阅读说明：本技术 一种复方干粉吸入剂及其应用 (Compound dry powder inhalant and application thereof ) 是由 刘志东 祁东利 彭辉 皮佳鑫 郭盼 邓秀平 李佳玮 于 2021-08-25 设计创作，主要内容包括：本申请提供了一种复方干粉吸入剂,包含黄芩苷、盐酸氨溴索、L-亮氨酸和磷酸盐,以复方干粉吸入剂的质量计,L-亮氨酸占0-50％,磷酸盐占15-35％,黄芩苷与盐酸氨溴索的总质量占15-85％,其中黄芩苷与盐酸氨溴索的质量比为1:(0.2-2)；所述复方干粉吸入剂的Dv90≤5μm。黄芩苷与盐酸氨溴索联合用药,可有效降低肺部组织的炎症和氧化损伤,缓解肺水肿和组织病理改变,减轻肺功能障碍和纤维化；复方干粉吸入剂通过肺部给药,明显提高药物在血浆中的半衰期和体内滞留时间,提高药物在肺组织中的生物利用度,降低药物在肺组织中的清除率,延长药物在肺部的滞留时间,有利于药物在肺组织中充分发挥作用。(The application provides a compound dry powder inhalant, which comprises baicalin, ambroxol hydrochloride, L-leucine and phosphate, wherein the L-leucine accounts for 0-50% of the mass of the compound dry powder inhalant, the phosphate accounts for 15-35% of the mass of the compound dry powder inhalant, and the total mass of the baicalin and the ambroxol hydrochloride accounts for 15-85%, wherein the mass ratio of the baicalin to the ambroxol hydrochloride is 1 (0.2-2); the Dv90 of the compound dry powder inhalant is less than or equal to 5 mu m. The combination of baicalin and ambroxol hydrochloride can effectively reduce the inflammation and the oxidative damage of lung tissues, relieve pulmonary edema and histopathological changes and relieve lung dysfunction and fibrosis; the compound dry powder inhalant is administrated through the lung, so that the half-life period and the in-vivo retention time of the medicine in plasma are obviously improved, the bioavailability of the medicine in lung tissues is improved, the clearance rate of the medicine in the lung tissues is reduced, the retention time of the medicine in the lung is prolonged, and the full play of the medicine in the lung tissues is facilitated.)

1. The compound dry powder inhalant comprises 0-50% of L-leucine, 15-35% of phosphate and 15-85% of total mass of baicalin and ambroxol hydrochloride, wherein the mass ratio of baicalin to ambroxol hydrochloride is 1: (0.2-2), preferably 1: (0.8-1.2); the Dv90 of the compound dry powder inhalant is less than or equal to 5 mu m.

2. The compound dry powder inhaler according to claim 1, wherein the L-leucine accounts for 10-40% by mass of the compound dry powder inhaler.

3. The compound dry powder inhaler according to claim 2, wherein the L-leucine accounts for 15-25% by mass of the compound dry powder inhaler.

4. The compound dry powder inhalant according to claim 1, wherein Dv90 is less than or equal to 3 μm.

5. The compound dry powder inhaler according to claim 1, wherein the fine particle fraction of baicalin is 30-60%; the fine particle fraction of the ambroxol hydrochloride is 25-50%.

6. The compound dry powder inhaler of claim 1, wherein the mass median aerodynamic particle size of baicalin is 2-3.5 μ ι η; the mass intermediate aerodynamic particle size of the ambroxol hydrochloride is 2-3.5 mu m.

7. The compound dry powder inhaler according to any one of claims 1-6, wherein the moisture content is less than 7% by mass of the compound dry powder inhaler.

8. The dry powder inhaler formulation according to any one of claims 1 to 6, which is prepared by spray drying.

9. The dry powder inhaler formulation according to claim 8, wherein the spray drying process comprises an inlet temperature of 75-85 ℃, an air flow of 80-120L/min, a pump speed of 20-25%, a spray rate of 50-70%, and an internal pressure of 30-35 Mbar.

10. Use of a dry powder inhaler formulation according to any one of claims 1-9 in the manufacture of a medicament for the treatment of idiopathic pulmonary fibrosis.

Technical Field

The application relates to the technical field of pharmacy, in particular to a compound dry powder inhalant and application thereof.

Background

Idiopathic pulmonary interstitial fibrosis (IPF) is a pulmonary disease with an undefined etiology, prone to chronic and irreversibly developing diffuse alveolitis and alveolar structural disorder in the elderly, characterized by extensive pulmonary remodeling due to abnormal deposition of extracellular matrix, ultimately leading to pulmonary interstitial fibrosis (PF). Most pulmonary fibrosis is of unknown etiology, and idiopathic pulmonary fibrosis is characterized by diffuse alveolitis and alveolar structural disorders, ultimately leading to pulmonary interstitial fibrosis. The development of IPF includes lung injury, inflammation, myofibroblast formation and extracellular matrix accumulation, ultimately leading to lung structural dysfunction. The pathogenesis is currently unknown, and it is widely believed that inflammatory responses and oxidative stress may be important factors involved in the initiation and progression of IPF. The clinical symptoms are manifested as hidden onset, unobvious early symptoms, cough and expectoration at the beginning, and dyspnea at the later stage is aggravated, which leads to respiratory failure and death. IPF mortality is high and is known as a neoplastic-like disease. Because IPF is prone to occur in middle-aged and elderly people, the incidence of the disease is higher and higher globally with the aging of the population of each country, and about 320 thousands of people suffer from IPF and 122 thousands of cases are newly increased every year according to the statistics of the world IPF association.

The treatment regimen proposed by the IPF clinical guidelines suggests that lung transplantation is the most direct and effective method of treating IPF. However, due to their high cost, few donors, and greater technical risk, only a few patients are acceptable; the glucocorticoid is clinically used for treating by combining an immunosuppressant and an antioxidant, but the result is not ideal and various toxic and side effects appear, so the glucocorticoid is not recommended to be used; in the latest guidelines, the chemical drugs nintedanib and pirfenidone are listed as drugs that conditionally recommend the clinical treatment with IPF. However, long-term administration may have some adverse reactions, such as gastrointestinal discomfort, impaired liver function, skin allergy, etc. Therefore, while the pathophysiological mechanism of pulmonary fibrosis is elucidated, the continuous search for therapeutic drugs and therapeutic means with definite curative effect, safety and effectiveness is urgently needed.

In addition, at present, the clinical prevention and treatment of IPF mainly comprises oral administration and intravenous injection, gastrointestinal tract stimulation and liver first-pass effect exist in oral administration, and the medicine inflow concentration is low, so that the bioavailability is low, and the curative effect is not obvious; because the treatment period is long, the selected injection has the defects of poor patient compliance, low drug targeting, large systemic toxicity and the like. Therefore, how to improve the targeting property and bioavailability of the drug and achieve multiple treatment effects becomes a long-term common effort target for medical researchers.

Disclosure of Invention

In the research of the application, the Baicalin (Baicalin, BA, molecular formula shown in formula I) and Ambroxol Hydrochloride (AH, molecular formula shown in formula II) are combined and prepared into a Dry Powder Inhaler (DPI) dosage form, and the pulmonary inhalation administration mode is adopted, so that the effect of improving and treating pulmonary fibrosis can be achieved by reducing the expression of inflammatory factors, improving lung injury, improving lung function and the like, and the application is completed based on the discovery.

The application provides a compound dry powder inhalant, which comprises baicalin, ambroxol hydrochloride, L-leucine and phosphate, wherein the L-leucine accounts for 0-50% of the mass of the compound dry powder inhalant, the phosphate accounts for 15-35% of the mass of the compound dry powder inhalant, the total mass of the baicalin and the ambroxol hydrochloride accounts for 15-85%, and the mass ratio of the baicalin to the ambroxol hydrochloride is 1: (0.2-2), preferably 1: (0.8-1.2); the Dv90 of the compound dry powder inhalant is less than or equal to 5 mu m.

In a second aspect, the present application provides the use of the compound dry powder inhalant provided by the first aspect of the present application in the preparation of a medicament for treating pulmonary interstitial fibrosis.

The application provides a compound dry powder inhalant and application thereof in preparing a medicament for treating pulmonary interstitial fibrosis, wherein baicalin and ambroxol hydrochloride are used in a combined manner, so that inflammation and oxidative damage of lung tissues can be effectively reduced, pulmonary edema and histopathological changes are relieved, and pulmonary dysfunction and pulmonary fibrosis are relieved. Furthermore, the compound dry powder inhalant is adopted and administered through the lung, so that the half-life period and the in-vivo retention time of the medicine in plasma are obviously improved, the bioavailability of the medicine in lung tissue is also improved, the clearance rate of the medicine in the lung tissue is effectively reduced, the retention time of the medicine in the lung is prolonged, and the full play of the medicine in the lung tissue is facilitated.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only one embodiment of the present application, and other embodiments can be obtained by those skilled in the art according to the drawings.

FIG. 1 is a graph showing the moisture content of BA/AH-DPI dry powder particles at different ratios.

FIG. 2 is a graph showing the moisture profiles of different ratios of BA/AH-DPI dry powder particles.

FIG. 3 is a scanning electron micrograph of BA, AH and L-leu starting materials.

FIG. 4 is the scanning electron microscope image of different ratios of BA/AH-DPI dry powder particles.

FIG. 5 is the particle size distribution diagram of BA, AH, L-Leu raw materials and BA/AH-DPI dry powder particles with 7 different mixture ratios.

FIG. 6 is a DSC analysis pattern of AH, BA, L-leu, physical mixture and BA/AH-DPI dry powder of example 4.

FIG. 7 is a schematic XRD representation of AH, BA, L-leu and physical mixtures thereof.

Figure 8 is a schematic XRD of different ratios of BA/AH-DPI dry powder particles.

FIG. 9A is a graph showing FPF comparison of BA in different ratios of BA/AH-DPI dry powder.

Figure 9B is a FPF comparison of AH in different formulations of BA/AH-DPI dry powder.

FIG. 10A shows the deposition rate of BA in various grades of NGI in different formulations of BA/AH-DPI dry powder.

Figure 10B shows deposition rates of AH at each grade of NGI for different formulations of BA/AH-DPI dry powder.

Figure 11 shows the body weight change of the rats in each group over the 1, 7, 14, 21, 28 day period.

Fig. 12 shows the lung coefficients of the rats in each group after 28 days.

FIG. 13 shows the HE staining results of lung tissues of rats in each group.

FIG. 14 shows the results of total protein concentration in alveolar lavage fluid of rats in each group.

FIG. 15 shows the expression of the inflammatory factors IL-4, IL-6, IL-8, IL-1. beta. in BALF of 8 groups of rats.

FIG. 16 shows the expression of SOD, MDA, LDH and Hyp in rat lung tissue.

FIG. 17 shows the comparison of MPO content in serum of rats of each group.

FIG. 18 is a curve of the concentration of BA in plasma (ng/mL, Mean. + -. SD, n ═ 6)

FIG. 19 is a graph showing AH concentration curves in plasma (ng/mL, Mean. + -. SD, n ═ 6)

FIG. 20 is a graph showing the concentration of BA in lung homogenate (ng/mL, Mean. + -. SD, n ═ 6)

FIG. 21 is a graph showing AH concentration curves in lung homogenate (ng/mL, Mean. + -. SD, n ═ 6)

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail below by referring to the accompanying drawings and examples. It is to be understood that the embodiments described are only a few embodiments of the present application and not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in this application are within the scope of protection of this application.

The application provides a compound dry powder inhalant (BA/AH-DPI) which comprises baicalin, ambroxol hydrochloride, L-leucine and phosphate, wherein the L-leucine accounts for 0-50%, the phosphate accounts for 15-35%, and the total mass of the baicalin and the ambroxol hydrochloride accounts for 15-85% of the mass of the compound dry powder inhalant, wherein the mass ratio of the baicalin to the ambroxol hydrochloride is 1: (0.2-2), preferably 1: (0.8-1.2); the Dv90 of the compound dry powder inhalant is less than or equal to 5 mu m.

The inventor finds in research that the combination of baicalin and ambroxol hydrochloride can effectively reduce inflammation and oxidative damage of lung tissues, relieve pulmonary edema and histopathological changes, and relieve lung dysfunction and fibrosis.

In some embodiments of the first aspect of the present application, the L-leucine is present in an amount of 10-40% by mass of the dry powder inhaler.

In some embodiments of the first aspect of the present application, the L-leucine is present in an amount of 15-25% by mass of the dry powder inhaler.

The inventors have found that when the content of L-leucine is within the above preferred range, it is advantageous to further improve the particle uniformity and the aerosolization performance of the dry powder inhaler.

In certain embodiments of the first aspect of the present application, the total content of BA and AH is from 15% to 85%, preferably from 40% to 60%, by mass of the dry powder inhaler.

In the application, in the preparation process of the compound dry powder inhaler, PBS (phosphate buffered saline) is used as a solvent, the obtained method comprises phosphate, and the content of the phosphate is 15-35% by mass of the compound dry powder inhaler.

The PBS solution described herein is a PBS buffer commonly used in the art and can be formulated by one skilled in the art based on existing formulations, and typically includes Na₂HPO₄、KH₂PO₄NaCl and KCl, and thus the phosphate salts described herein may include Na₂HPO₄、KH₂PO₄NaCl and KCl.

Unavoidable impurities may be additionally included within a range not to impair the effects of the present application.

In some embodiments of the first aspect of the present application, the compound dry powder inhaler Dv90 is less than or equal to 3 μm.

In some embodiments of the first aspect of the present application, the baicalin has a fine particle fraction of 30-60%; the fine particle fraction of the ambroxol hydrochloride is 25-50%.

In some embodiments of the first aspect of the present application, the baicalin has a mass median aerodynamic particle size of 2-3.5 μm; the mass intermediate aerodynamic particle size of the ambroxol hydrochloride is 2-3.5 mu m.

In some embodiments of the first aspect of the present application, the moisture content is less than 7% by mass of the dry powder inhaler.

In some embodiments of the first aspect of the present application, the dry powder inhaler is prepared by spray drying.

In some embodiments of the first aspect of the present application, the spray drying process comprises an inlet temperature of 75-85 ℃, an air flow of 80-120L/min, a pump speed of 20-25%, a spray rate of 50-70%, and an internal pressure of 30-35 Mbar.

The inventor finds that the compound dry powder inhalant prepared by the method is beneficial to reducing the cohesive force of the powder, improving the flowing and atomizing performance of medicine particles and ensuring the consistency of the aerosol performance of the medicine and auxiliary materials.

In a second aspect, the present application provides the use of the compound dry powder inhaler provided in the first aspect of the present application in the preparation of a medicament for the treatment of idiopathic pulmonary fibrosis.

The inventor finds in research that in the compound dry powder inhalant, baicalin and ambroxol hydrochloride are combined, so that inflammation and oxidative damage of lung tissues can be effectively reduced, pulmonary edema and histopathological changes can be relieved, and lung dysfunction and fibrosis can be alleviated, therefore, the compound dry powder inhalant can be used for treating idiopathic pulmonary fibrosis, and further can be used for preparing a medicament for treating idiopathic pulmonary fibrosis.

Preparation example of Compound Dry powder inhalant

A B-90 nanometer spray dryer is adopted to produce the compound dry powder inhalant. PBS solutions of baicalin, ambroxol hydrochloride and L-leucine with different mass contents are respectively prepared as feed solutions, wherein in each example, the composition of the feed solutions is shown in the following table 1, and each feed solution is spray-dried under the same parameters: inlet temperature 80 deg.C, air flow 100L min^-1Pump speed 23%, spray rate 60%, internal pressure 34 Mbar. And after the spray drying is finished, collecting the DPI dry powder in a normal-temperature drying environment, weighing, sealing and storing in a normal-temperature dryer.

TABLE 1

Quality evaluation of compound dry powder inhalant

Determination of component content

The dry powders prepared in examples 1-7 in different proportions of the L-leu formulation were weighed to about 10mg, placed in a 25mL volumetric flask and diluted to the desired volume with methanol, shaken up, and used as the test sample. And (3) adopting high performance liquid chromatography to carry out content determination analysis, and calculating the drug loading amount in the BA/AH-DPI dry powder with different proportions. The drug loading of the BA/AH-DPI dry powders obtained in examples 1-7 in different ratios are shown in Table 2.

Preparing a BA reference substance stock solution: accurately weighing 8.46mg of BA reference substance, placing in a 25mL volumetric flask, dissolving in methanol, diluting to scale, shaking to obtain reference substance solution with concentration of 338.4 μ g/mL, and refrigerating at 4 deg.C for storage.

Preparation of AH control stock solution: accurately weighing 8.73mg AH reference substance, placing in a 25mL volumetric flask, dissolving in methanol, diluting to scale, shaking to obtain reference substance solution with concentration of 349.2 μ g/mL, and cold preserving at 4 deg.C.

Preparing a standard solution: precisely measuring 2mL of BA and AH reference stock solution, placing the BA and AH reference stock solution into a 10mL volumetric flask, diluting the methanol solution to a constant volume to a scale, shaking up, and refrigerating and storing at 4 ℃. A standard solution containing 67.68. mu.g/mL BA and 69.84. mu.g/mL AH per 1mL was prepared.

The calculation method is as follows:

the drug loading rate (peak area of the sample/peak area of the standard substance) per standard substance concentration 25ml per dry powder weight

TABLE 2 BA and AH contents in different proportions of BA/AH-DPI dry powders (Mean + -SD, n ═ 3)

As can be seen from the table, the dosage range meets the requirements for administration. In addition, since PBS is used as a solvent in the present application, a certain amount of phosphate exists in the dry powder after spray drying, and the content of phosphate in the BA/AH-DPI dry powder prepared in the examples of the present application is about 15% -35%.

Determination of the yield

Yield (%) — mass of spray-dried powder-amount/total mass of spray-dried PBS dried powder

And (3) quality of the spray dry powder: the mass of DPI dry powder with different formulation ratios was prepared with 500mL PBS according to the methods of examples 1-7;

quality of spray-dried PBS dry powder: the mass obtained after spraying 500mL of PBS solution;

the total mass is as follows: adding the masses of BA, AH and L-leucine, and preparing 500mL PBS solution, wherein the sum of the masses of the added components is equal to the total mass of the added components.

The yield results of BA/AH-DPI prepared by a spray drying method are shown in the following table 3, and the yield of DPI particles with different formula proportions is 75-85%, so that the requirement of DPI micropowder is met.

TABLE 3 yield table for spray dry powder of different formulation ratio

Moisture determination

The moisture content in the BA/AH-DPI dry powders with different mixture ratios in examples 1 to 7 was measured by a thermogravimetric analyzer (TGA), and the moisture content in the micro powder was calculated according to the total loss of the micro powder. The moisture measurements for different formulation ratios of the BA/AH-DPI dry powder samples are shown in table 4 and the moisture trends are shown in figures 1 and 2.

TABLE 4 moisture data for different L-leu ratio formulas (Mean + -SD, n-3)

As can be seen from Table 4, the moisture content of the BA/AH-DPI microparticles was found to be less than 7.0% for all formulations. It can be seen from FIGS. 1 and 2 that the moisture content gradually decreases with the addition of L-leu, indicating that co-spray drying the resulting particles with L-leu can reduce the moisture content to prevent moisture effects.

Observation of particle morphology

Respectively detecting the surface morphology of the BA, AH and L-Leu raw materials and BA/AH-DPI dry powders with different proportions by adopting a Scanning Electron Microscope (SEM), uniformly fixing the sample powders on a conductive adhesive tape, spraying the conductive adhesive tape on an ion sputtering instrument for 30s, and then placing the conductive adhesive tape on the SEM to observe and collect images, wherein the image collection voltage is 10 kV. BA. The SEM image results of AH and L-leu raw materials are shown in FIG. 3, wherein the a-1 and a-2 images are SEM images of BA at different magnifications, the b-1 and b-2 images are SEM images of AH at different magnifications, and the c-1 and c-2 images are SEM images of L-leu at different magnifications; SEM images of particles in BA/AH-DPI dry powders of examples 1-7 at different formulation ratios are shown in FIG. 4, in which the A-1 and A-2 plots are SEM images of BA/AH-DPI particles with an L-leu content of 0% at different magnifications, and correspondingly the B-G plots are SEM images of particles with an L-leu content of 10%, 15%, 20%, 25%, 40%, 50% at different magnifications.

From the results in fig. 3, it can be seen that the BA, AH and L-leu raw materials have irregular, strip-like and non-spherical shapes, while the different ratios of BA/AH-DPI dry powders in fig. 4 are spherical particles with monodisperse size range, irregular folding flocculation, rough surface and wrinkle shape, consistent with the literature reports. As can also be seen from FIG. 4, the presence of L-leu in the range of 10% to 40% gives BA/AH-DPI with a better surface wrinkle morphology and a spherical shape with uniform size.

Without being bound by any theory, the inventors have analyzed that due to the higher P clet number (Pe) of L-leu, a hydrophobic layer forms on the surface of the DPI particles obtained by spray drying, resulting in the formation of moire particles, increasing the surface roughness of the BA/AH-DPI particles, and decreasing the surface energy of the particles, which is precisely a result of the enrichment of L-leu on the particle surface.

Particle size distribution test

The particle size distribution of the DPI dry powder was measured by a dry dispersion method using a laser particle size analyzer to determine Dv10, Dv50, Dv90, VMD (Volume mean diameter) and SMD (Sauter mean diameter), respectively, and the results are shown in table 5. The particle size distribution curves plotted against VMD and SMD are shown in FIG. 5, where A is BA; figure B is AH; the diagram C is L-leu raw material; panels D-J are BA/AH-DPI with L-leu contents of 0%, 10%, 15%, 20%, 25%, 40% and 50% (examples 1-7) in that order.

TABLE 5 particle size determination results for BA/AH-DPI dry powders of different compounding ratios

As can be seen from the results in Table 5 and FIG. 5, the particle size of the crude drug is large, and the particle size of each formulation after spray drying is obviously in accordance with the requirement of the inhalation preparation, the particle size Dv90 is within 5 μm, and when the content of L-leu is 10% -40%, the Dv90 of BA/AH-DPI is within 3 μm.

Differential Scanning Calorimetry (DSC) test

Accurately weighing BA, AH, L-leu, and the three components in a ratio of 2: 2: 1 mass ratio of 5mg of BA/AH-DPI dry powder (L-leu 20%) from example 4, pressed in an aluminum sample pan, placed in the sample chamber of a DSC and a blank reference pan, kept dry N₂And (3) detecting the environment (30mL/min) at a temperature rise range of 30-350 ℃ at a speed of 10 ℃/min after the system is stable, and drawing an endothermic and exothermic curve of the sample.

BA. The DSC patterns of AH, L-leu, the physical mixture of the three and BA/AH-DPI dry powder are shown in figure 6. As can be seen from FIG. 6, BA (curve C) has a melting endotherm at 216.49 deg.C, AH (curve D) has a melting endotherm at 246.26 deg.C, and L-leu (curve E) has a melting endotherm at 301.28 deg.C; the physical mixture of the three components (curve B) has an endothermic peak at 196.45 ℃, and the melting temperature of BA is reduced under the influence of the auxiliary material L-leu presumably; meanwhile, the physical mixture of the three can see an endothermic peak of AH at 255.19 ℃ and an exothermic peak of L-leu at 302.26 ℃. In DPI (curve A), small endothermic peaks of melting appear at 105.51 ℃ and 218.19 ℃, while the melting peak of BA is weakened, the melting and endothermic peaks of AH and L-leu disappear, and no characteristic peaks of BA and AH appear in the graph, which indicates that the crystal form forms of BA and AH are converted into the amorphous form of DPI. Without being bound by any theory, the inventors believe that this may be due to the molecular entrapment of BA and AH in L-leu after the formation of the dry powder of BA/AH-DPI, transforming from a crystalline form to an amorphous form.

X-ray diffraction (XRD) analysis

The physical mixture of BA, AH, L-leu and BA, AH and L-leu in different proportions in examples 1-7 was weighed separately and ground to a uniform powder, and the powder was prepared into a flat sample specimen. The XRD curve was recorded by Ni filtered Cu-K alpha radiation source, and the detection conditions were set: the XRD patterns are respectively drawn by the voltage of 40kV, the voltage of 200mA, the scanning range of 5-60 degrees (2 theta), the scanning speed of 4 degrees/min and the sampling time of 1 s. BA. The XRD patterns of AH, L-leu and the physical mixture of the three are shown in figure 7, and the XRD results of different ratios of BA/AH-DPI dry powders of examples 1-7 are shown in figure 8.

As can be seen from FIG. 7, the BA, AH, L-leu and the physical mixture of the three are all in crystal morphology, the BA has strong diffraction peaks at 8.52 °, 10.28 °, 12.32 °, 14.60 °, 16.90 °, 20.59 °, 23.70 °, 25.32 °, 27.90 ° and 29.34 °, the AH also has strong diffraction peaks at 12.91 °, 15.71 °, 17.47 °, 20.45 °, 22.50 °, 23.28 ° and 25.09 °, and the L-leu also has strong diffraction peaks at 6.13 °, 12.15 °, 24.37 °, 30.53 ° and 36.82 °. The physical mixture of the three shows the same diffraction peak, and the diffraction peaks with strong crystallization characteristics are found at 6.09 °, 8.58 °, 12.15 °, 12.95 °, 15.75 °, 16.96 °, 17.51 °, 20.49 °, 22.54 °, 23.32 °, 24.37 °, 25.09 °, 30.57 ° and 36.88 ° to indicate that the medicines are all crystals and the crystal forms are not changed after mixing.

The results in FIG. 8 show that the dry powder BA/AH-DPI is in a non-crystalline state. Although the fine powder of BA/AH-DPI has small and weak diffraction peaks with wide peaks at 19.14 degrees, 31.65 degrees and 45.39 degrees, the intensity of the diffraction peaks is weakened to disappear and the diffraction peaks show irregular electromagnetic radiation diffraction with the addition of L-leu, which indicates that the powder is mainly amorphous; further, the micropowder obtained after spray drying was transformed from a crystalline form to an amorphous form and the DSC results were confirmed.

In vitro aerodynamic analysis

The field generally measures the in vitro deposition of DPI under the conditions of normal temperature and low relative humidity to simulate the deposition of DPI in the lung.

The present application employs a new generation of drug impactor (the nex)t generation pharmaceutical Impactor, NGI) the in vitro deposition properties of the dry powders of BA/AH-DPI of examples 1-7 were determined. The various components of the instrument were connected in sequence, including the hinged lid of NGI, preseparator (Presep.), artificial throat (I.P.), and inlet adapter (M.A.), and an ethanol solution containing a quantity of benzyl and glycerol was added to the surface of each collection cup and left to evaporate for 0.5h to avoid particle bounce or re-entrainment. The BA/AH-DPI fine particles were excessively charged into an Easyhaler multidose powder inhaler to conduct a test (inhalation time: 4s, inhalation speed: 60L/min), the drug-containing fine powder in all stages was uniformly and sufficiently dissolved by rinsing with a predetermined volume of methanol after atomization, and the collected solution was analyzed by HPLC under the following chromatographic conditions and gradient elution conditions shown in Table 6. By passingThe software calculates the parameters of the aerosol properties: fine Particle Fraction (FPF), Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD), the results are shown in table 7. FPF measurements of BA and AH in different L-leu ratios of BA/AH-DPI dry powders are shown in FIGS. 9A and 9B; the deposition of BA and AH at each level of NGI is shown in fig. 10A and 10B.

Liquid chromatography conditions: the column was an Agilent ZORBAX Eclipse Plus C18 (4.6X 250mm, 5 μm), mobile phase: methanol-0.1% phosphoric acid water; detection wavelength: 248 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction amount: 10 μ L

TABLE 6 measurement of BA/AH content elution gradient conditions

TABLE 7 measurement results of in vitro deposition Properties of BA/AH-DPI

As can be seen from the results in Table 7 and FIGS. 9A and 9B, the FPF values for BA and AH were higher for the L-leu-supplemented formulations compared to DPI without L-leu; the FPF of the dry powder gradually increased with the increase of the L-leu content and reached the highest value at 15-25%, especially 20%, indicating that the in vitro deposition property of the BA/AH-DPI dry powder is the best at this time, and the FPF value is reduced on the contrary by further increasing the L-leu content. The fact that the addition of L-leu carrier in the DPI in proper amount can improve the FPF and other in-vitro deposition properties of DPI dry powder to a certain extent is demonstrated.

In-vitro aerodynamic research shows that the addition of L-leu obviously improves the FPF (53.95 +/-0.17%) of the micropowder compared with the addition of no L-leu, has an important effect on improving the dispersibility of BA/AH-DPI, and shows that the BA/AH-DPI has excellent aerosol performance and physical atomization stability due to the addition of the L-leu in a proper amount.

The above experimental results show that the in vitro aerodynamic determination results are optimal when the L-leu ratio is 15-25%. And then, the optimal prescription is selected when the L-leu ratio is 20%, and the in-vivo drug effect of BA/AH-DPI is further examined.

Study of pharmacodynamics

Experimental animal and medicine

The research is approved by medical ethics committee of Tianjin Chinese medicine university and is carried out in an animal experiment center, and animals used in the research strictly carry out research work according to approved research schemes.

Bleomycin (Bloe): zhejiang Haizheng pharmaceutical Co., Ltd; and dissolving the bleomycin in a normal saline solution to prepare a bleomycin solution with the concentration of 12.5mg/mL for trachea cannula administration.

BA-DPI: BA-DPI (BA content 0.638mg/mg in dry powder) was prepared under the same spray-drying conditions as in example 4 by dissolving 400mg of BA and 200mg of L-leucine in 500mL of PBS.

AH-DPI: 400mg of AH and 200mg of L-leucine were dissolved in 500mL of PBS, and AH-DPI (AH content in dry powder of 0.571mg/mg) was prepared under the same spray-drying conditions as in example 4.

BA/AH-DPI: the dry powder BA/AH-DPI of example 4 of the present application was used, wherein in the following pharmacodynamic studies and results, the dry powder BA/AH-DPI means the dry powder BA/AH-DPI of example 4, unless otherwise specified.

Tail vein injection formulation solution: the dry powder of BA/AH-DPI of example 4 containing 20% L-Leu was dissolved in physiological saline to prepare a tail vein injection solution of 3 mg/ml.

Pirfenidone: the manufacturer Beijing Contini pharmaceutical industry Co., Ltd, specification 100mg, product number national drug standard H20133376; pirfenidone is a currently common clinical drug for treating pulmonary fibrosis, and is used as a positive control in the application; dissolving the pirfenidone in a normal saline solution to prepare a pirfenidone solution with the concentration of 11.6mg/ml for intragastric administration.

Grouping and processing mode for experimental animals

48 SPF healthy SD male rats (body weight 180-220g) were randomly divided into 8 groups of 6 rats: (1) normal group (control group): normally feeding in an animal center; (2) the sham operation group: the group was given an equivalent dose of air by pulmonary administration; (3) bleo model group: administering 100 mu L of a Bleo solution with the concentration of 12.5mg/mL through a tracheal cannula, and rotating and shaking the rat right after administration for 5min, wherein the Bleo solution can be also referred to as a Bleo group or a model group in the application for short; (4) bleo + BA-DPI group: adopting a molding method same as that of the Bleo model group, starting to perform BA-DPI (BA-DPI) administration by a DP-4 rat dry powder lung administration device (manufacturer: Beijing Yuan Sen Kaided biotechnology, Co., Ltd.) from the second day of molding, wherein the dosage is 9mg/kg, the daily administration is 3mg, and the total administration lasts for 28 days; (5) bleo + AH-DPI group (same method as 4); (6) bleo + BA/AH-DPI group (same method as 4); (7) bleo + tail vein injection group: starting tail vein injection preparation solution from the second day of molding, and administering 1ml of the preparation solution at the concentration of 3mg/ml in 9mg/kg every day for 28 days; (8) bleo + pirfenidone group: the gavage was started on the second day after the successful molding with a daily dose of 2ml, 11.6mg/ml, for a total of 28 days.

Pulmonary DPI dry powder dosing procedure: precisely weighing DPI micropowder and placing the DPI micropowder in a storage chamber of a dosing device, installing a DP-4 dry powder dosing device, connecting an injector with 1.5-2ml of air, anesthetizing a rat, lying on an endotracheal intubation platform, exposing an endotracheal tube opening by using a rat laryngoscope, opening the endotracheal tube opening while the rat is in the state of being in the state of being in the rat. The sham group was pushed with 1.5-2ml of air (no drug included) as described above.

Data analysis

The results of the experiment are expressed as (Mean ± SD). Differences between groups were tested using one-way anova and Tukey multiple comparisons. p <0.05 is significant difference. All data calculations and analyses were performed using GraphPad Prism 8.0.

Rat body weight change analysis

The rats were observed daily for respiration, activity, feeding and body weight, and the body weight of each group was recorded for 1, 7, 14, 21, 28 days on the first day of the Bleo model and statistically analyzed, and the results are shown in fig. 11. The rats in the normal group and the sham operation group were normally bred, had good mental status, had normal diet, and had continuously increased body weight, and as can be seen from fig. 11, there was no difference between the body weight increase of the rats in the sham operation group and the rats in the normal group. The weight of the rats in the Bleo group is basically kept unchanged within 14 days, the rats in the Bleo + AH-DPI group are poor and slightly cacheable in mental state after being modeled, the amount of drinking water is reduced, movement is not favored, the rats are easy to gather, the complexion of ears and limbs is dull, the weight is gradually increased after 7 days, but the weight increase is not obvious as compared with that in the Bleo group. The body weight of the rats in the Bleo + BA-DPI group and the rats in the Bleo + BA/AH-DPI group are slightly increased in the first 7 days, the activity is less, the rats move more after 7 days, drinking water and eating are normal, and the body weight is rapidly increased; the body weights of rats in the Bleo + tail vein injection group and the Bleo + pirfenidone group are in a growing trend, the animal state is slightly improved, but the animal state is still poorer than that of the Bleo + BA/AH-DPI group; wherein the body weight of the rats in the normal group, the Bleo + BA/AH tail vein injection group and the Bleo + BA/AH-DPI group is obviously higher than that in the Bleo group (p <0.01 or p <0.05) from the 7 th day, and the body weight of the rats in the Bleo + pirfenidone group is obviously different from that in the Bleo group (p <0.01) from the 14 th day. After 28 days of tail vein injection, rats have certain discomfort in state, and the tail part of the rat is peeled to a certain degree. In conclusion, BA/AH-DPI can obviously improve the weight loss of the rats with the Bleo-induced pulmonary fibrosis.

Pulmonary function index testing

After 28 days of molding, the rat in free activity was examined by using an EMKA lung function detector for various respiratory indices including inspiratory duration (Ti), expiratory duration (Te), maximum inspiratory flow (PIF), maximum expiratory flow (PEF), Tidal Volume (TV), Relaxation Time (RT), Expiratory Volume (EV), etc., and the results are shown in table 8.

Pulmonary resistance and compliance are generally considered gold criteria for assessing lung function. The lung function parameter data are shown in table 8. The values of Ti, Te, PIF, PEF, RT and TV, EV in the lung function parameters of the rats in the Bleo group were all lower compared to the normal group (p <0.01, p < 0.05); compared with the Bleo group, the Bleo + AH-DPI group has obvious increase of PIF and PEF in lung function parameters (p < 0.01). The Bleo + BA-DPI group showed significant increases in PIF, PEF, RT among the lung function parameters. The Bleo + BA/AH tail vein injection group, the Bleo + BA/AH-DPI group and the Bleo + pirfenidone group all have higher Ti, Te, PIF, PEF, RT in lung function parameters than the Bleo group (p <0.01), and the Bleo + BA/AH-DPI group has significant difference in parameters TV and EV compared with the Bleo group (p <0.01, p < 0.05). The above results show that the BA/AH-DPI of the present application provides a more significant improvement in lung function compared to DPI administration of BA or AH alone, as well as administration by tail vein injection.

Rat pulmonary factor determination

Pulmonary edema is a recognized pulmonary fibrosis index, and the pulmonary coefficient is one of indexes reflecting the damage of lung tissues, pulmonary edema and the degree of fibrosis, and the higher the value of the pulmonary edema is, the more serious the degree of lung lesion is. The lung coefficients were calculated and statistically analyzed using the following formula.

The formula is as follows:

after 8 groups of rats (6 per group) were sacrificed, alveolar lavage fluid (for subsequent experiments) was collected from the rats, lung tissue was extracted and weighed, and the obtained lung coefficient values were as shown in table 9 and fig. 12, and it can be seen from the results that the lung coefficients of the normal group and the sham operation group were both between 4 and 5; the lung coefficient of the Bleo group is more than 8, which indicates that the index meets the requirement of the pulmonary fibrosis model. The lung coefficients of the Bleo + AH-DPI group and the Bleo + BA-DPI group are more than 7, and the lung coefficients of the rats of the Bleo + BA/AH-DPI group, the Bleo + BA/AH tail vein injection group and the Bleo + pirfenidone group are all in the range of 6-7, which indicates that the BA/AH combined drug has better effect on improving pulmonary edema compared with the single drug.

Table 9 lung tissue coefficient data for each group of rats (n ═ 6)

HE staining observation of rat lung tissue

The weighed left lung is stored in 10% neutral formalin fixing solution to be soaked for 24h, dehydrated and embedded in paraffin. The slice thickness is 5 μm, and the slice specimen is observed under a microscope and photographed after the steps of hematoxylin and eosin staining (HE staining), dehydration and transparency, neutral gum sealing and the like. The results are shown in FIG. 13.

As can be seen from fig. 13, the lung tissue pathology was less changed in the normal group and the sham group, the sections were normal, and the alveolar spaces were thin; while the lung tissue of rats in the Bleo model group showed severe fibrosis, with a typical fibrotic appearance of structural disorders, including inflammatory cell infiltration and alveolar bleeding, with arrows indicating increased alveolar volume and increased alveolar interstitium. Compared with the Bleo model group, the rats in the Bleo + tail vein injection group, the Bleo + pirfenidone group and the Bleo + BA/AH-DPI group have thinner alveolar interstitium, less fibrosis characteristics and less inflammatory infiltration; pathological results of the Bleo + BA-DPI group and the Bleo + AH-DPI group are similar to those of the Bleo group, and alveolar interstitium is slightly thinner than that of the Bleo group, inflammatory exudates are contained in the alveolar interstitium, but no obvious difference exists. This experiment demonstrates that the BA/AH-DPI of the present application can ameliorate histopathological changes caused by Bleo.

Analysis of Lung bronchoalveolar lavage fluid (BALF)

The alveolar surface lining liquid obtained by bronchoalveolar lavage is alveolar lavage fluid (BALF), and the BALF is mainly clinically used for detecting cytokines, oxidative stress related enzymes and soluble substances, and is used for judging, diagnosing and treating lung related diseases (such as pulmonary inflammation, IPF and the like), treating the lung related diseases and improving the prognosis of the lung related diseases^[63]。

Bronchoalveolar lavage (BAL) procedures performed in this experiment were as follows: after the rats were sacrificed by abdominal aorta blood removal, the thoracic cavity was opened, the excess adipose tissue on the airway was removed, and the outlet tube was exposed. Inserting a retention needle from the front end of the trachea to enable the needle head to reach the right lung bronchus, fixing the front and back of the needle head, slowly filling 2ml of precooled normal saline into the lung, staying for 2min for suction, repeating the operation for 3 times, merging the 3 times of BALF, placing the BALF into a 10ml EP tube, and storing at low temperature, wherein the lavage recovery rate of the method is more than 80%.

Subjecting the obtained BALF to temperature of 4 deg.C and 4000r min^-1Centrifuging for 10min under the condition to obtain supernatant. The total protein content is determined by colorimetry according to the steps of the instruction manual of a total protein determination kit (manufacturer: Nanjing institute of bioengineering, Co., Ltd.; product number: A045-4-2). The results of the total protein content measurement are shown in Table 10 and FIG. 14, where the total protein content of both the normal group and the sham-operated group was about 8mg/mL, and there was no difference (p)>0.05); compared with a sham operation group, the total protein content of the Bleo group is (17.04 +/-1.87) mg/mL, and is remarkably increased (p<0.01). Bleo + pirfenidone group (p) compared to Bleo group<0.01) and Bleo + BA/AH-DPI group (p)<0.05) rat BALF was low in total protein; and the total protein content of the Bleo + tail vein injection group, the Bleo + AH-DPI group and the Bleo + BA-DPI group is reduced (p)<0.05). The determination result shows that the effect of reducing the total protein content by combining the two medicines is more obvious than that of a single medicine, and the pirfenidone has no difference (p)>0.05)。

Table 10 determination of total protein concentration in BALF (Mean ± SD, n ═ 6)

To verify whether BA/AH-DPI can regulate the inflammatory process by regulating the secretion of cytokines, the application also detects the concentrations of inflammatory factors IL-4, IL-6, IL-8 and IL-1 beta in BALF to determine the protection and treatment effects of BA/AH-DPI on the pathogenesis of Bleo-induced idiopathic pulmonary fibrosis.

And (3) measuring and detecting the concentration levels of inflammatory factors IL-4, IL-6, IL-8, IL-1 beta, IFN-gamma and TGF-beta 1 in the BALF supernatant by adopting an enzyme-linked immunosorbent assay (ELISA). The specific experimental procedures were performed according to the procedures of the ELISA kit (IL-4, IL-6, IL-1. beta. manufacturers: WU Han Dr. Biotech Co., Ltd.; products Nos. EK0406, EK0412, EK0393. IL-8 manufacturers: Shanghai Kexing Biotech Co., Ltd.; product No. F8655-A), and the experimental results are shown in FIG. 15.

As can be seen from FIG. 15, the levels of the concentrations of the inflammatory factors IL-4, IL-6, IL-8, IL-1. beta. in the normal and sham rats BALF were comparable (p > 0.01). IL-4, IL-6, IL-8 and IL-1 beta levels in BALF of the Bleo group rats were significantly increased compared to the normal group (# # p < 0.01); IL-4, IL-6, IL-8, and IL-1 β were all significantly reduced in the Bleo + pirfenidone, Bleo + BA/AH-DPI, and Bleo + tail vein injection groups compared to the Bleo group (p <0.01 or p < 0.05); whereas the IL-4, IL-6, IL-8 and IL-1. beta. concentration values in BALF were reduced in the Bleo + BA-DPI group and the Bleo + AH-DPI group, but none were different (p > 0.05). The result shows that the BA/AH-DPI can obviously reduce the level of inflammatory factors and has an inhibition effect on the inflammatory response of the rats induced by Bleo.

Hydroxyproline (Hyp) and oxidative stress index determination analysis in lung tissue homogenate

The weighed right lung was rinsed with normal saline, and then the lung tissue was homogenized by blotting water with filter paper.

Oxidative stress plays an important role in the progression of Bleo-induced pulmonary fibrosis. The MDA expression level and SOD activity can reflect the balance state of oxidation and antioxidation. SOD was therefore co-assayed with MDA in the present application to achieve the effect observed in pulmonary fibrosis. The highest content of Hyp in collagen, and the high content of Hyp in lung homogenate shows the metabolic condition of collagen in lung tissue and the degree of pulmonary fibrosis.

The present application uses ELISA to detect the Hyp level according to the procedures of the ELISA kit (manufactured by Shanghai Kouxing Biotechnology Co., Ltd.; product No. F3609-A). SOD concentration level is detected by adopting a superoxide dismutase WST-1 method, MDA concentration level is detected by adopting a thiobarbituric acid (TBA) method, and Lactate Dehydrogenase (LDH) concentration level is detected by adopting a microplate method. The experimental procedures were carried out according to the instructions of the SOD, MDA and LDH assay kits (SOD, MDA and LDH manufacturers: Nanjing Bioengineer institute Co., Ltd.; Cat: A001-3, A003-1, A020-2) of the manufacturers. The results of Hyp, SOD, MDA and LDH detection are shown in FIG. 16.

As shown in fig. 16, after administration of Bleo induction, rat lung tissue oxidative metabolites MDA and LDH were significantly increased (p <0.01) and SOD was significantly decreased (p <0.01) compared to the normal group. Compared with the Bleo group, after 28 days of continuous administration, MDA levels in lung tissues of rats in the Bleo + pirfenidone group, the Bleo + BA/AH-DPI group and the Bleo + tail vein injection group are all remarkably reduced (p is less than 0.01, p is less than 0.05), SOD antioxidant indexes in lung tissues of rats in the Bleo + pirfenidone group and the Bleo + BA/AH-DPI group are remarkably increased (p is less than 0.01), LDH contents are remarkably reduced (p is less than 0.01 or p is less than 0.05), and SOD and LDH in the Bleo + tail vein injection group have certain rising and falling trends but have no difference (p is more than 0.05); the levels of MDA and LDH in the lung tissues of rats in the Bleo + BA-DPI group and the Bleo + AH-DPI group are reduced (p is more than 0.05), and the antioxidant index of SOD is increased (p is more than 0.05). Compared with the normal group, the Bleo + BA-DPI group and the Bleo + AH-DPI group have higher MDA level (p <0.05) and lower SOD antioxidant index (p <0.05) in the lung tissues of rats. Thus, the BA/AH-DPI of the application can effectively improve the oxidative damage in the lung tissue of the rat with pulmonary fibrosis.

The Hyp content in the lung tissues of rats in the normal group and the sham-operated group is 76.43pg/ml and 77.57pg/ml respectively, the Hyp content in the Bleo group is 118.33pg/ml, and is obviously higher than that in the normal group and the sham-operated group (p < 0.01). Compared with the Bleo group, the levels of Hyp in the lung tissues of rats in the Bleo + pirfenidone group (content: 83.40pg/ml), the Bleo + BA/AH-DPI group (content: 84.06pg/ml) and the Bleo + tail vein injection group (content: 85.65pg/ml) were significantly reduced (p <0.01, p <0.05), and were close to the levels in the normal group and the sham group (as shown in FIG. 16). The lung tissue Hyp content of the Bleo + AH-DPI group and the Bleo + BA-DPI group is 100.17pg/ml and 102.97pg/ml respectively, which are both significantly higher than that of the normal group and the sham group (p <0.05), but lower than that of the Bleo group, and no significant difference exists between the three groups (p > 0.05). Thus, the BA/AH-DPI of the application can improve pulmonary fibrosis of rats.

Analysis of myeloperoxidase content (MPO) in serum

The rats were bled via the abdominal aorta before sacrifice and the whole blood was at 4000 r.min^-1Centrifuging for 10min, collecting supernatant to obtain serum, and centrifuging at 4 deg.C for 4000r min^-1Centrifuging for 10min under the condition, taking supernatant, and detecting the MPO content by adopting a colorimetric method. Experimental procedures MPO levels were measured using a kit (manufacturer: Shanghai Koxing Biotech Co., Ltd.; product number: F3213-A) according to the manufacturer's instructions.

The MPO content in the serum reflects the accumulation of neutrophils in lung tissues, and is a reliable index for judging the infiltration of inflammatory cells of the lung. The results of MPO detection in rat serum of the present application are shown in FIG. 17, and show that MPO levels in rat serum are increased by Bleo induction. The serum MPO levels in rats of the Bleo group, the Bleo + BA-DPI group and the Bleo + AH-DPI group were significantly increased (p <0.01) compared to the normal group. Compared with the Bleo group, the Bleo + pirfenidone group, the Bleo + BA/AH-DPI group and the Bleo + tail vein injection group all obviously inhibit MPO activity (p <0.01 or p < 0.05); whereas the Bleo + BA-DPI group and the Bleo + AH-DPI group slightly inhibited MPO activity, but did not differ significantly (p > 0.05). Thus, it was demonstrated that the BA/AH-DPI of the present application was able to inhibit MPO activity and thereby improve lung inflammatory cell infiltration.

The BA/AH-DPI can improve the lung function of rats with pulmonary fibrosis and has a good improvement effect on pulmonary edema and lung pathological changes, and meanwhile, the inventor also finds that the BA/AH-DPI can improve lung verification and oxidative damage and reduce the content of Hyp in the lung, so that the BA/AH-DPI can improve the symptoms of pulmonary fibrosis.

In vivo pharmacokinetic Studies

Laboratory animal

SPF-grade healthy SD male rats weighing 180-: (23 ± 2) ° c, humidity: (50 +/-10)%, and feeding normally. The study was approved by the animal experiment center of Tianjin Chinese medicine university.

Collection of plasma samples and Lung tissue

108 SPF-grade SD male rats were randomly divided into a tail vein injection group and a pulmonary administration group, each of which was divided into 9 time points of 2min, 4min, 10min, 15min, 30min, 1h, 2h, 4h and 6h, and each of which was divided into 6 rats per time point. Rats were fasted 12h prior to the experiment without water deprivation. Weighing rats (calculating dosage) and numbering, wherein two groups are subjected to intraperitoneal injection of 10% chloral hydrate according to the dosage of 2mL/kg, and the 1 st group is subjected to tail vein injection of BA/AH-DPI normal saline solution according to the dosage of 9 mg/kg; group 2 was administered with 3mg of BA/AH-DPI micropowder (1.5 mL of air per push, 3-4 times per push) by spraying with DP-4 tracheal insufflator at the same dose; two groups of rats were sacrificed by abdominal aorta bleeding at the time points indicated above after drug administration. Placing the obtained whole blood in a centrifuge tube containing heparin sodium, centrifuging at 4000r/min for 10min, separating out upper layer plasma, and storing in a refrigerator at-80 deg.C. After blood is taken, the thoracic cavity is opened rapidly, the lung of a rat is separated, a small amount of floating blood on the surface of the lung tissue is cleaned by ice-bath normal saline, the surface moisture of the lung tissue is sucked dry by filter paper, the trachea is cut off, and the lung tissue is wrapped by tin foil paper and placed in a 10mL centrifuge tube. Accurately weighing lung weight, and storing in-80 deg.C refrigerator.

Treatment of plasma samples and lung tissue

Taking lung tissues according to the weight-volume ratio of 1: 4 (g: mL) is added with normal saline, and is scattered and ground evenly by a homogenizer under ice water bath to obtain homogenate, and is centrifuged for 20min at 6500r/min to obtain lung tissue homogenate supernatant.

Precisely absorbing 100 mu L of plasma sample or lung tissue supernatant, adding 200 mu L (100ng/mL) of methanol solution containing an internal standard, uniformly mixing for 3min by vortex, centrifuging for 10min at 12000r/min to obtain supernatant, namely the processed sample, and determining the BA and AH contents by using a UPLC-MS/MS method.

Liquid condition

A chromatographic column: waters ACQUITYBEH C18(100 mm. times.2.1 mm, 1.7 μm); mobile phase: 0.1% formic acid (a) -acetonitrile (B), gradient elution as shown in table 11, column temperature: 30 ℃, flow rate: 0.3mL/min, detection wavelength: sample introduction amount: 5 μ L, chamber temperature: 4 ℃ is prepared.

TABLE 11 gradient elution

Electrospray ion source (ESI source); the scanning mode is as follows: positive ion (ESI +) detection; the detection mode is as follows: multiple reactive ion monitoring (MRM). Electrospray voltage: 4 kV; atomizing gas: n is a radical of₂(ii) a Spray airflow rate (Gas 1): 15 psi; ion source temperature: at 300 ℃. Specific mass spectrometry parameters are shown in table 12 below.

TABLE 12 Mass Spectrometry parameter settings for determination of BA and AH content in rat plasma and lung homogenate

The results of the drug concentrations in rat plasma and lung homogenate after pulmonary and tail vein administration of BA/AH-DPI of the present application are shown in tables 13 and 14; the time-of-use curve results are shown in fig. 18-21, in which fig. 18 is the concentration curve of BA in plasma and fig. 19 is the concentration curve of AH in plasma; FIG. 20 is a graph of BA concentration in lung homogenate; FIG. 21 is a graph of AH concentration in lung homogenate; the results of the relevant pharmacokinetic parameters are shown in tables 15 and 16.

TABLE 13 plasma concentrations in rats administered pulmonary and tail vein (ng/mL, Mean + -SD, n ═ 6)

TABLE 14 concentration of drug in lung homogenate for pulmonary and tail vein injection in rats (ng/mL, Mean + -SD, n-6)

Pharmacokinetic parameters mean: HLz is biological half-life, the time required for the drug in vivo or blood concentration to decrease by half; t is_maxThe time to peak, the time required to reach the peak concentration of the drug after administration. C_maxThe peak concentration is the highest value of the blood concentration after administration. AUC is the area enclosed by the plasma concentration curve versus time axis. Vz is apparent distribution volume, and the proportionality constant of the in-vivo dosage and the blood concentration when the medicine achieves dynamic balance in vivo. CL/F is the clearance rate, the apparent volume of drug removed from the body per unit time. MRTlast is the average residence time, the average of the residence time of the drug molecule in vivo.

As can be seen from the results, the same dose of BA/AH-D was administered to the lung as compared with the tail vein injectionAfter dry PI powder, AH reached maximum plasma concentrations at 4min in plasma (Table 13), drug retention time in vivo was slightly greater but not statistically different (Table 15), AUC of AH in the pulmonary group_0-6hRelatively small but no statistical difference (table 15), 53.5% absolute bioavailability and higher bioavailability (AUC) were achieved. BA reached maximum plasma concentration (C) in plasma about 9min after pulmonary administration_max)(p<0.01) (Table 15), a significant increase in half-life in vivo compared to tail vein injection (p)<0.01), mean residence time in vivo (MRTlast) is significantly prolonged (p)<0.01) (table 15), all with significant differences. The half-life (HLz) of BA administered in lung is larger, AUC_0-6hSlightly smaller but no statistical difference (table 15), the absolute bioavailability reaches 60.7%, and the bioavailability is higher. It is known that pulmonary administration can significantly improve the bioavailability and mean residence time in vivo (MRTlast) of the drug in plasma and prolong the half-life.

AUC of BA in lung tissue in lung homogenate compared with tail vein injection_0-6hStatistically very significant difference (p)<0.01), indicating that pulmonary administration significantly improved the bioavailability (AUC) of BA in pulmonary tissues. The low clearance of BA in lung tissue and the relatively long in vivo retention time (MRTlast) (table 16), all with significant differences (p)<0.05). AH retention time in lung tissue (MRTlast) was relatively long in pulmonary administration compared to tail vein injection group (table 16) with no significant difference (p) evident>0.05), but AUC_0-6hIs significantly higher (p)<0.01) (Table 16), half-life (HLz) extension (p)>0.05), decreased in vivo clearance (CL/F) (p)<0.01) (table 16), with significant differences. Therefore, the lung administration can obviously improve the bioavailability of BA and AH in the lung, has certain lung targeting property and brings a potential prospect for treating lung diseases.

The above description is only for the preferred embodiment of the present application, and is not intended to limit the scope of the present application. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application are included in the protection scope of the present application.