Pressure-sensitive multivesicular liposomes for intervention of high altitude pulmonary edema and preparation method and application thereof

Abstract

The present application relates to a kind of pressure-sensitive multivesicular liposomes for intervening high altitude pulmonary edema and its preparation method and application.The multivesicular liposome is composed of cholesterol, amphiphilic phospholipid, negative charge phospholipid, neutral lipid, and calcium ion channel inhibitor and phosphoric acid in the water phase in the multivesicular liposome.The multivesicular liposome of the present application effectively solves the problem of high altitude pulmonary edema onset and timely intervention, and can also effectively relieve the pathological state of high altitude pulmonary edema.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pressure-sensitive multivesicular liposome for intervening in high-altitude pulmonary edema, its preparation method, and its application. Background Technology

[0002] High-altitude pulmonary edema (HAPE) is a common disease among long-term residents of high-altitude areas and those entering such areas for the first time. It has a high incidence and rapid onset. Conventional treatments typically involve supplemental oxygen or evacuation from the high-altitude area, or prevention with calcium channel blockers such as nifedipine. These treatments are often delayed, usually initiated after the patient develops obvious symptoms. Even a delay of just half an hour can lead to death. Therefore, timely treatment of HAPE is crucial for its prevention and control.

[0003] Calcium channel inhibitors can suppress the influx of calcium ions into cells under hypoxic conditions, thereby reducing vascular tone, alleviating the elevation of pulmonary artery pressure under hypoxic conditions, and subsequently alleviating the occurrence and progression of HAPE. Multivesicular liposomes, with particle sizes ranging from 5-100 μm, can induce a pulmonary embolism effect upon intravenous injection, thus enriching calcium channel inhibitors in the lungs. However, ordinary multivesicular liposomes release drugs slowly in vivo; for example, the sustained-release time of commercially available bupivacaine liposomes is 72 hours; the sustained-release time of ketorolac multivesicular liposomes disclosed in Chinese patent application CN113509440A is as long as 150 hours. During acute HAPE attacks, a large amount of drug needs to be rapidly released from liposomes to achieve rapid intervention, but the sustained-release effect of current multivesicular liposomes is insufficient for achieving rapid intervention.

[0004] Currently, there is an urgent need for a multivesicular liposome that can rapidly release drugs during a HAPE attack. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this application provides a pressure-sensitive multivesicular liposome for intervening in high-altitude pulmonary edema. This liposome is sensitive to hydrostatic pressure, unlike previously disclosed multivesicular liposomes. Under normal physiological conditions, the multivesicular liposome disclosed in this invention can slowly release calcium channel inhibitors, playing a preventative role in the occurrence of high-altitude pulmonary edema (HAPE). In the early stages of HAPE, the multivesicular liposome can spontaneously collapse under elevated pulmonary artery pressure (greater than 20 mmHg), thereby releasing the drug it encapsulates, achieving immediate relief of elevated pulmonary artery pressure and preventing high-altitude pulmonary edema caused by pulmonary vascular rupture due to high pulmonary artery pressure. Therefore, by using this pulmonary artery pressure-sensitive multivesicular liposome to carry a calcium channel blocker with rapid vasodilatory effects, immediate intervention for the onset of high-altitude pulmonary edema can be achieved.

[0006] The present invention is achieved by the following technical solution.

[0007] On one hand, the present invention provides a pressure-sensitive multivesicular liposome for intervention in high-altitude pulmonary edema, which consists of a lipid phase and an inner aqueous phase, wherein the lipid phase consists of cholesterol, amphiphilic phospholipids, negatively charged phospholipids, and neutral lipids, and the inner aqueous phase consists of calcium ion channel inhibitors and phosphate.

[0008] Preferably, the amphiphilic phospholipids include one or more of synthetic phospholipids and natural phospholipids;

[0009] Preferably, the synthetic phospholipid is disorhodophosphatidylcholine, and the natural phospholipid is egg yolk lecithin, soybean lecithin, hydrogenated lecithin, or cephalin natural phospholipid.

[0010] Preferably, the amphiphilic phospholipids account for 50 w / w%-70 w / w of the lipid phase (total lipid composition) of the multivesicular liposomes.

[0011] Preferably, the negatively charged phospholipids include one or more of egg yolk phosphatidylglycerol, distearate phosphatidylglycerol, 1,2-dipalmitoyl phosphatidylglycerol, and dilauroyl phosphatidylglycerol.

[0012] Preferably, the negatively charged phospholipids account for 1 w / w%-10 w / w% of the lipid phase (total lipid component) of the multivesicular liposomes, more preferably 3 w / w%-7 w / w%.

[0013] Preferably, the neutral lipids include one or more of tricaprylic acid glyceride, trioleic acid glyceride, 1,3-dicapryloylglycerol, monooleic acid glyceride, dioleic acid glyceride, and triolein, with tricaprylic acid glyceride being the most preferred.

[0014] Preferably, the neutral lipids account for 1.9 w / w%-3.7 w / w of the lipid phase (total lipid component) of the multivesicular liposomes.

[0015] Preferably, the calcium channel inhibitor includes one or more of nifedipine, amlodipine, felodipine, verapamil, and diltiazem, and more preferably one or more of nifedipine, amlodipine, felodipine, and verapamil. The above drugs may be present in various salt forms.

[0016] Preferably, the mass ratio of the calcium ion channel inhibitor to the total mass of the cholesterol, the amphiphilic phospholipid, the negatively charged phospholipid, and the neutral lipids is 1:5-1:20, and more preferably 1:7-1:15.

[0017] Preferably, the multivesicular liposome is composed of multiple vesicles arranged in a honeycomb pattern. The areas where the multiple vesicle chambers meet and accumulate are composed of neutral lipids (i.e., the neutral lipids fill the gaps between the vesicles in the multivesicular liposome to maintain the stability of the multivesicular structure). The amphiphilic phospholipids and cholesterol are the main components forming the vesicle walls. The negatively charged phospholipids are used to form a charge barrier for the vesicle walls to inhibit the fusion of the inner vesicle walls.

[0018] Preferably, the particle size distribution of the multivesicular liposomes is 5-50 μm.

[0019] The multivesicular liposomes of the present invention can slowly release calcium ion channel inhibitors under normal physiological conditions, playing a preventive role in the occurrence of HAPE. In the early stage of HAPE, the multivesicular liposomes can spontaneously collapse under the action of elevated hydrostatic pressure or pulmonary artery pressure (greater than 20 mmHg), thereby releasing the drugs encapsulated inside, such as calcium ion channel blockers, to achieve the effect of relieving elevated pulmonary artery pressure in the first instance and avoid high-altitude pulmonary edema caused by pulmonary vascular rupture due to high pulmonary artery pressure.

[0020] The vesicle liposomes of the present invention can take effect rapidly during HAPE attacks.

[0021] On the other hand, the method for preparing the above-mentioned multivesicular liposomes of the present invention includes the following steps:

[0022] Preparation of colostrum: Amphiphilic phospholipids, cholesterol, negatively charged phospholipids, and neutral lipids were dissolved in an organic solvent to obtain an oil phase; calcium ion channel inhibitors were dissolved in an aqueous phosphoric acid solution to obtain an inner aqueous phase; the oil phase and the inner aqueous phase were mixed and homogenized by high-speed shearing to obtain the colostrum.

[0023] Preparation of double emulsion: L-lysine is dissolved in water to obtain an external aqueous phase containing L-lysine aqueous solution. The primary emulsion is injected into the external aqueous phase and homogenized by high-speed shearing to obtain a double emulsion solution.

[0024] Formation of multivesicular liposomes: The double emulsion solution was placed in a 37°C water bath, nitrogen gas was introduced to remove the organic solvent, and L-lysine in the external aqueous phase was removed by ultrafiltration or dialysis to obtain multivesicular liposomes.

[0025] Preferably, the organic solvent is selected from dichloromethane, trichloromethane, and acetone, with dichloromethane being the most preferred.

[0026] Preferably, in the preparation of colostrum, the total concentration of lipid components (i.e., amphiphilic phospholipids, cholesterol, negatively charged phospholipids, and neutral lipids) in the oil phase is 3 w / v %-10 w / v %;

[0027] Preferably, in the preparation of colostrum, the concentration of the phosphoric acid aqueous solution is 1 w / v%-10 w / v%.

[0028] Preferably, in the preparation of the colostrum, the oil phase and the internal aqueous phase are mixed at a volume ratio of 1:2.

[0029] Preferably, in the preparation of the re-emulsion, the colostrum is injected into the external aqueous phase at a volume ratio of 1:20.

[0030] In one specific implementation, for example, the colostrum is sheared at a shearing speed of 35,000 rpm for 5 minutes; the re-emulsion is sheared at a shearing speed of 5,000-7,500 rpm for 1-2 minutes.

[0031] In another aspect, the present invention also provides the use of the multivesicular liposomes in the preparation of a drug for intervening in high-altitude pulmonary edema.

[0032] Compared with existing technologies, the multivesicular liposomes formed in this application can slowly release calcium ion channel inhibitors under normal physiological conditions, playing a preventive role in the occurrence of HAPE. In the early stage of HAPE, the multivesicular liposomes can spontaneously collapse under the action of elevated pulmonary artery pressure (greater than 20 mmHg), thereby releasing the drug encapsulated inside, achieving the effect of relieving elevated pulmonary artery pressure in the first instance, and avoiding high-altitude pulmonary edema caused by pulmonary vascular rupture due to high pulmonary artery pressure.

[0033] The multivesicular liposomes of this invention effectively solve the problem of the difficulty in timely intervention for the onset of high-altitude pulmonary edema, and can also effectively alleviate the pathological state of high-altitude pulmonary edema. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0035] Figure 1 Optical microscope images of AB-PSMVLs prepared in Example 1 and AB-MVLs prepared in Comparative Example 1;

[0036] Figure 2 Particle size distributions of AB-PSMVLs prepared in Example 1 and AB-MVLs prepared in Comparative Example 1;

[0037] Figure 3 Zeta potential diagrams of AB-PSMVLs prepared in Example 1 and AB-MVLs prepared in Comparative Example 1;

[0038] Figure 4 The in vitro release results of AB-PSMVLs prepared in Example 1 and AB-MVLs prepared in Comparative Example 1 are shown, with the left side showing the results over 60 hours and the right side showing a scaled-up version of the results from the left side over 0-8 hours.

[0039] Figure 5 Example 1: Inhibitory effect of AB-PSMVLs prepared on intracellular calcium ion channels;

[0040] Figure 6 Distribution of AB-PSMVLs prepared in Example 1 (labeled with rhodamine B), AB-MVLs prepared in Comparative Example 1, and amlodipine besylate solution (labeled as amlodipine in the figure) in the lung tissue of HAPE mice;

[0041] Figure 7 Effects of AB-PSMVLs prepared in Example 1, AB-MVLs prepared in Comparative Example 1, and amlodipine besylate solution (marked as amlodipine in the figure) on CT imaging of lung tissue in HAPE mice;

[0042] Figure 8 The effects of AB-PSMVLs prepared in Example 1, AB-MVLs prepared in Comparative Example 1, and amlodipine besylate solution (labeled as amlodipine in the figure) on blood oxygen saturation and lung water content in HAPE mice; and

[0043] Figure 9 The HE pathological sections of HAPE mouse lung tissue prepared by AB-PSMVLs in Example 1, AB-MVLs prepared by Comparative Example 1, and amlodipine besylate solution (marked as amlodipine in the figure) were observed under microscopic examination and immunofluorescence observation of HIF-α. Figure A shows the HE pathological section of HAPE mouse lung tissue, Figure B shows the immunofluorescence observation of HIF-α, and Figure C shows the quantitative results of Figure B. Detailed Implementation

[0044] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0045] Example 1: Pressure-sensitive multivesicular liposomes of the present invention for intervention in high-altitude pulmonary edema

[0046] Preparation of pressure-sensitive multivesicular liposomes (AB-PSMVLs) of the present invention, containing the drug amlodipine besylate, for intervention in high-altitude pulmonary edema: Cholesterol (42.8 mg), disqualyl phosphatidylcholine (DEPC, 74.4 mg), tricaprylic acid glyceride (2.4 mg), and 1,2-dipalmitoyl phosphatidylglycerol (DPPG, 8.6 mg) were dissolved in 2 mL of dichloromethane. Amlodipine besylate (15 mg) was dissolved in 1 mL of an aqueous solution containing 4% phosphoric acid. The two solutions were mixed and homogenized by high-speed shearing (15000 rpm) to obtain a colostrum. The colostrum was added to 60 mL of an external aqueous phase containing L-lysine (200 mM) and sheared (7500 RPM) for 1 minute to prepare a secondary emulsion. Nitrogen gas was introduced into the secondary emulsion to remove the dichloromethane, thus obtaining the multivesicular liposomes. The morphology of the prepared multivesicular liposomes is shown in [Figure 1]. Figure 1 Particle size distribution is shown in Figure 2 Potential see Figure 3 The results showed that the liposome particle size was 26.58 ± 7.52 μm and the zeta potential was -38.28 ± 2.32 mV.

[0047] Comparative Example 1:

[0048] Preparation of stable amlodipine besylate multivesicular liposomes (AB-MVLs): Cholesterol (42.8 mg), disqualyl phosphatidylcholine (DEPC, 74.4 mg), caprylic acid glyceride (9.6 mg), and 1,2-dipalmitoyl phosphatidylglycerol (DPPG, 8.6 mg) were dissolved in 2 mL of dichloromethane. Amlodipine besylate (15 mg) was dissolved in 1 mL of an aqueous solution containing 4% phosphoric acid. The two solutions were mixed thoroughly and homogenized by high-speed shearing (15000 rpm) to obtain a colostrum. The colostrum was added to 60 mL of an external aqueous phase containing L-lysine (200 mM), stirred, and nitrogen gas was introduced to remove the dichloromethane, thus obtaining the multivesicular liposomes. The morphology of the prepared multivesicular liposomes is shown in [Figure 1]. Figure 1 Particle size distribution is shown in Figure 2 Potential see Figure 3 The results showed that the liposome particle size was 23.91 ± 3.18 μm and the Zeta potential was -34.74 ± 1.77 mV.

[0049] Experimental Example 1: Determination of Encapsulation Efficiency of Multivesicular Liposomes

[0050] After ventilation, 0.5 mL of the liposome sample solution was placed in a 10 mL volumetric flask. 2 mL of methanol was added to break the emulsion, and then the mobile phase was added to bring the volume to 10 mL. The mixture was sonicated for 1 min to ensure complete demulsification of the liposomes. 20 μL of the solution was injected by high-performance liquid chromatography (HPLC) to determine the total drug concentration of the multivesicular liposome solution, C. 总Take 1 mL of the liposome sample solution after aeration, dilute it with an equal volume of physiological saline, centrifuge at 3500 rpm for 10 min, take 0.5 mL of the precipitate and place it in a 10 mL volumetric flask, add 2 mL of methanol to demulsify, then add mobile phase to make up to 10 mL, sonicate for 1 min to completely demulsify the liposomes, inject 20 μL by high performance liquid chromatography, and determine the free drug concentration of the multivesicular liposome solution as C. 包载 The encapsulation efficiency (%) of multivesicular liposomes is calculated using the following formula: Encapsulation efficiency (%) = (C 包载 / C 总 )×100%.

[0051] The encapsulation efficiencies of the amlodipine besylate multivesicular liposomes prepared in Example 1 and Comparative Example 1 were calculated to be 97.60% and 95.41%, respectively.

[0052] Experimental Example 2: In vitro release of amlodipine besylate multivesicular liposomes

[0053] After adding the AB-PSMVLs prepared in Example 1 or the AB-MVLs suspension prepared in Comparative Example 1 to the pressurization system, the system was pressurized at pressures of 25 ± 5 mmHg, 35 ± 5 mmHg and 45 ± 5 mmHg, respectively, and the drug release rate was measured within 0.5-6 h. Figure 4 The results show that the stable multivesicular liposomes (AB-MVLs) prepared in Comparative Example 1 maintained slow drug release even under a pressure of 45 mmHg. In contrast, the AB-PSMVLs prepared in Example 1 exhibited similar sustained-release behavior to AB-MVLs under no pressure. When pressures of 20-45 mmHg were applied, the drug release rate of AB-PSMVLs increased progressively with increasing pressure; even at 25 mmHg, the drug release within 30 minutes remained close to 40%. These data indicate that, compared to the stable multivesicular liposomes prepared in Comparative Example 1, the liposomes prepared in Example 1 exhibit significantly pressure-sensitive drug release characteristics.

[0054] Experiment Example 3: Effect of Neutral Lipid Types on the Stress Sensitivity of Multivesicular Liposomes

[0055] In Example 1, tricaprylic acid glyceride was replaced with trioleic acid glyceride, 1,3-dicapryloylglyceride, monooleic acid glyceride, dioleic acid glyceride, and triolein to prepare multivesicular liposomes. The prepared liposome solution was placed in a closed loop consisting of a peristaltic pump, a pressure gauge, a plastic tubing, and a beaker. The pressure inside the tubing was increased to 25 mmHg and 35 mmHg, respectively. After circulating in the tubing for 30 min, the liposome structure fragmentation was observed, and the liposome particle size and drug release rate were measured to screen for the neutral lipid type most sensitive to pressure-triggered drug release. The results are shown in Table 1. It can be seen that when tricaprylic acid glyceride is used as the neutral lipid, the liposomes exhibit the best pressure response, and structural fragmentation and rapid drug release can occur under pressure. Therefore, the pressure-sensitive amlodipine besylate multivesicular liposomes disclosed in this invention preferably use tricaprylic acid glyceride as the neutral lipid.

[0056] Table 1. Effects of neutral lipid types on the pressure sensitivity of multivesicular liposomes.

[0057] Types of neutral lipids Encapsulation efficiency of amlodipine besylate Drug release at 25 mmHg pressure Drug release at 35 mmHg pressure Drug release at 45 mmHg pressure Trioctyl glyceride 54.27% 43.97% 45.28% 59.52% Trioleic acid glycerides 97.60% 10.24% 13.75% 14.73% 1,3-Dioctanoylglycerol 39.42% 5.12% 5.63% 11.20% Glyceryl monooleate 70.18% 13.84% 17.24% 19.41% Dioleoglyceride 73.58% 4.57% 7.58% 9.75% Three oils 65.24% 14.57% 13.27% 21.57%

[0058] Experimental Example 4: Effect of the percentage of caprylic / capric triglyceride in total lipid components on the pressure sensitivity of multivesicular liposomes

[0059] By varying the percentage of tricaprylic acid glyceride in the total lipids in Example 1, the drug release behavior of liposomes under different pressure levels was determined, and the results are shown in Table 2. The results show that within the range of 25-45 mmHg, as the amount of tricaprylic acid glyceride decreased, the liposomes released more encapsulated drug under pressure. However, when the tricaprylic acid glyceride content was reduced to 1% of the total lipids, the encapsulation efficiency decreased sharply, and the pressure sensitivity also decreased sharply. Therefore, to ensure good drug release from the liposomes at the corresponding pressure, the preferred concentration of tricaprylic acid glyceride is 1.9 w / w%-3.7 w / w of the total lipids.

[0060] Table 2 Effect of Trioctyl Trityl Acetate Dosage on Pressure Sensitivity of Multivesicular Liposomes

[0061] The proportion of trioctyl glycerol in total lipids Encapsulation efficiency of amlodipine besylate Drug release at 25 mmHg pressure Drug release at 35 mmHg pressure Drug release at 45 mmHg pressure 1% 12.4% 10.24% 13.75% 14.73% 1.9% 62.3% 35.9% 41.3% 46.7% 2.8% 97.6% 43.97% 45.28% 59.52% 3.7% 95.7% 51.57% 59.52% 63.27% 5.4% 95.6% 10.44% 11.75% 13.24% 7.1% 96.3% 5.74% 5.72% 6.25% 7.9% 98.2% 3.31% 3.47% 5.97%

[0062] Experimental Example 5: Effect of Amphiphilic Phospholipid Dosage on the Pressure Sensitivity of Multivesicular Liposomes

[0063] By varying the amount of amphiphilic phospholipid (DEPC) used in Example 1, the drug encapsulation efficiency of the liposomes was determined, and the results are shown in Table 3. When the proportion of DEPC in the total lipid composition was less than 50% or greater than 70%, the encapsulation efficiency of amlodipine besylate decreased significantly. However, when the proportion of DEPC in the total lipid composition was within the range of 50%-70%, the prepared multivesicular liposomes exhibited a higher drug encapsulation efficiency. Therefore, the amlophilic phospholipid used in the pressure-sensitive amlodipine besylate multivesicular liposomes disclosed in this invention preferably has an amphiphilic phospholipid content of 50 w / w%-70 w / w in the total lipid composition.

[0064] Table 3 Effect of DEPC dosage on encapsulation efficiency of amlodipine besylate in multivesicular liposomes

[0065] The proportion of DEPC in total lipids 30% 40% 50% 60% 70% 80% Encapsulation efficiency of amlodipine besylate 30.23% 52.10% 89.56% 94.01% 87.45% 49.32%

[0066] Experimental Example 6: Effect of the amount of negatively charged phospholipids on the encapsulation efficiency of amlodipine besylate in multivesicular liposomes

[0067] By varying the amount of negatively charged phospholipid (DPPG) used in Example 1, the drug encapsulation efficiency of the liposomes was determined, and the results are shown in Table 4. The results show that when the proportion of DPPG in the total lipids is less than 3% or greater than 7%, the encapsulation efficiency of amlodipine besylate decreases significantly. Therefore, the pressure-sensitive amlodipine besylate multivesicular liposomes disclosed in this invention preferably use negatively charged phospholipids with a proportion of 3 w / w%-7 w / w in the total lipids.

[0068] Table 4. Effect of DPPG dosage on encapsulation efficiency of amlodipine besylate in multivesicular liposomes

[0069] DPPG percentage in total lipids 1% 3% 5% 7% 10% Encapsulation efficiency of amlodipine besylate 35.24% 91.76% 80.52% 94.11% 59.06%

[0070] Experimental Example 7: Inhibitory effect of amlodipine besylate multivesicular liposomes prepared in Example 1 on hypoxia-induced intracellular calcium ion influx.

[0071] Human vascular endothelial cells (HUVEC cells) were incubated with amlodipine besylate multivesicular liposomes (5 μg / ml) prepared in Example 1 under different pressures. The cells were then cultured for 1 h with the fluorescent probe Fluo-3 AM (1 μg / ml, 1 μL) to observe intracellular calcium. After removing the culture medium, the HUVEC cells were washed with calcium-free buffer, and then 200 μL of calcium-free medium was added. The cells were stored in the dark for 15 min. Intracellular calcium ion concentration was observed using a laser scanning confocal microscope (STELLARIS 5, USA). Figure 5The study showed that intracellular calcium ion content increased significantly under hypoxic conditions, while free amlodipine besylate effectively inhibited the influx of calcium ions into cells under hypoxic conditions. The amlodipine besylate multivesicular liposomes prepared in Example 1 could not inhibit the increase in intracellular calcium ion concentration under hypoxic conditions without pressure. This is because, under pressureless conditions, the multivesicular liposomes are structurally stable and can only slowly release amlodipine besylate. However, when a pressure of 25 mmHg was applied for 10 or 30 minutes, the intracellular calcium ion concentration decreased significantly. This is because, under pressure, amlodipine besylate in the multivesicular liposomes was rapidly released, effectively inhibiting hypoxia-induced influx of calcium ions into cells.

[0072] Experimental Example 8: Distribution of pressure-sensitive amlodipine besylate multivesicular liposomes prepared in Example 1 in HAPE mouse lung tissue

[0073] HAPE mice were injected via tail vein with a solution of rhodamine B-labeled amlodipine besylate, a stable multivesicular liposome of rhodamine B-labeled form (Comparative Example 1), and a pressure-sensitive multivesicular liposome of rhodamine B-labeled amlodipine besylate (Example 1). Two hours later, the mice were sacrificed, and their lungs were harvested, frozen sections were prepared, and cell nuclei were stained with DAPI. The distribution of rhodamine B-labeled amlodipine besylate in the mouse lung tissue was observed under a fluorescence inverted microscope.

[0074] Figure 6 This study demonstrates that after intravenous injection of rhodamine B-labeled amlodipine besylate solution, the drug is distributed both within the pulmonary blood vessels and in the interstitial spaces outside the vessels. The vast majority of the rhodamine B-labeled amlodipine besylate stable multivesicular liposomes (Comparative Example 1) are distributed within the vascular wall. This is because rhodamine B-labeled amlodipine besylate is present within multivesicular liposomes, and the particle size of these liposomes makes them difficult to penetrate the vascular wall, thus resulting in most drug distribution within the blood vessels. Following intravenous administration of rhodamine B-labeled amlodipine besylate pressure-sensitive multivesicular liposomes, significant drug distribution was observed in the interstitial spaces outside the blood vessels. This is because, under the elevated pulmonary artery pressure in HAPE mice, the pressure-sensitive liposomes ruptured, releasing a large amount of drug into the bloodstream, which then permeated the vascular wall and entered the interstitial spaces. Compared with the stable multivesicular liposomes prepared in Comparative Example 1, the pressure-sensitive multivesicular liposomes prepared in Example 1 release drugs under the influence of HAPE-induced pulmonary artery pressure, which is beneficial to the interaction between the drug and vascular endothelial cells and can effectively improve the timely intervention effect of the drug on HAPE.

[0075] Experimental Example 9: The therapeutic effect of pressure-sensitive amlodipine besylate multivesicular liposomes prepared in Example 1 on lung pathology in HAPE mice.

[0076] Eighteen KM mice were randomly divided into six groups: negative control group, HAPE model group, amlodipine besylate group, comparative example 1 group, example 1 group, and blank example 1 group. The negative control group received no treatment. The HAPE model group received 100 μL of physiological saline via tail vein injection before entering the hypobaric chamber. Amlodipine, example 1, and comparative example 1 received the corresponding free drug or liposomes via tail vein injection before entering the hypobaric chamber at a dose of 2 mg / kg and an injection volume of 100 μL. Blank example 1 received the same dose of blank liposomes (prepared according to example 1, liposomes without amlodipine) via tail vein injection before entering the hypobaric chamber.

[0077] Hypobaric oxygen chamber (HAPE) modeling method: Animals are placed in a hypobaric oxygen chamber, and the instrument parameters are adjusted to raise the internal environment to a simulated altitude of 5000 m and a temperature of 10℃ within 10 minutes. The mouse HAPE model is obtained by maintaining this simulated 5000 m environment for 72 hours.

[0078] Figure 7 The results of lung CT scans of mice in each group show that the lungs of the negative control group were clear, with no enlargement of the pulmonary hilum, patent trachea and bronchi, and no pleural effusion or pleural thickening. In contrast, the HAPE model group showed ground-glass opacities on CT scans, thickening of the bronchovascular bundles, and diffuse smooth thickening of the interlobular septa. The amlodipine group showed similar results to Comparative Example 1, with partial ground-glass opacities and interlobular septal thickening. Example 1 showed significant improvement compared to the HAPE model group, with clear lungs and a small amount of bronchial wall thickening. This indicates that compared to the same dose of free amlodipine besylate and stable amlodipine besylate polyvesicular liposomes (Comparative Example 1), the pressure-sensitive amlodipine besylate polyvesicular liposomes disclosed in this invention have a better effect on improving the pathological changes in the lungs of mice with high-altitude pulmonary edema.

[0079] Figure 8 The results showed that the blood oxygen saturation and lung water content of mice in each group were measured. The results also showed that the blood oxygen saturation of the HAPE model group was significantly lower than that of the normal group. The blood oxygen saturation values ​​of the blank Example 1 group and the comparative example 1 group were similar, slightly higher than those of the HAPE model group. The blood oxygen saturation of the amlodipine and Example 1 groups was higher than that of other groups. Lung water content and lung coefficient reflected the degree of pulmonary edema in mice. As shown in the figure, the lung water content of the HAPE model group and the comparative example 1 group was higher than that of the negative control group, while the lung water content of Example 1 group was similar to that of the blank group, with no significant increase. This further demonstrates that compared with the same dose of free amlodipine besylate and stable amlodipine besylate multivesicular liposomes, the pressure-sensitive amlodipine besylate multivesicular liposomes disclosed in this invention have a better effect on improving pulmonary edema in HAPE mice.

[0080] Figure 9 The results of HE staining pathological examination and immunohistochemical analysis of mice in each group are presented. HE staining pathological examination showed that the lung tissue of mice in the negative control group was healthy, with no thickening of blood vessel walls or infiltration of inflammatory cells between alveoli. Mice in the HAPE model group and the blank Example 1 group showed significant thickening of blood vessel walls in their lung tissue, with a large number of inflammatory cells infiltrating between alveoli, and the alveolar walls were not obvious. Mice in the amlodipine group showed improvement compared to the HAPE model group, but still had a large number of inflammatory cells. Mice in the Comparative Example 1 group showed thickening of blood vessel walls, but the condition was milder than that in the HAPE model group. The pathological condition of mice in the Example 1 group was significantly reduced, with fewer inflammatory cells between alveoli, obvious alveolar walls, and no thickening of blood vessel walls. This further demonstrates that compared with the same dose of free amlodipine besylate and stable amlodipine besylate multivesicular liposomes, the pressure-sensitive amlodipine besylate multivesicular liposomes disclosed in this invention have a better effect on improving lung tissue damage in mice with high-altitude pulmonary edema.

[0081] HIF-α is a nucleoprotein produced by the body in hypoxic environments to adapt to low oxygen levels. HIF-α expression can increase pulmonary vascular permeability, thereby exacerbating alveolar fluid exudation and leading to pulmonary edema. According to... Figure 9 As shown, no obvious red fluorescence was observed in the lung tissue of the negative control group mice. The highest HIF-α fluorescence intensity and widest distribution were observed in the lung tissue of the HAPE model group, the blank Example 1 group, and the amlodipine group mice. The HIF-α fluorescence of the comparative Example 1 group mice was significantly weakened, but some fluorescence could still be observed. The Example 1 group mice showed almost no red fluorescence. This indicates that both Comparative Example 1 and Example 1 have a significant inhibitory effect on the expression of HIF-α in the lung tissue of mice with high-altitude pulmonary edema, and Example 1 has a stronger effect.

[0082] Experiment Example 10: Effect of different types of calcium channel blockers on lung water content in mice with high-altitude pulmonary edema caused by pressure-sensitive multivesicular liposomes.

[0083] By changing the types of drugs in Example 1, multivesicular liposomes were prepared using nifedipine hydrochloride, amlodipine besylate, felodipine hydrochloride, verapamil hydrochloride, and diltiazem hydrochloride, respectively.

[0084] Multivesicular liposomes containing the aforementioned drugs were administered to mice with a high-altitude pulmonary edema model, and lung water content was measured after administration. Table 5 shows that all five drugs were effective in improving lung function in the mice with high-altitude pulmonary edema. To enhance the therapeutic effect of liposomes on high-altitude pulmonary edema, the preferred drugs should be nifedipine, amlodipine, diltiazem, felodipine, and verapamil.

[0085] Table 5. Therapeutic effects of different drug types on lung water content in mice with high-altitude pulmonary edema caused by multivesicular liposomes.

[0086] Types of drugs Drug encapsulation rate mouse lung water content negative control group - 74.21% Model group - 82.63% Nifedipine hydrochloride 89.42% 74.25% Amlodipine besylate 91.24% 77.82% Felodipine Hydrochloride 87.42% 75.71% Verapamil Hydrochloride 85.47% 73.78% Diltiazem Hydrochloride 87.51% 76.29%

[0087] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A pressure-sensitive multivesicular liposome for intervention in high-altitude pulmonary edema, the multivesicular liposome comprising a lipid phase and an internal aqueous phase, wherein the lipid phase comprises cholesterol, amphiphilic phospholipids, negatively charged phospholipids, and neutral lipids, and the internal aqueous phase comprises calcium ion channel inhibitors and phosphate. in, The amphiphilic phospholipids comprise 50 w / w%-70 w / w% of the lipid phase of the multivesicular liposomes. The calcium channel inhibitors include one or more of nifedipine, amlodipine, felodipine, verapamil, and diltiazem, and these drugs may exist in various salt forms; The method for preparing the multivesicular liposomes includes the following steps: Preparation of colostrum: Amphiphilic phospholipids, cholesterol, negatively charged phospholipids, and neutral lipids were dissolved in an organic solvent to obtain an oil phase; calcium ion channel inhibitors were dissolved in an aqueous phosphoric acid solution to obtain an inner aqueous phase; the oil phase and the inner aqueous phase were mixed and homogenized by high-speed shearing to obtain the colostrum. Preparation of double emulsion: L-lysine is dissolved in water to obtain an external aqueous phase containing L-lysine aqueous solution. The primary emulsion is injected into the external aqueous phase and homogenized by high-speed shearing to obtain a double emulsion solution. Formation of multivesicular liposomes: The double emulsion solution was placed in a 37°C water bath, nitrogen gas was introduced to remove the organic solvent, and L-lysine in the external aqueous phase was removed by ultrafiltration or dialysis to obtain multivesicular liposomes; The negatively charged phospholipids account for 3 w / w%-7 w / w% of the lipid phase of the multivesicular liposomes. The neutral lipid is caprylic acid glyceride; The neutral lipids comprise 1.9 w / w% to 3.7 w / w of the lipid phase of the multivesicular liposomes.

2. The multivesicular liposome according to claim 1, wherein, The amphiphilic phospholipids include one or more of synthetic phospholipids and natural phospholipids.

3. The multivesicular liposome according to claim 2, wherein, The artificially synthesized phospholipid is disorhodophosphatidylcholine, and the natural phospholipid is egg yolk lecithin, soybean lecithin, hydrogenated lecithin, or cephalin natural phospholipid.

4. The multivesicular liposome according to any one of claims 1 to 3, wherein, The negatively charged phospholipids include one or more of egg yolk phosphatidylglycerol, distearate phosphatidylglycerol, 1,2-dipalmitoyl phosphatidylglycerol, and dilauroyl phosphatidylglycerol.

5. The multivesicular liposome according to any one of claims 1 to 3, wherein, The mass ratio of the calcium ion channel inhibitor to the total mass of the cholesterol, the amphiphilic phospholipids, the negatively charged phospholipids, and the neutral lipids is 1:5 to 1:

20.

6. The multivesicular liposome according to claim 5, wherein, The mass ratio of the calcium ion channel inhibitor to the total mass of the cholesterol, the amphiphilic phospholipids, the negatively charged phospholipids, and the neutral lipids is 1:7 to 1:

15.

7. The multivesicular liposome according to any one of claims 1 to 3, wherein, The multivesicular liposome is composed of multiple vesicles arranged in a honeycomb pattern. The areas where the multiple vesicle chambers meet and accumulate are composed of neutral lipids. The amphiphilic phospholipids and cholesterol are the main components forming the vesicle walls. The negatively charged phospholipids are used to form a charge barrier for the vesicle walls to inhibit the fusion of the inner vesicle walls.

8. The multivesicular liposome according to any one of claims 1 to 3, wherein, The particle size distribution of the multivesicular liposomes is 5-50 μm.

9. A method for preparing multivesicular liposomes according to any one of claims 1 to 8, the method comprising the following steps: Preparation of colostrum: Amphiphilic phospholipids, cholesterol, negatively charged phospholipids, and neutral lipids were dissolved in an organic solvent to obtain an oil phase; calcium ion channel inhibitors were dissolved in an aqueous phosphoric acid solution to obtain an inner aqueous phase; the oil phase and the inner aqueous phase were mixed and homogenized by high-speed shearing to obtain the colostrum. Preparation of double emulsion: L-lysine is dissolved in water to obtain an external aqueous phase containing L-lysine aqueous solution. The primary emulsion is injected into the external aqueous phase and homogenized by high-speed shearing to obtain a double emulsion solution. Formation of multivesicular liposomes: The double emulsion solution was placed in a 37°C water bath, nitrogen gas was introduced to remove the organic solvent, and L-lysine in the external aqueous phase was removed by ultrafiltration or dialysis to obtain multivesicular liposomes.

10. The preparation method according to claim 9, wherein, The organic solvent is selected from dichloromethane, trichloromethane, and acetone.

11. The preparation method according to claim 10, wherein, The organic solvent is dichloromethane.

12. The preparation method according to claim 9, wherein, In the preparation of colostrum, the total concentration of lipid components in the oil phase is 3w / v%-10w / v.

13. The preparation method according to claim 9, wherein, In the preparation of colostrum, the concentration of the phosphoric acid aqueous solution is 1 w / v%-10 w / v.

14. The preparation method according to claim 9, wherein, In the preparation of colostrum, the oil phase and the internal aqueous phase are mixed at a volume ratio of 1:

2.

15. The preparation method according to claim 9, wherein, In the preparation of the re-emulsion, the colostrum is injected into the external aqueous phase at a volume ratio of 1:

20.

16. The use of the multivesicular liposomes according to any one of claims 1 to 8 in the preparation of a medicament for the intervention of high-altitude pulmonary edema.

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