CN115300635A - Bionic pulmonary surfactant carrier and medicine of inhalant for treating pulmonary hypertension - Google Patents

Abstract

The invention relates to a bionic lung surfactant carrier, which is prepared from total lipid and mimic peptide, wherein in the total lipid, the mass ratio of dipalmitoyl phosphatidylcholine to dipalmitoyl phosphatidylglycerol to dipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000 to cholesterol is 8-12:1:1:1; the lung surfactant mimic peptide is a mimic peptide of SP-B protein and/or a mimic peptide of SP-C protein. The bionic lung surfactant carrier has the advantages of high loading rate and high stability. The invention also relates to an inhalant dosage form which is loaded with the medicine for treating the pulmonary hypertension by the bionic pulmonary surfactant carrier, the carried medicine is distributed in the lung more uniformly, the inhalant medicine is closer to the natural pulmonary surfactant in terms of components and physiological functions, the degradation of the product can be delayed, the utilization rate of the medicine can be improved, and a good pulmonary hypertension treatment effect can be achieved.

Description

Biomimetic pulmonary surfactant carrier and drug for inhalation in the treatment of pulmonary arterial hypertension

technical field

The invention relates to the field of pharmaceutical dosage forms, in particular to a carrier of an inhalant for treating pulmonary arterial hypertension, a medicine and a preparation method thereof.

Background technique

Common lung diseases include asthma, bronchitis, tuberculosis, infectious and non-infectious pneumonia, chronic obstructive pulmonary disease, pulmonary hypertension, lung cancer, etc. Pulmonary arterial hypertension (PAH) is a malignant lung disease characterized by increased pulmonary artery pressure and pulmonary vascular remodeling. PAH can lead to right heart failure and even death in patients, and the morbidity and mortality rates are extremely high. Traditionally, supportive treatments such as anticoagulants, diuretics, digoxin, digitalis, and oxygen inhalation are mainly used for PAH, and the curative effect is very limited. In recent years, by studying the pathogenesis of PAH, targeted drugs targeting various signaling pathways such as prostacyclin, nitric oxide, and endothelin-1 have been developed. These targeted drugs are mainly oral, including prostacyclin receptor agonists (treprostinil, beraprost, selexipag), type 5 phosphodiesterase inhibitors (sildenafil, tadala non), endothelin receptor antagonists (bosentan, ambrisentan, macitentan), soluble guanylate cyclase agonists (riociguat), etc., and a small amount of injections (epoprostenol, Quqian Prostinier). The above-mentioned targeted drugs can reduce the pulmonary artery pressure and improve the symptoms of PAH, but the long-term prognosis of patients is still unsatisfactory, and the mortality rate remains high.

Combination therapy refers to the combination of two or more drugs with different pathways of action for treatment. Combined medication can maximize the efficacy of drugs, reduce toxicity, and reduce drug doses. For PAH, a disease with multiple pathogenic pathways, theoretically, combination therapy is better than monotherapy. There are two strategies for the combined application of PAH targeted drugs: sequential combination therapy and initial combination therapy. The results of a number of randomized controlled trials released in recent years have shown that both sequential combination therapy and initial combination therapy can significantly reduce the occurrence of clinical deterioration in PAH patients. Therefore, the Chinese Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension (2021 Edition) recommend that, except for patients with PAH risk stratification as low risk or elderly patients, patients with risk stratification as intermediate or high risk are recommended to use combination therapy. At present, the most commonly used combined targeted therapy for PAH is the combination of phosphodiesterase type 5 inhibitors and endothelin receptor antagonists, such as bosentan + sildenafil, ambrisentan + tadalafil, etc.

At the same time, the toxic side effects and drug-induced adverse reactions of these oral and injection drugs cannot be eliminated all the time. For example, as the first-line clinical drug for PAH, bosentan has teratogenic effects and liver toxicity, and sildenafil can cause severe cardiac events and abnormal vision. In view of the limitations of the existing PAH treatment drugs, such as many adverse reactions, low bioavailability, and poor prognosis, changing the dosage form of existing drugs, or adjusting the dosage form to make some difficult-to-drug targets into drugs has become an important idea for the treatment of PAH.

Compared with oral and injection, inhaled drug delivery has huge advantages in the treatment of PAH and other lung diseases. Firstly, the lung is the direct lesion site of PAH, and inhalation administration can deliver the drug directly to the diseased tissue; secondly, the lung has a large absorption area and has a fast onset of action; thirdly, inhalation administration avoids the first-pass effect and has high bioavailability , can reduce the dosage of drugs and reduce the distribution of drugs in other tissues of the human body and related adverse reactions. Therefore, inhalation administration has become the most effective treatment method for pulmonary diseases such as asthma and chronic obstructive pulmonary disease, and it is also the theoretically best route of administration for the treatment of PAH. However, the inhaled drugs for PAH are very limited at present, only a few types such as iloprost and treprostinil have inhaled dosage forms, and most of the commonly used targeted drugs such as bosentan and sildenafil have not achieved the function. Inhalation dosage form for effective application.

Although inhalation administration has the above-mentioned advantages, it also faces three key issues of drug effectiveness, immunogenicity and safety, that is, how to improve the residence of the drug in the lungs, and how to prevent the drug from being released by the lung immune system. Identify and remove, and how to avoid drugs (mainly excipients in preparations) from damaging the lungs and the human body. The first two problems involve the clearance of foreign substances by the lung defense system, while the third problem is mainly determined by the composition of pharmaceutical preparations. The lung defense system is mainly composed of immune cells and pulmonary surfactant. Pulmonary surfactant is a lipoprotein complex synthesized and secreted by alveolar type II epithelial cells, covering the alveolar surface. After foreign substances enter the lungs, they will be blocked by pulmonary surfactant, excreted through ciliary movement, or recognized and cleared by immune cells. Therefore, in order to improve lung residence and reduce immunogenicity of inhaled drugs, it is necessary to overcome the blocking of pulmonary surfactant and the recognition of immune cells.

Lipids in lung surfactant account for about 90%, and proteins account for about 10%. The lipid component is mainly phosphatidylcholine (about 70-80%), and also includes a small amount of phosphatidylglycerol (about 8%), phosphatidylethanolamine (about 8%), cholesterol (about 8%) and the like. The protein component is mainly surfactant-associated protein (SP protein). There are four kinds of SP proteins, including hydrophilic SP-A and SP-D proteins and hydrophobic SP-B and SP-C proteins. Although the content of the latter two is small (about 1-2%), they play an important role in maintaining alveolar structure, especially SP-B is a key component in maintaining alveolar surface tension in pulmonary surfactant, and can prevent pulmonary surfactant Substances are degraded by phospholipids (Hite RD, et al. Surfactantprotein B inhibits secretory phospholipase A2 hydrolysis of surfactantphospholipids. Am J Physiol Lung Cell Mol Physiol.2012). Based on the amino acid sequence and structural characteristics of SP-B, a polypeptide containing 21 amino acids can mimic the physiological function of SP-B (Cochrane CG, et al.Pulmonary surfant protein B (SP-B):structure-function relationships.Science.1991) . The amino acid sequence of this polypeptide is KLLLLKLLLLKLLLLKLLLLK (KL4 polypeptide), or RLLLRLLLLLRLLLLLRLLLLR (RL4 polypeptide), and the KL4 polypeptide is also called sinapultide (Sinapultide). There are several other peptides that also mimic the function of SP-B or SP-C.

Pulmonary surfactant is widely used clinically in the treatment of neonatal respiratory distress syndrome. Pulmonary surfactant as a drug is divided into two types: natural type and synthetic type. Natural pulmonary surfactants are extracted from the lung tissue of animals such as cattle or pigs, such as Survanta (Shoufeijia) and Infasurf in the United States, Curosurf in Italy, and Kelisu and Feihuotong in China. Synthetic pulmonary surfactants are formulated according to the main components and proportions of natural pulmonary surfactants, such as Exosurf and Lucinactant (Luxinatam, trade name Surfaxin) in the United States. Among them, Exosurf only contains phospholipids, while Lucinatan also adds KL4 polypeptide in addition to phospholipids, which is closer to natural lung surfactant than Exosurf in terms of composition, function and curative effect. Compared with natural surfactants, synthetic pulmonary surfactants are basically the same in terms of clinical efficacy and safety, with smaller batch-to-batch differences and no potential risk of spreading animal diseases, so they have unique advantages. In the animal model of PAH, the SP protein content decreased, reducing lung function (Gutierrez JA, et al. Decreased surfactant proteins inlambs 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 mimetic peptide can directly improve lung function in patients with PAH.

It has been reported to prepare liposomes as drug carriers by using phospholipids, sphingolipids, cholesterol and other materials. However, liposomes are mainly used for injection treatment of diseases such as tumors, and are rarely used for inhalation administration. At present, the representative liposome inhalation drug is the amikacin liposome developed by Insmed of the United States for the treatment of infectious lung diseases (such as refractory non-tuberculous mycobacterium avium infection). bacillary lung disease). In this product, amikacin is loaded with liposomes consisting of dipalmitoylphosphatidylcholine (DPPC) and cholesterol. Compared with intravenous amikacin, inhalation of amikacin liposomes prolongs the release of star in the lungs while reducing systemic exposure, thereby reducing systemic toxicity. In China, there is an attempt to add fatty alcohol, surfactant, unsaturated phospholipid and other components to DPPC and use it as an inhalation drug carrier to load insulin to lower blood sugar levels (patent application number CN200610025706). There are foreign reports of using glyceryl distearate, beeswax or stearic acid in combination with oleic acid and emulsifiers to make nano-liposomes loaded with sildenafil for inhalation 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, liposomes have their own composition, and there is a big difference from the natural lung surfactant components, and the key SP protein in the lung surfactant is missing. Therefore, no matter in terms of composition, physical and chemical properties, or physiological In terms of function, they are very different from natural lung surfactant.

Contents of the invention

Based on this, the object of the present invention is to provide a kind of bionic pulmonary surfactant carrier and medicine of the inhalant for treating pulmonary hypertension, the medicine prepared by the bionic pulmonary surfactant carrier of the present invention has high loadability, good stability, Good biological activity characteristics.

In order to achieve the above object, the present invention is realized by including the following technical solutions.

The first aspect of the present invention provides a bionic lung surfactant carrier.

A bionic pulmonary surfactant carrier, which is prepared from total lipids and pulmonary surfactant mimic peptides, in the total lipids, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidyl The mass ratio of acetylethanolamine-polyethylene glycol 2000 and cholesterol is 8-12:1:1:1; the mass ratio of the total lipid to the mimic peptide is 100:0.8-1.2.

In some of these embodiments, the bionic lung surfactant carrier is prepared from total lipids and mimic peptides, wherein the total lipids are composed of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidyl Ethanolamine-polyethylene glycol 2000 and cholesterol are composed of four components. Among the total lipids, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, The mass ratio of cholesterol is 9-11:1:1:1; more preferably dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol mass ratio is 10:1:1:1.

In some of the embodiments, the mass ratio of the total lipid to the mimetic peptide is 100:0.9-1.1, most preferably 100:1.

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

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

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

The second aspect of the present invention is to provide the application of bionic lung surfactant carrier as drug carrier in the preparation of inhalation drug for treating pulmonary arterial hypertension.

The third aspect of the present invention is to provide an inhalation medicine for treating pulmonary hypertension.

An inhalation drug for treating pulmonary hypertension, which is prepared by loading the drug for treating pulmonary hypertension on the bionic lung surfactant carrier.

In some of these embodiments, the drug is one or both of sildenafil and bosentan.

In some preferred embodiments, the drugs are sildenafil and bosentan.

In some of these embodiments, the ratio of drug to lipid in the inhalation drug is 1:20-40.

In some preferred embodiments, the drug-to-lipid ratio in the inhalation drug is 1:30-40, and the optimal ratio is 1:30.

The fourth aspect of the present invention is to provide a preparation method of the bionic lung surfactant carrier.

A preparation method of the bionic lung surfactant carrier, comprising the following steps:

Add the mimetic peptide to the chloroform solution of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol, mix well, and rotate in a constant temperature water bath until the chloroform The solvent evaporates to form a uniform lipid film layer;

Water is added to fully hydrate the lipid film layer, and after the hydration is complete, the cellulose acetate membrane used in the hydration solution is repeatedly extruded to obtain the bionic lung surfactant.

The fifth aspect of the present invention is to provide a preparation method of the inhalation medicine for treating pulmonary hypertension.

A preparation method of the inhalation medicine for the treatment of pulmonary hypertension, comprising the following steps:

Add the mimetic peptide to the chloroform solution of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol, and the treatment of pulmonary hypertension Drugs, mixed, rotary evaporation in a constant temperature water bath until the chloroform solvent evaporates, forming a uniform lipid film layer;

Water is added to fully hydrate the lipid film layer, and after the hydration is complete, the cellulose acetate film used in the hydration solution is repeatedly extruded to obtain an inhalant drug for treating pulmonary hypertension.

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

Using bionic pulmonary surfactant (bPS) as a drug carrier for the treatment of pulmonary hypertension and other pulmonary diseases has multiple advantages. In addition to the disclosed bPS itself having a therapeutic effect, it can reduce alveolar surface tension and improve lung function. ( 1) The bPS described in the present invention is similar to the composition of the natural pulmonary surfactant, and is an ideal pharmaceutical preparation adjuvant, which avoids damage to the lungs; (2) The bPS of the present invention is similar in function to the natural pulmonary surfactant, so that The carried drugs can escape immune cell recognition and natural pulmonary surfactant blocking, which improves the residence and penetration of the drugs in the lungs; (3) the inventors found that the bPS of suitable composition makes the carried drugs (sildenafil and bosentan ) to obtain a more uniform distribution in the lung. Compared with the existing way of using liposomes for drug loading, the bPS provided by the present invention adds SP-B protein and/or SP-C protein mimics (such as KL4 polypeptide or RL4 polypeptide), which is closer to natural lung surfactant in composition and physiological function, reduces the surface tension of the product in the lung, improves the particle size stability of the product, can increase the distribution of the product in the lung, delay the degradation of the product, Improve drug utilization.

Description of drawings

Figure 1. The particle size and dispersion coefficient stability analysis of bPS-SF-BT prepared under different drug-to-lipid ratios.

Figure 2. Particle size potential and TEM images of bPS-SF-BT.

Figure 3. Schematic diagram of the cell viability results of drugs and bPS under different dosage conditions.

Figure 4. Schematic diagram of the pulmonary artery pressure waveforms and analysis results of PAH rats before and after treatment.

Figure 5. Schematic diagram of the therapeutic effect of bPS-SF-BT on pulmonary artery lumen stenosis and occlusion.

Figure 6. Schematic diagram of the therapeutic effect of bPS-SF-BT on pulmonary hypertension-induced right heart hypertrophy.

Figure 7. Comparison of contact angle test results of bPS-SF-BT without and with mimic peptides and hydrophobic solids.

Detailed ways

In order to facilitate the understanding of the present invention, the following will describe the present invention more fully. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the present disclosure more thorough and comprehensive.

The experimental method that does not indicate specific condition in the following examples is generally according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacture conditions recommended by the manufacturer. Various commonly used chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the technical field of the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Some embodiments of the present invention relate to a bionic pulmonary surfactant carrier (bPS), which is prepared from total lipids and pulmonary surfactant mimic peptides, in the total lipids, dipalmitoylphosphatidylcholine, Dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol mass ratio is 8-12:1:1:1; the mass ratio of the total lipid and the mimic peptide is 100: 0.8-1.2. At the same time, it also relates to the drug for treating pulmonary arterial hypertension loaded with the bionic lung surfactant carrier and prepared into inhalation.

Compared with oral or injection administration, modifying the inhalation dosage form of drugs is a new idea for the treatment of pulmonary hypertension. The method for loading drugs on bPS provided by the present invention can realize the effective encapsulation and delivery of one or more drugs. The highest encapsulation efficiency can reach 93.15% and 74.57%, respectively, realizing the good encapsulation of the drug. After bPS is loaded with drugs, the way of inhalation to treat lung diseases can avoid the liver first-pass effect of drugs, improve bioavailability, achieve sustained release, and reduce the frequency of administration. The sildenafil and bosentan loaded with bPS are firstly distributed to the lungs after inhalation, thereby inhibiting the proliferation of pulmonary artery smooth muscle cells, dilating pulmonary artery vessels, reducing the average pulmonary artery pressure, further reducing right heart hypertrophy, improving cardiac function and improving prognosis, reaching pulmonary artery The effect of hyperbaric therapy.

Studies have shown that drug particles with a diameter greater than 5 μm are mainly deposited in the upper respiratory tract, and drug particles with a diameter of 1–5 μm can be deposited in the central and distal airways where the air velocity is low, while drug particles with a diameter of less than 1 μm can be deposited in large quantities in the respiratory tract. and eventually reach the alveolar region. The average particle size of the bPS prepared in the present invention after loading is about 123.37nm, and the particle size meets the theoretical requirement for alveolar deposition.

Studies have shown that the cytotoxicity of nanomaterials is one of the serious and urgent problems for their clinical translation. Each component of bPS used in the present invention is the main component of natural lung surfactant, which avoids the toxicity of preparation materials to cells.

In addition to drug safety, reducing drug immunogenicity and preventing drug elimination in vivo is another problem that needs to be solved. Studies have found that many macromolecular drugs such as peptides, proteins, and nucleic acids are easily aggregated and cleared by macrophages. The average particle size of the bPS prepared in the present invention is about 123.37nm after drug loading, and the particle size can avoid the capture and phagocytosis of alveolar macrophages; at the same time, bPS has alveolar affinity, which reduces immune stimulation and enables the drug to reside for a long time lungs, prolonging drug stability and bioavailability.

The amino acid sequences of the analogues of the present invention are shown in Table 1 and Table 2.

The present invention will be described in further detail below in conjunction with specific examples.

Embodiment 1: Preparation of bionic lung surfactant containing SP-B mimetic peptide by thin film dispersion method

The preparation method comprises the following steps:

Dissolve dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG2000), and cholesterol in chloroform respectively to a concentration of 100mg/ml, and use chloroform:methanol=1:1( v:v) Dissolve dipalmitoylphosphatidylglycerol (DPPG) and SP-B mimetic peptide KL4 to a concentration of 10mg/ml respectively. Add 5ml chloroform in 50ml round bottom flask, add DPPC 3.923ml (392.3mg), DPPG 3.923ml (39.23mg), DPPE-PEG2000 0.3923ml (39.23mg), cholesterol 0.3923ml (39.23mg) successively, total lipid mass is 510 mg. Then add 510ul (5.1mg) of KL4 polypeptide with 1% total lipid mass, and ultrasonically shake for 30s to fully mix the ingredients in the bottle, and then rotate and evaporate in a constant temperature water bath at 50°C and 180rpm for 2h until the chloroform solvent evaporates. A uniform lipid film layer forms at the bottom.

Add 5-10ml of water to the bottle after rotary evaporation, and ultrasonically hydrate in a water bath at 40°C for 15 minutes to fully hydrate the lipid layer. The bionic lung surfactant was obtained after repeated extrusion 21 times with a cellulose acetate membrane with a pore size of 200 nm, which is abbreviated as bPS in the following text. Finally, the solution was centrifuged several times at 5000 rpm for 20 min with a 50 kDa ultrafiltration tube until it was concentrated to the desired concentration, and collected and stored at 4°C for use.

Example 2: Bionic pulmonary surfactant loaded with two PAH therapeutic drugs, sildenafil and bosentan

Dissolve the raw material components of bPS in the manner provided in Example 1, then dissolve sildenafil (Sildenafil, SF) with chloroform:methanol=1:1 (v:v) to a concentration of 10mg/ml, bosentan ( Bosentan, BT) was dissolved in chloroform to a concentration of 10 mg/ml. Add each raw material composition that embodiment 1 provides in the round bottom flask, then according to the ratio (i.e. medicine lipid ratio) of total amount of medicine and total lipid composition, be 1:10 to 1:50 (w:w) corresponding ratio input The steps of sildenafil, bosentan, ultrasonic vibration and mixing, and subsequent rotary evaporation are the same as those in Example 1. The obtained bionic pulmonary surfactant contains two PAH therapeutic drugs, sildenafil and bosentan, which are abbreviated as bPS-SF-BT hereinafter.

In order to test the stability of bPS-SF-BT, the above-mentioned bPS-SF-BT prepared according to different drug-to-lipid ratios was stored at 4°C for 5 days, diluted with water every day, and measured by a nano laser particle size analyzer (model Nano ZS90). The particle size and dispersion coefficient are analyzed according to the change of particle size and dispersion coefficient, and the results are shown in Figure 1. The bionic pulmonary surfactant of the present invention has good stability when prepared in a relatively large range of drug-to-lipid ratio, and the product prepared at a drug-to-lipid ratio of 1:30 has the best stability.

Embodiment 3: Measuring the encapsulation efficiency of medicine in bPS-SF-BT

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

The results showed that when the drug-to-lipid ratio was 1:30, the drug encapsulation efficiency of bPS-SF-BT was relatively optimal, which were 93.15% for sildenafil and 74.57% for bosentan, which confirmed that bionic lung surfactant had good drug encapsulation efficiency. sealing efficiency. In Example 4-Example 9 of the present invention, unless otherwise specified, bPS-SF-BT with a drug-to-lipid ratio of 1:30 was used. Table 3.1 to Table 3.5: The encapsulation efficiency of sildenafil and bosentan loaded alone, and the encapsulation efficiency of sildenafil and bosentan loaded simultaneously.

chart 3.1

Table 3.2

Table 3.3

Table 3.4

Table 3.5

Example 4: Identification of physical properties of bPS-SF-BT

After the bionic lung surfactant bPS-SF-BT loaded with sildenafil and bosentan was obtained according to Example 2, take an appropriate amount of bPS-SF-BT and dilute it with water and use a nano laser particle size analyzer (model Nano ZS90) It was measured that the average particle size was about 123.37nm, the average potential was about -31.67mV, and the dispersion coefficient was 0.08 (Fig. 2A). Take 10 μl of diluted bPS-SF-BT and drop it on the copper grid coated with carbon film in advance, stain with 2% phosphotungstic acid solution (pH 7.0), let it stand and dry naturally at room temperature, and use a transmission electron microscope (model JEM -1400) to observe and measure the bPS-SF-BT particles, showing that the particle size is basically consistent with that measured by the nano-laser particle size analyzer (Fig. 2B). Therefore, the prepared bPS-SF-BT has the basic physical properties of nanomedicine.

Example 5: Detection of cytotoxicity of bPS-SF-BT

Mouse type II alveolar epithelial cells MLE-12 were cultured with DMEM/F12 medium (containing 10% fetal bovine serum, 1% penicillin/streptomycin double antibody solution) and used for cytotoxicity test. The cells were evenly seeded in a 96-well plate at a density of 1×10 ⁴ cells/well, and fresh medium was replaced after 24 hours, and sildenafil (SF) and bosentan with a concentration range of 20-200ug/ml were added (BT), sildenafil and bosentan (SF-BT) and bPS-SF-BT, with 5 replicate wells for each concentration. Add 10% CCK-8 reagent 24 hours after dosing, place it in a cell incubator to react for 1.5-2 hours, and then use a microplate reader (model Synergy H1) to measure the absorbance at a wavelength of 450 nm. The calculation formula of cell viability is: Vitality percentage (%)=(OD plus drug-OD blank)/(OD cell-OD blank)*100.

From the results in Figure 3, it can be seen that sildenafil alone, bosentan alone or the combination of the two drugs have the cytotoxicity of inhibiting cell proliferation at a certain dose, while the cell viability of bPS is above 90% in the test dose range, showing a great Low cytotoxicity. The cell viability of bPS-SF-BT is above 70% within the test dose range, which has good safety.

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

Sprague Dawley rats (male, 200 g) were selected to establish an animal model of pulmonary hypertension. The PAH model was established by one-time injection of monocrotaline (MCT) 50 mg/kg in the back of the neck. 7 days after the injection, the rats were given drug treatment. The sildenafil and bosentan loaded without bPS were given by oral gavage, and the bPS-SF-BT used a small animal atomization drug delivery device (model YLS- 8B) Administration by inhalation. The dosages are: sildenafil 6 mg/kg, bosentan 11 mg/kg, and the frequency of administration is once every 3 days until the 25th day after MCT injection.

Hemodynamic evaluation was performed on the 25th day after MCT injection, and the right ventricular systolic pressure (RVSP) of each group was measured by catheter method. The specific measurement method is: anesthetize the rat with 4% tribromoethanol intraperitoneal injection and fix it on the operating table in the supine position. After the abdominal hair is shaved, find and separate the external jugular vein on the chest, and use pre-filled 1000U/ml heparin sodium solution The PE50 catheter directly enters the right ventricle through the external jugular vein, and the other end is connected to the blood pressure transducer. After the right ventricle pressure signal appears, use the physiological signal recording and analysis system (model BL-420N) to record the real-time RVSP pressure waveform and calculate RVSP.

The results in Figure 4 show that the RVSP (equivalent to mean pulmonary arterial pressure) of the normal group was about 13mmHg, and the RVSP of the pulmonary hypertension group was about 47mmHg. Oral administration of sildenafil and bosentan had a certain antihypertensive effect. Inhalation administration of bPS-SF-BT has a better antihypertensive effect, and the RVSP is about 17mmHg, indicating that the inhalation administration of bPS as the drug carrier of sildenafil and bosentan can achieve a very good therapeutic effect on PAH.

Example 7: Evaluation of the therapeutic effect of bPS-SF-BT on rats with pulmonary hypertension II (determination of the degree of pulmonary artery lumen hyperplasia)

Pulmonary artery remodeling is the main pathological feature of PAH. It mainly refers to the progressive stenosis and occlusion of the pulmonary artery lumen due to intima injury, medial hypertrophy, adventitial fibrosis, and basement membrane sclerosis. The pressure keeps building.

At the end of Example 6, that is, after the hemodynamic evaluation was completed on the 25th day after the MCT injection, the lung tissue was dissected, rinsed with normal saline and fixed with 4% paraformaldehyde for 24 to 48h, then paraffin sectioned the tissue and carried out Hematoxylin and eosin (HE) staining was used to observe the morphology of pulmonary arterioles. The specific steps are:

(1) The fixed lung tissue was gradually dehydrated from low-concentration alcohol to high-concentration alcohol, soaked in wax and embedded, and then displayed after sectioning. Paraffin sections were dewaxed in xylene for 5-10 minutes, and then anhydrous , 95% ethanol, 85% ethanol, 75% ethanol for 5 minutes each, rinse with water for 1 minute;

(2) Dyeing with hematoxylin solution for 3-5 minutes, washing with water for 1-2 minutes;

(3) Alcohol differentiation with 0.8%-1% hydrochloric acid, return to blue with dilute lithium carbonate aqueous solution, and then wash with water for 1-2min;

(4) Dyeing with eosin dye solution for 1-2 sec, without washing, directly put into 95% ethanol, and dehydrate in absolute ethanol for 1-2 min;

(5) Xylene transparent, sealed, microscopic examination.

Through the HE staining results of hilar cross-section of lung tissue in Fig. 5, we can observe the changes of pulmonary blood vessels in each test group. PAH can cause severe stenosis and occlusion of the pulmonary artery lumen in rats. Oral administration of sildenafil and bosentan has a certain inhibitory effect on the stenosis and occlusion of the pulmonary artery lumen of PAH rats, while aerosol inhalation of bPS-SF- The inhibitory effect of BT is more obvious, indicating that the inhalation administration of bPS as the drug carrier of sildenafil and bosentan can better inhibit the stenosis and occlusion of the pulmonary artery lumen, thereby inhibiting the increase of pulmonary artery pressure.

Example 8: Evaluation of the therapeutic effect of bPS-SF-BT on rats with pulmonary hypertension III (determination of right heart hypertrophy index)

Pulmonary hypertension can cause compensatory hypertrophy of the right ventricle, leading to decreased cardiac function and advanced right heart failure. At the end of Example 6, that is, after the hemodynamic evaluation was completed on the 25th day after injection, the heart tissue was dissected and rinsed with normal saline, the atrium, auricle and great blood vessels were removed, the left and right ventricles were separated, and the blood and excess water were blotted with filter paper , using an analytical balance to measure the weight of the right ventricle (right ventricle, RV) and left ventricle+septum (left ventricle+septum, LV+S), and thus obtain the right ventricular hypertrophy index, which is calculated as: RV/(LV+ S).

From the results in Figure 6, it can be seen that PAH has caused severe right heart hypertrophy in rats. Although oral administration of sildenafil and bosentan (oral preparations) has a certain inhibitory effect on right heart hypertrophy in PAH rats, the inhibitory effect is not statistically significant. difference. However, the inhibitory effect of bPS-SF-BT administered by nebulization inhalation is very obvious, and there is a statistical difference compared with the PAH group, indicating that the inhaled administration of bPS as the drug carrier of sildenafil and bosentan has a better inhibitory effect. The right heart hypertrophy caused by PAH was eliminated, and the heart function was improved.

Example 9: Comparison of contact angle test results between bPS-SF-BT without and with mimic peptides and hydrophobic solids

According to the method provided in Example 2, two types of bPS-SF-BT were prepared with a ratio of drug to lipid of 1:30, one of which was completely composed of the ingredients in Example 2, that is, bPS-SF containing KL4 mimetic peptide -BT; the second is bPS-SF-BT without KL4 mimetic peptide, and the composition of other components remains unchanged, that is, bPS-SF-BT without KL4 mimetic peptide. The above two kinds of bPS-SF-BT were dropped onto the hydrophobic solid surface on which Parafilm was laid, and measured and compared using a contact angle meter (model Kruss DSA-100). When the properties of the solid surface are known, the hydrophilic and hydrophobic properties of the liquid can be obtained through the contact angle test, that is, the properties of the solid surface and the liquid are of the same nature and attract each other (small contact angle), and opposite sexes repel each other (large contact angle).

It can be seen from the results in Figure 7 that the contact angle of bPS-SF-BT containing KL4 mimic peptide is smaller than that of bPS-SF-BT without KL4 mimic peptide on the hydrophobic solid, confirming that the bPS-SF-BT containing KL4 mimic peptide has a more hydrophobic. Therefore, the bPS provided by the present invention is closer to the natural lung surfactant in composition and physiological function by adding the polypeptide mimetic (such as KL4 polypeptide or RL4 polypeptide) of SP-B protein and/or SP-C protein, and reduces the production rate. The surface tension in the lung can increase the distribution of the product in the lung and improve the utilization rate of the drug.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements 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 for the present invention should be based on the appended claims.

Claims (14)

1. A biomimetic lung surfactant vector prepared from total lipid in which dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, dipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol in a mass ratio of 8-12:1:1:1; the mass ratio of the total lipid to the mimetic peptide is 100:0.8-1.2, wherein the lung surfactant mimetic peptide is a mimetic peptide of SP-B protein and/or a mimetic peptide of SP-C protein.

2. The biomimetic lung surfactant carrier according to claim 1, wherein, in the total lipid, the mass ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol is 9-11:1:1:1; more preferably dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, wherein the mass ratio of cholesterol is 10:1:1:1.

3. the biomimetic lung surfactant vector according to claim 1, wherein the mass ratio of total lipid to mimetic peptide is 100:0.9-1.1, most preferably 100:1.

4. the biomimetic lung surfactant carrier according to any one of claims 1-3, wherein the peptidomimetic of the SP-B protein is selected from at least one of SEQ ID nos. 1-7.

5. The biomimetic lung surfactant vector according to claim 4, wherein the mimetic peptide of SP-B protein is SEQ ID No.1 and/or SEQ ID No.2.

6. The biomimetic lung surfactant vector according to any of claims 1-3, wherein the mimetic peptide of SP-C protein is selected from at least one of SEQ ID No. 8-10.

7. Use of a biomimetic lung surfactant vector according to any of claims 1-6 as a drug carrier in the preparation of an inhalant medicament for the treatment of pulmonary hypertension.

8. An inhalant drug for treating pulmonary hypertension, which is prepared by loading a drug for treating pulmonary hypertension on the bionic lung surfactant vector according to any one of claims 1 to 6.

9. The inhalant medicament for treating pulmonary arterial hypertension according to claim 8, wherein the medicament is one or both of sildenafil and bosentan.

10. The inhalant medicament for the treatment of pulmonary hypertension according to claim 9, wherein the medicament is sildenafil and bosentan.

11. The inhalant drug for the treatment of pulmonary arterial hypertension according to claim 8, wherein the ratio of drug to lipid in the inhalant drug is 1:20-40.

12. The inhalant drug for the treatment of pulmonary arterial hypertension according to claim 11, wherein the ratio of drug to lipid in the inhalant drug is 1:30-40, preferably 1:30.

13. the inhalant medicament for treating pulmonary hypertension according to claim 9, wherein the mass ratio of sildenafil to bosentan is 1:4 to 4:1, preferably, 1:2 to 2:1, most preferably 1:2.

14. a method for preparing an inhalant drug for the treatment of pulmonary hypertension according to any one of claims 8 to 13, comprising the steps of:

adding the mimic peptide and the drug for treating pulmonary arterial hypertension into a chloroform solution of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 and cholesterol, uniformly mixing, and performing rotary evaporation in a constant-temperature water bath until a chloroform solvent is evaporated to form a uniform lipid film layer; and adding water to fully hydrate the lipid film layer, and repeatedly extruding the hydration solution by using a cellulose acetate film after the hydration is completed to obtain the inhalant medicine for treating the pulmonary hypertension.

Images