Sivelestat sodium suspension, sivelestat sodium freeze-dried powder, sivelestat sodium atomization inhalant and preparation method thereof

Abstract

The invention discloses a sivelestat sodium suspension, freeze-dried powder, an aerosol inhalant and a preparation method thereof, the sivelestat sodium suspension is prepared by shearing, stirring and dispersing a mixed solution containing an osmotic pressure regulator and a surfactant, sivelestat sodium and porous phospholipid particles, and homogenizing; the mass ratio of the sivelestat sodium to the porous phospholipid particles is (1.2-2): 1. The sivelestat sodium suspension disclosed by the invention is stable in system and long in uniform dispersion time, and the sivelestat sodium freeze-dried powder prepared by spray freeze-drying can still keep a suspension state after being redissolved and is excellent in redispersion performance; after the aerosol inhalant is atomized by an atomization device, the percentage of particles with the particle size of less than 5.0 microns is high, the fine particle fraction FPF exceeds 70%, the aerosol inhalant has excellent aerodynamic characteristics, more particles are deposited at the 4-5 level, and the aerosol inhalant is more suitable for going deep into pulmonary alveoli and lung tiny blood vessels and is used for treating acute lung injury / acute respiratory distress syndrome.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and more specifically, relates to a sivelestat sodium suspension, a lyophilized powder, an aerosol inhalation agent and a preparation method thereof. Background Art

[0002] ALI / ARDS refers to acute, progressive hypoxic respiratory failure caused by various pathogenic factors inside and outside the lungs other than cardiogenic factors. ALI is a serious clinical symptom that can develop into ARDS. The main pathological features of ALI / ARDS are protein-rich pulmonary edema and hyaline membrane formation in alveolar exudate caused by increased pulmonary microvascular permeability, accompanied by pulmonary interstitial fibrosis. Clinically, it manifests as refractory hypoxemia, respiratory rate and respiratory distress. Chest X-ray shows diffuse infiltration shadows in both lungs, and multiple organ dysfunction is often complicated in the later stage.

[0003] Sivelestat sodium is the only drug used for ALI / ARDS in the world. As a highly specific neutrophil elastase inhibitor, it can directly inhibit the core of the inflammatory response. Neutrophil elastase is a proteolytic enzyme that is freed from neutrophils that gather in the lungs. It can decompose lung connective tissue, increase lung vascular permeability, and induce ALIARDS. It can also promote the production of neutrophil chemokines, aggravate the inflammatory response, and is an important injury factor associated with ALIARDS of systemic inflammatory response syndrome. Clinical studies have shown that Sivelestat sodium can effectively reduce the lung injury score of ARDS patients, improve lung function, shorten the duration of mechanical ventilation and intensive care unit hospitalization, and reduce mortality.

[0004] Currently, only injections of sildenastat sodium are available on the market, which require continuous intravenous administration for 24 hours. It is prone to adverse reactions such as abnormal liver function and has the potential risk of inducing systemic toxic side effects. Preparing sildenastat sodium into an inhalation preparation increases the local concentration of the drug in the lungs, accelerates drug absorption through the dense capillary network in the lungs, and thus reduces the impact on other tissues throughout the body. It is the preferred method of administration for treating lung diseases. However, sildenastat sodium has poor water solubility and the clinical dosage is extremely large. Therefore, a nebulizer inhaler with a high drug loading and no solvent is the preferred dosage form for preparing sildenastat sodium into an inhalation preparation. For example, CN116115589A discloses a pharmaceutical composition of silivelestat sodium for atomization inhalation, wherein silivelestat sodium and the amphiphilic material distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in an organic solvent respectively, the two are mixed in proportion, and the organic solvent is removed by reduced pressure evaporation to obtain a drug-containing lipid film, which is dissolved in water and then a certain amount of mannitol is added, and then freeze-dried. When used, it is first re-dissolved with water, and then atomized and inhaled through an inhalation dosing device. CN116635018A discloses a silivelestat sodium lyophilized powder for atomization inhalation, which contains excipient mannitol, a pH regulator, an osmotic pressure regulator and a surfactant, and the solution is prepared at low temperature, and then freeze-dried. When used, the lyophilized powder is dissolved in water or a carrier, and a conventional inhalation device is used for atomization inhalation. CN117982414B discloses sivelestat sodium nanocrystals, dry powder, inhalation powder and preparation method thereof. Vitamin E polyethylene glycol succinate (TPGS) is used as a stabilizer, which is homogenized with an aqueous dispersion of sivelestat sodium under high pressure to prepare a nanocrystal suspension, which is solidified by spray freeze drying technology to obtain sivelestat sodium nanocrystal dry powder, which is then loaded into capsules.

[0005] However, the current nebulizer inhalation of sivelestat sodium still has the following problems: first, the uniform dispersion time of sivelestat sodium suspension is short, it is easy to settle after shaking, and after being made into lyophilized powder, the redispersion performance is poor, and the uniformity of droplets after atomization is poor, which will affect its dispersion and deposition during inhalation; second, the percentage of particles below 5 μm (fine particle fraction, FPF) in the suspension is low. When used for the treatment of ALI / ARDS, the aerodynamic characteristics are not enough, and more particles are deposited at levels 2 to 3, which cannot penetrate into the alveoli and tiny blood vessels of the lungs; third, the drug is released quickly in the body and needs to be administered continuously, which also has the potential risk of toxicity. Summary of the invention

[0006] Based on this, the object of the present invention is to provide a sivelestat sodium suspension with a long uniform dispersion time. The freeze-dried powder prepared using the suspension has good redispersion performance, a high percentage of fine particles in the prepared aerosol inhalation, excellent aerodynamic properties, and a moderate drug release rate.

[0007] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0008] The first aspect of the present invention provides a sivelestat sodium suspension, which is obtained by shearing, stirring and dispersing a mixed solution containing an osmotic pressure regulator and a surfactant with sivelestat sodium and porous phospholipid particles, and then homogenizing; the mass ratio of sivelestat sodium to porous phospholipid particles is 1.2 to 2:1.

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned sivelestat sodium suspension, comprising the following steps: adding sivelestat sodium and porous phospholipid particles to a mixed solution containing an osmotic pressure regulator and a surfactant, shearing and stirring to disperse, and then homogenizing.

[0010] The third aspect of the present invention provides a sildenastat sodium lyophilized powder, which is obtained by spray freeze-drying the sildenastat sodium suspension.

[0011] The fourth aspect of the present invention provides a sivelestat sodium nebulizer inhalation preparation, which comprises the above-mentioned sivelestat sodium lyophilized powder.

[0012] The present invention uses light porous phospholipid particles as carriers to load silvestat sodium drug crystals, and after shearing and stirring and dispersing with a mixed solution containing an osmotic pressure regulator and a surfactant, homogenization is performed to prepare a silvestat sodium suspension with a stable system and a uniform dispersion time of more than 5 minutes, and the longest dispersion time can exceed 20 minutes. The silvestat sodium suspension is spray-freeze-dried to obtain a silvestat sodium freeze-dried powder, and the silvestat sodium freeze-dried powder is made into an aerosol inhalation agent. After the freeze-dried powder is redissolved, it can still maintain a suspended state, does not settle for at least 24 hours, and has excellent redispersibility. After the aerosol inhalation agent is atomized by an atomizer, the fine particle fraction FPF (the percentage of particles with a particle size of less than 5.0 μm) exceeds 70%, has excellent aerodynamic characteristics, and more particles are deposited at levels 4 to 5, which is more suitable for penetrating into alveoli and pulmonary microvessels, and is used for the treatment of acute lung injury (ALI) / acute respiratory distress syndrome (ARDS).

[0013] The present invention controls the ratio of the sivelestat sodium drug to the light porous phospholipid particles within a certain range, and cooperates with a suitable shear dispersion process and a homogenization (especially microfluidization homogenization) process, so that the prepared sivelestat sodium suspension has a small PDI, a large Zata potential, high stability, and a long uniform dispersion time. After being prepared into an aerosol inhaler, it has a suitable particle size and a moderate drug release rate, thereby overcoming the problem of the need for continuous drug administration and the potential risk of toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is the in vitro release rate result of the sivelestat sodium suspension in Test Example 6 of the present invention.

[0015] Figure 2 The deposition results of silvelastin in each layer in Test Example 8 of the present invention are shown. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0017] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0018] Unless otherwise specified, the examples are based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions, and the raw materials and reagents used in the present invention are all commercially available.

[0019] In some embodiments of the present invention, a sivelestat sodium suspension is disclosed, which is obtained by shearing, stirring and dispersing a mixed solution containing an osmotic pressure regulator and a surfactant with sivelestat sodium and porous phospholipid particles, and then homogenizing; the mass ratio of sivelestat sodium to porous phospholipid particles is 1.2 to 2:1.

[0020] In some embodiments, the mass ratio of sivelestat sodium to porous phospholipid particles is 1.2-1.5:1, more preferably 1.2-1.4:1.

[0021] In some embodiments, the pH of the mixed solution is 6.5-8.5, preferably 6.5-7.5, and more preferably 6.5-7.0.

[0022] In some embodiments, the osmotic pressure regulator is sodium chloride and / or glucose, preferably sodium chloride.

[0023] In some embodiments, the surfactant is sodium dodecyl sulfate and / or Tween 80, preferably Tween 80.

[0024] In some embodiments, in the mixed solution, the concentration of sodium chloride is 8 mg / mL to 10 mg / mL, preferably 8.5 mg / mL to 9.5 mg / mL, and the concentration of Tween 80 is 0.01 mg / mL to 10.0 mg / mL, preferably 0.01 mg / mL to 5.0 mg / mL, and more preferably 1.5 mg / mL to 1.7 mg / mL.

[0025] In some embodiments, the mass volume ratio of the sivelestat sodium to the mixed solution is 40 mg to 60 mg: 1 mL, preferably 50 mg to 60 mg: 1 mL, and more preferably 55 mg to 60 mg: 1 mL.

[0026] In some embodiments, the stirring speed of the shear stirring is 10000 rpm to 12000 rpm, preferably 10000 rpm to 11000 rpm, and more preferably 10000 rpm to 10050 rpm.

[0027] In some embodiments, the shear stirring time is 10 min to 15 min, preferably 10 min to 13 min, and more preferably 10 min to 11 min.

[0028] In some embodiments, the homogenization is high-pressure homogenization or microfluidization, more preferably microfluidization. The sildenafil sodium and the porous phospholipid particles collide with each other in the Y-shaped homogenization chamber of the microfluidizer to ensure that the drug is effectively adsorbed to the surface and inside of the porous phospholipid particles to achieve efficient loading, and then the high shear force and turbulence of the Z-shaped homogenization chamber are used to achieve homogenization.

[0029] In some embodiments, the homogenization pressure of the microfluidizer is 15000psi to 20000psi, preferably 18000psi to 20000psi, and more preferably 19000psi to 20000psi.

[0030] In some embodiments, the microfluidizer is homogenized 2 to 3 times, more preferably 2 times.

[0031] In some embodiments, the d90 particle size of the porous phospholipid particles is 2 μm to 4 μm.

[0032] In some embodiments, the pore size of the porous phospholipid particles is 0.2 μm to 1 μm.

[0033] In some embodiments, the density of the porous phospholipid particles is 0.4 g / cm 3 ~0.5g / cm 3 .

[0034] In some of the embodiments, the porous phospholipid particles are prepared by the following method: DSPC and CaCl2 are added to 70°C to 85°C injection water in a molar ratio of 1 to 3:1 for dispersion, a porogen is added while stirring, shear stirring, high pressure homogenization is performed, and then spray drying is performed.

[0035] In some embodiments, the molar ratio of DSPC to CaCl2 is 1.5-2.5:1, more preferably 1.8-2.2:1.

[0036] In some of the embodiments, the mass and volume ratio of the DSPC and CaCl2 to the water for injection is 3-15 g:100 mL, preferably 3.6-13.5 g:100 mL, preferably 4-6 g:100 mL, and more preferably 4.5-5.5 g:100 mL.

[0037] In some embodiments, the porogen is perfluorooctyl bromide (PFOB) or perfluoropropane, preferably PFOB.

[0038] In some embodiments, the volume ratio of the injection water to the porogen is 1 to 4:1, preferably 2 to 4:1, and more preferably 3 to 4:1.

[0039] In some embodiments, the shear stirring speed is 8000 rpm to 10000 rpm.

[0040] In some embodiments, the pressure of the high-pressure homogenization is 140MPa-180MPa, preferably 150MPa-170MPa, and more preferably 160MPa-170MPa; the number of high-pressure homogenization is 3 to 6 times, preferably 3 to 5 times, and more preferably 4 to 5 times.

[0041] In some of the embodiments, the process parameters of the spray drying include: inlet temperature 90°C to 135°C; outlet temperature 68°C to 72°C; feed rate 2mL / min to 8mL / min, total gas flow rate 425L / min to 525L / min.

[0042] In other embodiments of the present invention, a method for preparing the above-mentioned sivelestat sodium suspension is disclosed, comprising the following steps: adding sivelestat sodium and porous phospholipid particles to a mixed solution containing an osmotic pressure regulator and a surfactant, dispersing by shearing and stirring, and then homogenizing.

[0043] In some embodiments, the stirring speed of the shear stirring is 10000 rpm to 12000 rpm, preferably 10000 rpm to 11000 rpm, and more preferably 10000 rpm to 10050 rpm.

[0044] In some embodiments, the shear stirring time is 10 min to 15 min, preferably 10 min to 13 min, and more preferably 10 min to 11 min.

[0045] In some embodiments, the homogenization is high pressure homogenization or microfluidization homogenization.

[0046] In some embodiments, the homogenization pressure of the microfluidizer is 15000psi to 20000psi, preferably 18000psi to 20000psi, and more preferably 19000psi to 20000psi.

[0047] In some embodiments, the pressure of the high-pressure homogenization is 1000bar~1400bar, and the time of high-pressure homogenization is 10min~30min; preferably, the pressure of the high-pressure homogenization is 1100bar~1300bar, and the time of high-pressure homogenization is 10min~20min; more preferably, the pressure of the high-pressure homogenization is 1150bar~1250bar, and the time of high-pressure homogenization is 10min~15min.

[0048] In other embodiments of the present invention, a sildenastat sodium lyophilized powder is disclosed, which is obtained by spray freeze-drying the sildenastat sodium suspension.

[0049] In other embodiments of the present invention, a silvestat sodium aerosol inhalation is disclosed, comprising the above-mentioned silvestat sodium lyophilized powder. The silvestat sodium aerosol inhalation is mixed with water for injection, and then atomized by a nebulizer (preferably a vibrating mesh nebulizer) and then inhaled for administration. According to the existing daily dosage of injection, the dosage is 5 mL, and the drug specification is 60 mg / mL.

[0050] The porous phospholipid particles for loading silvestat sodium described in the present invention can be prepared according to the suspension particle preparation method in the prior art (CN105193773 B). In the following examples, the specific preparation steps of the porous phospholipid particles used are as follows: 18.7g DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and 1.3g calcium chloride are homogenized in 400mL hot water (75°C) using a high shear agitator (8000rpm); 100mL PFOB is slowly added during the homogenization process; the crude emulsion is further homogenized 5 times using a high pressure homogenizer at a pressure of 170MPa; the emulsion is spray dried under nitrogen, with an inlet temperature of 95°C, an outlet temperature of 72°C, an emulsion feed rate of 2.4mL / min, and a total gas flow rate of 498L / min. The prepared porous phospholipid particles have a d90 particle size of about 2 μm, a pore size of 0.5 μm, and a density of 0.5 g / cm3 The density after loading sivelestat sodium is close to 1.0g / cm 3 , which can ensure good suspension performance and is not easy to settle.

[0051] The microfluidizer used in the present invention is manufactured by Noze Fluid Technology (Shanghai) Co., Ltd. and its model is nano-LA 75Y.

[0052] The present invention is further described in detail below with reference to specific embodiments.

[0053] Example 1 Sivelestat sodium suspension, lyophilized powder

[0054] This embodiment first provides a sivelestat sodium phospholipid complex suspension, which is prepared by the following steps:

[0055] (1) Sodium chloride and Tween 80 were added to 1 mL of water for injection in order to a concentration of 9 mg / mL and 1.6 mg / mL, respectively, and stirred to dissolve. The pH of the above solution was adjusted to 7.5 with a buffer solution made of sodium dihydrogen phosphate and sodium hydroxide (pH value was 12.5);

[0056] (2) adding sivelestat sodium (60 mg) and porous phospholipid particles (46.15 mg) to the above solution at a mass ratio of 1.3:1, and mechanically stirring at a speed of 10000 rpm for 10 min to achieve preliminary dispersion;

[0057] (3) The mixture was homogenized at 1200 bar for 15 min using a high pressure homogenizer to prepare a 60 mg / mL sivelestat sodium suspension.

[0058] In this embodiment, the prepared sivelestatin suspension is made into sivelestatin lyophilized powder by spray freeze drying, and the method is as follows:

[0059] The sildenafil sodium suspension was atomized by a 0.7 mm dual-fluid nozzle and then dispersed in liquid nitrogen for rapid freezing to form small ice crystal particles. The liquid nitrogen was continuously stirred during the dispersion process at a stirring speed of 500 rpm / min. The small ice crystal particles were then transferred to a freeze dryer (Buch LyovaporTM L-200pro) for spray freeze drying for 48 hours. The spray freezing parameters were: compressed air flow rate of 473 L / h, feed rate of 20% (about 4 mL / min), spray pressure of 5 bar, and freezing vacuum of 0.4 mbar.

[0060] Example 2 Sivelestat sodium suspension, lyophilized powder

[0061] This embodiment first provides a sivelestat sodium phospholipid complex suspension, which is prepared by the following steps:

[0062] (1) to (2) are the same as in Example 1;

[0063] (3) Using the Y-shaped interactive chamber or Z-shaped auxiliary chamber of the microfluidizer, homogenization was performed twice at a homogenization pressure of 20,000 psi, a homogenization speed of 53 mm / s, and a condensation cycle temperature of 25° C. to prepare a 60 mg / mL sivelestat sodium suspension.

[0064] In this example, the sivelestatin suspension was prepared into sivelestatin lyophilized powder by the spray freeze drying method, and the method was the same as that in Example 1.

[0065] Comparative Example 1 Sivelestat Sodium Suspension for Inhalation

[0066] This comparative example provides a sivelestat sodium suspension for inhalation, and its preparation method is as follows:

[0067] 1. Weigh Tween 80, sorbitan laurel, and NaCl, add water for injection and stir to dissolve, and prepare mixed solutions with concentrations of 14 mg / mL, 1.8 mg / mL, and 9 mg / mL, respectively. Then adjust the pH of the above solution to 7.5 with a buffer solution (pH value of 12.5) made of sodium dihydrogen phosphate and sodium hydroxide, and add sivelestat sodium and stir to mix evenly;

[0068] 2. Use a high-pressure homogenizer with a homogenization pressure of 1000 bar and a feed rate of 120 L / h to homogenize to the desired particle size (X50 = 1.38; X90 = 2.41 μm), to obtain a 60 mg / mL sildenastat sodium suspension. Comparative Example 2: Suspension of sildenastat sodium nanocrystals for inhalation

[0069] This comparative example provides a sivelestat sodium nanocrystalline suspension for inhalation, and the preparation method thereof is as follows:

[0070] 1. Dissolve sivelestat sodium in water for injection to prepare a solution with a concentration of 60 mg / mL, adjust the pH of the solution to 7.5 with sodium dihydrogen phosphate-sodium hydroxide buffer (pH 12.5), and then add sodium chloride and Tween 80 with final concentrations of 9 mg / mL and 1 mg / mL, respectively;

[0071] 2. Use a high shear stirrer at 12000 rpm for 10 min to perform preliminary dispersion to obtain a dispersion;

[0072] 3. The obtained dispersion was homogenized by a high-pressure homogenizer at a homogenization pressure of 1500 bar for 15 min to obtain a 60 mg / mL sivelestat sodium nanocrystalline suspension.

[0073] Comparative Example 3: Sivelestat Sodium Nanocrystal Suspension for Inhalation

[0074] This comparative example provides a sivelestat sodium nanocrystalline suspension for inhalation, and the preparation method thereof is as follows:

[0075] 1. Dissolve sivelestat sodium in water for injection to prepare a solution with a concentration of 60 mg / mL, adjust the pH to 7.5 with sodium dihydrogen phosphate-sodium hydroxide buffer, and add sodium chloride and Tween 80 with final concentrations of 9 mg / mL and 1 mg / mL respectively;

[0076] 2. Use a high shear stirrer at 12000 rpm for 10 min to perform preliminary dispersion to obtain a dispersion;

[0077] 3. Using the Y-shaped interactive cavity or Z-shaped auxiliary cavity of the microfluidizer, the obtained dispersion was homogenized twice at a homogenization pressure of 20,000 psi, a homogenization speed of 53 mm / s, and a condensation cycle temperature of 25°C to obtain a 60 mg / mL sivelestat sodium nanocrystalline suspension.

[0078] Comparative Example 4: Sivelestat Sodium Liposomal Suspension for Inhalation

[0079] In this comparative example, a sivelestat sodium liposome suspension for inhalation was prepared by a thin film dispersion method, and the preparation method thereof is as follows:

[0080] 1. Weigh 1.44 g DSPC, 0.36 g cholesterol, and 0.03 g α-tocopherol and dissolve them in chloroform. Weigh 1.8 g sivelestat sodium and add it to an appropriate amount of methanol, and stir until dissolved. Combine the solutions, evaporate under reduced pressure at low temperature to remove the organic solvent, and obtain a phospholipid membrane.

[0081] 2. Add 0.27g sodium chloride and 0.48g Tween 80 to sodium dihydrogen phosphate-sodium hydroxide buffer with a pH of 7.5 to obtain an aqueous phase. Slowly add the aqueous phase to the phospholipid membrane and gently shake the flask to allow the phospholipid membrane to mix and hydrate with the aqueous phase to obtain an emulsion;

[0082] 3. Using the Y-shaped interactive chamber or Z-shaped auxiliary chamber of the microfluidizer, the obtained emulsion was homogenized twice under the conditions of a homogenization pressure of 15000 psi, a homogenization speed of 53 mm / s, and a condensation cycle temperature of 25°C;

[0083] 4. Remove the organic solvent by nitrogen gas, and use 0.9% NaCl solution to pass through a tangential flow ultrafiltration system (hollow fiber column diaphragm area 118cm 2 , fiber inner diameter 0.5 mm) to remove free drugs and concentrate the volume to 30 mL;

[0084] 5. Sterile filter with a sterile 0.22 μm membrane filter and refrigerate at 4°C for 2 h to obtain a 60 mg / mL sivelestat sodium liposome suspension.

[0085] Comparative Example 5: Sivelestat Sodium Liposomal Suspension for Inhalation

[0086] In this comparative example, a sivelestat sodium liposome suspension for inhalation was prepared by emulsification solidification method, and the preparation method thereof is as follows:

[0087] 1. Weigh 1.44 g DSPC, 0.36 g cholesterol, and 0.03 g α-tocopherol and dissolve them in chloroform. Weigh 1.8 g sivelestat sodium and add it to an appropriate amount of methanol, and stir until dissolved. Combine the solutions to obtain an organic phase solution.

[0088] 2. Add 0.27 g sodium chloride and 0.48 g Tween 80 to water for injection in sequence, stir to dissolve, and adjust the pH value of the above solution to 7.5 with sodium dihydrogen phosphate-sodium hydroxide buffer (pH value is 12.5) to obtain an aqueous phase;

[0089] 3. In a 45°C water bath and under continuous magnetic stirring, slowly drip the organic phase into the aqueous phase to form an O / W emulsion;

[0090] 4. Using the Y-shaped interactive chamber or Z-shaped auxiliary chamber of the microfluidizer, the obtained emulsion was homogenized twice under the conditions of a homogenization pressure of 15000 psi, a homogenization speed of 53 mm / s, and a condensation cycle temperature of 25°C;

[0091] 5. Remove the organic solvent by nitrogen gas, and use 0.9% NaCl solution to pass through a tangential flow ultrafiltration system (hollow fiber column diaphragm area 118cm 2 , fiber inner diameter 0.5 mm) to remove free drugs and concentrate the volume to 30 mL;

[0092] 6. Sterilize and filter with a sterile 0.22 μm membrane filter and refrigerate at 4°C for 2 h to obtain a 60 mg / mL sivelestat sodium liposome suspension.

[0093] Test Example 1 Investigation of Suspension Performance of Sivelestat Sodium Suspension

[0094] 1. Sedimentation and stratification conditions and time: The suspension samples of Examples 1 to 2 and Comparative Examples 1 to 5 were shaken for 30 seconds and then allowed to stand to examine the sedimentation and stratification conditions and time of the samples.

[0095] 2. Average particle size, polydispersity index (PDI), and Zeta potential: Take the suspension samples of Examples 1 to 2 and Comparative Examples 1 to 5, dilute them 100 times with pure water until they are clear and transparent, use a pipette to draw 1 mL into a quartz cuvette, place it in a Malvern nanoparticle sizer, and use dynamic light scattering to determine the particle size, polydispersity index (PDI) and Zeta potential of the sivelestat sodium suspension. The measurements were performed three times in parallel and the average value was taken.

[0096] The results are shown in Table 1.

[0097] Table 1 Suspension characteristics and particle size results of sivelestat sodium suspension

[0098]

[0099] From the results in Table 1, it can be seen that the silvestat sodium suspension (Comparative Example 1) prepared by conventional methods is very easy to settle, and PDI>0.3, the absolute value of Zeta potential is small (less than 30mV), and the system is unstable; silvestat sodium nanocrystalline suspension (Comparative Examples 2, 3), silvestat sodium liposome suspension (Comparative Examples 4, 5) and silvestat sodium phospholipid complex suspension (Examples 1, 2) can all maintain a uniform dispersion state of ≥3min. Among them, the silvestat sodium suspension (Examples 1, 2) using porous phospholipid particles as carrier load can be uniformly dispersed in an aqueous solution for more than 20min after shaking, which far exceeds the dispersion time of silvestat sodium nanocrystalline suspension (Comparative Examples 2, 3) and silvestat sodium liposome suspension (Comparative Examples 4, 5).

[0100] Except for comparative example 1, the PDI of suspensions of different forms (nanocrystals, liposomes, phospholipid complexes) can be maintained below 0.3, the absolute value of the Zeta potential is above 30mV, the system is stable, but the particle size difference is quite significant, and the particle size of the silvesta sodium phospholipid complex suspension (Examples 1 and 2) is significantly larger than that of the silvesta sodium nanocrystal suspension (Comparative Examples 2 and 3) and the silvesta sodium liposome suspension (Comparative Examples 4 and 5). The smaller the drug particle size, the larger its specific surface area, the faster the drug is released in the body, and continuous administration is required, and there is a potential risk of toxicity. Therefore, the silvesta sodium phospholipid complex suspension is more in line with medication needs.

[0101] Test Example 2 Redispersibility of Sivelestat Sodium Lyophilized Powder

[0102] 0.3 g of lyophilized powder of sivelestat sodium (preparation process is the same as that of Example 1) of Examples 1 to 2 and Comparative Examples 2 to 5 was accurately weighed and dispersed in 10 mL of pure water, and the changes in particle size, PDI and Zeta potential of lyophilized powder of sivelestat sodium after redispersion were investigated, and the redispersion index was calculated according to formula (2). The results are shown in Table 2.

[0103]

[0104] Table 2 Redispersion results after reconstitution of sildenafil sodium lyophilized powder

[0105] No. Average particle size (nm) PDI Zeta potential (mV) Rindex Example 1 2506 0.19 -42.29 1.05 Example 2 2108 0.17 -43.81 1.03 Comparative Example 2 962 0.27 -38.27 1.43 Comparative Example 3 695 0.28 -32.45 1.67 Comparative Example 4 283 0.26 -39.24 1.27 Comparative Example 5 252 0.23 -38.63 1.09

[0106] The closer the redispersion index is to 1, the better the redispersion performance is, the particle PDI is less than 0.3, and the absolute value of the Zeta potential is greater than 30, indicating that the particles are more stable. As can be seen from Table 2, after each lyophilized powder is redispersed, it can still maintain good stability, but the redispersion performance of the lyophilized powder made from the silvesta sodium phospholipid complex suspension (Examples 1 and 2) is significantly better than the redispersion performance of the lyophilized powder made from the silvesta sodium liposome suspension (Comparative Examples 4 and 5) and the silvesta sodium nanocrystalline suspension (Comparative Examples 2 and 3). Compared with the silvesta sodium phospholipid complex suspension (Example 1) prepared by high-pressure homogenization, the silvesta sodium phospholipid complex suspension (Example 2) prepared by microfluidization homogenization has a smaller particle size, better stability, and better dispersion performance.

[0107] Experimental Example 3 Comparison of the effects of the ratio of drug to porous phospholipid particles on sivelestat sodium suspension

[0108] The sivelestat sodium suspension was prepared according to the method of Example 2, except that in step (2), the mass ratios of sivelestat sodium to porous phospholipid particles were 2:1, 1.4:1, 1.3:1, 1.2:1, 1:1, and 1:2, respectively. The suspension properties of each suspension were investigated according to the method of Experimental Example 1, and the results are shown in Table 3.

[0109] Table 3 Dispersion state, particle size, PDI and Zeta potential of each suspension

[0110]

[0111] The mass ratio of sivelestat sodium to porous phospholipid particles has a significant effect on the suspension state of the suspension. If the proportion of sivelestat sodium added is too high, sedimentation and stratification will easily occur. If the proportion of porous phospholipid particles is too high, stratification and floating will easily occur. From the results in Table 3, it can be seen that when the mass ratio of sivelestat sodium to porous phospholipid particles is 1:1 or 1:2, PDI>0.3, or the absolute value of Zeta potential is less than 30mV, the suspension is unstable, the uniform dispersion time is short, and the dispersion performance is poor; when the mass ratio of sivelestat sodium to carrier particles exceeds 1:1, the mixture is shaken for 30s and then allowed to stand for ≥3min to maintain uniform dispersion, and PDI≤0.3, the absolute value of Zeta potential is greater than 30mV, and the stable state of the suspension can be achieved. Among them, when the mass ratio of sivelestat sodium to porous phospholipid particles is 1.3:1, the prepared suspension has the longest uniform dispersion time and the smallest particle size, and at the same time, PDI is lower than 0.3 and the absolute value of Zeta potential is larger.

[0112] Experimental Example 4 Comparison of the Effect of Preparation Process on Sivelestat Sodium Suspension

[0113] The sivelestat sodium suspension was prepared according to the method of Example 2, except that different dispersion or homogenization processes were used in step (2) or step (3):

[0114] Group 1: In step (2), the stirring speeds were 8000 rpm, 10000 rpm, and 12000 rpm, respectively, and the other steps were the same as in Example 1.

[0115] Group 2: In step (2), the stirring time is 5 min, 10 min, and 15 min, respectively, and the other steps are the same as in Example 1.

[0116] Group 3: In step (3), the microfluidization homogenization pressures were 15000psi, 20000psi, 25000psi, and 30000psi, respectively, and the other steps were the same as in Example 2.

[0117] Group 4: In step (3), the microfluidization homogenization was performed once, twice, and three times respectively, and the other steps were the same as those in Example 2.

[0118] The suspension performance of each suspension was investigated according to the method of Test Example 1. The results are shown in Table 4.

[0119] Table 4 Effects of different dispersion and homogenization processes on sivelestat sodium suspension

[0120]

[0121]

[0122] The results in Table 4 show that the speed and time of high shear stirring, the homogenization pressure of the microjet, and the number of homogenization times will have a great influence on the uniformity of the sildenafil suspension. When the speed of the high shear stirrer exceeds 10,000 rpm and the stirring time exceeds 10 minutes; the homogenization pressure of the microjet homogenization is lower than 20,000 psi, and the number of homogenization times is greater than 2 times, the stable state of the suspension can be achieved. Under the conditions of a stirring speed of 10,000 rpm and a stirring time of 10 minutes, the PDI is the smallest. When the microjet homogenization pressure is 20,000 psi and the homogenization is repeated twice, the particle size of the sildenafil sodium suspension is moderate, PDI is less than 0.3, and the absolute value of the Zeta potential is large. The sildenafil suspension prepared under this process condition is the most stable.

[0123] Experimental Example 5 Comparison of the Effect of pH Value on Sivelestat Sodium Suspension

[0124] The sivelestat sodium suspension was prepared according to the method of Example 2, except that in step (1), the pH of the solution was adjusted to 6.5, 7.0, 7.5, 8.0, and 8.5, respectively, using a buffer solution made of sodium dihydrogen phosphate and sodium hydroxide (pH value is 12.5).

[0125] Considering that sildenastat sodium is easily hydrolyzed, pH value has a great influence on the stability of sildenastat sodium nebulizer inhalation. In order to ensure the stability during the freeze-drying process and use, the sildenastat sodium suspension prepared at different pH values ​​was placed for different time periods and then tested for related substances. HPLC was used for quantitative analysis, the chromatographic column was Diamonsil C18 (200mm×4.6mm, 5μm), the mobile phase was 0.02mol / L potassium dihydrogen phosphate buffered aqueous solution (pH=3.5)-acetonitrile (55:45), and the detection wavelength was 208nm.

[0126] The temperature of the water for injection was controlled to be 20°C, and the sildenastat sodium suspension was placed at 20°C for 24 hours. The changes in impurity A (impurities produced by hydrolysis) and total impurities of the sildenastat sodium suspension were investigated within 0, 0.5h, 1h, 2h, 4h, 6h, 12h, and 24h. The results are shown in Table 5.

[0127] Table 5 Changes of related substances in sivelestat sodium suspension after 24 hours of storage

[0128]

[0129] From the results in Table 5, it can be seen that impurity A and total impurities of the sivelestat sodium suspension gradually increase during the storage process. When the pH of the solution is between 6.5 and 7.5, the impurity A and total impurities of the prepared sivelestat sodium suspension increase slowly and are relatively stable.

[0130] Test Example 6 In vitro release study of sivelestat sodium suspension

[0131] The in vitro release of the sivelestat sodium suspension was investigated using a circulation cell and a dialysis bag. The in vitro dissolution rate of the raw material drug in the lyophilized powder prepared from the sivelestat sodium suspensions of Examples 1 to 2 and Comparative Examples 2 to 5 (the preparation method is the same as that of Example 1) 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).

[0132] Weigh 3 portions of freeze-dried powder equivalent to 0.3g of sivelestat sodium accurately, reconstitute with 5mL of water for injection, transfer into a dialysis bag with MWCO=3000Da (cellulose ester), put into a circulation pool, add 200mL of artificial lung fluid, and conduct drug release investigation by closed-loop method at 37°C. Take 1mL of liquid at 0, 1min, 5min, 10min, 15min, 20min, 30min, 60min, 120min, 240min, and 360min, and add 1mL of artificial lung fluid to the release medium. Determine the content of sivelestat sodium by HPLC (parameters are the same as those in Test Example 5).

[0133] The results are as follows Figure 1 As shown. Figure 1 It can be seen that in the artificial lung fluid, the sildenastat sodium in the sildenastat sodium nanocrystalline suspension (Comparative Examples 2-3), the sildenastat sodium liposome suspension (Comparative Examples 4-5), and the sildenastat sodium phospholipid complex suspension (Examples 1-2) can be gradually released, among which the sildenastat sodium nanocrystalline suspension has the fastest release rate, and more than 90% of the drug can be released in about 30 minutes (Comparative Examples 2-3), the sildenastat sodium liposome suspension has a release rate of more than 90% within 6 hours, and the release is relatively sufficient (Comparative Examples 4-5), and the sildenastat sodium phospholipid complex suspension has a moderate drug release rate, and the release rate reaches 90% in 2 hours (Examples 1-2).

[0134] Test Example 8 Investigation of the atomization characteristics of silvestat sodium lyophilized powder

[0135] 1. Total delivery volume and delivery efficiency

[0136] The lyophilized powder samples of silvestat sodium of Examples 1 to 2 and Comparative Examples 2 to 5 (the preparation methods are the same as those of Example 1) were reconstituted with water for injection and atomized with a PARI eflow nebulizer. The operation was carried out in accordance with the 0111 of the General Rules of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia, and the nebulizer was connected. The respiratory characteristics of the respiratory simulator were set to the adult mode. The first atomization lasted for 60 seconds, the first filter paper was taken out, and the second filter paper was replaced and the atomization was continued. The atomization time was 5 minutes. Each filter paper was taken out, and the first filter paper, the second filter paper, the exhaled filter paper, the atomization cup, the filter paper device, and the adapter were fully washed with methanol, and the active substances were quantitatively collected. Three groups were made in parallel for each atomization time. HPLC was used for quantitative analysis, the chromatographic column was Diamonsil C18 (200mm×4.6mm, 5μm), the mobile phase was 0.02mol / L potassium dihydrogen phosphate buffered aqueous solution (pH=3.5)-acetonitrile (55:45), and the detection wavelength was 208nm. Accurately measure 10 μL of the reference solution and the test solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms, calculate the mass of sivelestat sodium in each device and filter paper by the external standard method, and calculate the total delivery amount and delivery efficiency. The results are shown in Table 6.

[0137] 2. Aerodynamic particle size distribution investigation

[0138] Referring to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Rules 0951, a new generation of pharmaceutical cascade impactors (NGI) were used to determine the aerodynamic particle size of silivelestat sodium lyophilized powder: 0.3 g of silivelestat sodium lyophilized powder was weighed, reconstituted with 5 mL of water for injection, and atomized with a PARI eflow nebulizer. The assembled impactor and L-type connecting tube were precooled in a cooling device (5 ° C) for at least 90 minutes, and the measurement was started within 5 minutes after being taken out of the cooling device, and the air tightness was tested. The flow rate was set to 15L / min for nebulization for 3 minutes. After the atomization was completed, it was fully washed with methanol, and the active substances at each level were collected, and 3 groups were made in parallel. HPLC was used for quantitative analysis, and the chromatographic conditions were the same as above. Copley's data analysis software version 3.10 was used to analyze the data, calculate the fine particle dose (Fine Particle Does, FPD), fine particle fraction (Fine Particle Fraction, FPF), and mass median aerodynamic diameter (Median Mass Aerodynamic Diameter, MMAD), and all samples were tested 3 times. The results are shown in Table 6, and the percentage of deposition at each level is shown in Figure 2 .

[0139] Table 6 Atomization characteristics investigation results

[0140]

[0141] From the results in Table 6, it can be seen that the freeze-dried powders prepared by using the silicene sodium nanocrystalline suspension (Comparative Examples 2-3), the silicene sodium liposome suspension (Comparative Examples 4-5), and the silicene sodium phospholipid complex suspension (Examples 1-2) have no significant differences in atomization characteristics such as delivery rate, total delivery amount, fine particle dose, and mass median aerodynamic diameter. However, the freeze-dried powder prepared by the silicene sodium phospholipid complex suspension (Examples 1-2) has a significantly higher percentage of particles below 5 μm, and the fine particle fraction FPF can reach more than 70%. And from Figure 2 It can be seen that the lyophilized powder made from the silivrestat sodium nanocrystalline suspension (Comparative Examples 2-3) has more particles deposited at levels 2-3, the lyophilized powder made from the silivrestat sodium liposome suspension (Comparative Examples 4-5) has more particles deposited at levels 2-4, and the lyophilized powder made from the silivrestat sodium phospholipid complex suspension (Examples 1-2) has more particles deposited at levels 4-5, indicating that the lyophilized powder made from the silivrestat sodium phospholipid complex suspension of the present invention has better aerodynamic properties, is more suitable for penetrating into the alveoli and pulmonary microvessels, and is used for the treatment of ALI / ARDS.

[0142] Test Example 9 Stability Study of Sivelestat Sodium Lyophilized Powder

[0143] This test example investigated the stability of the lyophilized powders prepared from the sivelestat sodium suspensions of Example 2, Comparative Example 3 and Comparative Example 5 (the preparation methods were the same as those of Example 1) at 0, 1, 2, 3 and 6 months, respectively. The results are shown in Table 7.

[0144] Table 7 Stability results of sildenafil sodium lyophilized powder

[0145]

[0146] It can be seen from Table 7 that the lyophilized powders made from the sivelestat sodium nanocrystalline suspension, the sivelestat sodium liposome suspension, and the sivelestat sodium phospholipid complex suspension had impurity A less than 1.0% and total impurities less than 2% within 6 months, and all had good stability.

[0147] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0148] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A sivelestat sodium suspension, characterized in that, The invention is prepared by shearing, stirring and dispersing a mixed solution containing an osmotic pressure regulator and a surfactant, sivelestat sodium and porous phospholipid particles, and then homogenizing the mixture; the mass ratio of sivelestat sodium to porous phospholipid particles is 1.2 to 2:

1.

2. The sivelestat sodium suspension according to claim 1, characterized in that The mass ratio of sivelestat sodium to porous phospholipid particles is 1.2-1.5:1, more preferably 1.2-1.4:

1.

3. The sivelestat sodium suspension according to claim 1, characterized in that The pH of the mixed solution is 6.5 to 8.5, preferably 6.5 to 7.5, more preferably 6.5 to 7.0; And / or, the osmotic pressure regulator is sodium chloride and / or glucose, preferably sodium chloride; And / or, the surfactant is sodium dodecyl sulfate and / or Tween 80, preferably Tween 80; And / or, the mass volume ratio of the sivelestat sodium to the mixed solution is 40 mg to 60 mg: 1 mL, preferably 50 mg to 60 mg: 1 mL, and more preferably 55 mg to 60 mg: 1 mL; And / or, the d90 particle size of the porous phospholipid particles is 2 μm to 4 μm; the pore size of the porous phospholipid particles is 0.2 μm to 1 μm; the density of the porous phospholipid particles is 0.4 g / cm 3 ~0.5g / cm 3 ; And / or, the porous phospholipid particles are prepared by the following method: DSPC and CaCl2 are added to 70°C to 85°C injection water in a molar ratio of 1 to 3:1 for dispersion, a porogen is added while stirring, shear stirring, high pressure homogenization is performed, and then spray drying is performed.

4. The sivelestat sodium suspension according to claim 3, characterized in that In the mixed solution, the concentration of sodium chloride is 8 mg / mL to 10 mg / mL, preferably 8.5 mg / mL to 9.5 mg / mL; the concentration of Tween 80 is 0.01 mg / mL to 10.0 mg / mL, preferably 0.01 mg / mL to 5.0 mg / mL, more preferably 1.5 mg / mL to 1.7 mg / mL; and / or, the molar ratio of DSPC to CaCl2 is 1.5 to 2.5:1, more preferably 1.8 to 2.2:1; and / or, the mass and volume ratio of the DSPC and CaCl2 to the water for injection is 3-15 g:100 mL, preferably 3.6-13.5 g:100 mL, preferably 4-6 g:100 mL, more preferably 4.5-5.5 g:100 mL; And / or, the porogen is perfluorooctyl bromide or perfluoropropane, preferably perfluorooctyl bromide; the volume ratio of the water for injection to the porogen is 1 to 4:1, preferably 2 to 4:1, and more preferably 3 to 4:

1. And / or, the shear stirring speed is 8000rpm~10000rpm; And / or, the pressure of the high-pressure homogenization is 140MPa to 180MPa, preferably 150MPa to 170MPa, more preferably 160MPa to 170MPa; the number of high-pressure homogenization is 3 to 6 times, preferably 3 to 5 times, more preferably 4 to 5 times; And / or, the process parameters of the spray drying include: inlet temperature 90°C to 135°C; outlet temperature 68°C to 72°C; feed rate 2mL / min to 8mL / min, total gas flow rate 425L / min to 525L / min.

5. The sivelestat sodium suspension according to claim 1, characterized in that The stirring speed of the shear stirring is 10000rpm to 12000rpm, preferably 10000rpm to 11000rpm, more preferably 10000rpm to 10050rpm; And / or, the stirring time of the shear stirring is 10 min to 15 min, preferably 10 min to 13 min, more preferably 10 min to 11 min; And / or, the homogenization is high pressure homogenization or microfluidization, more preferably microfluidization.

6. The sivelestat sodium suspension according to claim 5, characterized in that The homogenization pressure of the microfluidizer is 15000psi to 20000psi, preferably 18000psi to 20000psi, more preferably 19000psi to 20000psi; And / or, the speed of the microfluidization homogenization is 20 mm / s to 70 mm / s, preferably 40 mm / s to 60 mm / s; And / or, the microfluidization homogenization is performed 2 to 3 times, preferably 2 times.

7. A method for preparing the sivelestat sodium suspension according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: adding sivelestat sodium and porous phospholipid particles into a mixed solution containing an osmotic pressure regulator and a surfactant, dispersing by shearing, stirring and then homogenizing.

8. The method for preparing the sivelestat sodium suspension according to claim 7, characterized in that: The stirring speed of the shear stirring is 10000rpm to 12000rpm, preferably 10000rpm to 11000rpm, more preferably 10000rpm to 10050rpm; And / or, the stirring speed of the shear stirring is 10 min to 15 min, preferably 10 min to 13 min, more preferably 10 min to 11 min; And / or, the homogenization is high pressure homogenization or microfluidization homogenization; Preferably, the pressure of the high-pressure homogenization is 1000 bar to 1400 bar, and the time of the high-pressure homogenization is 10 min to 30 min; preferably, the pressure of the high-pressure homogenization is 1100 bar to 1300 bar, and the time of the high-pressure homogenization is 10 min to 20 min; more preferably, the pressure of the high-pressure homogenization is 1150 bar to 1250 bar, and the time of the high-pressure homogenization is 10 min to 15 min; Preferably, the homogenization pressure of the microjet homogenization is 15000psi to 20000psi, preferably 18000psi to 20000psi, and more preferably 19000psi to 20000psi; the speed of the microjet homogenization is 20mm / s to 70mm / s, preferably 40mm / s to 60mm / s; the homogenization times of the microjet homogenization are 2 to 3 times, preferably 2 times.

9. A lyophilized powder of sivelestat sodium, characterized in that: The sivelestat sodium suspension is obtained by spray freeze drying the sivelestat sodium suspension according to any one of claims 1 to 6.

10. A sivelestat sodium aerosol inhalation agent, characterized in that: The invention comprises the lyophilized powder of sivelestat sodium as claimed in claim 9.

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