Inhalable bronchodilator sustained release compositions for the treatment of pulmonary disorders

Abstract

This invention provides a liposomal sustained-release composition of a bronchodilator for treating lung diseases. The liposomal bronchodilator comprises liposomes encapsulating the bronchodilator. The bronchodilator is stably encapsulated in the liposomes, and the resulting liposomal bronchodilator has been shown to be stably nebulized or aerosolized for administration via inhalation to treat individuals in need.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 847,613, filed May 14, 2019, the contents of which are incorporated herein by reference in their entirety. Background of the Invention Technical Field

[0003] This invention relates to an inhalable drug delivery system for delivering sustained-release liposome compositions. This invention also relates to a method for preparing said drug delivery system. Furthermore, this invention relates to sustained-release drug compositions suitable for pulmonary delivery systems, having an extended duration of efficacy.

[0004] Related technical descriptions

[0005] Unwanted lung diseases, triggered by various external factors, are becoming an unavoidable issue in an aging society. Chronic obstructive pulmonary disease (COPD) is an extremely serious and debilitating lung disease that can lead to premature death. COPD is characterized by airway inflammation and thickening of the mucous membranes, alveolar breakdown, and deterioration of lung function. Common symptoms of COPD include chronic cough, shortness of breath, and chest tightness.

[0006] There are four distinct categories of potential medications for treating lung diseases, particularly COPD: corticosteroids; bronchodilators, especially anticholinergic agents (particularly muscarinic antagonists) and β2-adrenergic receptor agonists; antibiotics; and mucolytics. Currently, inhalation therapy is the preferred route of administration for COPD treatment. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) recommends bronchodilators as the first-line standard of care for most COPD patients. Furthermore, GOLD recommends nebulized inhalation therapy for specific patient groups, such as the elderly and those with low inspiratory flow rates.

[0007] Two long-acting muscarinic antagonists (LAMAs) already used and currently being used to treat COPD are tiotropium bromide and glycopyrrolate. Tiotropium bromide is available in dry powder form, with a recommended dose of 18 μg twice daily. NDA number 21395, Boehringer Ingelheim; or as an inhalation solution, the dose is 2.5 μg, twice daily. NDA number 207070, Boehringer Ingelheim. Glycerin bromide is available as a dry powder, with a recommended dose of 15.6 μg twice daily. NDA number 207923, Novartis. Furthermore, glycopyrronium bromide inhalation solution via aerosolization has recently been approved by the U.S. Food and Drug Administration (FDA) with a recommended dose of 25 μg twice daily. NDA number 208437, Sunovion.

[0008] In addition, many drug combinations are widely used to treat COPD. One such dual COPD medication combines glycopyrronium bromide and indacaterol (an ultra-long-acting β2-adrenergic receptor agonist, or ultra-LABA), with recommended doses of 15.6 μg and 27.5 μg, respectively, twice daily. NDA number 207930, Novartis. Another COPD medication combination contains tiotropium bromide and olodaterol (another ultra-LABA), with recommended doses of 3 μg and 2.7 μg, respectively, twice daily. NDA number 206756 (Boehringer Ingelheim). Side effects of using these medications to treat COPD include tremor, tachycardia, dizziness, allergic reactions, blurred vision, and throat irritation.

[0009] Liposomes have been used as drug carriers for sustained drug delivery for decades. Encapsulating drugs in liposomes alters the pharmacokinetic properties of free drugs, providing slow drug release, either systemically or locally, allowing for higher doses and lower dosing frequencies, and potentially reducing side effects and toxicity. High drug encapsulation in liposomes can be achieved through remote loading (also known as active loading), which relies on transmembrane pH and ion gradients to allow free, uncharged drugs to diffuse into the liposome. Within the liposome, free drugs can form complexes with trapping agents (counterions in the aqueous interior) or even precipitate as drug-counterion salts that remain within the liposome. Liposome drug formulations can be designed to achieve sustained in vivo drug release, which prolongs the therapeutic effect. This can be achieved by adjusting the liposome formulation and optimizing specific liposome properties, such as the phospholipids used (different chain lengths, phase transition temperatures), the ratio of lipids to cholesterol, the amount of polyethylene glycol (PEG) on the liposomes (to avoid clearance by macrophages), the scavenging agents used to encapsulate drug substances, and possibly the lamellarity of the liposomes.

[0010] Even when drugs are stably encapsulated in liposomes, it remains uncertain whether the resulting liposomal drug formulation can be aerosolized or nebulized for inhalation delivery. It is currently unclear whether reconstituted bronchodilators using liposomal technology can produce therapeutic doses of inhaled liposomal drug formulations for the treatment of COPD and other related lung diseases.

[0011] Currently, there are no practically usable inhaled liposomal drug formulations for the treatment of COPD and other related lung diseases. Regarding liposomal drug formulations currently used for inhalation therapy, two inhalable liposomal drugs are under development and have reached the clinical trial stage: liposomal amikacin (Insmed Inc.) and liposomal ciprofloxacin (Aradigm Corporation). These two inhaled liposomal antibiotics are currently being investigated to treat various respiratory diseases, such as cystic fibrosis (CF), non-cystic fibrosis bronchiectasis, nontuberculous mycobacterial lung disease, and other virulent infections. Recently, Amikacin liposomal inhalation suspension has received accelerated approval from the FDA for the treatment of Mycobacterium avium complex lung disease. These two liposomal drug formulations for inhalation therapy are designed to reduce the attractiveness of the antibiotic to macrophages by modifying the lipid component to be electrically neutral (see US 8,226,975) or by adjusting the particle size and amount of free ciprofloxacin (US 8,071,127), thereby facilitating the antibiotic's access to microorganisms or infected tissue.

[0012] Unfortunately, existing inhalable liposomal compositions fail to meet unmet needs for treating other lung diseases, such as COPD, which may require pharmaceutical products with different target product characteristics, such as deep lung deposition, enhanced mucus penetration, prolonged drug retention in the lungs, increased liposomal drug stability, etc. To date, no studies have reported effective inhalable drugs in the form of lipid-based sustained-release compositions for the treatment of COPD or similar diseases. Therefore, there remains an unmet need for sustained-release formulations with predetermined encapsulation efficiency to balance reducing the dosing frequency of bronchodilators (such as anticholinergics and / or β2-adrenergic receptor agonists) with achieving the desired therapeutic window. Furthermore, formulations suitable for treating COPD and other related lung diseases should be inhalable, possess improved stability or resistance to damage caused by local pulmonary surfactant, and further have the desired dose strength to ensure the potential to achieve the desired efficacy in the pulmonary environment. This invention addresses this and other needs. Invention Overview

[0014] The present invention provides an inhalable liposomal pharmaceutical formulation comprising one or more phospholipids, optionally present sterols, and / or PEG-modified phospholipids, and a bronchodilator, particularly an anticholinergic agent, and more particularly a quaternary ammonium muscarinic antagonist, such as tiotropium bromide, encapsulated in the aqueous interior of the liposome.

[0015] To improve existing treatment modalities for lung diseases (such as COPD) and to take advantage of the benefits of slow, sustained drug release, we have developed a sustained-release composition of a bronchodilator comprising a liposomal bronchodilator and a predetermined amount of free bronchodilator in an aqueous suspension, which can be nebulized and inhaled for intensive treatment of lung diseases. Specifically, there is a need for an inhalable sustained-release formulation for the treatment of COPD.

[0016] This invention provides a liposomal bronchodilator for treating lung diseases (particularly COPD), which has the following advantages: 1) achieving a longer therapeutic effect compared to inhaled free drug substances; 2) delivering the drug directly to the disease site; 3) faster onset of action; 4) reducing adverse drug reactions and systemic effects; 5) bypassing the first-pass metabolism observed in oral administration, thereby improving the bioavailability of the drug substance (and potentially reducing hepatotoxicity); 6) increasing the residence time of the drug substance in the lungs through sustained release from the liposomal drug; 7) reducing the frequency of drug administration; 8) non-invasive inhalation delivery; and 9) improving patient treatment outcomes and compliance.

[0017] The bronchodilator of the present invention is encapsulated in liposomes via remote loading using a trapping agent composed of an ammonium compound and an anionic counterion to achieve a sustained-release composition with preferred release characteristics and reduced toxicity.

[0018] The liposome bronchodilator of the present invention optionally includes a large amount of PEG moiety on the vesicle surface to achieve a longer sustained drug release, which is safe, effective and suitable for administration once daily or even less frequently.

[0019] In one specific embodiment, the liposome bronchodilator comprises phosphatidylcholine (PC):cholesterol in a molar ratio of 1:1 to 3:2, wherein the PC may be hydrogenated soyphosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or mixtures thereof, such as DSPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) in a molar ratio of 1:1.

[0020] In one specific embodiment, the PEG-modified phosphorylethanolamine (PE) may be DSPE-PEG2000 and range from 0.0001 mol% to 40 mol% of the total lipid content of the liposomes.

[0021] In some embodiments, the lipid concentration of the liposomal bronchodilator in the sustained-release composition is from 10 mM to 25 mM, and the drug / lipid ratio (D / L) is from 0.01 mol / mol to 5 mol / mol.

[0022] In some embodiments, the liposome bronchodilator has an average particle size of 50 nm to 1,000 nm.

[0023] In one specific embodiment, the liposomal bronchodilator comprises an anticholinergic agent or a β2-adrenergic receptor agonist.

[0024] In some embodiments, the liposomal bronchodilator comprises a bronchodilator selected from the group consisting of tiotropium bromide, glycopyrronium bromide, umeclidinium bromide, aclidinium bromide, ipratropium bromide, oxitropium bromide, revefenacin, indacaterol, artoformoterol, formoterol, olodaterol, salbutamol, salmeterol, vilanterol, and combinations thereof.

[0025] In some embodiments, the liposomal bronchodilator includes a quaternary ammonium muscarinic antagonist.

[0026] In another aspect, the present invention also provides an atomized particulate composition of a liposomal bronchodilator for treating lung diseases, having a drug / lipid ratio of at least 0.1 mol / mol.

[0027] In another aspect, the present invention also provides an atomized particulate composition for treating lung diseases, comprising a liposomal bronchodilator, the atomized particulate composition comprising a sustained-release composition used according to the present invention.

[0028] Other objects, advantages, and novel features of the invention will become clearer from the following detailed description taken in conjunction with the accompanying drawings. Brief description of the attached diagram

[0030] Figure 1 A graph showing the in vitro release characteristics of liposomal tiotropium bromide sustained-release formulations with different compositions.

[0031] Figure 2To show the changes in body weight in three groups of mice treated with lipopolysaccharide (LPS), the mice were either infused with liposomal tiotropium bromide with different trapping agents or untreated.

[0032] Figure 3 Line graphs showing the survival rates of three groups of mice treated with LPS, which were either infused with liposomal tiotropium bromide with different trapping agents or untreated. Invention Details

[0034] definition

[0035] Unless otherwise stated, the following terms, as used above and throughout this disclosure, shall be understood to have the following meanings.

[0036] The singular forms “a,” “a,” “the,” and “this” used in this article include plural referents unless the context clearly indicates otherwise.

[0037] All figures in this document are to be understood as being modified by “about”, and when referring to measurable values ​​such as amount, duration and the like, unless otherwise stated, they mean a variation of ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1% of the specified value, as such variation is suitable for obtaining the desired amount of liposomal drug.

[0038] As used herein, the term "treating, treated, or treatment" includes preventative (e.g., prophylactic), palliative, and curative uses or outcomes. The term "subject" includes vertebrates suffering from cancer or other diseases. Preferably, the subject is a warm-blooded animal, including mammals, and preferably humans.

[0039] As used herein, the term "drug to lipid ratio" ("D / L") refers to the ratio of bronchodilator content to total phospholipid content. The bronchodilator content of free drug and / or liposomal drug was determined by ultraviolet-visible (UV-Vis) absorbance measurement. The phospholipid content or concentration of liposomes and liposomal drug was determined by measuring the phosphorus content in liposome and liposomal drug samples using a phosphorus assay (adapted from G. Rouuser et al., Lipids 1970, 5, 494-496).

[0040] Lung diseases

[0041] The lung diseases described in this invention include, but are not limited to: chronic obstructive pulmonary disease (COPD), COPD-related diseases (such as chronic bronchitis and emphysema), asthma, exercise-induced bronchospasm, cystic fibrosis, and atelectasis. Symptoms typically include chronic cough, dyspnea, chest tightness, and gradually developing shortness of breath. Complications include pulmonary hypertension, heart failure, pneumonia, or pulmonary embolism.

[0042] Liposomes

[0043] As used herein, the term "liposome" refers to a particle characterized by having an aqueous internal space isolated from an external medium by one or more vesicle-forming bilayer membranes. The bilayer membrane of a liposome is typically formed of one or more lipids, i.e., synthetic or naturally derived amphiphilic molecules containing spatially separated hydrophobic and hydrophilic domains. In some embodiments of the invention, the term "liposome" refers to a small unilamellarvesicle (SUV) in which one lipid bilayer forms a membrane.

[0044] Generally, liposomes contain a mixture of lipids, which typically include one or more lipids selected from the following: diliphatic chain lipids, such as phospholipids, diglycerides, and diliphatic glycolipids; monolipids, such as sphingomyelin and lycosphingolipids; sterols, such as cholesterol and its derivatives; and combinations thereof.

[0045] Examples of phospholipids according to the present invention include, but are not limited to: 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (DPPC). Sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soybean phosphatidylcholine (hydrogenated soybean phosphatidylcholine) Soy phosphatidylcholine (HSPC), 1,2-dimyristoyl-sn-glycero-3-phosphate-(1'-rac-glycerol)(sodium salt), DMPG, 1,2-dipalmitoyl-sn-glycero-3-phosphate-(1'-rac-glycerol)(sodium salt), DPPG, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphate-(1'-rac-glycerol)(sodium salt) (salt), PSPG), 1,2-distearyl-sn-glycerol-3-phosphate-(1'-racemic glycerol) (sodium salt) (1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol)(sodium salt), DSPG; 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol), DOPG; 1,2-dimyristoyl-sn-glycero-3-phosphate-L-serine (sodium salt), DMPS; 1,2-dipalmitoyl-sn-glycero-3-phosphate-L-serine (sodium salt), DMPS. DPPS), 1,2-distearoyl-sn-glycero-3-phosphate-L-serine (sodium salt, DSPS), 1,2-dioleoyl-sn-glycero-3-phosphate-L-serine (DOPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt, DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt, DMPA). Salt), DPPA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt), DOPA), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt), DOPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol)(ammonium salt) (DPPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol(ammonium salt) (DPPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol(ammonium salt) (e.g., DOPI), cardiolipin, L-α-phosphatidylcholine (EPC), and L-α-phosphatidylethanolamine (EPE).

[0046] Lipids modified with polyethylene glycol (PEG)

[0047] The polyethylene glycol-modified lipid comprises a polyethylene glycol moiety conjugated to the lipid. In some embodiments, the polyethylene glycol moiety has a molecular weight of about 1,000 to about 20,000 Daltons. In one specific embodiment, the polyethylene glycol-modified lipid is mixed with phospholipids to form liposomes having one or more bilayer membranes. In some embodiments, the amount of the polyethylene glycol-modified lipid, based on total phospholipids and sterols, is 0.0001 mol% to 40 mol%, optionally 0.001 mol% to 30 mol%, optionally 0.01 mol% to 20 mol%, particularly not exceeding 6 mol%, optionally 5 mol%, 3 mol%, or 2 mol%. In some embodiments, the polyethylene glycol-modified lipid has a PEG moiety with an average molecular weight of 1,000 g / mol to 5,000 g / mol. In one specific embodiment, the polyethylene glycol-modified lipid is phosphatidylethanolamine (PE-PEG) linked to a polyethylene glycol group. In a further embodiment, the polyethylene glycol-modified phosphatidylethanolamine is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)](DSPE-PEG).

[0048] Liposome bronchodilators

[0049] The terms "liposomal bronchodilator" or "liposomal drug" are used interchangeably in this disclosure. The liposomal bronchodilator according to the invention comprises liposomes encapsulating a bronchodilator, prepared by encapsulating the bronchodilator within the aqueous interior of the liposomes via a transmembrane pH gradient-driven remote loading method. In some embodiments, a transmembrane pH gradient is generated by using a trapping agent to remotely load the bronchodilator into the liposomes, and said trapping agent is composed of an ammonium compound and an anionic counterion.

[0050] The term "ammonium compound" includes unsubstituted or substituted ammonium, which is in the form of NR4. + The cation represents a hydrogen or organic residue, wherein each R is independently hydrogen or an organic residue, and said organic residue is independently alkyl, alkylene, heterocyclic alkyl, cycloalkyl, aryl, alkenyl, cycloalkenyl, or a hydroxylated derivative thereof, optionally including a sulfur, oxygen, or nitrogen atom in its hydrocarbon chain to form an ether, ester, thioether, amine, or amide bond. In one embodiment, the ammonium compound is ammonium.

[0051] The term "anionic counterion" refers to an anion or an entity covalently bonded to an anionic functional group. This anion or anionic functional group carries a negative charge in the physiological environment.

[0052] The anion or anionic functional group may be selected from one or more of the following: sulfate, citrate, sulfonate, phosphate, pyrophosphate, tartrate, succinate, maleate, borate, carboxylate, bicarbonate, glucuronide, chloride, hydroxide, nitrate, cyanate, or bromide.

[0053] In one embodiment, the anion or anionic functional group is selected from one or more of the following: citrate, sulfate, sulfonate, phosphate, pyrophosphate, or carboxylate.

[0054] In another embodiment, the entity linked to the anionic functional group can be a natural or synthetic organic or inorganic compound. Examples of such entities include, but are not limited to, non-polymeric substances selected from alkyl or aryl groups, such as benzene, nucleotides, and sugars. The alkyl group refers to a saturated hydrocarbon group having a specified number of carbon atoms. For example, the alkyl group is selected from: alkyl groups having 1 to 4 carbon atoms (C... 1-4 Alkyl groups, having 1 to 6 carbons (C 1-6 Alkyl groups, having 1 to 8 carbons (C 1-8 Alkyl groups, having 1 to 10 carbon atoms (C 1-10 Alkyl groups, having 1 to 12 carbon atoms (C 1-12 Alkyl groups, having 1 to 14 carbons (C 1-14 Alkyl groups, having 1 to 16 carbons (C 1-16 Alkyl groups, having 1 to 18 carbons (C 1-18 Alkyl groups and alkyl groups having 1 to 20 carbon atoms (C 1-20 alkyl).

[0055] In some embodiments, the anionic counterion is selected from sulfate, phosphate, citrate, and combinations thereof.

[0056] In some embodiments, the capturing agent is selected from ammonium sulfate, ammonium phosphate, ammonium citrate, dimethylammonium sulfate, dimethylammonium phosphate, dimethylammonium citrate, diethylammonium sulfate, diethylammonium phosphate, diethylammonium citrate, trimethylammonium sulfate, trimethylammonium phosphate, trimethylammonium citrate, triethylammonium sulfate, triethylammonium phosphate, triethylammonium citrate, and combinations thereof.

[0057] In some embodiments, in the sustained-release compositions of the present invention, the liposome bronchodilator has an average particle size of 50 nm to 1,000 nm. Non-limiting examples of liposomes have average particle sizes of 50 nm to 20 μm, 50 nm to 10 μm, 50 nm to 1000 nm, 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 250 nm, or 50 nm to 200 nm.

[0058] The term "bronchodilator" refers to a substance that dilates the bronchi or bronchioles, thereby increasing airflow into the lungs.

[0059] In some implementations, bronchodilators refer to anticholinergic agents and β2-adrenergic receptor agonists. Among bronchodilators, quaternary ammonium muscarinic antagonists, including tiotropium bromide, glycopyrronium bromide, roxithromycin bromide, adecyl bromide, ipratropium bromide, and oxaprium bromide, are commonly used anticholinergic agents. They bind to muscarinic receptors on airway smooth muscle and block cholinergic contractile action. Because quaternary ammonium muscarinic antagonists are completely ionized, they are difficult to absorb into the bloodstream and cannot cross the blood-brain barrier, thus limiting their anticholinergic effect to the site of delivery and avoiding systemic adverse reactions.

[0060] In some embodiments, the bronchodilator according to the invention is a β2-adrenergic receptor agonist selected from the following: indacaterol, afortrol, formoterol, olodaterol, salbutamol, salmeterol, and vilanterol.

[0061] In one aspect, the liposome bronchodilator comprises:

[0062] A lipid bilayer comprising: one or more phospholipids, sterols, and optionally, polyethylene glycol (PEG)-modified phospholipids, particularly PEG-modified phosphatidylethanolamine (PEG-PE); and

[0063] The aqueous interior, which is covered by the lipid bilayer, contains one or more bronchodilators.

[0064] In one embodiment, the one or more phospholipids are neutral phospholipids, and the polyethylene glycol (PEG) modified phospholipid is DSPE-PEG. The amount of DSPE-PEG, based on total phospholipids and sterols, is from 0.001 mol% to 5 mol%.

[0065] Atomized particles of the sustained-release composition

[0066] The sustained-release composition according to the invention is suitable for preparing atomized particulate compositions. In one embodiment, the liposomal bronchodilator comprises: a lipid bilayer containing phospholipids, sterols, and PEG-modified phosphatidylethanolamine; and an aqueous interior encapsulated by the lipid bilayer containing the bronchodilator, wherein less than 10% of the liposomal bronchodilator leaks from the liposomes after atomization.

[0067] In one embodiment, the sustained-release composition of the bronchodilator used according to the invention has a lipid concentration of 1 mM to 25 mM. In one embodiment, the bronchodilator concentration of the sustained-release composition is 0.1 mg / mL to 30 mg / mL, 0.5 mg / mL to 20 mg / mL, 1 mg / mL to 15 mg / mL, and 2 mg / mL to 10 mg / mL. In one embodiment, the drug / phospholipid ratio of the sustained-release composition of the bronchodilator used according to the invention is at least 0.1 mol / mol, preferably 0.05 mol / mol to 1 mol / mol, optionally 0.1 mol / mol to 0.7 mol / mol, optionally 0.15 mol / mol to 0.6 mol / mol, and optionally 0.15 mol / mol to 0.2 mol / mol. In some embodiments, the amount of free bronchodilator in the sustained-release composition is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, based on the total amount of bronchodilator in the sustained-release composition.

[0068] In some embodiments, the atomized particulate composition of the sustained-release composition of this disclosure is generated by an atomizer selected from air-jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers.

[0069] In some embodiments, the mass median aerodynamic diameter of the atomized particulate composition is 0.5 μm to 5 μm, and optionally 1 μm to 3 μm.

[0070] In one specific embodiment, the nebulized particulate composition is delivered via the lungs to an individual in need, exhibiting a release rate of approximately 0.5% to 25% per hour based on the administered drug dose, and complete release of the bronchodilator after a minimum of approximately 12 to 24 hours.

[0071] This disclosure is further described with reference to the following specific, non-limiting embodiments. Example

[0072] The following examples illustrate the preparation and properties of certain embodiments of the present invention.

[0073] Example 1: Stability of liposome bronchodilators

[0074] Preparation of liposomal tiotropium bromide

[0075] I. Preparation of empty liposomes

[0076] Liposomes are prepared using either thin-film hydration or solvent injection methods. The method for preparing empty liposomes via thin-film hydration specifically includes the following steps:

[0077] 1. Weigh the lipid mixture containing phospholipids and cholesterol, including DSPE-PEG2000, to the predetermined molar ratio, and add them to 10 mL of chloroform in a round-bottom flask;

[0078] 2. Place the flask in a rotary evaporator at 60°C, stir the flask to dissolve the lipid mixture, and then place the flask under vacuum while stirring to evaporate the chloroform, thereby obtaining a dry lipid membrane;

[0079] 3. A trapping agent solution (e.g., ammonium sulfate (AS)) is prepared by adding the trapping agent to distilled water and vortexing the solution to dissolve the powder;

[0080] 4. Add the capture agent solution to the dried lipid membrane and stir at 60°C for 30 minutes to form a proliposome solution;

[0081] 5. The precursor liposome solution was freeze-thawed five times using liquid nitrogen and a 60°C water bath to obtain liposome samples;

[0082] 6. The obtained liposome sample was extruded 10 times through a 0.2 μm polycarbonate membrane at 60 °C, and then extruded 10 times through a 0.1 μm polycarbonate membrane.

[0083] 7. Dialyze the extruded liposome sample to remove free trapping agent, then add the sample to a dialysis bag (molecular weight cutoff (MWCO): 25 kDa), seal the bag, and stir the dialysis bag in 100 times its volume of 9.4% (w / v) sucrose solution; further replace the sucrose solution after 1 hour and 4 hours, and stir overnight; and

[0084] 8. The dialyzed liposome sample was sterilized by filtration through a 0.45 μm polytetrafluoroethylene (PTFE) membrane to obtain empty liposomes.

[0085] II. Loading tiotropium bromide into liposomes to obtain liposomal tiotropium bromide.

[0086] The following method represents a typical protocol for encapsulating tiotropium bromide in liposomes via remote loading, comprising the following steps:

[0087] 1. Prepare a 9.4% (w / v) sucrose solution and a 9.4% (w / v) sucrose buffer solution containing 200 mM L-histidine (L-His) at pH 7.68 to maintain the pH under loading conditions;

[0088] 2. Prepare a 10 mg / mL tiotropium bromide solution in 9.4% (w / v) sucrose and briefly heat the solution at 60 °C to obtain a stock solution containing tiotropium bromide (hereinafter referred to as Tio stock solution);

[0089] 3. In a conical tube, empty liposomes prepared by the method in Section I of Example 1 [in a typical embodiment, having the following conditions: HSPC:cholesterol:DSPE-PEG2000 in a molar ratio of 3:2:0.045, 300 mM ammonium sulfate (AS) and 20 mM lipid concentration], 9.4% (w / v) sucrose solution, 9.4% (w / v) sucrose buffer at pH 7.68, ethanol (finally 10% by volume in the mixture) and TiO2 stock solution were mixed to obtain a loading solution with a D / L ratio of 100 g / mol;

[0090] 4. Shake the loading solution continuously at 60°C for 60 minutes to form a liposomal drug sample, then place the liposomal drug sample on ice for a few minutes;

[0091] 5. The loading solution was diluted 10-fold in a 9.4% sucrose solution containing 40 mM L-His, and the diluted sample was then loaded into an Amicon Ultra-15 centrifuge filter (molecular weight cutoff (MWCO): 100 kD). The sample was centrifuged at 3,800 g for 80 minutes to remove ethanol and free drug. Finally, the concentration of tiotropium bromide was adjusted to 1 mg / mL with a 9.4% sucrose solution containing 40 mM L-His.

[0092] 6. Size-exclusion column chromatography and HPLC were used to determine the drug encapsulation (i.e., loading efficiency) of the final sample (the drug concentration of all samples in liposome or all forms was determined by absorbance measurement at a wavelength of 237 nm).

[0093] B. The effects of different trapping agents on drug loading characteristics

[0094] Different trapping agents were used to coat bronchodilators, including anticholinergics and β2-adrenergic receptor agonists. Liposome drug formulations were prepared according to Section A above (using the same lipid bilayer composition, with a molar ratio of HSPC:cholesterol:DSPE-PEG2000 of 3:2:0.045), except that the following trapping agents were used: (1) 75 mM triethylammonium sucrose octasulfate (TEA-SOS) and (2) 300 mM ammonium sulfate. The results of the drug loading experiments are shown in Table 1.

[0095] Table 1. Drug loading characteristics of different trapping agents

[0096]

[0097]

[0098] C. Storage stability of liposomal drugs

[0099] The stability of liposomal tiotropium bromide stored at 4°C was monitored for at least two months. Tiotropium bromide was loaded into empty liposomes using 75 mM sucrose octaethylammonium sulfate (TEA-SOS) as trapping agent 1 to obtain liposomal drug samples. After storing the liposomal drug samples at 4°C for more than two months, no drug leakage from the liposomes was observed. Regarding the average particle size, the liposomal drug composed of HSPC:cholesterol:DSPE-PEG2000 in a molar ratio of 3:2:0.045 (0.9 mol% PEG) maintained approximately 140 nm over time. In terms of encapsulation stability, the D / L ratio of the liposomal drug maintained approximately 0.19 (mol / mol) over time. For the encapsulation stability of the liposomal drug composed of HSPC:cholesterol in a 3:2 ratio, its D / L ratio maintained approximately 0.14 (mol / mol).

[0100] Example 2: Release characteristics of liposome bronchodilators

[0101] A. Preparation of liposomal tiotropium bromide

[0102] I. Preparation of empty liposomes

[0103] Liposomes are prepared using either thin-film hydration or solvent injection methods. The method for preparing empty liposomes via solvent injection specifically includes the following steps:

[0104] 1. Weigh the lipid mixture of phospholipids, cholesterol and DSPE-PEG2000 in a predetermined molar ratio (details of the composition are shown in Table 2), add them to ethanol placed in a glass test tube, and dissolve the lipid mixture at 60°C;

[0105] 2. Preheat the specified capture agent solution ((1) 75 mM sucrose octaethylammonium sulfate, (2) 300 mM ammonium sulfate) at 60 °C for at least 30 minutes;

[0106] 3. With stirring, add the dissolved lipid mixture to the preheated capture agent solution using a syringe to form a precursor liposome sample, and then continue stirring the precursor liposome sample at 60°C for 5 minutes.

[0107] 4. The precursor liposome sample was extruded through a 0.2 μm polycarbonate membrane at 60 °C, and then extruded through a 0.1 μm polycarbonate membrane at 60 °C to obtain the liposome sample;

[0108] 5. Using a dialysis bag (MWCO: 25 kDa) and 9.4% (w / v) sucrose (100 times the volume of the liposome sample as the dialysis solution), dialyze the extruded liposome sample to remove the external medium from the liposomes; replace the sucrose solution twice between 4-hour and 8-hour stirring intervals; and

[0109] 6. The dialyzed liposome sample was sterilized by filtration through a 0.2 μm polytetrafluoroethylene (PTFE) membrane to obtain empty liposomes.

[0110] II. Loading tiotropium bromide into liposomes to obtain liposomal tiotropium bromide.

[0111] The following method represents an exemplary scheme for encapsulating tiotropium bromide in liposomes via remote loading, comprising the following steps:

[0112] 1. Prepare a 9.4% (w / v) sucrose solution and a 9.4% (w / v) sucrose buffer solution at pH 7.68 containing 200 mM L-histidine (L-His);

[0113] 2. Prepare a 10 mg / mL tiotropium bromide solution in 9.4% (w / v) sucrose;

[0114] 3. In a conical tube, empty liposomes prepared by the aforementioned method (in a typical embodiment, under the following conditions: HSPC:cholesterol, 300 mM ammonium sulfate (AS), and 20 mM lipid concentration in a molar ratio of 3:2), 9.4% (w / v) sucrose solution, 9.4% (w / v) sucrose buffer at pH 7.68 with a final L-His concentration of 40 mM, ethanol (final volume in the mixture is 10%), and TiO2 stock solution are mixed to obtain a loading solution with a D / L ratio of 100 g / mol; and

[0115] 4. Shake the loading solution continuously at 60°C for 60 minutes to form a liposomal drug sample, then place the liposomal drug sample on ice for a few minutes.

[0116] III. Optionally, DSPE-mPEG can be post-inserted onto the liposome tiotropium bromide.

[0117] 1. Prepare 11.25 mM DSPE-mPEG stock solution in 9.4% (w / v) sucrose;

[0118] 2. Mix the DSPE-mPEG stock solution with liposomal tiotropium bromide (obtained from Section II) at a predetermined total lipid concentration of 3%; and

[0119] 3. Shake the liposome solution containing DSPE-mPEG continuously at 60°C for 5 minutes, then place the liposome drug sample on ice for a few minutes.

[0120] IV. Removal of free drug and ethanol

[0121] 1. Dilute the liposomal drug sample (with or without DSPE-mPEG) 10-fold in a centrifuge tube (Amicon Ultra-15 centrifuge filter (MWCO: 100kD)) with a 9.4% sucrose solution containing 40 mM L-His, and centrifuge the sample at 3,800 g for 80 minutes to remove ethanol and free drug. Finally, adjust the concentration of tiotropium bromide to 1 mg / mL with a 9.4% sucrose solution containing 40 mM L-His; and

[0122] 2. The drug encapsulation (i.e., loading efficiency) of the final samples was determined using a UV-Vis plate reader (the drug concentration of all samples, in liposome form or in all forms, was determined by absorbance measurement at a wavelength of 237 nm). The results are shown in Table 2 below.

[0123] Table 2. Drug loading characteristics of different trapping agents

[0124]

[0125] B. In vitro drug release in simulated lung fluid

[0126] Release characteristics of liposomal tiotropium bromide with and without PEG content were demonstrated in simulated lung fluid (SLF) to prove their sustained-release properties. Test samples (prepared liposomal tiotropium bromide samples) were prepared with approximately equal amounts of tiotropium bromide (1 mg / mL drug), each containing only a small amount of free drug (0.01% to 10% of total drug content). The in vitro release (IVR) assay protocol is as follows:

[0127] 1. The test sample was diluted 10-fold by mixing 0.5 mL of each liposomal tiotropium bromide sample with 4.5 mL of SLF (preheated at 37°C), and the diluted sample was placed in a 15 mL centrifuge tube.

[0128] 2. Place the centrifuge tube containing the diluted sample onto the sample well of the Inteli-mixer, rotate at 20 rpm, and incubate at 37°C; and

[0129] 3. Take 1 mL of diluted sample at predetermined time points (0, 4 and 24 hours) to analyze the coating efficiency.

[0130] The analytical method for determining the coating efficiency of tiotropium bromide is as follows:

[0131] a. Fill and wash a 2 mL G50 column with 9.4% sucrose solution (less than 5 mL);

[0132] b. Add 0.2 mL of sample to the column, then add 0.15 mL of 9.4% sucrose solution, repeating this process three times, and wait for the solution to elute from the column.

[0133] c. Add 0.7 mL of 9.4% sucrose solution to the column and collect the eluent (as liposomes) in a 1.5 mL microcentrifuge (Eppendorf) tube. Then transfer 0.24 mL of the eluent and mix it with 0.96 mL of methanol.

[0134] d. Place 0.2 mL of the unpurified sample in another 1.5 mL microcentrifuge tube, add 0.5 mL of 9.4% sucrose solution and mix thoroughly, then transfer 0.24 mL of this solution and mix with 0.96 mL of methanol (as total).

[0135] e. Centrifuge the pre- and post-column samples (liposome form and all forms) at 20,600g for 10 minutes; and

[0136] f. The absorbance of the final supernatant of the samples was measured at a wavelength of 237 nm using a UV-Vis plate reader to determine the drug concentration of each sample.

[0137] The encapsulation efficiency (EE) of liposomal bronchodilators is calculated using the following formula: liposomal form of the drug (LF) divided by the total form of the drug (TF):

[0138] EE (%) = LF / TF * 100%.

[0139] Release characteristics are drawn as Figure 1 The graph illustrates the relationship between release rate (%) and time. The release rate is calculated using the following formula: initial liposome form minus the liposome form at each time point, then divided by the initial liposome form:

[0140] (LF t0 -TF t ) / LF t0 *100%.

[0141] Liposome tiotropium bromide with either trapping agent 1 or 2 exhibited slow release characteristics in simulated lung fluid. Liposome tiotropium bromide with trapping agent 1 and 0.9% and 3% PEG showed stable and slow release characteristics, releasing up to 50% of the initial drug content in the SLF after 24 hours. On the other hand, compared to liposomal tiotropium bromide containing 3% PEG-DSPE, liposomal tiotropium bromide containing trapping agent 2 and containing no or only a small amount (0.9%) of PEG showed slower release characteristics (slow drug release, releasing only up to 30% of the initial drug content in the SLF after 24 hours). Prolonged release characteristics of the drug substance are desired for improved efficacy and treatment with low dosing frequency. Therefore, we selected two liposomal tiotropium bromide formulations with slower release characteristics from all formulations and used them for the following toxicity studies. The two selected formulations (A and C in Table 2) had low or zero DSPE-mPEG content, respectively, and used two different trapping agents.

[0142] Example 3: Toxicity assessment of inhaled liposomal tiotropium bromide in an LPS-induced acute COPD animal model

[0143] The toxicity of two liposomal tiotropium bromide formulations was investigated in mice with lipopolysaccharide (LPS)-induced acute COPD. The experimental design is described in Table 3. In short, 12 mice were divided into 3 groups (N=4). The untreated control group only simulated symptoms, and the mice in the control group did not receive any treatment.

[0144] Each composition is described below:

[0145] Group 1: Liposome tiotropium bromide formulation of Example 2: Liposomes of tiotropium bromide loaded with 300 mM ammonium sulfate as a trapping agent. This formulation contains 20 mM lipid concentration and 1 mg / mL tiotropium bromide concentration;

[0146] Group 2: Liposome tiotropium bromide formulation of Example 2: Liposomes loaded with tiotropium bromide using 75 mM TEA-SOS as a capture agent. This formulation contains 20 mM lipid concentration and 1 mg / mL tiotropium bromide concentration;

[0147] Group 3: Untreated control group, with LPS induction only.

[0148] An acute COPD animal model was established by intratracheal (IT) infusion of LPS at 1 mg / kg on day 0. Twenty-four hours later, 25 μL of liposomal tiotropium bromide with different trapping agents (Groups 1 and 2, N=4) were infused intratracheally into LPS-treated mice to assess toxicity compared to untreated controls (Group 3, N=4). Dosing regimens are shown in Table 3. Both formulations were administered once at a dose of 1 mg / kg. Body weight and survival status were recorded during the study period.

[0149] Table 3 Study design for liposomal tiotropium bromide toxicity in acute COPD animal models

[0150] Group number Composition Animal number Dosage frequency dose 1 AS-Tio 1.06 mg / mL 4 Single injection 1mg / kg 2 TEA-Tio 1.07 mg / mL 4 Single injection 1mg / kg 3 Untreated control group 4 N / A N / A

[0151] Toxicity is determined by the animal's body weight and survival status at the experimental endpoint. (Reference) Figure 2 As stated, compared with group 3 (control group), a weight loss of more than 20% was observed in both groups 1 and 2. Figure 3 As illustrated, the survival rate in group 2 (25%) was lower than that in group 1 (75%), indicating that the toxicity was less and the treatment was more tolerable when ammonium sulfate was used as a trapping agent. Therefore, in the treatment of COPD, although an equal total amount of tiotropium was administered via intratracheal infusion, the liposomal tiotropium formulation containing ammonium sulfate showed reduced side effects compared to the TEA-SOS formulation group.

Claims

1. A sustained-release composition of a bronchodilator for treating lung diseases, comprising a liposomal bronchodilator, wherein the liposomal bronchodilator comprises: A lipid bilayer comprising: one or more phospholipids and sterols, wherein the molar ratio of total phospholipids to sterols in the lipid bilayer is 1:1 to 3:2; and The aqueous interior, encapsulated by the lipid bilayer, contains a bronchodilator encapsulated in the liposome via remote loading using a trapping agent; The bronchodilator mentioned above is selected from tiotropium bromide, glycopyrronium bromide, rodiflubenzuron bromide, adecyl bromide, and oxytropium bromide. The capturing agent is selected from ammonium sulfate, ammonium phosphate, ammonium citrate, dimethyl ammonium sulfate, dimethyl ammonium phosphate, dimethyl ammonium citrate, diethyl ammonium sulfate, diethyl ammonium phosphate, diethyl ammonium citrate, trimethyl ammonium sulfate, trimethyl ammonium phosphate, trimethyl ammonium citrate, triethyl ammonium sulfate, triethyl ammonium phosphate, triethyl ammonium phosphate, triethyl ammonium citrate, and combinations thereof. The sustained-release composition of the bronchodilator has a drug / phospholipid ratio of 0.01 mol / mol to 1 mol / mol.

2. The sustained-release composition of the bronchodilator according to claim 1, wherein the lung disease is chronic obstructive pulmonary disease (COPD).

3. The sustained-release composition of the bronchodilator according to claim 1, wherein the lung disease is a COPD-related disease or symptom.

4. The sustained-release composition of the bronchodilator according to claim 1, wherein the lung disease is a complication of COPD.

5. The sustained-release composition of the bronchodilator according to claim 1, wherein the sterol is cholesterol.

6. The sustained-release composition of the bronchodilator according to claim 1, wherein the one or more phospholipids are selected from hydrogenated soybean phosphatidylcholine (HSPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), and combinations thereof.

7. The sustained-release composition of the bronchodilator according to claim 1, wherein the lipid bilayer comprises polyethylene glycol (PEG) modified lipids, and the amount of the PEG modified lipids is from 0.0001 mol% to 40 mol% based on total phospholipids and sterols.

8. The sustained-release composition of the bronchodilator according to claim 7, wherein the PEG-modified lipid has a PEG portion having an average molecular weight of 1,000 g / mol to 5,000 g / mol.

9. The sustained-release composition of the bronchodilator according to claim 7, wherein the PEG-modified lipid is 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG).

10. The sustained-release composition of the bronchodilator according to claim 9, wherein the one or more phospholipids are neutral phospholipids, and the amount of DSPE-PEG in the liposomes is from 0.001 mol% to 5 mol% based on total phospholipids and sterols.

11. The sustained-release composition of the bronchodilator according to claim 1, wherein the liposome bronchodilator has an average particle size of 50 nm to 1,000 nm.

12. The sustained-release composition of the bronchodilator according to any one of claims 1 to 11, having a lipid concentration of 1 mM to 25 mM.

13. The sustained-release composition of the bronchodilator according to any one of claims 1 to 11, having a bronchodilator concentration of 1 mg / mL to 15 mg / mL.

14. The sustained-release composition of the bronchodilator according to claim 1, wherein the drug / phospholipid ratio is from 0.1 mol / mol to 0.7 mol / mol.

15. The sustained-release composition of the bronchodilator according to claim 14, wherein the drug / phospholipid ratio is from 0.15 mol / mol to 0.6 mol / mol.

16. The sustained-release composition of the bronchodilator according to claim 15, wherein the drug / phospholipid ratio is from 0.15 mol / mol to 0.2 mol / mol.

17. An atomized particle composition for treating lung diseases, comprising a liposomal bronchodilator, said atomized particle composition comprising a plurality of particles of a sustained-release composition of a bronchodilator for treating lung diseases as described in any one of claims 1 to 16.

18. The atomized particulate composition according to claim 17, wherein the median aerodynamic diameter of the plurality of particles is 0.5 μm to 5 μm.

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