness and cough. Treatment of COPD typically comprises maintenance treatment in which a subject is administered a drug on a regular basis (for instance once or twice daily) to improve lung function and ameliorate the symptoms of COPD.

[0005] Two questionnaires commonly used to to evaluate symptoms and evaluate the severity of the disease are the COPD Assessment Test (CAT) and Medical Research Council Breathlessness Scale (MRC). A CAT score can range from 0-40. A higher CAT score requires greater impact of COPD on a patient.

[0006] There are a number of drugs disclosed for use in treating COPD. It would be clinically advantageous to administer specific drugs which are particularly effective in improving CAT score, to optimize the therapeutic effect of the pharmacological intervention.SUMMARY OF THE INVENTION

[0007] An embodiment is a method of treating COPD, comprising administering by inhalation to a patient in need thereof two or more doses per day of a composition comprising a therapeutically effective amount of ensifentrine or a pharmaceutically acceptable salt thereof, wherein the patient is already being administered a combination of a muscarinic antagonist and a beta-2-antagonist and has a CAT score of greater than or equal to 10 before starting administration of the composition.

[0008] Provided herein is a method to improve the CAT score of a subject suffering from COPD, the method comprising administering to the subject via inhalation two or more doses of a composition comprising a therapeutically effective amount of ensifentrine or a pharmaceutically acceptable salt thereof, wherein the CAT baseline score in the subject is at least 10, and wherein the CAT score improve by at least 1 point from baseline following the administration of the composition. In one aspect, the CAT score improves at least 2 points from baseline.

[0009] In some aspects, the subject has improved breathlessness, phlegm, and / or chest tightness following administration of the composition.

[0010] In some aspects, the method further comprises administering to the subject a muscarinic receptor antagonist, a beta-adrenergic receptor agonist, an inhaled corticosteroid, or a combination thereof. In one aspect, the method further comprises administering to the subject a muscarinic receptor antagonist, a beta-adrenergic receptor agonist, and an inhaled corticosteroid. In one aspect, the muscarinic receptor agonist is a long-acting muscarinic receptor antagonist (LAMA), optionally wherein the LAMA is aclidinium, darotropium, tiotropium, glycopyrrolate, or umeclidinium. In one aspect, the beta-adrenergic receptor agonist is a long-acting beta-adrenergic receptor agonist (LABA), optionally wherein the LABA is salmeterol, formoterol, indacaterol, vilanterol, olodaterol, abediterol or carmoterol. In one aspect, the inhaled corticosteroid is beclomethosone, budesonide, fluticasone propionate, ciclesonide, mometasone, or fluticasone furoate.BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1 shows synergistic relaxation of airway smooth muscle with ensifentrine treatment on top of dual or triple therapy. FF is formoterol fumarate, GB is glycopyrronium bromide, and BDP is beclomethasone dipropionate.DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein and in the claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, “comprise,” “comprises” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers. The term “or” is inclusive unless modified, for example, by “either.” Thus, unless context indicates otherwise, the word “or” means any one member of a particular list and also includes any combination of members of that list. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.”

[0013] Headings are provided for convenience only and are not to be construed to limit the invention in any way. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. In order that the present disclosure can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0014] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 0.05%, 1%, 2%, 5%, 10% or 20%. It is to be understood, although not always explicitly stated that all numerical designations are optionally preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0015] As used herein, “subject” and “patient” are used interchangeably.

[0016] A subject is typically receiving the compound as a maintenance therapy. The compound may be administered to the subject once, twice or three times daily. The compound is preferably administered as a twice-daily maintenance therapy.

[0017] The compound is ensifentrine or a pharmaceutically acceptable salt thereof.Pharmaceutically acceptable salts are well known to the skilled person. Typically, the compound is ensifentrine (i.e. ensifentrine free base).

[0018] Crystalline Form

[0019] In some embodiments, the compound (e.g., ensifentrine) provided herein is in crystalline form. In some embodiments, at least 90% of the ensifentrine by weight is in the form of ensifentrine free base Form I. In some embodiments, at least 90% of the particles comprise ensifentrine free base Form I. In some embodiments, Ensifentrine free base Form I is a crystalline polymorph of ensifentrine (crystalline polymorph Form I) which has a powder X-ray diffraction pattern comprising characteristic peaks at 10.1° and 12.9°±0.1° 29. As used herein, values of °29 may be measured using an X-ray wavelength of Cu Ka radiation ( = 1.5406 A). In some embodiments, the powder x-ray diffraction pattern of Form I further comprises characteristic peaks at 15.3° and 17.6°±0.1° 29. In some embodiments, Form I of ensifentrine may have a powder X-ray diffraction pattern comprising at least 5 characteristic peaks selected from 6.4°, 10.1 °, 12.6°, 12.9°, 13.6°, 14.2°, 14.7°, 15.3°, 15.4°, 15.8°, 17.0°, 17.6°, 18.9°, 20.9°, 22.4 °, 22.8° and 28.7° ±0.1° 29. In some embodiments, crystalline polymorph Form I has a differential scanning calorimetry trace showing a maximum at 248°C.

[0020] In some embodiments, the powder x-ray diffraction pattern of Form I of ensifentrine comprises at least 5 (e.g., at least 3, 4, 6, 7, or 8) characteristic peaks, in terms of 29, selected from about 6.4°, about 10.1°, about 12.6°, about 12.9°, about 13.6°, about 14.2°, about 14.7°, about 15.3°, about 15.4°, about 15.8°, about 17.0°, about 17.6°, about 18.9°, about 20.9°, about 22.4°, about 22.8°, and about 28.7°.

[0021] In some embodiments, the particles comprise at least 98% by weight of ensifentrine crystalline polymorph Form I. In some embodiments, the (e.g., ensifentrine) particles comprise at least 99% by weight of ensifentrine crystalline polymorph Form I.

[0022] In some embodiments, ensifentrine crystalline polymorph Form I has the following structural parameters obtained by single crystal analysis:• Wavelength 0.71073 A• Crystal System Triclinic• Space Group P-1• Unit Cell Dimensions a = 8.1246(4) A a = 91.583(4)°• b b = 11.4573(5) A 13 =90.299(4)°• c c = 13.2398(6) A y = 99.628(4)°• Volume 1214.56(10) A 3• C 2.

[0023] In some instances, the crystalline polymorph Form I is the most thermodynamically stable polymorphic form of ensifentrine and is also expected to have the longest shelflife storage. It is expected that this advantage should extend further to the potential shelflife of any commercial ensifentrine drug product comprising the stable crystalline form I as an API. Furthermore, crystalline polymorph I is more amenable for the development of a reproducible uniform micronized dry solid powder of ensifentrine for certain pharmaceutical formulation applications, such as preparation of formulations described herein.

[0024] Ensifentrine Pharmaceutical Compositions and Methods of Use

[0025] Described herein are compositions comprising a therapeutically effective amount of ensifentrine or a pharmaceutically acceptable salt thereof. Also described herein are methods to improve the COPD Assessment Test (CAT) scores of subjects suffering from COPD.

[0026] The method may comprise administering to the subject via inhalation two or more doses of a composition comprising a therapeutically effective amount of ensifentrine or a pharmaceutically acceptable salt thereof, wherein the CAT baseline score in the subject is at least 10, and wherein the CAT score improve by at least 1 point from baseline following the administration of the composition. In one aspect, the CAT score improves by at least 2 points from baseline following the administration of the composition. In some aspects, the subject has improved breathlessness, cough, and / or phlegm following the administration of the composition.

[0027] The subject may be human. The subject may be male. The subject may be female. The subject may have an age of greater than or equal to 65 years. The subject may have an age of less than 65 years. The subject may be taking a background medication selected from one or more of a LAMA, a LABA and an inhaled corticosteroid ICS.

[0028] The method typically comprises administering the compound to the subject by inhalation. A pharmaceutical composition comprising the compound and one or more pharmaceutically acceptable excipients or diluents is typically administered to the subject by inhalation, for instance by nebuliser, pressurised metered dose inhaler (pMDI) or dry powder inhaler (DPI).

[0029] Preferably, the method comprises administering the compound to the subject by inhalation from a nebuliser. Nebulisers aerosolise a liquid pharmaceutical composition into an aerosol that is inhaled into a subject’s respiratory tract. Examples of nebulisers include a soft mist nebuliser, a vibrating mesh nebuliser, a jet nebuliser and an ultrasonic wave nebuliser. Suitable nebuliser devices include the Philips I-neb™ (Philips), the Philips SideStream (Philips), the AeroNeb® (Philips), the Philips InnoSpire Go (Philips), the Pari LC Sprint (Pari GmbH), the AERxR™ Pulmonary Delivery System (Aradigm Corp) and the Pari LC Plus Reusable Nebuliser (Pari GmbH). The nebulizer may for instance be a PARI LC Sprint jet nebulizer with a PARI Vios® PRO Aerosol Delivery System PARI BOY® compressor. The compound may be inhaled via the nebuliser for from 1 to 15 minutes.

[0030] Typically, the method comprises administering the compound to the subject once, twice or three times per day, for instance twice or three times per day. The compound may be administered to the subject by inhalation once, twice or three times a day. Preferably the method comprises administering the compound to the subject by inhalation twice a day. The method may comprise administering a first dose of the compound in the morning (for instance within 3 hours following waking) and a second dose of the compound in the evening (for instance within 3 hours before bed). Typically, the morning and evening doses are administered from 10 to 14 hours apart, for instance about 12 hours apart.

[0031] The compound may be used in any suitable therapeutically effective amount.Typically, the daily dose of the compound is from 0.1 to 20 mg. Typically, the method comprises administering a total daily dose of the compound of from 0.5 to 10 mg.Preferably, the total daily dose of the compound (e.g. ensifentrine free base) is from 5 to 7 mg, for instance about 6 mg per day. The total daily dose of the compound may be 6.0 mg.

[0032] Typically, the compound is administered twice a day in two separate doses which are the same or similar. For instance, the method may comprise administering the compound to the subject twice a day in a first dose of from 1 to 5 mg and a second dose of from 1 to 5 mg. Typically, the method may comprise administering the compound to the subject twice a day in a first dose of from 2 to 4 mg and a second dose of from 2 to 4 mg.

[0033] Preferably, the method comprises administering two doses of about 3 mg ensifentrine free base to the subject per day by inhalation. The method preferably comprises administering a dose of about 3 mg of the compound to the subject twice a day (3 mg BID) by inhalation. More preferably, the method comprises administering by nebuliser a dose ofabout 3 mg the compound to the subject twice a day. Each dose may be 3.0 mg free base ensifentrine administered by nebulizer.

[0034] The compound is typically used as a maintenance therapy. Typically, the method comprises administering the compound to the subject at least once per day for at least 8 weeks. The compound may be administered to the subject at least once per day for at least 16 weeks, preferably for at least 24 weeks. The compound may be administered daily to the subject for at least 1 year. The method may comprise administering the compound to the subject at least once every 24 hours, preferably at least twice every 24 hours, for at least 8 weeks, preferably for at least 16 weeks, more preferably for at least 24 weeks.

[0035] The compound is preferably administered as a suspension formulation, i.e. a suspension of particles comprising the compound in a diluent. The compound may alternatively be delivered as a dry powder, for instance a dry powder comprising particles comprising the compound and particles of a carrier such as lactose.

[0036] The method typically comprises administering an inhalable pharmaceutical composition comprising a suspension of particles of the compound in a diluent. The particles comprising the compound typically have a particle size distribution with a Dv50 of from 0.5 pm to 5.0 pm. The particles preferably have a Dv50 of from 1.0 pm to 2.0 pm.

[0037] Particle sizes are described herein by reference to the Dv50 value, which is the median particle size for a volume distribution. Thus, half the volume of the particles have diameters of less than the Dv50 value and half the volume of the particles have diameters of greater than the Dv50 value. This is a well-known manner in which to describe particle size distributions.

[0038] The technique used to measure the Dv50 values as stated herein is typically laser diffraction. For instance, the particle size distribution can be measured by laser diffraction using a Malvern Mastersizer 3000. Typically, the instrument parameters for the Malvern Mastersizer 3000 are as follows:• Non-spherical particle mode: Yes• Refractive index: 1.500Absorption index: 0.500Particle density: 1.00 g / cm3Dispersant name: 0.02% Polysorbate 20 in water• Refractive index: 1.330• Stirrer; 1000 rpm• Sample measurement: 10 s• Obscuration: 10-20 %.

[0039] Particle sizes are described herein by reference to the Dv50 value, which is the median particle size for a volume distribution. Thus, half the volume of the particles have diameters of less than the Dv50 value and half the volume of the particles have diameters of greater than the Dv50 value. This is a well-known manner in which to describe particle size distributions.

[0040] The particles comprising the compound typically comprise ensifentrine (i.e. ensifentrine free base). The particles may comprise at least 90 wt% ensifentrine free base relative to the total weight of the particles. The particles may comprise at least 99 wt% ensifentrine. The particles may consist of ensifentrine.

[0041] The concentration of particles comprising the compound in the inhalable pharmaceutical composition is typically from 0.1 to 5.0 mg / mL, preferably from 0.1 to 2.5 mg / mL, more preferably from 1.0 to 2.0 mg / mL.

[0042] The inhalable pharmaceutical composition typically further comprises a carrier. The carrier may be a solid or a liquid, or both. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. In one aspect the carrier is suitable for administration with an inhalation device. The carrier may be included in the formulation to be aerosolized, atomized, or nebulized. In one aspect, the carrier may be sterile water.

[0043] In one aspect the carrier is a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material. Pharmaceutically acceptable carriers include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers are well known in the art.

[0044] The inhalable pharmaceutical composition typically further comprises one or more tonicity adjusters, one or more buffers and one or more surfactants. The tonicity adjuster is typically sodium chloride.

[0045] Examples of buffers include a citrate buffer, a phosphate buffer, an acetate buffer, and a bicarbonate buffer. Preferably, the buffer is a phosphate buffer, for instance sodium dihydrogen phosphate dihydrate and / or disodium phosphate dihydrate.

[0046] Examples of surfactants include lecithin, oleic acid, polyoxyethylene glycol alkyl ethers (for instance PEG 300, PEG 600, PEG 1000, Brij 30, Brij 35, Brij 56, Brij 76 and Brij 97), polypropylene glycol (for instance PPG 2000), glucoside alkyl ethers, polyoxyethylene glycol octylphenol ethers, polyoxyethylene glycol alkylphenol ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters (polysorbates, for instance polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80), sorbitan alkyl esters (for instance sorbitan monolaurate (Span 20), sorbitan monooleate (Span 80) and sorbitan trioleate (Span 85)), cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, block copolymers of polyethylene glycol and polypropylene glycol (poloxamers), block copolymers of polyethylene glycol and polypropylene oxide (for instance Pluronic surfactants), polyvinyl pyrrolidone K25, polyvinyl alcohol, oligolactic acid, sodium dioctyl sulfosuccinate and polyethoxylated tallow amine (POEA).

[0047] Preferably, the one or more surfactants comprise a polysorbate and / or a sorbitan alkyl ester. The one or more surfactants may for instance comprise polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate) or polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). The one or more surfactants may for instance comprise sorbitan monolaurate (Span 20), sorbitan monooleate (Span 80) or sorbitan trioleate (Span 85). Preferably, the sterile liquid vehicle comprises polysorbate 20 and / or sorbitan monolaurate (Span 20).

[0048] For instance, the method may comprise administering to the subject an inhalable liquid pharmaceutical composition comprising:• water;particles consisting of ensifentrine free base at a concentration of from 0.1 to 20 mg / mL; one or more tonicity adjusters at a total concentration of from 1.0 to 15 mg / mL;• one or more buffers at a total concentration of from 0.1 to 4 mg / mL; and • one or more surfactants at a total concentration of from 0.05 to 3 mg / mL.

[0049] The inhalable liquid pharmaceutical composition may comprise:• water;• particles consisting of ensifentrine free base at a concentration of from 0.5 to 6 mg / mL;• sodium chloride at a concentration of from 5 to 12 mg / mL;• sodium dihydrogen phosphate dihydrate at a concentration of from 0.3 to 2 mg / mL;• disodium phosphate dihydrate at a concentration of from 0.3 to 2 mg / mL;• polysorbate 20 at a concentration of from 0.1 to 1.5 mg / mL; and• sorbitan monolaurate at a concentration of from 0.01 to 0.5 mg / mL.

[0050] The compound may be used in combination with a second active agent. The compound may be administered separately or simultaneously with the second active agent. The subject may already be taking a second active agent as a background therapy for COPD. Alternatively, treatment with the second active agent may start at around the same time as treatment with the compound. The compound and the second active agent may be administered in a fixed combination.

[0051] The second active agent is typically a muscarinic receptor antagonist, a beta-adrenergic receptor agonist or an inhaled corticosteroid. The compound may accordingly be used in combination with muscarinic receptor antagonist, a beta-adrenergic receptor agonist or an inhaled corticosteroid. The second active agent may be a long-acting muscarinic receptor antagonist (LAMA) or a long-acting beta-adrenergic receptor agonist (LABA).

[0052] Examples of LAMAs include aclidinium, darotropium, tiotropium, glycopyrrolate and umeclidinium. Examples of LABAs include salmeterol, formoterol, indacaterol, vilanterol, olodaterol, abediterol and carmoterol. Examples of inhaled corticosteroids include beclomethosone, budesonide, fluticasone propionate, ciclesonide, mometasone and fluticasone furoate.

[0053] The subject may be using a beta-agonist (for instance salbutamol) as a rescue medication.

[0054] The invention is described in more detail by the following Example.EXAMPLES

[0055] Example 1

[0056] A clinical study was conducted to determine the efficacy of twice-daily nebulized ensifentrine at a dose of 3 mg on COPD Assessment Test (CAT) scores over 12 weeks in subjects with moderate to severe COPD. Enrollment statistics are presented in Table 1.

[0057] CAT scores are determined using the COPD Assessment Test questionnaire. The questionnaire is used to assess the impact of COPD on health status. CAT scores can range from 0-40. More information about the CAT score questionairre can be found at “ COPD Assessment Test (CAT),” American Thorasic Society, thoracic. org / members / assemblies / assemblies / sm / questionaires / copd.php (last accessed November 5, 2024), P.W. Jones, G. Harding, P. Berry, I. Wiklund, W-H. Chen and N. Kline Leidy. Development and first validation of the COPD Assessment Test. Eur Respir J 2009, 34: 648-654, and Jones PW, Brusselle G, Dal Negro RW, Ferrer M, Kardos P, Levy ML, et al. Properties of the COPD assessment test in a cross-sectional European study. European Respiratory Journal. 2011 July 1, 2011 ;38(l):29-35, the entire contencts of which are incorporated herein by reference.

[0058] Table 1. Clinical study participants. Open-label study. Subjects enrolled on stable dual (LAMA / LABA) or triple (LAMA+LABA+ICS) with symptoms CAT > / =10 units at baseline for treatment with ensifentrine over 12 weeks. SAE is a serious adverse event. ICS is inhaled corticosteroid.

[0059] Results

[0060] Treatment with twice-daily ensifentrine over in the entrolled trial participants resulted in decreased mean CAT scores. Further, subjects had inproved breathlessness, chest tightness and phlegm (Table 2).

[0061] Table 2. CAT Clinical Study Results (Weeks 6 and 12). A CAT responder is a subjet who has an improvement from the baseline CAT score by at least two points.

[0062] Example 2

[0063] Applicant has demonstrated a synergistic relaxation of airway smooth muscle (FIG. 1) and synergistic reduction in inflammatory cytokine release with ensifentrine treatment on top of dual or triple therapy. This finding, that ensifentrine on top of dual or triple therapy would have a synergistic effect, is surprising.

[0064] FF is formoterol fumarate, GB is glycopyrronium bromide, and BDP is beclomethasone dipropionate. Additional information on FF, GB, and BDP is included below.

[0065] Tissue preparation

[0066] Human bronchial tissue is collected from COPD donors undergoing lobectomy and refrigerated in Krebs-Henseleit buffer solution (KH) (NaCl, 119.0 mmol; KC1, 5.4 mmol; CaC12, 2.5 mmol; KH2PO4 mmol, 1.2 mmol; MgSO4, 1.2 mmol; NaHCO3, 25.0 mmol; glucose, 11.7 mmol; indomethacin 5 pM; pH 7.4; 37°C) during transport. COPD diagnosis is performed according to spirometry; all the donors have no history of asthma and the serum immunoglobulin E (IgE) levels are in the normal range (<100 lU / ml) (Stanojevic et al., 2022; GOLD, 2023).

[0067] In the laboratory, medium bronchi are cut into rings (sub-segmental bronchi: thickness 1-2 mm, diameter 4-5 mm) and transferred into a 10-ml High Tech 8 Channels Manual Compact Organ Bath system (Panlab Harvard Apparatus, Spain) containing KH buffer solution (37°C) and aerated with O2 / CO2 (95:5%). Tissues are allowed to equilibrate and the KH buffer solution is constantly changed (Calzetta et al., 2023b).

[0068] Small isolated airways are prepared as precision lung cut slices (PCLS). Bronchioles (inner diameter ~1 mm) are cut by a motorised vibratome equipped with ceramic blades and refined via microsurgery under a stereo microscope to obtain PCLS. PCLS are mounted into a visual imaging and patching chamber connected to a proportional integral derivative temperature controller with dual thermistor feedback containing KH buffer solution (37°C) and continuously aerated with O2 / CO2 (95:5%) (Calzetta et al., 2023b).

[0069] Contraction measurement

[0070] Isolated airways are mounted on isometric force transducers Fort25 (WPI, UK) connected to PowerLab 8 / 36 and an Octal Bridge Amp system (ADInstruments, UK). Raw data are recorded and analyzed by using the LabChart 7 interface software (ADInstruments, UK). Tissues are mounted on hooks, with one end attached with a thread to a stationary rod while the other was tied with a thread to the isometric force displacement transducer.Airways are allowed to equilibrate by flushing with fresh KH buffer solution. Passive tension is determined by gentle stretching of tissue (0.5-1.0 g) during equilibration. The isometric change in tension is measured by the transducers and the tissue vitality assessed by transmural stimulation (also called electrical field stimulation, EFS) at 10 Hz; when theresponse reached the plateau, rings are washed three times and allowed to equilibrate for 45 min (Calzetta et al., 2023b).

[0071] Epithelium removal and cells harvesting

[0072] In medium bronchi epithelium is mechanically removed from isolated airways using a cotton-tipped applicator gently rubbed for 5 sec on the luminal surface. Epithelium removal is an effective procedures confirmed by histology that does not penetrate the basal membrane and leave the lamina propria intact (Calzetta et al., 2023b).

[0073] Primary bronchial epithelial cells are harvested by gently scraping the luminal airway surface with a convex scalpel blade #10. Collected epithelial cells are then pooled in PBS, centrifuged at 500 g for 5 min at 4 °C and then directly used for experiments in the visual imaging and patching chamber system (Calzetta et al., 2023b).

[0074] Experiments on contractile response in epithelium-intact and epithelium-denuded medium bronchi

[0075] After LPS challenge, epithelium-intact and epithelium-denuded medium bronchial rings are sub-maximally pre-contracted by concentrations of carbachol eliciting 70% maximal contractility (EC70) in order to stimulate the cholinergic tone. When the contractile plateau is reached (usually 5 - 10 min), semi -logarithmic concentration-response curve (CRC) to ensifentrine is constructed. Ensifentrine is administered at concentrations that cover all the CRC, according to solubility characteristics of the compound. Each drug administration is performed after that the relaxant plateau induced by the previous administration is reached. At the end of the experiments, papaverine and atropine (both 100 pM) are added to determine the maximal relaxant response (Emax) achievable for each tissue specimen. Semi -logarithmic CRC to ensifentrine is also constructed on the contractile response to EFS at 3, 10, and 25 Hz. These EFS frequencies reproduce ex vivo the vagal firing of non -myelinated C-fibers that are involved in the neurogenic airway inflammation (3 Hz), small myelinated B-fibers (10 Hz), and large myelinated A-fibers (25 Hz) (Calzetta et al., 2017).

[0076] In some experiments and after LPS challenge, ensifentrine is combined with a LABA (i.e. formoterol), a LAMA (i.e. glycopyrronium), a dual LABA / LAMA combination (i.e. formoterol / glycopyrronium), a dual ICS / LABA combination (i.e. beclomethasone / formoterol), and a triple ICS / LABA / LAMA combination (i.e. beclomethasone / formoterol / glycopyrronium) at isoeffective concentrations (EC30 and EC50) and tested on the contractile response induced by carbachol and EFS.

[0077] Tissue treated with vehicle are used as negative control.

[0078] Experiments on inflammatory response in epithelium intact and epithelium-denuded medium bronchi, PCLS, and primary bronchial epithelial cells

[0079] Epithelium-intact and epithelium-denuded medium bronchi, precision lung cut slices (PCLS), and primary bronchial epithelial cells are pre-treated with concentration response curves (CRCs) to ensifentrine according to solubility characteristics of the compound. After that, bronchial tissue is challenged with lipopolysaccharide (LPS).

[0080] In some experiments and before LPS challenge, ensifentrine is combined with a LABA (i.e. formoterol), a LAMA (i.e. glycopyrronium), a dual LABA / LAMA combination (i.e. formoterol / glycopyrronium), a dual ICS / LABA combination (i.e. beclomethasone / formoterol), and a triple ICS / LABA / LAMA combination (i.e. beclomethasone / formoterol / glycopyrronium) at isoeffective concentrations (EC30 and EC50) and then the LPS challenge performed.

[0081] At the end of experiments, supernatant is collected and stored at -80°C for the assessment of inflammatory panel. LPS challenge

[0082] Isolated airways are challenged 2 h with lipopolysaccharide (LPS) at 300 ng / ml, a validated ex vivo model that mimics the inflammatory response and ASM contractility typical of COPD in vivo (Calzetta et al., 2023b).

[0083] Inflammatory profile quantification

[0084] Luminex multiplex assays are used to detect and quantify cytokines, chemokines, and growth factors secreted by bronchial tissue and respiratory epithelium using ProcartaPlex assays via xMAP bead-based technology according to manufacturer’s instructions (Ozger et al., 2021) (thermofisher.com). A wide panel of analytes is investigated, including, but not limited to, IL-1 , IL-6, IL-8, IL-10, IL-33, MCP-1, TSLP, TNF-a, GM-CSF, TGF-0, EGF. References CitedCalzetta, L., Page, C., Matera, M.G., Cazzola, M., and Rogliani, P. (2023b). Use of human airway smooth muscle in vitro and ex vivo to investigate drugs for the treatment of chronic obstructive respiratory disorders. Br. J. Pharmacol.Calzetta, L., Rogliani, P., Facciolo, F., Rendina, E., Cazzola, M., and Matera, M.G. (2017). Pharmacological characterization of the interaction between umeclidinium and vilanterol in human bronchi. Eur. J. Pharmacol. 812: 147-154.GOLD (2023). 2023 GOLD Reports - Global Initiative for Chronic Obstructive Lung Disease - GOLD.Ozger, H.S., Karakus, R., Kuscu, E.N., Bagriacik, U.E., Oruklu, N., Yaman, M., et al. (2021). Serial measurement of cytokines strongly predict COVID-19 outcome. PLoS One 16:.Stanojevic, S., Kaminsky, D.A., Miller, M.R., Thompson, B., Aliverti, A., Barjaktarevic, I., et al. (2022). ERS / ATS technical standard on interpretive strategies for routine lung function tests. Eur. Respir. J. 60:.* * *

[0085] Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.

[0086] All of the publications, patent applications and patents cited in this specification are incorporated herein by reference in their entirety.The following are additional aspects of the invention.1. A method of treating COPD, comprising administering by inhalation to a patient in need thereof two or more doses per day of a composition comprising a therapeutically effective amount of ensifentrine or a pharmaceutically acceptable salt thereof, wherein the patient is already being administered a combination of a muscarinic antagonist and a beta-2 -antagonist and has a CAT score of greater than or equal to 10 before starting administration of the composition.2. The method of aspect 1, wherein the patient is already being administered an inhaled corticosteroid in addition to the combination of a muscarinic antagonist and a beta-2 -antagonist before starting administration of the composition.3. The method of aspect 1, wherein the administration of the composition improves the CAT score of the patient by at least 1 point from baseline.4. The method of aspect 3, wherein the CAT score improves by at least 2 points from baseline.5. The method of aspect 1, wherein the patient has improved breathlessness following the administration of the composition.6. The method of aspect 1, wherein the patient has improved cough following the administration of the composition.7. The method of aspect 1, wherein the patient has improved phlegm following the administration of the composition.8. The method of aspect 2, wherein the muscarinic receptor agonist is a long-acting muscarinic receptor antagonist (LAMA) selected from aclidinium, darotropium, tiotropium, glycopyrrolate, or umeclidinium.9. The method of aspect 2, wherein the beta-adrenergic receptor agonist is a long-acting beta-adrenergic receptor agonist (LABA) selected from salmeterol, formoterol, indacaterol, vilanterol, olodaterol, abediterol or carmoterol.10. The method of aspect 3, wherein the inhaled corticosteroid is selected from beclomethosone, budesonide, fluticasone propionate, ciclesonide, mometasone, or fluticasone furoate.11. The method of aspect 3, wherein the improvement of CAT score occurs within 6 weeks of beginning administration of the composition.12. The method of aspect 3, wherein the improvement of CAT score occurs within 12 weeks of beginning administration of the composition.

Claims

WHAT IS CLAIMED IS:

1. A method of treating COPD, comprising administering by inhalation to a patient in need thereof two or more doses per day of a composition comprising a therapeutically effective amount of ensifentrine or a pharmaceutically acceptable salt thereof, wherein the patient is already being administered a combination of a muscarinic antagonist and a beta-2 -antagonist and has a CAT score of greater than or equal to 10 before starting administration of the composition.

2. The method of claim 1, wherein the patient is already being administered an inhaled corticosteroid in addition to the combination of a muscarinic antagonist and a beta-2 -antagonist before starting administration of the composition.

3. The method of claim 1 or claim 2, wherein the administration of the composition improves the CAT score of the patient by at least 1 point from baseline.

4. The method of claim 3, wherein the CAT score improves by at least 2 points from baseline.

5. The method of any one of claims 1 to 4, wherein the patient has improved breathlessness following the administration of the composition.

6. The method of any one of claims 1 to 5, wherein the patient has improved cough following the administration of the composition.

7. The method of any one of claims 1 to 6, wherein the patient has improved phlegm following the administration of the composition.

8. The method of claim 2, wherein the muscarinic receptor agonist is a long-acting muscarinic receptor antagonist (LAMA) selected from aclidinium, darotropium, tiotropium, glycopyrrolate, or umeclidinium.

9. The method of claim 2 or claim 8, wherein the beta-adrenergic receptor agonist is a long-acting beta-adrenergic receptor agonist (LABA) selected from salmeterol, formoterol, indacaterol, vilanterol, olodaterol, abediterol or carmoterol.

10. The method of claim 3, wherein the inhaled corticosteroid is selected from beclomethosone, budesonide, fluticasone propionate, ciclesonide, mometasone, or fluticasone furoate.

11. The method of claim 3 or claim 10, wherein the improvement of CAT score occurs within 6 weeks of beginning administration of the composition.

12. The method of any one of claims 3, 10 or 11, wherein the improvement of CAT score occurs within 12 weeks of beginning administration of the composition.

13. A compound for use in a method of treating COPD in a patient in need thereof, which compound is ensifentrine or a pharmaceutically acceptable salt thereof, wherein the method comprises administering by inhalation to the patient two or more doses per day of a composition comprising the compound, wherein the patient is already being administered a combination of a muscarinic antagonist and a beta-2-antagonist and has a CAT score of greater than or equal to 10 before starting administration of the composition.

14. Use of a compound in the manufacture of a medicament for a method of treating COPD in a patient in need thereof, which compound is ensifentrine or a pharmaceutically acceptable salt thereof, wherein the method comprises administering by inhalation to the patient two or more doses per day of a composition comprising the compound, wherein the patient is already being administered a combination of a muscarinic antagonist and a beta-2 -antagonist and has a CAT score of greater than or equal to 10 before starting administration of the composition.

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