Biomimetic lung surfactant carriers and drugs for inhalation for the treatment of pulmonary hypertension

Abstract

The present application relates to a kind of biomimetic lung surfactant carrier, which is prepared from total fat and simulation peptide, in the total fat, the mass ratio of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol is 8-12:1:1:1;The lung surfactant simulation peptide is the simulation peptide of SP-B protein and / or the simulation peptide of SP-C protein.The biomimetic lung surfactant carrier has the advantages of high loading rate and high stability.The present application also relates to inhalation dosage form of drug for treating pulmonary arterial hypertension loaded by the biomimetic lung surfactant carrier, which makes the loaded drug obtain more uniform intra-pulmonary distribution, the inhalation drug is more close to natural lung surfactant in composition and physiological function, can delay the degradation of product, improve the utilization rate of drug, achieve good pulmonary arterial hypertension treatment effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical dosage forms, specifically to carriers and drugs for inhaled medications for treating pulmonary hypertension, and methods for their preparation. Background Technology

[0002] Common lung diseases include asthma, bronchitis, tuberculosis, infectious and non-infectious pneumonia, chronic obstructive pulmonary disease (COPD), pulmonary hypertension (PAH), and lung cancer. Pulmonary arterial hypertension (PAH) is a malignant lung disease characterized by elevated pulmonary artery pressure and pulmonary vascular remodeling. PAH can lead to right ventricular failure and even death, with extremely high rates of disability and mortality. Traditionally, PAH has been treated primarily with supportive therapies such as anticoagulants, diuretics, digoxin, digitalis, and oxygen therapy, with very limited efficacy. In recent years, research into the pathogenesis of PAH has led to the development of targeted drugs targeting multiple signaling pathways, including prostacyclin, nitric oxide, and endothelin-1. These targeted therapies are primarily oral, including prostacyclin receptor agonists (treprostine, beraprost, selexipag), phosphodiesterase type 5 inhibitors (sildenafil, tadalafil), endothelin receptor antagonists (bosentan, ambrisentan, macitentan), and soluble guanylate cyclase agonists (riociguat), with a small number available in injectable formulations (eprostol, treprostine). While these targeted therapies can reduce pulmonary artery pressure and improve PAH symptoms, long-term prognosis remains unsatisfactory, and the mortality rate remains high.

[0003] Combination therapy refers to the use of two or more drugs with different pathways of action. Combination therapy can maximize the efficacy of drugs, reduce toxicity, and decrease drug dosage. For PAH, a disease with clearly defined multiple pathogenic pathways, combination therapy is theoretically more effective than monotherapy. The combined application of targeted drugs for PAH involves two strategies: sequential combination therapy and initial combination therapy. Several randomized controlled trials published in recent years have shown that both sequential and initial combination therapies can significantly reduce the incidence of clinical exacerbations in PAH patients. Therefore, the Chinese Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension (2021 edition) recommend combination therapy for all PAH patients, except for those with low-risk PAH risk stratification or elderly patients, and for those with intermediate- or high-risk PAH risk stratification. Currently, the most commonly used combination targeted therapy for PAH is the combination of a phosphodiesterase type 5 inhibitor and an endothelin receptor antagonist, such as bosentan + sildenafil, ambrisentan + tadalafil, etc.

[0004] Meanwhile, the toxic side effects and drug-induced adverse reactions of these oral and injectable drugs have not been eliminated. For example, bosentan, a first-line clinical drug for PAH, has teratogenic effects and hepatotoxicity, while sildenafil can cause serious cardiac events and visual abnormalities. Given the limitations of existing PAH treatments, such as numerous adverse reactions, low bioavailability, and poor prognosis, modifying the dosage form of existing drugs or adjusting the dosage form to make some difficult-to-drug targets available as drugs has become an important approach to treating PAH.

[0005] Compared to oral and injectable medications, inhaled drug delivery offers significant advantages for treating PAH and other lung diseases. First, the lungs are the direct site of PAH lesions, allowing for direct drug delivery to the affected tissues. Second, the lungs have a large absorption area, resulting in rapid onset of action. Third, inhaled delivery avoids the first-pass effect, has high bioavailability, reduces drug dosage, and minimizes drug distribution and related adverse reactions in other tissues. Therefore, inhaled drug delivery has become the most effective treatment for lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), and is theoretically the optimal route of administration for PAH treatment. However, currently, inhaled medications for PAH are very limited, with only a few, such as iloprost and treprostol, available in inhaled formulations. Most commonly used targeted therapies, such as bosentan and sildenafil, do not yet have effectively usable inhaled formulations.

[0006] While inhaled drug delivery offers the advantages mentioned above, it also faces three key challenges: drug efficacy, immunogenicity, and safety. These challenges involve improving drug retention in the lungs, preventing the pulmonary immune system from recognizing and clearing the drug, and avoiding damage to the lungs and the body from the drug (primarily excipients in the formulation). The first two issues relate to the lung's defense system's clearance of exogenous substances, while the third is mainly determined by the drug's formulation components. The lung's defense system primarily consists of immune cells and pulmonary surfactant. Pulmonary surfactant is a lipoprotein complex synthesized and secreted by type II alveolar epithelial cells, covering the alveolar surface. Exogenous substances entering the lungs are either blocked by pulmonary surfactant, expelled through ciliary movement, or recognized and cleared by immune cells. Therefore, to improve lung retention and reduce immunogenicity, inhaled drugs need to overcome the barriers of pulmonary surfactant and the recognition by immune cells.

[0007] Lipids comprise approximately 90% of pulmonary surfactant, while proteins comprise approximately 10%. The lipid component is primarily phosphatidylcholine (approximately 70-80%), with smaller amounts of phosphatidylglycerol (approximately 8%), phosphatidylethanolamine (approximately 8%), and cholesterol (approximately 8%). The protein component mainly consists of surfactant-associated proteins (SP proteins). There are four types of SP proteins: hydrophilic SP-A and SP-D proteins, and hydrophobic SP-B and SP-C proteins. Although the latter two are present in smaller amounts (approximately 1-2%), they play a crucial role in maintaining alveolar structure. In particular, SP-B is a key component in pulmonary surfactant that maintains alveolar surface tension and prevents pulmonary surfactant from being degraded by phospholipases (Hite RD, et al. Surfactant protein B inhibits secretory phospholipase A2 hydrolysis of surfactant phospholipids. Am J Physiol Lung Cell Mol Physiol. 2012). Based on the amino acid sequence and structural characteristics of SP-B, a 21-amino acid polypeptide can mimic the physiological function of SP-B (Cochrane CG, et al. Pulmonary surfactant protein B (SP-B): structure-function relations. Science. 1991). The amino acid sequence of this polypeptide is KLLLLKLLLLKLLLLKLLLLK (KL4 polypeptide) or RLLLLRLLLLRLLLLRLLLLR (RL4 polypeptide). The KL4 polypeptide is also known as sinapultide. Several other polypeptides can also mimic the functions of SP-B or SP-C.

[0008] Pulmonary surfactant is widely used clinically to treat neonatal respiratory distress syndrome. As a pharmaceutical product, pulmonary surfactant is divided into two types: natural and synthetic. Natural pulmonary surfactant is extracted from the lung tissue of animals such as cattle or pigs, such as Survanta and Infasurf from the United States, Curosurf from Italy, and domestically produced products like Kelisu and Feihuotong. Synthetic pulmonary surfactant is formulated based on the main components and proportions of natural pulmonary surfactant, such as Exosurf and Lucinactant (trade name Surfaxin) from the United States. Exosurf contains only phospholipids, while Lucinactant, in addition to phospholipids, also contains KL4 peptides, making it closer to natural pulmonary surfactant in terms of composition, function, and efficacy. Compared with natural surfactant, synthetic pulmonary surfactant is essentially consistent in clinical efficacy and safety, with smaller batch-to-batch variations and no potential risk of transmitting animal diseases, giving it unique advantages. In PAH animal models, reduced SP protein levels impair lung function (Gutierrez JA, et al. Decreased surfactant proteins in lambs with pulmonary hypertension secondary to increased blood flow. Am JPhysiol Lung Cell Mol Physiol. 2001). Therefore, supplementation with pulmonary surfactant containing SP protein or its mimic peptides can directly improve lung function in PAH patients.

[0009] Liposomes prepared from materials such as phospholipids, sphingolipids, and cholesterol have been reported as drug carriers, but liposome-loaded drugs are mainly used for injection therapy of diseases such as tumors, and rarely for inhalation. Currently, a representative liposome inhalation drug is amikacin liposome developed by Insmed in the United States for the treatment of infectious lung diseases (such as refractory nontuberculous mycobacterial lung disease caused by avian mycobacterium infection). In this product, amikacin is loaded into liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol. Compared with intravenous amikacin, inhaled amikacin liposomes prolong the release of amikacin in the lungs while reducing systemic exposure, thereby reducing systemic toxicity. In China, there have been attempts to add fatty alcohols, surfactants, unsaturated phospholipids, etc., to DPPC and use it as an inhalation drug carrier to load insulin to lower blood glucose levels (patent application number CN200610025706). There are reports from abroad of using distearate, beeswax, or stearic acid combined with oleic acid and emulsifiers to create nanoliposomes loaded with sildenafil for inhalation therapy in the treatment of PAH (Nafee N, et al. Nanostructured lipid carriers versus solid lipid nanoparticles for the potential treatment of pulmonary hypertension via nebulization. Eur JPharmSci, 2018). However, in the above studies, the liposomes each have their own composition and differ significantly from the components of natural pulmonary surfactant, and they lack the key SP protein in pulmonary surfactant. Therefore, they differ greatly from natural pulmonary surfactant in terms of composition, physicochemical properties, and physiological function. Summary of the Invention

[0010] Based on this, the purpose of the present invention is to provide a biomimetic pulmonary surfactant carrier and drug for treating pulmonary hypertension inhalation. The drug prepared from the biomimetic pulmonary surfactant carrier of the present invention has the characteristics of high loading capacity, good stability and good biological activity.

[0011] To achieve the above objectives, the present invention is implemented through the following technical solutions.

[0012] In a first aspect, the present invention provides a biomimetic lung surfactant carrier.

[0013] A biomimetic pulmonary surfactant carrier is prepared from total lipids and pulmonary surfactant mimic peptides. In the total lipids, the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 8-12:1:1:1; and the mass ratio of the total lipids to the mimic peptides is 100:0.8-1.2.

[0014] In some embodiments, the biomimetic lung surfactant carrier is prepared from total lipids and mimic peptides, wherein the total lipids consist of four components: dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol. In the total lipids, the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 9-11:1:1:1; more preferably, the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 10:1:1:1.

[0015] In some embodiments, the mass ratio of the total lipids to the mimic peptides is 100:0.9-1.1, most preferably 100:1.

[0016] In some embodiments, the pulmonary surfactant mimic peptide is a mimic peptide of SP-B protein and / or a mimic peptide of SP-C protein.

[0017] In some preferred embodiments, the mimic peptide of the SP-B protein is selected from at least one of SEQ ID NO. 1-7. More preferably, it is SEQ ID NO. 1 and / or SEQ ID NO. 2.

[0018] In some preferred embodiments, the mimic peptide of the SP-C protein is selected from at least one of SEQ ID NO. 8-10.

[0019] A second aspect of the present invention is to provide the use of a biomimetic pulmonary surfactant carrier as a drug carrier in the preparation of an inhaled medicament for treating pulmonary hypertension.

[0020] A third aspect of the present invention is to provide an inhaled drug for treating pulmonary hypertension.

[0021] An inhaled drug for treating pulmonary hypertension, which is prepared by loading the drug for treating pulmonary hypertension onto the aforementioned biomimetic pulmonary surfactant carrier.

[0022] In some embodiments, the drug is one or both of sildenafil and bosentan.

[0023] In some preferred embodiments, the drug is sildenafil and bosentan.

[0024] In some embodiments, the drug-to-lipid ratio in the inhaled medication is 1:20-40.

[0025] In some preferred embodiments, the drug-to-lipid ratio in the inhaled medication is 1:30-40, with the optimal ratio being 1:30.

[0026] A fourth aspect of the present invention is to provide a method for preparing the biomimetic lung surfactant carrier.

[0027] A method for preparing the biomimetic lung surfactant carrier includes the following steps:

[0028] Add the simulated peptide to the chloroform solution of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol, mix well, and then rotate and evaporate in a constant temperature water bath until the chloroform solvent evaporates to form a uniform lipid film layer.

[0029] Water is added to fully hydrate the lipid film layer. After complete hydration, the cellulose acetate membrane used in the hydration solution is repeatedly extruded to obtain biomimetic lung surfactant.

[0030] A fifth aspect of the present invention is a method for preparing the inhaled medicament for treating pulmonary hypertension.

[0031] A method for preparing an inhaled medicament for treating pulmonary hypertension includes the following steps:

[0032] The mimic peptide and the drug for treating pulmonary hypertension are added to a chloroform solution of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol. The mixture is stirred and then evaporated in a constant-temperature water bath until the chloroform solvent evaporates, forming a uniform lipid film layer.

[0033] Water is added to fully hydrate the lipid film layer. After complete hydration, the cellulose acetate membrane used in the hydration solution is repeatedly squeezed out to obtain an inhaled drug for treating pulmonary hypertension.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] Using biomimetic pulmonary surfactant (bPS) as a drug carrier for the treatment of pulmonary hypertension and other lung diseases has many advantages. In addition to the fact that bPS itself has therapeutic effects, can reduce alveolar surface tension and improve lung function, (1) the composition of the bPS is similar to that of natural pulmonary surfactant, making it an ideal pharmaceutical excipient and avoiding damage to the lungs; (2) the function of the bPS of the present invention is similar to that of natural pulmonary surfactant, which allows the carried drugs to evade recognition by immune cells and the blockage of natural pulmonary surfactant, thereby improving the retention and penetration of drugs in the lungs; (3) the inventors have found that a bPS with a suitable composition can make the carried drugs (sildenafil and bosentan) more evenly distributed in the lungs. Compared with existing methods of drug delivery using liposomes, the bPS provided by this invention, by adding polypeptide mimics of SP-B protein and / or SP-C protein (such as KL4 polypeptide or RL4 polypeptide) to a suitable total lipid component, is closer to the natural pulmonary surfactant in terms of composition and physiological function. This reduces the surface tension of the product in the lungs, improves the particle size stability of the product, increases the distribution of the product in the lungs, delays the degradation of the product, and improves the utilization rate of the drug. Attached Figure Description

[0036] Figure 1 Stability analysis of particle size and dispersion coefficient of bPS-SF-BT prepared under different drug-lipid ratios.

[0037] Figure 2 Particle size potential and transmission electron microscopy image of .bPS-SF-BT.

[0038] Figure 3 Schematic diagram of cell viability results of drug and bPS under different dosage conditions.

[0039] Figure 4 .Schematic diagram of pulmonary artery pressure waveforms and analysis results before and after PAH treatment in PAH rats.

[0040] Figure 5 Schematic diagram of the therapeutic effects of .bPS-SF-BT on pulmonary artery stenosis and occlusion.

[0041] Figure 6 Schematic diagram of the therapeutic effect of .bPS-SF-BT on right ventricular hypertrophy caused by pulmonary hypertension.

[0042] Figure 7 Contact angle test results of bPS-SF-BT with and without mimetic peptides compared with hydrophobic solids. Detailed Implementation

[0043] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0044] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0045] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0046] Some embodiments of the present invention relate to a biomimetic pulmonary surfactant carrier (bPS), which is prepared from total lipids and pulmonary surfactant mimic peptides. In the total lipids, the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 8-12:1:1:1; the mass ratio of the total lipids to the mimic peptides is 100:0.8-1.2. The invention also relates to the preparation of an inhaler by loading a drug for treating pulmonary hypertension onto the aforementioned biomimetic pulmonary surfactant carrier.

[0047] Compared to oral or injectable administration, inhalation formulation modification of drugs represents a novel approach for treating pulmonary hypertension. The bPS-loaded drug delivery method provided by this invention enables effective encapsulation and delivery of one or more drugs. In the experiments described in the following examples, the encapsulation rates of the pulmonary hypertension-targeting drugs sildenafil and bosentan, respectively, reached a maximum of 93.15% and 74.57%, achieving excellent drug encapsulation. Treatment of lung diseases via inhalation after bPS loading avoids the first-pass effect in the liver, improves bioavailability, and achieves sustained release and reduced dosing frequency. After inhalation, the bPS-loaded sildenafil and bosentan are first distributed to the lungs, thereby inhibiting pulmonary artery smooth muscle cell proliferation, dilating pulmonary artery vessels, reducing mean pulmonary artery pressure, further alleviating right ventricular hypertrophy, improving cardiac function, and improving prognosis, thus achieving the therapeutic effect for pulmonary hypertension.

[0048] Studies show that drug particles larger than 5 μm in diameter mainly deposit in the upper respiratory tract, while those between 1 and 5 μm can deposit in the central and distal airways where airflow velocity is lower. Drug particles smaller than 1 μm in diameter can deposit extensively in the respiratory tract and eventually reach the alveoli. The average particle size of the bPS prepared in this invention after drug loading is approximately 123.37 nm, meeting the theoretical requirements for alveolar deposition.

[0049] Studies have shown that the cytotoxicity of nanomaterials is one of the serious and urgent problems to be solved in their clinical translation. The bPS used in this invention comprises all major components of natural pulmonary surfactant, thus avoiding the cytotoxicity of the formulation materials.

[0050] Besides drug safety, reducing drug immunogenicity and preventing drug elimination from the body are other issues that need to be addressed. Studies have found that many large molecular drugs, such as peptides, proteins, and nucleic acids, tend to aggregate and be cleared by macrophages. The average particle size of the drug-loaded bPS prepared in this invention is approximately 123.37 nm, a particle size that avoids capture and phagocytosis by alveolar macrophages; at the same time, bPS has alveolar affinity, reducing immune stimulation, allowing the drug to remain in the lungs for a longer period, thus prolonging drug stability and bioavailability.

[0051] The amino acid sequences of the analogues of this invention are shown in Tables 1 and 2.

[0052]

[0053]

[0054]

[0055] The present invention will be further described in detail below with reference to specific embodiments.

[0056] Example 1: Preparation of biomimetic lung surfactant containing SP-B mimic peptides by thin-film dispersion method

[0057] The preparation method includes the following steps:

[0058] Dissolve dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG2000), and cholesterol in chloroform to a concentration of 100 mg / ml. Dissolve dipalmitoylphosphatidylglycerol (DPPG) and SP-B mimic peptide KL4 in chloroform:methanol at a ratio of 1:1 (v:v) to a concentration of 10 mg / ml. Add 5 ml of chloroform to a 50 ml round-bottom flask, then add 3.923 ml (392.3 mg) of DPPC, 3.923 ml (39.23 mg) of DPPG, 0.3923 ml (39.23 mg) of DPPE-PEG2000, and 0.3923 ml (39.23 mg) of cholesterol in sequence, for a total lipid mass of 510 mg. Add 510ul (5.1mg) of KL4 peptide (1% of total lipid mass), sonicate for 30s to thoroughly mix all components in the flask, and rotary evaporate at 50°C in a constant temperature water bath and 180rpm for 2 hours until the chloroform solvent evaporates and a uniform lipid film layer is formed at the bottom of the round-bottom flask.

[0059] Add 5-10 ml of water to the flask after rotary evaporation, and sonicate in a 40°C water bath for 15 min to fully hydrate the lipid layer. After complete hydration, repeatedly extrude the hydrated solution through a 400 nm pore size cellulose acetate membrane 21 times, and then repeatedly extrude it through a 200 nm pore size cellulose acetate membrane 21 times to obtain the biomimetic lung surfactant, abbreviated as bPS in the following text. Finally, centrifuge the solution several times at 5000 rpm for 20 min using a 50 kDa ultrafiltration tube until the desired concentration is achieved, and collect and store at 4°C for later use.

[0060] Example 2: Biomimetic lung surfactant encapsulating two PAH treatment drugs, sildenafil and bosentan

[0061] Dissolve the raw materials of bPS as described in Example 1, then dissolve sildenafil (SF) in chloroform:methanol at a ratio of 1:1 (v:v) to a concentration of 10 mg / ml, and dissolve bosentan (BT) in chloroform to a concentration of 10 mg / ml. Add the raw materials provided in Example 1 to a round-bottom flask, then add sildenafil and bosentan in a ratio of 1:10 to 1:50 (w:w) of total drug to total lipid components (i.e., drug-lipid ratio), mix by ultrasonication, and follow the same steps as in Example 1, including rotary evaporation. The obtained biomimetic pulmonary surfactant encapsulates two PAH treatment drugs, sildenafil and bosentan, and will be abbreviated as bPS-SF-BT in the following text.

[0062] To conduct stability testing on bPS-SF-BT, the bPS-SF-BT prepared at different drug-lipid ratios was stored at 4°C for 5 days. Each day, an appropriate amount was diluted with water and its particle size and dispersion coefficient were measured using a nanolaser particle size analyzer (Nano ZS90). Stability analysis was performed based on the changes in particle size and dispersion coefficient. The results are shown below. Figure 1 The biomimetic lung surfactant described in this invention exhibits good stability across a wide range of drug-to-lipid ratios, with the product prepared at a drug-to-lipid ratio of 1:30 showing the best stability.

[0063] Example 3: Determination of drug encapsulation efficiency in bPS-SF-BT

[0064] The contents of sildenafil and bosentan in bPS-SF-BT under different drug-lipid ratios were determined by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: ACE 5C18-AR column (250 mm × 4.6 mm, 5 μm), column temperature 40 °C, mobile phase acetonitrile:0.1% trifluoroacetic acid aqueous solution (70:30, v / v), detection wavelength 254 nm, flow rate 1 mL / min, and sample volume 10 μL. Concentration standard curves of sildenafil and bosentan standards were first established by HPLC, and then the contents of the two drugs in bPS-SF-BT under different drug-lipid ratios were calculated (Tables 3.1-3.5).

[0065] The results showed that bPS-SF-BT had the best drug encapsulation efficiency at a drug-to-lipid ratio of 1:30, with 93.15% for sildenafil and 74.57% for bosentan, confirming that the biomimetic pulmonary surfactant has good drug encapsulation efficiency. In Examples 4-9 of this invention, unless otherwise specified, bPS-SF-BT with a drug-to-lipid ratio of 1:30 was used. Tables 3.1 to 3.5: Encapsulation efficiency of sildenafil and bosentan loaded alone, and encapsulation efficiency of sildenafil and bosentan loaded simultaneously.

[0066] Table 3.1

[0067]

[0068]

[0069] Table 3.2

[0070]

[0071] Table 3.3

[0072]

[0073] Table 3.4

[0074]

[0075]

[0076] Table 3.5

[0077]

[0078] Example 4: Identification of the physical properties of bPS-SF-BT

[0079] After obtaining the biomimetic pulmonary surfactant bPS-SF-BT loaded with sildenafil and bosentan according to Example 2, an appropriate amount of bPS-SF-BT was diluted with water and its average particle size was measured using a nanolaser particle size analyzer (Nano ZS90). The average particle size was approximately 123.37 nm, the average potential was approximately -31.67 mV, and the dispersion index was 0.08. Figure 2 A). 10 μl of diluted bPS-SF-BT was dropped onto a copper grid pre-coated with a carbon film, stained with 2% phosphotungstic acid solution (pH 7.0), and allowed to dry naturally at room temperature. The bPS-SF-BT particles were then observed and measured using a transmission electron microscope (model JEM-1400), showing that the particle size was basically consistent with that measured by a nano-laser particle size analyzer. Figure 2 B). Therefore, the prepared bPS-SF-BT possesses the basic physical properties of nanomedicines.

[0080] Example 5: Detection of the cytotoxicity of bPS-SF-BT

[0081] Mouse type II alveolar epithelial cells (MLE-12) were cultured in DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin solution) and used for cytotoxicity testing. Cells were cultured at 1 × 10⁶ cells / year. 4 Cells were evenly seeded at a density of [number] cells / well in 96-well plates. After 24 hours, the medium was replaced with fresh medium, and sildenafil (SF), bosentan (BT), sildenafil and bosentan (SF-BT), and bPS-SF-BT were added at concentrations ranging from 20-200 μg / ml, with five replicates for each concentration. 24 hours after drug addition, 10% CCK-8 reagent was added, and the plates were incubated in a cell culture incubator for 1.5-2 hours. The absorbance was then measured at 450 nm using a Synergy H1 microplate reader. Cell viability was calculated using the formula: Viability percentage (%) = (OD of drug addition - OD of blank) / (OD of cells - OD of blank) * 100.

[0082] from Figure 3The results showed that sildenafil alone, bosentan alone, or the combination of both drugs exhibited cytotoxicity that inhibited cell proliferation at certain doses, while bPS maintained cell viability above 90% within the tested dose range, demonstrating very low cytotoxicity. Furthermore, bPS-SF-BT maintained cell viability above 70% within the tested dose range, indicating good safety.

[0083] Example 6: Evaluation of the therapeutic effect of bPS-SF-BT on pulmonary hypertension rats (pulmonary artery pressure measurement)

[0084] A pulmonary hypertension (PAH) animal model was established using male Sprague Dawley rats (200g). PAH was induced by a single injection of 50mg / kg of methyl thiocyanate (MCT) into the neck and back. Seven days after injection, the rats were treated with medication. Sildenafil and bosentan (without bPS loading) were administered orally by gavage, while bPS-SF-BT was administered via inhalation using a small animal nebulizer (model YLS-8B). The dosage was 6mg / kg for sildenafil and 11mg / kg for bosentan, administered every 3 days until day 25 following MCT injection.

[0085] Hemodynamic evaluation was performed on day 25 after MCT injection, and right ventricular systolic pressure (RVSP) was measured in each group using a catheter method. The specific measurement method was as follows: rats were anesthetized with 4% tribromoethanol via intraperitoneal injection and fixed in a supine position on the operating table. After shaving the hair on the abdomen, the external jugular vein was located and isolated at the chest. A PE50 catheter pre-filled with 1000 U / ml heparin sodium solution was directly inserted into the right ventricle through the external jugular vein. The other end was connected to a blood pressure transducer. After a right ventricular pressure signal was observed, the real-time RVSP pressure waveform was recorded using a physiological signal recording and analysis system (model BL-420N), and the RVSP was calculated.

[0086] Figure 4 The results showed that the RVSP (equivalent to mean pulmonary artery pressure) in the normal group was about 13 mmHg, and the RVSP in the pulmonary hypertension group was about 47 mmHg. Oral sildenafil and bosentan had a certain antihypertensive effect, with RVSP at 35 mmHg. However, the nebulized inhalation of bPS-SF-BT had a better antihypertensive effect, with RVSP at about 17 mmHg. This indicates that inhaled administration of bPS as a drug carrier for sildenafil and bosentan can achieve excellent PAH treatment results.

[0087] Example 7: Evaluation of the therapeutic effect of bPS-SF-BT on pulmonary hypertension rats (II) (Determination of pulmonary artery lumen hyperplasia)

[0088] Pulmonary artery remodeling is the main pathological feature of PAH, which mainly refers to the progressive narrowing and occlusion of the pulmonary artery lumen caused by intimal damage, media thickening, adventitia fibrosis and basement membrane sclerosis, resulting in continuously increasing pulmonary vascular resistance and pulmonary artery pressure.

[0089] At the end of Example 6, i.e., 25 days after MCT injection, after completing the hemodynamic evaluation, lung tissue was dissected, rinsed with physiological saline, and fixed with 4% paraformaldehyde for 24 to 48 hours. The tissue was then sectioned in paraffin and stained with hematoxylin and eosin (HE) to observe the morphology of the pulmonary arterioles. The specific steps are as follows:

[0090] (1) The fixed lung tissue was dehydrated stepwise by low concentration alcohol to high concentration alcohol, and then embedded in paraffin and embedded. After sectioning, the sections were spread out. The paraffin sections were dewaxed in xylene for 5-10 minutes, and then rinsed with anhydrous, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes each, and then rinsed with water for 1 minute.

[0091] (2) Stain with hematoxylin solution for 3-5 min, then wash with water for 1-2 min;

[0092] (3) Differentiate with 0.8%-1% hydrochloric acid alcohol, turn blue with dilute lithium carbonate aqueous solution, and then wash with water for 1-2 min;

[0093] (4) Stain with eosin solution for 1-2 seconds, without rinsing with water, and directly immerse in 95% ethanol or anhydrous ethanol for 1-2 minutes to dehydrate.

[0094] (5) Xylene clearing, sealing, and microscopic examination.

[0095] pass Figure 5 HE staining of transverse sections of lung tissue at the hilum allowed us to observe changes in pulmonary vessels in each test group. PAH causes severe stenosis and occlusion of the pulmonary artery lumen in rats. Oral administration of sildenafil and bosentan showed some inhibitory effect on pulmonary artery stenosis and occlusion in PAH rats, while the inhibitory effect of nebulized bPS-SF-BT was more significant. This indicates that inhaled administration using bPS as a drug carrier for sildenafil and bosentan can better inhibit pulmonary artery stenosis and occlusion, thereby inhibiting the increase in pulmonary artery pressure.

[0096] Example 8: Evaluation of the therapeutic effect of bPS-SF-BT on pulmonary hypertension rats (right heart hypertrophy index measurement)

[0097] Pulmonary hypertension can cause compensatory hypertrophy of the right ventricle, leading to decreased cardiac function and late-stage right heart failure. At the end of Example 6, i.e., 25 days after injection, after completing the hemodynamic evaluation, the heart tissue was dissected and rinsed with saline. The atria, auricles, and great vessels were removed. The left and right ventricles were separated, and blood and excess water were blotted dry with filter paper. The weights of the right ventricle (RV) and left ventricle + septum (LV + S) were measured using an analytical balance to obtain the right ventricular hypertrophy index, calculated as: RV / (LV + S).

[0098] from Figure 6 The results showed that PAH induced severe right ventricular hypertrophy in rats. While oral administration of sildenafil and bosentan (oral formulations) had some inhibitory effect on right ventricular hypertrophy in PAH rats, the difference in inhibitory effect was not statistically significant. However, the nebulized inhalation of bPS-SF-BT showed a very significant inhibitory effect, with a statistically significant difference compared to the PAH group. This indicates that inhaled administration using bPS as a drug carrier for sildenafil and bosentan better inhibited PAH-induced right ventricular hypertrophy and improved cardiac function.

[0099] Example 9: Comparison of contact angle test results between bPS-SF-BT (with and without mimetic peptides) and hydrophobic solids.

[0100] Following the method provided in Example 2, two types of bPS-SF-BT were prepared at a drug-to-lipid ratio of 1:30. One type contained the KL4 mimic peptide, with the composition exactly as described in Example 2. The other type contained the KL4 mimic peptide but with the other components remaining unchanged. Both types of bPS-SF-BT were dropped onto a hydrophobic solid surface coated with Parafilm, and their contact angles were measured and compared using a Kruss DSA-100 contact angle meter. When the properties of the solid surface are known, the hydrophilicity / hydrophobicity of the liquid can be determined by the contact angle test; that is, if the properties of the solid surface and the liquid are similar, they attract each other (small contact angle); if they are dissimilar, they repel each other (large contact angle).

[0101] Depend on Figure 7 The results show that bPS-SF-BT containing the KL4 mimic peptide has a smaller contact angle on hydrophobic solids than bPS-SF-BT without the KL4 mimic peptide, confirming that bPS-SF-BT containing the KL4 mimic peptide is more hydrophobic. Therefore, the bPS provided by this invention, by incorporating peptide mimics of SP-B protein and / or SP-C protein (e.g., KL4 peptide or RL4 peptide), is closer to the natural pulmonary surfactant in composition and physiological function, reducing the surface tension of the product in the lungs, thereby increasing the distribution of the product in the lungs and improving drug utilization.

[0102] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a biomimetic pulmonary surfactant carrier as a drug carrier in the preparation of an inhaled drug for treating pulmonary hypertension; wherein the biomimetic pulmonary surfactant carrier is prepared from total lipids and mimic peptides, wherein the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol in the total lipids is 10:1:1:1; the mass ratio of the total lipids to the mimic peptides is 100:0.8-1.2, the pulmonary surfactant mimic peptides are mimic peptides of SP-B protein and / or SP-C protein, the drug is sildenafil and bosentan, the drug-to-lipid ratio in the inhaled drug is 1:30, the SP-B protein mimic peptides are selected from at least one of SEQ ID NO. 1-7, and the SP-C protein mimic peptides are selected from at least one of SEQ ID NO. 8-10.

2. The application according to claim 1, characterized in that, The mass ratio of total lipids to the mimic peptides is 100:0.9-1.

1.

3. The application according to claim 2, characterized in that, The mass ratio of total lipids to the mimic peptides is 100:

1.

4. The application according to claim 1, characterized in that, The mimic peptides of the SP-B protein are SEQ ID NO.1 and / or SEQ ID NO.

2.

5. The application according to any one of claims 1-4, characterized in that, The mass ratio of sildenafil to bosentan is 1:4 to 4:

1.

6. An inhaled medication for treating pulmonary hypertension, characterized in that, It is prepared by loading a drug for treating pulmonary hypertension onto a biomimetic pulmonary surfactant carrier; the biomimetic pulmonary surfactant carrier is prepared from total lipids and mimic peptides, wherein the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol in the total lipids is 10:1:1:1; the mass ratio of the total lipids to the mimic peptides is 100:0.8-1.2, the pulmonary surfactant mimic peptides are mimic peptides of SP-B protein and / or SP-C protein, the drug is sildenafil and bosentan, the drug-to-lipid ratio in the inhaled drug is 1:30, the SP-B protein mimic peptide is selected from at least one of SEQ ID NO. 1-7, and the SP-C protein mimic peptide is selected from at least one of SEQ ID NO. 8-10.

7. The inhaled medication for treating pulmonary hypertension according to claim 6, characterized in that, The mass ratio of sildenafil to bosentan is 1:4 to 4:

1.

8. The inhaled medication for treating pulmonary hypertension according to claim 7, characterized in that, The mass ratio of sildenafil to bosentan is 1:2 to 2:

1.

9. The inhaled medication for treating pulmonary hypertension according to any one of claims 6-8, characterized in that, The mass ratio of total lipids to the mimic peptides is 100:

1.

10. A method for preparing an inhaled medicament for treating pulmonary hypertension according to any one of claims 6-9, characterized in that, The process includes the following steps: adding the mimic peptide and the drug for treating pulmonary hypertension to a chloroform solution of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol; mixing well; and rotating the mixture in a constant-temperature water bath until the chloroform solvent evaporates to form a uniform lipid film layer. Water is added to fully hydrate the lipid film layer. After complete hydration, the hydrated solution is repeatedly extruded through a cellulose acetate membrane to obtain an inhaled drug for treating pulmonary hypertension.

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