A nanoliposome suspension for soft fogging and its preparation method

Abstract

A nanoliposome suspension for soft aerosol spray and its preparation method are disclosed. The active ingredient of the nanoliposome suspension is selected from the following combinations: inhaled glucocorticoids, long / short-acting β2 receptor agonists and long / short-acting anticholinergic drugs (LAMA / SAMA), inhaled glucocorticoids, long / short-acting β2 receptor agonists and long / short-acting anticholinergic drugs, inhaled glucocorticoids and long / short-acting β2 receptor agonists, inhaled glucocorticoids and long / short-acting anticholinergic drugs; the active ingredient is encapsulated in nanoliposomes, which are prepared using cholesterol and phospholipids.

Description

Technical Field

[0001] This invention relates to an inhaled pharmaceutical composition. Background Technology

[0002] Inhaled drug products (IDPs) are preparations that deliver medications to the respiratory tract and / or lungs via inhalation to exert local or systemic effects. They are primarily used for respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). COPD is a heterogeneous lung disease and the most serious chronic respiratory disease in China. It is characterized by airflow limitation due to airway narrowing and / or obstruction, loss of lung elastic recoil, or both, affecting the central and peripheral airways, lung parenchyma and alveoli, and causing chronic inflammation of the pulmonary vessels. COPD patients are prone to respiratory infections, pulmonary hypertension, lung infections, weight loss, and other complications, which can accelerate the process of lung tissue destruction. Asthma is a common chronic lung disease affecting millions of people worldwide. It is characterized by recurrent episodes of wheezing, coughing, shortness of breath, and chest tightness. Besides asthma and COPD, other respiratory diseases can also occur, such as bronchiectasis, pneumonia, or interstitial lung disease.

[0003] Inhaled corticosteroids (ICS) have a strong local anti-inflammatory effect, acting directly on the respiratory tract. Regular use of ICS can improve symptoms, lung function, and quality of life. Compared to oral medication, ICS has fewer systemic adverse reactions. Bronchodilators relax bronchial smooth muscle, dilate bronchi, and relieve airflow limitation, thereby alleviating COPD symptoms. They are the cornerstone of COPD treatment. The main bronchodilators include SABA / LABA (short / long-acting β2 receptor agonists), SAMA / LAMA (short / long-acting anticholinergic drugs), and theophylline, which can be selected based on the drug's effects and the patient's treatment response. For asthma treatment, initial treatment usually begins with short-acting inhaled medications, with low to moderate daily doses of ICS added as needed for better asthma control. Subsequently, if necessary, a bronchodilator called a LABA is usually added to the inhaled corticosteroid, which causes airway dilation and relaxation, thereby reducing dyspnea. The guidelines recommend that when asthma is not controlled with a combination of moderate-dose inhaled ICS and LABA, a high-dose ICS or the addition of another bronchodilator called LAMA (i.e., triple inhaled therapy) should be used.

[0004] Based on the patient's clinical characteristics, such as symptoms, frequency and severity of acute exacerbations, inhaled medications can be classified as: single bronchodilators (single bronchodilators), such as LAMA; dual bronchodilators (dual bronchodilators), such as LABA+LAMA; and triple therapy of LABA+LAMA+ICS.

[0005] BudiGelfide Inhalation Aerosol It is an innovative triple inhaled drug developed by AstraZeneca for the treatment of COPD. It delivers three drug components in combination: budesonide (inhaled corticosteroid ICS), glycopyrronium bromide (long-acting anticholinergic drug LAMA), and formoterol fumarate (long-acting β2 receptor agonist LABA). It provides an important treatment option for patients with stable COPD. It was first approved in Europe and the United States and was approved for marketing in China in May 2020.

[0006] Fluticasone Propionate Inhalation Powder, November 2019 Approved for marketing in China, this is GSK's first triple therapy drug for COPD approved in China. It is suitable for the treatment of stable COPD and is the world's first triple therapy drug for COPD that can be administered once a day. The overall safety profile of this triple therapy is good, and its safety data is similar to those of ICS / LABA and LAMA / LABA drugs.

[0007] Inhalable nanoliposomes show promising applications in respiratory diseases. They overcome the pharmacokinetic limitations of traditional drug formulations, no longer constrained by limited drug dispersion and rapid degradation. They can directly penetrate the alveolar membrane and act on the lesion site, effectively avoiding the clearance effects of blood circulation and the first-pass clearance effect of the liver, thus achieving continuous treatment of the lesion site. Inhaled nanoliposomes are closed vesicles with a bilayer structure composed of amphiphilic molecules. The internal hydrophilic core effectively encapsulates hydrophilic / lipophilic molecules, improving drug stability. Simultaneously, the structural characteristics of liposomes are similar to cell membranes, enabling drug delivery into cells through membrane fusion or endocytosis, thereby improving drug membrane permeability. They are typically composed of amphiphilic phospholipids and cholesterol; the amphiphilic phospholipids form the bilayer structure, while cholesterol supports and maintains it. Liposomes exhibit good biocompatibility with the alveoli and are unlikely to induce toxicity or immune responses after administration.

[0008] Pulmonary surfactant (PS) is distributed on the surface of alveoli and is a complex mixture composed of 90% lipids and 10% proteins. It reduces alveolar surface tension, maintains the relative stability of alveolar and alveolar volume, prevents fluid from filtering out of alveolar capillaries, and provides the structural and functional basis for gas exchange in the lungs. It also possesses innate immune function, helping to control inflammation and prevent lung infections. Studies have shown that DPPC (dispalmitoylphosphatidylcholine), the most abundant phospholipid, is a saturated phospholipid, accounting for more than 60% of PS. POPG-NA (phosphatidylglycerol), the second most abundant lipid, is an innate immunomodulatory factor in the lungs, inhibiting the activation of TLR2 and TLR4 in pro-inflammatory Toll-like receptors (TLRs) and exhibiting antiviral properties.

[0009] Compared to pressure-controlled metered-dose inhalers (pMDI), soft mist inhalers (SMI) offer superior delivery performance. Developed by Boehringer Ingelheim, the SMI device is a groundbreaking new-generation propellant-free inhaler, a pioneering innovation in inhalation therapy. Powered by the mechanical energy generated by a compressed spring in the base, it forms and releases a drug aerosol. Employing precise capillary dosing, it delivers a fixed dose of medication through fine droplets. Its unique design causes two drug jets to collide at a specific angle, creating a "soft mist." Achieving a slower operating speed of approximately 0.8 m / s and a slower aerosol spray (approximately 1.5 seconds), it reduces oropharyngeal deposition and achieves higher pulmonary deposition, ensuring effective drug delivery even for patients with poor inhalation technique. Patents CN106132466A and CN11789942A disclose the structure and principles of the SMI device, with a delivery volume of approximately 15 μL and a droplet size of approximately 3–10 μm. Currently, the FDA has approved four SMI products using the Respimat device: Combivent Respimat, Striverdi Respimat, Stiolto Respimat, and Spiriva Respimat. All use sterile solutions, packaged in 4.5mL plastic vials. While SMI devices offer significant advantages in drug delivery, many ICS and LABA drugs used to treat respiratory diseases are poorly soluble, and their water solubility is insufficient to achieve therapeutic doses. Therefore, combining SMI with nanoliposome technology improves the bioavailability of poorly soluble drugs and enables targeted lung therapy.

[0010] Existing patent publications report on inhaled nanoliposomes in powder inhalers, inhalation solutions, and aerosols, but there is no information on nanoliposomes for soft inhalers. Among them, CN117510570A discloses a modification of budesonide to budesonide-21-succinate to improve its water solubility for use in soft inhalers. CN118634207A discloses a soft inhaler patent for nucleic acid lipid nanoparticles, which are solid lipid nanoparticles formed by complexing nucleic acid drugs (negatively charged) with cationic lipids, administered via a soft inhaler device. Because triple-combination inhaled formulations require coordinating the release characteristics of various active ingredients, it is even more difficult to modify existing aerosol and powder inhaler formulations into soft inhalers based on nanoliposome formulations.

[0011] Given the shortcomings of existing technologies, the main problem that needs to be solved in the existing technologies is to provide a compound inhalation drug based on nanoliposomes suitable for soft inhalers using liposome technology, thereby improving the solubility of poorly soluble drugs in compound inhalation formulations and thus achieving direct and coordinated drug delivery. Summary of the Invention

[0012] To solve the aforementioned problem, the technical solution adopted by the present invention is as follows:

[0013] A nanoliposome suspension for soft inhaler is provided, wherein the active ingredient of the nanoliposome suspension is selected from the following combinations: inhaled corticosteroids (ICS), long / short-acting β2 receptor agonists (LABA / SABA), and long / short-acting anticholinergic drugs (LAMA / SAMA); inhaled corticosteroids, long / short-acting β2 receptor agonists and long / short-acting anticholinergic drugs, inhaled corticosteroids and long / short-acting β2 receptor agonists, inhaled corticosteroids and long / short-acting anticholinergic drugs, preferably inhaled corticosteroids (ICS), long / short-acting β2 receptor agonists (LABA / SABA), and long / short-acting anticholinergic drugs (LAMA / SAMA); the active ingredient is encapsulated in nanoliposomes. The nanoliposomes are prepared using cholesterol and phospholipids, wherein the phospholipids are selected from one or more of the following: dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG-NA), dimyristoylphosphatidylcholine (DMPC), disearylphosphatidylcholine (DEPC), distearylphosphatidylcholine (DSPC), dioleoyllecithin (DOPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), egg yolk phosphatidylglycerol (EPG), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), and phosphatidylethanolamine (DSPE-mPEG2000). The aforementioned nanoliposome suspension for soft mist further includes a stabilizer selected from one of Tween 80, poloxamer 407, L-ascorbyl palmitate, vitamin E polyethylene glycol succinate (TPGS), and polyvinylpyrrolidone (PVP 30), preferably vitamin E polyethylene glycol succinate. The mass ratio of the stabilizer to the active ingredient is 1:50-70.

[0014] The aforementioned nanoliposome suspension for soft mist further includes an osmotic pressure regulator, wherein the osmotic pressure regulator is glucose or sodium chloride; the molar osmotic concentration of the nanoliposome suspension is 240-360 mOsm / kg, preferably 270-320 mOsm / kg; the pH value is 5.0-8.0, preferably 6.0-7.5.

[0015] The aforementioned nanoliposome suspension for soft inhaler further preferably contains inhaled corticosteroids (ICS), long / short-acting β2 receptor agonists (LABA / SABA), and long / short-acting anticholinergic drugs (LAMA / SAMA), wherein the ICS content is 5–20 mg / mL; the LABA / SABA content is 0.1–1 mg / mL; and the LAMA / SAMA content is 0.1–1 mg / mL. The inhaled corticosteroids are selected from budesonide, cicosone, fluticasone propionate, fluticasone furoate, beclomethasone propionate, and mometasone furoate, with budesonide being preferred; the long / short-acting β2 receptor agonists are selected from formoterol fumarate, indacaterol, salmeterol, aterol, olodaterol, and salbutamol, with formoterol fumarate being preferred; the long / short-acting anticholinergic drugs are selected from glycopyrronium bromide, umemetronium bromide, tiotropium bromide, and ipratropium bromide, with glycopyrronium bromide being preferred. The method for preparing the nanoliposome suspension is as follows: first, prepare nanoliposome suspensions containing inhaled corticosteroids (ICS) and long / short-acting anticholinergic drugs; then, add long / short-acting β2 receptor agonists to the nanoliposome suspensions containing the two active ingredients; incubate at 35℃~40℃ for 40min~80min; and concentrate the drug solution to a suitable volume using tangential flow technology. The prepared nanoliposomes have a particle size of 50~300nm; the drug-liposome ratio is 1:1~6, preferably 1:3.

[0016] The preparation method of the nanoliposome suspension of the inhaled corticosteroid (ICS) and long / short-acting anticholinergic drugs can employ a thin-film dispersion-pH gradient method, specifically...

[0017] (1) Phospholipids, cholesterol, stabilizers, and inhaled glucocorticoids were dispersed and dissolved in an organic solvent to obtain a lipid organic phase solution; the organic phase was evaporated to remove the organic solvent, and a drug-containing phospholipid film was obtained.

[0018] (2) Dissolve long-acting / short-acting anticholinergic drugs in an aqueous solution with pH adjusted to 3.8-4.5 using a citrate-sodium citrate-NaOH buffer solution, add an osmotic pressure regulator, and stir until completely dissolved to form the aqueous phase;

[0019] (3) The aqueous phase was poured into the drug-containing phospholipid film for hydration and dispersion. The resulting mixed solution was homogenized and dispersed using a microfluidic homogenizer at a homogenization pressure of 10,000 to 25,000 psi and for 1 to 5 homogenization cycles. The external aqueous phase was replaced with a phosphate buffer solution of pH 7.3 to 7.6 to obtain an ICS+LAMA nanoliposome suspension.

[0020] The preparation method of the nanoliposome suspension of the inhaled corticosteroid (ICS) and long / short-acting anticholinergic drugs can also employ a thin-film dispersion-ammonium sulfate gradient method, specifically as follows:

[0021] (1) Phospholipids, cholesterol, stabilizers, and inhaled glucocorticoids are dispersed and dissolved in an organic solvent to obtain a lipid organic phase solution; the organic phase is evaporated to remove the organic solvent, and a drug-containing phospholipid film is obtained;

[0022] (2) Dissolve long / short-acting anticholinergic drugs in an aqueous solution containing ammonium sulfate, add an osmotic pressure regulator, and stir until completely dissolved to form the aqueous phase;

[0023] (3) The aqueous phase is poured into the drug-containing phospholipid film for hydration and dispersion. The resulting mixed solution is homogenized and dispersed using a microfluidic homogenizer at a homogenization pressure of 10,000 to 25,000 psi and homogenization times of 1 to 5. The ammonium sulfate solution of the original liposome in vitro phase is replaced by an external aqueous phase solution with a pH of 7.3 to 7.6 using column chromatography to obtain a nanoliposome suspension of inhaled corticosteroids (ICS) and long / short-acting anticholinergic drugs.

[0024] The preparation method of the nanoliposome suspensions of inhaled corticosteroids (ICS) and long / short-acting anticholinergic drugs can also employ the reverse evaporation-ammonium sulfate gradient method, specifically...

[0025] 1) Phospholipids, cholesterol, stabilizers, and inhaled glucocorticoids were dispersed and dissolved in an organic solvent to obtain a lipid organic phase solution;

[0026] 2) Dissolve long-acting / short-acting anticholinergic drugs in an aqueous ammonium sulfate solution as the aqueous phase. Slowly add the aqueous phase to the lipid organic phase solution and sonicate under a water bath at 35℃~40℃ to form a stable W / O emulsion. Then, evaporate under reduced pressure to remove the solvent and form a viscous gel state. Add an aqueous ammonium sulfate solution containing an osmotic pressure regulator and continue to concentrate under reduced pressure to remove the solvent until the system is fully hydrated and collapses from the gel state to become a liposome suspension. Then, homogenize and disperse the suspension using a microfluidic homogenizer at a homogenization pressure of 10000~25000psi and homogenize 1~5 times. Use column chromatography to replace the original ammonium sulfate solution of the external phase of the liposome with an external aqueous phase solution at pH 7.3~7.6 to obtain a nano-liposome suspension of inhaled corticosteroids (ICS) and long-acting / short-acting anticholinergic drugs.

[0027] The preparation method of the nanoliposome suspension of inhaled corticosteroids (ICS) and long / short-acting anticholinergic drugs can also employ the reverse evaporation-pH gradient method, specifically...

[0028] 1) Phospholipids, cholesterol, stabilizers, and inhaled glucocorticoids were dispersed and dissolved in an organic solvent to obtain a lipid organic phase solution;

[0029] 2) Dissolve long-acting / short-acting anticholinergic drugs in an aqueous solution with pH adjusted to 3.8–4.5 using a citrate-sodium citrate-NaOH buffer solution and stir until completely dissolved to obtain the aqueous phase. Slowly add the aqueous phase to the lipid organic phase solution and sonicate under a water bath at 35–40°C to form a stable W / O emulsion. Remove the solvent by vacuum evaporation to form a viscous gel state. Add an osmotic pressure regulator solution and continue to concentrate under vacuum to remove the solvent until the system is fully hydrated and collapses from the gel state to become a liposome suspension. Then, homogenize and disperse the suspension using a microfluidic homogenizer at a homogenization pressure of 10,000–25,000 psi and homogenize 1–5 times. Replace the external aqueous phase with a sodium phosphate buffer solution at pH 7.3–7.6 using tangential flow technology to obtain a nano-liposome suspension of inhaled corticosteroids (ICS) and long-acting / short-acting anticholinergic drugs.

[0030] The method for preparing the nanoliposome suspension of inhaled corticosteroids (ICS) and long / short-acting anticholinergic drugs, wherein the homogenization and dispersion conditions of the microfluidic homogenizer are a homogenization pressure of 15000 psi and a homogenization cycle of 2.

[0031] This invention also provides a method for preparing a nanoliposome suspension for soft fogging, wherein the active ingredients are budesonide, glycopyrronium bromide, and formoterol fumarate, and the preparation method specifically includes the following steps:

[0032] (1) Phospholipids, cholesterol, stabilizers, and inhaled glucocorticoids are dispersed and dissolved in an organic solvent to obtain a lipid organic phase solution; the inhaled glucocorticoids are dissolved in an organic solvent to obtain a drug-containing solution; the lipid solution and the drug-containing solution are mixed evenly to form an organic phase; the organic phase is evaporated to remove the organic solvent to obtain a drug-containing phospholipid film.

[0033] (2) Dissolve glycopyrronium bromide in an aqueous solution with pH adjusted to 3.8-4.5 using a citric acid-sodium citrate-NaOH buffer solution, add an osmotic pressure regulator, and stir until completely dissolved to form the aqueous phase.

[0034] (3) The aqueous phase was poured into the drug-containing phospholipid film for hydration and dispersion. The resulting mixed solution was homogenized and dispersed using a microfluidic homogenizer at a homogenization pressure of 10,000 to 25,000 psi and for 1 to 5 homogenization cycles. The external aqueous phase was replaced with a phosphate buffer solution of pH 7.3 to 7.6 to obtain a budesonide-glycerin bromide nanoliposome suspension.

[0035] (4) Add formoterol fumarate to the nanoliposome suspension obtained in step (3), stir until dissolved, incubate at 35℃~40℃ for 1h, and concentrate the drug solution to a suitable volume using 0.9% NaCl tangential flow technology.

[0036] The method for preparing a nanoliposome suspension for a soft mist spray, further...

[0037] In step 1), the organic solvent is chloroform, and the organic phase is concentrated under reduced pressure at 50–55 °C.

[0038] Step 2) The osmotic pressure regulator is sodium chloride; the pH of the buffer solution is adjusted to 4;

[0039] Step 3) The homogenization pressure is 15000, the homogenization is performed twice, and the pH of the phosphate buffer is 7.4;

[0040] Step 4) The incubation temperature is 37℃.

[0041] The present invention also provides the application of the aforementioned nanoliposome suspension for soft fogging in drug delivery using a soft fogging device, wherein the nanoliposome suspension, after being atomized by the soft fogging device, has a particle size of 0.5–5.0 μm.

[0042] This invention provides a nano-liposome suspension for soft inhalers, in which active ingredients (phospholipids, cholesterol) are embedded within liposomes. The liposome particle size is controlled using a microfluidic homogenizer. Free drugs and residual organic solvents in the suspension are removed by a tangential flow ultrafiltration system. The drug is delivered via a soft inhaler (SMI) device to promote the treatment of respiratory diseases. It improves the bioavailability of poorly water-soluble drugs such as inhaled ICS and LABA. The SMI device achieves lung-targeting drug delivery. Droplets with an aerodynamic diameter >5 μm will settle in the oropharyngeal region, thus entering the gastrointestinal tract rather than the respiratory tract; while droplets of 1–5 μm will deposit in the lower respiratory tract, terminal bronchioles, and alveoli, thereby penetrating lung tissue. This is suitable for the treatment of diseases such as COPD. This invention provides a nanoliposome suspension that overcomes the shortcomings of traditional suspensions, such as easy sedimentation and uneven distribution. By controlling the ratio of drug to carrier, a uniform, stable dispersion with an encapsulation rate of over 80% is prepared. Simultaneously, the nanoliposome form also achieves sustained drug release, reducing the frequency of administration. It also provides a possibility for multi-nebulization synergistic drug delivery for soft inhalers and inhaled liquid formulations. This invention uses phospholipids (DPPC, POPG-NA) similar to endogenous pulmonary surfactant components as membrane materials to prepare liposomes, exhibiting good physiological compatibility and minimizing the risk of toxicity and immune responses. Combining nanoliposome technology with SMI technology enables direct drug delivery to lung lesions, prolonging the drug's action time in the lungs, reducing the frequency of administration, and avoiding gastrointestinal damage and the first-pass effect of the liver. Attached Figure Description

[0043] Figure 1 This is a comparison graph of the in vitro cumulative release rate curves of budesonide for Example 26 and the control drug;

[0044] Figure 2This is a comparison chart of the cumulative in vitro release rate curves of glycopyrronium bromide in Example 26 and the control drug;

[0045] Figure 3 This is a comparison of the in vitro cumulative release rate curves of formoterol fumarate for Example 26 and the control drug;

[0046] Figure 4 This is a comparison diagram of the aerodynamic particle size distribution of budesonide in Example 26 and the control drug;

[0047] Figure 5 This is a comparison diagram of the aerodynamic particle size distribution of glycopyrronium bromide in Example 26 and the control drug;

[0048] Figure 6 This is a comparison diagram of the aerodynamic particle size distribution of formoterol fumarate in Example 26 and the control drug; Detailed Implementation

[0049] Example 1

[0050] The preparation of an inhaled ICS liposome suspension includes the following steps:

[0051] ICS (278.3 mg budesonide), 180.0 mg DPPC, and 90.0 mg cholesterol were dissolved in 50 mL of chloroform. The organic solvent was removed by vacuum evaporation at 55 °C to obtain a drug-containing phospholipid membrane. 180 mg NaCl was dissolved in 20 mL of water for injection, and the pH of the solution was adjusted to 6 with a citrate-sodium citrate-NaOH buffer solution to form the aqueous phase. The aqueous phase was poured into the drug-containing phospholipid membrane for hydration and dispersion, and then homogenized twice using a microfluidic homogenizer (homogenization pressure 15000 psi). The mixture was then sterilized and filtered through a sterile 0.22 μm membrane filter to obtain an ICS (budesonide) nanoliposome suspension.

[0052] Example 2

[0053] The preparation of an inhaled LAMA liposome suspension includes the following steps:

[0054] 18.0 mg of DPPC and 9.0 mg of cholesterol were dissolved in 10 mL of anhydrous ethanol to obtain the organic phase solution. 180 mg of NaCl was dissolved in 20 mL of water for injection, and the pH of the solution was adjusted to 4 using a citrate-sodium citrate-NaOH buffer solution. LAMA (10.1 mg glycopyrronium bromide) was then added and dissolved with stirring to form the aqueous phase. The organic phase was slowly added to the aqueous phase, and then the solution was homogenized twice using a microfluidic homogenizer (homogenization pressure 15000 psi). Nitrogen gas was then purged to the bottom of the solution to remove the organic solvent. A 0.9% NaCl solution was then passed through a tangential flow ultrafiltration system (hollow fiber column diaphragm area 118 cm²). 2The fiber inner diameter is 0.5 mm (the same parameters apply to the following examples). The solution is washed and filtered to remove residual organic solvents and free drug, and then concentrated to 20 mL. It is then sterilized and filtered through a sterile 0.22 μm membrane filter to obtain the LAMA (glycopyrronium bromide) nanoliposome suspension.

[0055] Example 3

[0056] The preparation of an inhaled LABA liposome suspension includes the following steps:

[0057] 18.0 mg of DPPC and 9.0 mg of cholesterol were dissolved in 50 mL of chloroform. The organic solvent was removed by vacuum evaporation at 55 °C to obtain a drug-containing phospholipid membrane. 180 mg of NaCl was dissolved in 20 mL of water for injection. The pH of the solution was adjusted to 4 using a citrate-sodium citrate-NaOH buffer solution. The aqueous phase was poured into the drug-containing phospholipid membrane for hydration and dispersion. The membrane was then homogenized twice using a microfluidic homogenizer (homogenization pressure 15000 psi). The external aqueous phase was replaced with 100 mL of pH 7.4 sodium phosphate buffer using tangential flow technology. The membrane was then homogenized twice using a microfluidic homogenizer (homogenization pressure 15000 psi). The membrane was then sterilized and filtered through a sterile 0.22 μm membrane filter to obtain blank liposomes.

[0058] LABA (11.4 mg formoterol fumarate) was added to the above blank liposome suspension and magnetically stirred until dissolved. The mixture was then incubated in a 37°C water bath for 1 hour. The solution was then concentrated to 20 mL using 0.9% NaCl tangential flow technology to obtain the LABA (formoterol fumarate) nanoliposome suspension.

[0059] Example 4

[0060] The preparation of an inhaled ICS+LABA liposome suspension includes the following steps:

[0061] Weigh ICS (278.3 mg budesonide) and LABA (11.4 mg formoterol fumarate) and add them to 50 mL of a suitable organic solvent (methanol), and stir until dissolved; dissolve 180.0 mg DPPC and 90.0 mg cholesterol in 50 mL of chloroform; mix the above solutions and stir until clear, then evaporate the solution through the solvent under a gentle nitrogen flow to obtain a drug-containing phospholipid film;

[0062] Dissolve 180 mg of NaCl in 20 mL of water for injection, adjust the pH of the solution to 6 with citric acid-sodium citrate-NaOH buffer, and use this as the aqueous phase. Pour the aqueous phase into a drug-containing phospholipid membrane for hydration and dispersion, then pass it through a microfluidic homogenizer (homogenization pressure 15000 psi, homogenization twice), and sterilize and filter it with a sterile 0.22 μm membrane filter to obtain an ICS+LABA (budesonide + formoterol fumarate) nanoliposome suspension.

[0063] Example 5

[0064] The preparation of an inhaled ICS+LABA liposome suspension includes the following steps:

[0065] Weigh 278.3 mg budesonide, 180.0 mg DPPC, and 90.0 mg cholesterol and dissolve them in 50 mL of chloroform. Remove the organic solvent by vacuum evaporation at 55 °C to obtain a drug-containing phospholipid membrane. Dissolve 180.0 mg NaCl in 20 mL of water for injection and adjust the pH of the solution to 4 with a citrate-sodium citrate-NaOH buffer. Pour the aqueous phase into the drug-containing phospholipid membrane for hydration and dispersion, and then pass it through a microfluidic homogenizer (homogenization pressure 15000 psi, homogenization twice). Replace the external aqueous phase with 100 mL of pH 7.4 sodium phosphate buffer using tangential flow technology. Sterilize and filter the solution using a sterile 0.22 μm membrane filter to obtain the ICS (budesonide) liposome suspension.

[0066] LABA (11.4 mg formoterol fumarate) was added to the liposome suspension obtained above and magnetically stirred until dissolved. The mixture was then incubated in a 37°C water bath for 1 hour. The solution was then concentrated to 20 mL using 0.9% NaCl tangential flow technology to obtain ICS+LABA (budesonide + formoterol fumarate) nanoliposome suspension.

[0067] Example 6

[0068] The preparation of an inhaled ICS+LAMA liposome suspension includes the following steps:

[0069] Weigh out ICS (278.3 mg budesonide), 180.0 mg DPPC, and 90.0 mg cholesterol and dissolve them in 50 mL of chloroform. Remove the organic solvent by vacuum evaporation at 55 °C to obtain a drug-containing phospholipid membrane. Dissolve 180.0 mg NaCl in 20 mL of water for injection. Adjust the pH of the solution to 4 with a citrate-sodium citrate-NaOH buffer solution. Add LAMA (10.1 mg glycopyrronium bromide) to dissolve it as the aqueous phase. Pour the aqueous phase into the drug-containing phospholipid membrane for hydration and dispersion. Then, pass it through a microfluidic homogenizer (homogenization pressure 15000 psi, homogenization twice). Replace the external aqueous phase with 0.9% NaCl solution through a tangential flow ultrafiltration system to remove free drug. Concentrate the drug solution to 20 mL and sterilize it with a sterile 0.22 μm membrane filter to obtain an ICS+LAMA (budesonide+glycopyrronium bromide) nanoliposome suspension.

[0070] Example 7

[0071] The preparation of an inhaled ICS+LAMA liposome suspension includes the following steps:

[0072] Weigh out ICS (278.3 mg budesonide), 180.0 mg DPPC, and 90.0 mg cholesterol, dissolve them in 30 mL of anhydrous ethanol to obtain the organic phase solution; dissolve 450.0 mg NaCl in 50 mL of water for injection, adjust the pH of the solution to 4 with citrate-sodium citrate-NaOH buffer, and add LAMA (10.1 mg glycopyrronium bromide) to form the aqueous phase; slowly add the organic phase to the aqueous phase, then pass the solution through a microfluidic homogenizer (homogenization pressure 15000 psi, homogenization twice), and then purge the bottom of the solution with nitrogen to remove the organic solvent; wash the solution with 0.9% NaCl solution through a tangential flow ultrafiltration system to remove the organic solvent and free drug, and concentrate the solution to 20 mL. Then sterilize and filter the solution through a sterile 0.22 μm membrane filter to obtain the ICS+LAMA (budesonide + glycopyrronium bromide) nanoliposome suspension.

[0073] Example 8

[0074] The preparation of an inhaled LABA+LAMA liposome suspension includes the following steps:

[0075] Weigh LABA (11.4 mg formoterol fumarate) and add it to 20 mL of a suitable organic solvent (methanol), and stir until dissolved; dissolve 180.0 mg DPPC and 90.0 mg cholesterol in 50 mL of chloroform; mix the above solutions and stir until dissolved, then evaporate the solution through the solvent under a gentle nitrogen flow to obtain a drug-containing phospholipid film.

[0076] Dissolve 180.0 mg of NaCl in 100 mL of water for injection, adjust the pH of the solution to 4 with citrate-sodium citrate-NaOH buffer, and add LAMA (10.1 mg glycopyrronium bromide) to dissolve and prepare the aqueous phase.

[0077] The aqueous phase was poured into a drug-containing phospholipid membrane for hydration and dispersion, and then passed through a microfluidic homogenizer (homogenization pressure 15000psi, homogenization twice). The external aqueous phase was replaced with 0.9% NaCl solution through a tangential flow ultrafiltration system to remove free drug, and the drug solution was concentrated to 20 mL. The solution was then sterilized and filtered through a sterile 0.22 μm membrane filter to obtain a LABA+LAMA (formoterol fumarate + glycopyrronium bromide) nanoliposome suspension.

[0078] Example 9

[0079] The preparation of an inhaled LABA+LAMA liposome suspension includes the following steps:

[0080] Weigh 180.0 mg of DPPC and 90.0 mg of cholesterol and dissolve them in 50 mL of chloroform. Remove the organic solvent by evaporation under reduced pressure at 55 °C to obtain a drug-containing phospholipid film.

[0081] Dissolve 180.0 mg of NaCl in 100 mL of water for injection, adjust the pH of the solution to 4 with citrate-sodium citrate-NaOH buffer, and add LAMA (10.1 mg glycopyrronium bromide) to dissolve and prepare the aqueous phase.

[0082] The aqueous phase was poured into a drug-containing phospholipid membrane for hydration and dispersion, and then passed through a microfluidic homogenizer (homogenization pressure 15000psi, homogenization twice). The external aqueous phase was replaced by 100mL of pH7.4 sodium phosphate buffer using tangential flow technology. The mixture was then sterilized and filtered through a sterile 0.22μm membrane filter to obtain the LAMA (glycopyrronium bromide) liposome suspension.

[0083] LABA (11.4 mg formoterol fumarate) was added to the above liposome suspension and magnetically stirred until dissolved. The mixture was then incubated in a 37°C water bath for 1 hour. The solution was then concentrated to 20 mL using 0.9% NaCl tangential flow technology to obtain a LABA+LAMA (formoterol fumarate + glycopyrronium bromide) nanoliposome suspension.

[0084] Example 10

[0085] The preparation of an inhaled ICS+LABA+LAMA liposome suspension includes the following steps:

[0086] Weigh ICS (278.3 mg budesonide) and LABA (11.4 mg formoterol fumarate) and add them to 50 mL of methanol, stirring until dissolved; dissolve 180.0 mg DPPC and 90.0 mg cholesterol in 50 mL of chloroform; mix the above solutions and stir until dissolved, then evaporate the solution through a solvent under a gentle nitrogen flow to obtain a drug-containing phospholipid film;

[0087] Dissolve 180.0 mg of NaCl in 100 mL of water for injection, adjust the pH of the solution to 4 with citrate-sodium citrate-NaOH buffer, and add LAMA (1.01 mg glycopyrronium bromide) to dissolve and prepare the aqueous phase.

[0088] The aqueous phase was poured into a drug-containing phospholipid membrane for hydration and dispersion, and then passed through a microfluidic homogenizer (homogenization pressure 15000psi, homogenization twice). The external aqueous phase was replaced with 0.9% NaCl solution through a tangential flow ultrafiltration system to remove free drug, and the drug solution was concentrated to 20 mL. It was then sterilized and filtered through a sterile 0.22 μm membrane filter to obtain an ICS+LABA+LAMA (budesonide+formoterol fumarate+glycopyrronium bromide) nanoliposome suspension.

[0089] Example 11

[0090] The preparation of an inhaled ICS+LABA+LAMA liposome suspension includes the following steps:

[0091] Weigh out ICS (278.3 mg budesonide), 180.0 mg DPPC, and 90.0 mg cholesterol and dissolve them in 100 mL chloroform. Remove the organic solvent by vacuum evaporation at 55 °C to obtain a drug-containing phospholipid film.

[0092] Dissolve 180.0 mg of NaCl in 100 mL of water for injection, adjust the pH of the solution to 4 with citrate-sodium citrate-NaOH buffer, and add LAMA (10.1 mg glycopyrronium bromide) to dissolve and prepare the aqueous phase.

[0093] The aqueous phase was poured into a drug-containing phospholipid membrane for hydration and dispersion, and then passed through a microfluidic homogenizer (homogenization pressure 15000psi, homogenization twice). The external aqueous phase was replaced with 100mL of pH7.4 sodium phosphate buffer using tangential flow technology. The mixture was then sterilized and filtered through a sterile 0.22μm membrane filter to obtain the ICS+LAMA (budesonide + glycopyrronium bromide) liposome suspension.

[0094] LABA (11.4 mg formoterol fumarate) was added to the above liposome suspension and magnetically stirred until dissolved. The mixture was then incubated in a 37°C water bath for 1 hour. The solution was then concentrated to 20 mL using 0.9% NaCl tangential flow technology to obtain ICS+LABA+LAMA (budesonide + formoterol fumarate + glycopyrronium bromide) nanoliposome suspension.

[0095] Example 12 Effect of different stabilizers on the stability of nanoliposome suspensions

[0096] When dissolving phospholipids, different stabilizers were added to the solvent according to Table 1, and the remaining operation steps were the same as in Example 11. Budesonide + formoterol fumarate + glycopyrronium bromide nanoliposome suspensions with different stabilizers were prepared.

[0097] The particle size of the unstabilized nanoliposome suspension was measured at 0°C and after 1 hour of incubation. Simultaneously, the particle size of nanoliposomes with different stabilizers was measured after 1 hour of incubation. 50 μL of the prepared nanoliposome suspension was diluted 100 times with pure water until clear and transparent. 1 mL was pipetted into a quartz cuvette and placed in a Malvern nanoparticle size analyzer. The particle size, polydispersity index (PDI), and zeta potential of the nanoliposome suspension were determined using dynamic light scattering. Three parallel measurements were performed, and the average value was taken. See Table 1 for details.

[0098] The results are shown in Table 1. After 1 hour of standing, the particle size of the unstabilized nanoliposome suspension increased significantly, and the PDI > 0.3, indicating that it was prone to aggregation and uneven distribution. Therefore, a stabilizer was needed to prevent aggregation and improve dispersibility. The addition of the stabilizer improved the dispersibility of the nanoliposome suspension, significantly reducing its particle size and PDI. Different stabilizers showed significant differences in particle size and potential, with most having a PDI below 0.3. Nanoliposomes can improve the bioavailability of poorly soluble drugs and achieve slow release in vivo. Generally, a system is considered relatively stable when the absolute value of the Zeta potential is above 30 mV. Considering the results of particle size, PDI, and Zeta potential, vitamin E polyethylene glycol succinate (TPGS) with a suitable particle size and a large absolute value of Zeta potential was selected as the stabilizer. The specific specification of TPGS is vitamin E polyethylene glycol 1000 succinate.

[0099] Table 1. Particle size, PDI, and Zeta potential of nanoliposome suspensions with different stabilizers

[0100] Examples 13-18

[0101] According to the membrane material composition and dosage in Table 2, TPGS was added to dissolve the membrane material, and the remaining operation steps were the same as in Example 11, to prepare budesonide + formoterol fumarate + glycopyrronium bromide nanoliposome suspensions with different membrane materials.

[0102] Table 2 Composition and dosage of liposome membrane materials

[0103]

[0104] Example 19 Liposome Stability Study

[0105] A major challenge in liposome research is the physical stability issues such as drug leakage, aggregation, and fusion into large clumps. This study investigates liposome stability by observing the overall aggregation state of liposome formulations. The morphological changes of liposome samples from Examples 13–18 during aggregation and sedimentation were observed and recorded over 24 hours, and the stability of liposomes with different formulations was compared.

[0106] In terms of appearance, the liposome suspensions of Examples 13-18 showed no precipitation, no flocculation or stratification, were uniformly dispersed and had good fluidity, and exhibited obvious opalescence when exposed to light.

[0107] Take 50 μL of the nanoliposome suspension prepared in Examples 13-18, dilute it 100 times with pure water until clear and transparent, pipette 1 mL into a quartz cuvette, place it in a Malvern nanoparticle size analyzer, and determine the particle size, polydispersity index (PDI), and zeta potential of the nanoliposome suspension using dynamic light scattering method. Perform three parallel measurements and take the average value. See Table 3 for details.

[0108] Liposomes are composed of a flowing, dynamic phospholipid membrane. Phospholipids continuously and freely exchange positions across the membrane, causing spontaneous aggregation and sedimentation of liposome particles, thus altering liposome size and zeta potential. The size distribution and zeta potential of liposomes are important parameters for evaluating their stability. High-quality liposomes should have relatively stable size and distribution, exhibiting a normal distribution with a narrow range. Table 3 shows that the addition of pEG-modified long-circulating phospholipids (Examples 15 / 18) improves the stability of liposome suspensions. The addition of glycerophospholipids (PG type) (Examples 14 / 17) and ethanolamine phospholipids (PE type) (Examples 15 / 18) also increases the stability of nanoliposomes to some extent. Smaller liposome size is not necessarily better; excessively small particles are unstable and prone to sedimentation, while particles of 100–200 nm have higher membrane permeability and are less prone to sedimentation, potentially leading to greater stability. The smaller the drug particle size, the larger its specific surface area, and the faster the drug is released in the body. Generally, the PDI is mostly below 0.3, and the absolute value of the Zeta potential needs to be above 30mV for the system to be relatively stable.

[0109] Table 3. Particle size results of nanoliposome suspensions

[0110] name Particle size (nm) PDI Zeta potential (mV) Example 13 53.34 0.42 -26.53 Example 14 86.99 0.23 -35.57 Example 15 117.62 0.27 -31.19 Example 16 239.43 0.28 -35.95 Example 17 158.17 0.21 -39.32 Example 18 192.16 0.23 -37.28

[0111] The encapsulation efficiency of nanoliposomes prepared in Examples 13-18 (without a tangential flow ultrafiltration system) was determined. The specific procedure was as follows: 0.5 mL of nanoliposome suspension was transferred to a 10 mL volumetric flask, an appropriate amount of methanol was added, and the mixture was sonicated for 40 min to completely demulsify. The volume was then adjusted to the mark. The solution was filtered through a 0.22 μm filter membrane, and the concentration was determined by HPLC and denoted as C. 总 Take another 0.5 mL of the nanoliposome suspension and place it in a 100 KD ultrafiltration centrifuge tube. Centrifuge at 4000 rpm for 20 min, then collect the lower layer filtrate. Determine its concentration by HPLC and record it as Cfree. Calculate the encapsulation efficiency of the nanoliposomes using the following formula.

[0112] EE(%) = (1-W) 游离 / W 总 )×100% formula (1)

[0113] In the formula, W 游离Wtotal represents the mass of free drug in the nanoliposome suspension, and Wtotal represents the total mass of drug in the nanoliposome suspension.

[0114] HPLC chromatographic conditions: Waters 18RP column (250 mm × 4.6 mm, 5 μm); mobile phase A was 20 mmol / L sodium phosphate buffer (5.68 g of anhydrous disodium hydrogen phosphate was dissolved in 2000 ml of water and the pH was adjusted to 8.00 with phosphoric acid) - acetonitrile (95:5), and mobile phase B was acetonitrile, with gradient elution according to Table 4; flow rate was 1.0 mL / min, column temperature was 45 ℃; detection wavelength was 220 nm.

[0115] Table 4 Gradient Elution of Mobile Phase

[0116] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95.0 5.0 1 95.0 5.0 10 86.0 14.0 15 30.0 70.0 17 95 5.0 20 95.0 5.0

[0117] According to the encapsulation efficiency test in Section IV of the 2020 Chinese Pharmacopoeia, the encapsulation efficiency of liposomes should not be less than 80%.

[0118] The encapsulation efficiencies of the liposomes prepared in Examples 13-18 without a tangential flow ultrafiltration system were 83.26%, 90.45%, 87.29%, 84.78%, 91.73%, and 88.34%, respectively. This demonstrates that adding glycerophospholipid (PG) anionic phospholipids (Examples 14 / 17) and PEG-modified phospholipids (Examples 15 / 18) to the liposomes can improve the drug encapsulation efficiency. Based on the encapsulation efficiency results, the membrane material of Example 17 is preferred. Considering that unencapsulated drugs in the liposomes would affect the sustained-release effect of the formulation, a tangential flow ultrafiltration system was used in all examples to remove free drugs, achieving a near 100% encapsulation efficiency for the high-nano liposomes.

[0119] Examples 20-24

[0120] Compositions were prepared according to different drug-to-lipid ratios (mass ratio of active ingredient API to lipids) as shown in Table 5. TPGS was added to dissolve the lipids. In Examples 20-24, the lipids were dissolved in 20 mL of chloroform, and the remaining procedures were the same as in Example 11, resulting in different budesonide + formoterol fumarate + glycopyrronium bromide nanoliposome suspensions. The stability of the liposomes with different drug-to-lipid ratios was tested according to Example 19.

[0121] Table 5. Different drug-lipid ratios (API / lipid, w / w) in nanoliposome suspensions.

[0122]

[0123] From an appearance perspective, the liposome suspensions of Examples 17-24 showed no precipitation, no flocculation or stratification, were uniformly dispersed, and had good flowability, with a noticeable opalescence visible under light. Table 6 shows that the drug-to-lipid ratio significantly affects the liposome particle size and encapsulation efficiency. An optimal encapsulation efficiency was achieved with a drug-to-lipid ratio (w / w) of 1:3 (Example 23), resulting in smaller particle size, a PDI less than 0.3, and the highest absolute value of the Zeta potential. This indicates that the liposomes prepared at this drug-to-lipid ratio (w / w) are the most stable and have the best performance.

[0124] Table 6. Results of particle size and encapsulation efficiency of liposome suspensions prepared from compositions with different drug-liposome ratios.

[0125]

[0126] Example 25

[0127] Weigh out ICS (2783.0 mg budesonide), 6600.0 mg DPPC, 1920.0.0 mg POPG-NA, 480.0 mg cholesterol, and 50 mg TPGS, dissolve them in 1500 mL chloroform, and remove the organic solvent by evaporation under reduced pressure at 55 °C to obtain a drug-containing phospholipid film.

[0128] Dissolve 1800.0 mg of NaCl in 1000 mL of water for injection, adjust the pH of the solution to 4 with citrate-sodium citrate-NaOH buffer, and add LAMA (101.0 mg glycopyrronium bromide) to dissolve and prepare the aqueous phase.

[0129] The aqueous phase was poured into a phospholipid membrane containing the drug for hydration and dispersion. Nine portions of the mixture were taken, each containing 100 mL. The mixture was then passed through a microfluidic homogenizer (mean parameters are detailed in Table 7). The external aqueous phase was replaced with a sodium phosphate buffer solution at pH 7.4 using tangential flow technology. The mixture was then sterilized and filtered using a sterile 0.22 μm membrane filter to obtain the ICS+LAMA (budesonide + glycopyrronium bromide) liposome suspension.

[0130] Take the liposome suspensions prepared under the above different homogenization conditions, add LABA (11.4 mg formoterol fumarate) to each of the 9 portions, stir magnetically until dissolved, incubate in a 37°C water bath for 1 h, and concentrate the drug solution to 20 mL using 0.9% NaCl tangential flow technology to obtain ICS+LABA+LAMA (budesonide+formoterol fumarate+glycopyrronium bromide) nanoliposome suspension.

[0131] Homogenization was performed using different microfluidic homogenizers. The homogenization parameters and results are shown in Table 7. When the microfluidic homogenization pressure was 15000 psi and homogenization was performed twice, the nanoliposome suspension had a moderate particle size, PDI < 0.3, and a relatively large absolute value of Zeta potential. The suspension prepared by this process was the most stable.

[0132] Table 7. Effects of different homogenization pressures and homogenization cycles on the composition liposome suspension.

[0133]

[0134]

[0135] Example 26 (Thin-film dispersion method + pH gradient method)

[0136] Weigh 278.3 mg budesonide, 660.0 mg DPPC, 192.0 mg POPG-NA, 48.0 mg cholesterol, and 5.0 mg TPGS and dissolve them in 150 mL chloroform. Remove the organic solvent by vacuum evaporation at 55 °C to obtain a drug-containing phospholipid film.

[0137] Dissolve 180.0 mg of NaCl in 100 mL of water for injection, adjust the pH of the solution to 4 with citric acid-sodium citrate-NaOH buffer, and add 10.1 mg of glycopyrronium bromide to dissolve as the aqueous phase;

[0138] The aqueous phase was poured into a phospholipid membrane containing the drug for hydration and dispersion, and then homogenized twice using a microfluidic homogenizer at a pressure of 15000 psi. The external aqueous phase was replaced with 100 mL of pH 7.4 sodium phosphate buffer using tangential flow technology, and the mixture was sterilized and filtered through a sterile 0.22 μm membrane filter to obtain the budesonide + glycopyrronium bromide liposome suspension.

[0139] Add 11.4 mg of formoterol fumarate to the above liposome suspension and stir magnetically until dissolved. Incubate in a 37°C water bath for 1 h. Concentrate the solution to 20 mL using 0.9% NaCl tangential flow technology to obtain a budesonide + formoterol fumarate + glycopyrronium bromide nanoliposome suspension.

[0140] Example 27 (Reverse Evaporation Method + pH Gradient Method)

[0141] Weigh 278.3 mg budesonide, 660.0 mg DPPC, 192.0 mg POPG-NA, 48.0 mg cholesterol, and 5.0 mg TPGS and dissolve them in 150 mL chloroform to form a lipid-containing organic phase solution.

[0142] Weigh 10.1 mg of glycopyrronium bromide and dissolve it in 50 mL of water for injection. Adjust the pH of the solution to 4 using a citrate-sodium citrate-NaOH buffer solution.

[0143] The drug-containing aqueous solution was slowly added to the organic phase solution, and the mixture was sonicated in a 37°C water bath until a stable W / O (water-in-oil) emulsion was formed. This emulsion was then transferred to a round-bottom flask, and the solvent was removed by rotary evaporation to form a viscous gel. 100 mL of an aqueous solution containing 180.0 mg of NaCl was then added, and evaporation under reduced pressure continued until complete hydration. When sufficient organic solvent was removed, the gel collapsed, forming a liposome suspension. This suspension was then homogenized twice using a microfluidic homogenizer at a pressure of 15000 psi. The external aqueous phase was replaced with 10 mL of pH 7.4 sodium phosphate buffer using tangential flow technology, and the mixture was sterilely filtered through a 0.22 μm membrane filter to obtain the budesonide + glycopyrronium bromide liposome suspension.

[0144] Add 11.4 mg of formoterol fumarate to the above liposome suspension and stir magnetically until dissolved. Incubate in a 37°C water bath for 1 h. Concentrate the solution to 20 mL using 0.9% NaCl tangential flow technology to obtain a budesonide + formoterol fumarate + glycopyrronium bromide nanoliposome suspension.

[0145] Example 28 (Thin-film dispersion method + ammonium sulfate gradient method)

[0146] Weigh 278.3 mg budesonide, 660.0 mg DPPC, 192.0 mg POPG-NA, 48.0 mg cholesterol, and 5.0 mg TPGS and dissolve them in 150 mL chloroform. Remove the organic solvent by vacuum evaporation at 55 °C to obtain a drug-containing phospholipid film.

[0147] 396.4 mg of ammonium sulfate (300 mM), 180.0 mg of NaCl, and 10.1 mg of glycopyrronium bromide were dissolved in 100 mL of water for injection to form the aqueous phase. The aqueous phase was then poured into a drug-containing phospholipid membrane for hydration and dispersion, followed by homogenization twice using a microfluidic homogenizer at a pressure of 15000 psi. Using column chromatography, the ammonium sulfate solution in the original liposome in vitro phase was replaced with 200 mL of an external aqueous phase solution (pH 7.4) consisting of 250 mM sucrose solution and 50 mM histidine (pH adjusted with citrate-sodium citrate-NaOH buffer) to obtain a budesonide + glycopyrronium bromide liposome suspension. 11.4 mg of formoterol fumarate was added to the above liposome suspension and magnetically stirred until dissolved. The suspension was then incubated in a 37°C water bath for 1 h for drug loading. The drug solution was concentrated to 20 mL using 0.9% NaCl tangential flow technology to obtain a budesonide + formoterol fumarate + glycopyrronium bromide nanoliposome suspension.

[0148] Example 29 (Reverse Evaporation Method + Ammonium Sulfate Gradient Method)

[0149] Weigh 278.3 mg budesonide, 660.0 mg DPPC, 192.0 mg POPG-NA, 48.0 mg cholesterol, and 5.0 mg TPGS and dissolve them in 150 mL chloroform to form a lipid-containing organic phase solution.

[0150] Weigh 10.1 mg of glycopyrronium bromide and dissolve it in 50 mL of 300 mM ammonium sulfate aqueous solution as the aqueous phase. Slowly add the drug-containing aqueous solution to the lipid-containing organic phase solution, and sonicate in a water bath at 37°C until a stable W / O (water-in-oil) emulsion is formed. Then transfer it to a round-bottom flask, and remove the solvent by rotary evaporation to form a viscous gel. Then add 100 mL of 300 mM ammonium sulfate aqueous solution containing 180.0 mg NaCl, and continue to evaporate under reduced pressure until fully hydrated. When enough organic solvent is removed, the gel collapses and forms a liposome suspension. Then pass it through a microfluidic homogenizer at a homogenization pressure of 15000 psi for two homogenizations. Using column chromatography, 200 mL of an external aqueous phase solution (pH 7.4) consisting of 250 mM sucrose solution and 50 mM histidine (pH adjusted with citric acid-sodium citrate-NaOH buffer) was used to replace the ammonium sulfate solution in the original liposome external phase to obtain a budesonide + glycopyrronium bromide liposome suspension. 11.4 mg of formoterol fumarate was added to the above liposome suspension and magnetically stirred until dissolved. The mixture was incubated in a 37°C water bath for 1 h, and the solution was concentrated to 20 mL using 0.9% NaCl tangential flow technology to obtain a budesonide + formoterol fumarate + glycopyrronium bromide nanoliposome suspension.

[0151] As shown in Table 8, the liposome preparation of multi-drug combination drugs using conventional single passive drug loading methods, such as thin film dispersion, results in low drug encapsulation efficiency (Example 11). Combining passive and active drug loading methods according to the drug properties can greatly improve the drug encapsulation efficiency.

[0152] Table 8. Particle size distribution and encapsulation efficiency results of Examples 26-29

[0153]

[0154] Example 30: In vitro release study of nanoliposome suspension for nebulized inhalation

[0155] The in vitro release of the nanoliposome suspension was investigated using a flow cell and dialysis bag. The in vitro dissolution rate of the drugs in Example 26 and the control drugs was investigated in artificial simulated lung fluid. The composition of the artificial lung fluid included: MgCl2·6H2O (0.20 g / L), NaCl (6.02 g / L), KCl (0.30 g / L), Na2HPO4·12H2O (0.36 g / L), Na2SO4 (0.07 g / L), CaCl2 (0.28 g / L), CH3COONa (0.57 g / L), NaHCO3 (2.60 g / L), Na3H5C6O7·2H2O (0.10 g / L), and dipalmitoylphosphatidylcholine (0.02% w / v). Accurately weigh appropriate amounts of budesonide aerosol (batch number: 6101695C00, AstraZeneca) used as the control drug in Example 26 (lyophilized to remove propellant, reconstituted with water for injection), perform three parallel measurements, transfer to a MWCO=3000Da (cellulose ester) dialysis bag, place in a flow cell, add 200mL of artificial lung fluid, and conduct drug release studies using a closed-loop method at 37℃. Take 1mL of the solution at 5min, 10min, 15min, 30min, 60min, 120min, 240min, 360min, 420min, 540min, 600min, and 720min to add 1mL of artificial lung fluid to the release medium. Determine the content of the three active pharmaceutical ingredients by HPLC, calculate the cumulative release rate, and plot the cumulative release curves of budesonide, glycopyrronium bromide, and formoterol fumarate, as shown below. Figures 1-3 .

[0156] according to Figures 1-3 In artificial lung fluid, the in vitro release results of the three active ingredients in budesonide-formoterol fumarate aerosol showed that budesonide (BUD) achieved a release rate of over 50% within 60 minutes and over 80% within 4 hours; glycopyrronium bromide (GB) achieved a release rate of over 50% within 30 minutes and over 80% within 2 hours; and formoterol fumarate (FF) achieved a release rate of over 50% at approximately 65 minutes and over 80% within 4 hours. According to the 2020 edition of the Chinese Pharmacopoeia, Volume IV, 9014 Guidelines for Microparticle Formulations, the release rate within the first 0.5 hours should be less than 40%. The three active ingredients in the nanoliposome suspension can be released gradually. Within 30 minutes, the drug release is less than 40%. It takes 4 hours for BUD and FF to reach a release rate of more than 50%, and 3 hours for GB to reach a release rate of more than 50%. Compared with aerosol, the release of the three components is relatively slow, with the release time delayed by about 4 to 6 times. The release rate reaches more than 80% within 10 hours, which is relatively sufficient.

[0157] Note: Based on clinical trials of Bude Golgi Inhaler aerosol, it is known that...[1,2] In COPD subjects, after inhalation of this product, the Cmax of BUD is reached within 20-40 minutes, with a half-life of 5 hours; the Cmax of GB is reached within 6 minutes, with a half-life of 15 hours; and the Cmax of FF is reached within 40-60 minutes, with a half-life of 10 hours.

[0158] [1]P.Darken,P.DePetrillo,C.Reisner,E.StRose,P.Dorinsky,Thepharmacokinetics of three doses of budesonide / glycopyrronium / formoterolfumarate dihydrate metered dose inhaler compared with active controls:A PhaseI randomized,single-dose,crossover study in healthyadults.Pulm.Pharmacol.Ther.50(2018)11-18.

[0159] [2]Dunn LJ,Kerwin EM,DeAngelis K,et al.Pharmacokinetics ofbudesonide / glycopyrrolate / formoterol fumarate metered dose inhaler formulationusing co-suspension delivery technology after single and chronic dosing inpatients with COPD.Pulm Pharmacol Ther 2020;60:10187

[0160] Example 31: Investigation of Aerodynamic Particle Size Distribution

[0161] For Example 26, the control drug, budesonide aerosol (batch number: 6101695C00, AstraZeneca), was tested according to the Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0951. The aerodynamic particle size of the above samples was determined using a new generation pharmaceutical cascade impactor (NGI) at a flow rate of 15 L / min. The collection tray and adapter were used. Data were analyzed using Copley data analysis software version 3.10 to calculate the fine particle dose (FPD), fine particle fraction (FPF), and median mass aerodynamic diameter (MMAD). All samples were tested three times. The results are detailed in Table 9. A comparison chart of the deposition percentages at each level for the three active ingredients (aerodynamic particle size distribution comparison chart) is also provided. Figures 4-6 .

[0162] As shown in Table 9, the atomization characteristics of Examples 26 and 6101695C00 (Budigfa aerosol) have a higher percentage of particles smaller than 5μm, with an FPF approaching 60%. Figures 3-5 The aerodynamic particle size distribution shows that the soft mist (Example 26) has better aerodynamic characteristics than the aerosol (6101695C00), with less deposition in the oropharynx and more particles deposited at levels 3 to 5, indicating that it is more suitable for penetrating deep into the alveoli and pulmonary microvessels for the treatment of COPD.

[0163] Table 9 Results of Atomization Characteristics Evaluation

[0164]

[0165] Example 32 (Thin-film dispersion method + pH gradient method)

[0166] Weigh 160.1 mg fluticasone furoate, 660.0 mg DPPC, 192.0 mg POPG-NA, 48.0 mg cholesterol, and 5.0 mg TPGS and dissolve them in 150 mL chloroform. Remove the organic solvent by evaporation under reduced pressure at 55 °C to obtain a drug-containing phospholipid film.

[0167] Dissolve 180.0 mg of NaCl in 100 mL of water for injection, adjust the pH of the solution to 4 with citric acid-sodium citrate-NaOH buffer, and add 99.9 mg of umemetammonium bromide (calculated as umemetammonium) to dissolve and prepare the aqueous phase.

[0168] The aqueous phase was poured into a phospholipid membrane containing the drug for hydration and dispersion, and then homogenized twice using a microfluidic homogenizer at a pressure of 15000 psi. The external aqueous phase was replaced with 100 mL of pH 7.4 sodium phosphate buffer using tangential flow technology, and the mixture was sterilized and filtered through a sterile 0.22 μm membrane filter to obtain a fluticasone furoate + umemet bromide liposome suspension.

[0169] Add 40.0 mg of vilanterol triphenylacetic acid (calculated as vilanterol) to the above liposome suspension and stir magnetically until dissolved. Incubate in a 37°C water bath for 1 h. Concentrate the drug solution to 20 mL using 0.9% NaCl tangential flow technology to obtain a fluticasone furoate + umemetammonium bromide + vilanterol nanoliposome suspension.

[0170] Example 33 (Reverse Evaporation Method + Ammonium Sulfate Gradient Method)

[0171] Weigh out 160.1 mg fluticasone furoate, 660.0 mg DPPC, 192.0 mg POPG-NA, 48.0 mg cholesterol, and 5.0 mg TPGS, and dissolve them in 150 mL chloroform to form a lipid-containing organic phase solution.

[0172] 99.9 mg of umemebromide (calculated as umeme) was dissolved in 50 mL of 300 mM ammonium sulfate aqueous solution as the aqueous phase. This aqueous phase was slowly added to the lipid organic phase solution, and the mixture was sonicated in a 37°C water bath until a stable W / O (water-in-oil) emulsion was formed. The emulsion was then transferred to a round-bottom flask, and the solvent was removed by rotary evaporation to form a viscous gel. Then, 100 mL of 300 mM ammonium sulfate aqueous solution containing 180.0 mg NaCl was added, and evaporation under reduced pressure continued until complete hydration. When sufficient organic solvent was removed, the gel collapsed, forming a liposome suspension. The suspension was then homogenized twice using a microfluidic homogenizer at a pressure of 15000 psi. The original ammonium sulfate solution in the liposome in vitro phase was replaced by column chromatography with 200 mL of an external aqueous phase solution (pH 7.4) consisting of 250 mM sucrose solution and 50 mM histidine (pH adjusted with citric acid-sodium citrate-NaOH buffer).

[0173] Add 40.0 mg of vilanterol triphenylacetic acid (calculated as vilanterol) to the above liposome suspension, stir magnetically until dissolved, and incubate in a 37°C water bath for 1 h to load the drug. Concentrate the drug solution to 20 mL using 0.9% NaCl tangential flow technology to obtain the budesonide + formoterol fumarate + glycopyrronium bromide nanoliposome suspension.

[0174] Example 34 (Thin-film dispersion method + pH gradient method)

[0175] Weigh 157.4 mg beclomethasone propionate, 410.1 mg DPPC, 119.3 mg POPG-NA, 29.8 mg cholesterol, and 3.0 mg TPGS and dissolve them in 50 mL chloroform. Remove the organic solvent by evaporation under reduced pressure at 55 °C to obtain a drug-containing phospholipid film.

[0176] Dissolve 180.0 mg of NaCl in 100 mL of water for injection, adjust the pH of the solution to 4 using citric acid-sodium citrate-NaOH buffer, and add 19.9 mg of glycopyrronium bromide to dissolve it as the aqueous phase.

[0177] The aqueous phase was poured into a phospholipid membrane containing the drug for hydration and dispersion, and then homogenized twice using a microfluidic homogenizer at a pressure of 15000 psi. The external aqueous phase was replaced with 150 mL of pH 7.4 sodium phosphate buffer using tangential flow technology, and the mixture was sterilized and filtered through a sterile 0.22 μm membrane filter to obtain a beclomethasone + glycopyrronium bromide liposome suspension.

[0178] Add 9.1 mg of formoterol fumarate to the above liposome suspension and stir magnetically until dissolved. Incubate in a 37°C water bath for 1 h. Concentrate the solution to 20 mL using 0.9% NaCl tangential flow technology to obtain a beclomethasone + glycopyrronium bromide + formoterol fumarate nanoliposome suspension.

[0179] Example 35 (Reverse Evaporation Method + Ammonium Sulfate Gradient Method)

[0180] Weigh out 157.4 mg beclomethasone propionate, 410.1 mg DPPC, 119.3 mg POPG-NA, 29.8 mg cholesterol, and 3.0 mg TPGS, and dissolve them in 50 mL chloroform to form a lipid organic solvent.

[0181] 19.9 mg of glycopyrronium bromide was dissolved in 50 mL of 300 mM ammonium sulfate aqueous solution as the aqueous phase. This aqueous phase was slowly added to the lipid organic phase solution, and the mixture was sonicated in a 37°C water bath until a stable W / O (water-in-oil) emulsion was formed. The emulsion was then transferred to a round-bottom flask, and the solvent was removed by rotary evaporation to form a viscous gel. Then, 100 mL of 300 mM ammonium sulfate aqueous solution containing 180.0 mg NaCl was added, and evaporation under reduced pressure continued until complete hydration. When sufficient organic solvent was removed, the gel collapsed, forming a liposome suspension. The suspension was then homogenized twice using a microfluidic homogenizer at a pressure of 15000 psi. Using column chromatography, 200 mL of an external aqueous phase solution (pH 7.4) consisting of 250 mM sucrose solution and 50 mM histidine (pH adjusted with citric acid-sodium citrate-NaOH buffer) was used to replace the original ammonium sulfate solution in the external lipid phase, yielding a beclomethasone propionate + glycopyrronium bromide liposome suspension.

[0182] Add 9.1 mg of formoterol fumarate to the above liposome suspension and stir magnetically until dissolved. Incubate in a 37°C water bath for 1 h. Concentrate the solution to 20 mL using 0.9% NaCl tangential flow technology to obtain a beclomethasone + glycopyrronium bromide + formoterol fumarate nanoliposome suspension.

[0183] Example 36 (Thin-film dispersion method + pH gradient method)

[0184] Weigh out 246.2 mg mometasone furoate, 1244.6 mg DPPC, 362.1 mg POPG-NA, 90.5 mg cholesterol, and 8.9 mg TPGS, dissolve them in 100 mL chloroform, and remove the organic solvent by vacuum evaporation at 55 °C to obtain a drug-containing phospholipid film.

[0185] Dissolve 180.0 mg of NaCl in 100 mL of water for injection, adjust the pH of the solution to 4 using citric acid-sodium citrate-NaOH buffer, and add 83.3 mg of glycopyrronium bromide to dissolve it as the aqueous phase.

[0186] The aqueous phase was poured into a phospholipid membrane containing the drug for hydration and dispersion, and then homogenized twice using a microfluidic homogenizer at a pressure of 15000 psi. The external aqueous phase was replaced with 300 mL of pH 7.4 sodium phosphate buffer using tangential flow technology, and the mixture was sterilized and filtered through a sterile 0.22 μm membrane filter to obtain a mometasone furoate + glycopyrronium bromide liposome suspension.

[0187] Add 206.3 mg of indacaterol acetate to the above liposome suspension and stir magnetically until dissolved. Incubate in a 37°C water bath for 1 h. Concentrate the solution to 20 mL using 0.9% NaCl tangential flow technology to obtain a mometasone furoate + glycopyrronium bromide + indacaterol acetate nanoliposome suspension.

[0188] Example 37 (Reverse Evaporation Method + Ammonium Sulfate Gradient Method)

[0189] Weigh out 246.2 mg mometasone furoate, 1244.6 mg DPPC, 362.1 mg POPG-NA, 90.5 mg cholesterol, and 8.9 mg TPGS and dissolve them in 100 mL chloroform to form a lipid organic phase solution.

[0190] 83.3 mg of glycopyrronium bromide was dissolved in 50 mL of 300 mM ammonium sulfate aqueous solution as the aqueous phase. The aqueous phase was slowly added to the lipid organic phase solution, and the mixture was sonicated in a 37°C water bath until a stable W / O (water-in-oil) emulsion was formed. This emulsion was then transferred to a round-bottom flask, and the solvent was removed by rotary evaporation to form a viscous gel. Then, 200 mL of 300 mM ammonium sulfate aqueous solution containing 180.0 mg NaCl was added, and evaporation under reduced pressure continued until complete hydration. When sufficient organic solvent was removed, the gel collapsed, forming a liposome suspension. The suspension was then homogenized twice using a microfluidic homogenizer at a pressure of 15000 psi. Using column chromatography, 500 mL of an external aqueous phase solution (pH 7.4) consisting of 250 mM sucrose solution and 50 mM histidine (pH adjusted with citric acid-sodium citrate-NaOH buffer) was used to replace the original ammonium sulfate solution in the external lipid phase, yielding a mometasone furoate + glycopyrronium bromide liposome suspension.

[0191] Add 206.3 mg of indacaterol acetate to the above liposome suspension and stir magnetically until dissolved. Incubate in a 37°C water bath for 1 h. Concentrate the drug solution to 20 mL using 0.9% NaCl tangential flow technology to obtain a beclomethasone + glycopyrronium bromide + formoterol fumarate nanoliposome suspension.

[0192] The liposome suspensions prepared in Examples 32-37 were observed to be free of precipitation, flocculation, and stratification, exhibiting uniform dispersion and good flowability, and displaying a noticeable opalescence under light. Particle size and encapsulation efficiency (referring to those not subjected to tangential flow ultrafiltration) were measured, and the results are shown in Tables 10-12. It is evident that the preparation method of this liposome suspension is effective and can be used for encapsulating inhaled ICS, LABA, and LAMA to improve their solubility, providing a superior nebulized inhalation formulation for the treatment of lung diseases.

[0193] Table 10. Particle size distribution and encapsulation efficiency results of Examples 32 and 33

[0194]

[0195] Table 11. Particle size distribution and encapsulation efficiency results of Examples 34 and 35

[0196]

[0197] Table 12. Particle size distribution and encapsulation efficiency results of Examples 36 and 37

[0198]

[0199]

[0200] For those skilled in the art, modifications and refinements can be made without departing from the scope of the claims of this invention, and such modifications and refinements also fall within the protection scope of this invention.

Claims

1. A nanoliposome suspension for soft mist inhalation characterized in that The active ingredients of the nanoliposome suspension are selected from the following combinations: Inhaled glucocorticoids, long / short-acting β2 receptor agonists and long / short-acting anticholinergic drugs; The active ingredient is encapsulated in nanoliposomes, which are prepared using cholesterol and phospholipids. The phospholipids are selected from one or more of the following: dipalmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylglycerol, dimyristoyl phosphatidylcholine, disqualoyl phosphatidylcholine, distearyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and phosphatidylethanolamine.

2. A nano-liposomal suspension for soft mist inhalation according to claim 1, wherein The active ingredients are inhaled glucocorticoids, long / short-acting β2 receptor agonists, and long / short-acting anticholinergic drugs.

3. A nanoliposome suspension for soft mist inhalation according to claim 1 or 2, wherein It also includes a stabilizer selected from one of Tween 80, poloxamer 407, L-ascorbyl palmitate, vitamin E polyethylene glycol succinate, and polyvinylpyrrolidone PVP 30, with a mass ratio of stabilizer to active ingredient of 1:50~70.

4. The nanoliposome suspension for soft fogging as described in claim 3, characterized in that, The stabilizer is vitamin E polyethylene glycol succinate.

5. A nanoliposome suspension for soft fogging as described in any one of claims 1 to 4, characterized in that, It also contains an osmotic pressure regulator, which is glucose or sodium chloride; the molar osmotic pressure concentration of the nanoliposome suspension is 240-360 mOsm / kg, and the pH value is 5.0-8.

0.

6. The nanoliposome suspension for soft fogging as described in claim 5, characterized in that... The preferred molar osmotic pressure concentration is 270-320 mOsm / kg, and the pH value is 6.0-7.

5.

7. A nanoliposome suspension for soft mist as described in any one of claims 1 to 6, characterized in that, The active ingredients are preferably inhaled corticosteroids, long / short-acting β2 receptor agonists, and long / short-acting anticholinergic drugs. The concentration of inhaled corticosteroids is 5-20 mg / mL; the concentration of long / short-acting β2 receptor agonists is 0.1-1 mg / mL; and the concentration of long / short-acting anticholinergic drugs is 0.1-1 mg / mL. The inhaled corticosteroids are selected from budesonide, cicosone, fluticasone propionate, fluticasone furoate, beclomethasone propionate, and mometasone furoate. The long / short-acting β2 receptor agonists are selected from formoterol fumarate, indacaterol, salmeterol, aforterol, and olodacaterol. Tarceva and salbutamol; the long / short-acting anticholinergic drugs are selected from glycopyrronium bromide, umemet bromide, tiotropium bromide, and ipratropium bromide. The preparation method of the nanoliposome suspension is as follows: first, prepare a nanoliposome suspension of inhaled glucocorticoids and long / short-acting anticholinergic drugs; then, add a long / short-acting β2 receptor agonist to the nanoliposome suspension containing the two active ingredients; incubate at 35℃~40℃ for 40min~80min; and concentrate the drug solution to a suitable volume using tangential flow technology. The prepared nanoliposomes have a particle size of 50~300nm and a drug-liposome ratio of 1:1~6.

8. The nanoliposome suspension for soft fogging as described in claim 7, characterized in that... For example, the inhaled corticosteroid is budesonide, the long / short-acting β2 receptor agonist is formoterol fumarate, and the long / short-acting anticholinergic drug is glycopyrronium bromide; the drug-to-lipid ratio is 1:

3.

9. The method for preparing a nanoliposome suspension for soft fogging as described in claim 7, wherein the active ingredients are budesonide, glycopyrronium bromide, and formoterol fumarate, characterized in that... The preparation method specifically includes the following steps: (1) Phospholipids, cholesterol, stabilizers, and inhaled glucocorticoids are dispersed and dissolved in an organic solvent to obtain a lipid organic phase solution; the inhaled glucocorticoids are dissolved in an organic solvent to obtain a drug-containing solution; the lipid solution and the drug-containing solution are mixed evenly to form an organic phase; the organic phase is evaporated to remove the organic solvent to obtain a drug-containing phospholipid film. (2) Dissolve glycopyrronium bromide in an aqueous solution with pH adjusted to 3.8-4.5 using citric acid-sodium citrate-NaOH buffer, add an osmotic pressure regulator, and stir until completely dissolved to form the aqueous phase; (3) The aqueous phase was poured into the drug-containing phospholipid film for hydration and dispersion. The resulting mixed solution was homogenized and dispersed using a microfluidic homogenizer at a homogenization pressure of 10,000 to 25,000 psi and homogenized 1 to 5 times. The external aqueous phase was replaced with a phosphate buffer solution of pH 7.3 to 7.6 to obtain a budesonide-glycerin bromide nanoliposome suspension. (4) Add formoterol fumarate to the nanoliposome suspension obtained in step (3), stir until dissolved, incubate at 35℃~40℃ for 1h, and concentrate the drug solution to a suitable volume using 0.9% NaCl tangential flow technology.

10. The application of a nanoliposome suspension for soft fogging as described in any one of claims 1 to 8 in drug delivery using a soft fogging device, wherein the nanoliposome suspension has a particle size of 0.5 to 5.0 μm after being atomized by the soft fogging device.

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