Ambroxol ibuprofen eutectic dry powder inhaler, preparation method and application thereof

Abstract

The present invention relates to an ambroxol-ibuprofen cocrystal dry powder inhaler, a preparation method and application thereof, and belongs to the professional field of pharmaceutical preparations. The dry powder inhaler is mainly composed of ambroxol, ibuprofen and lactose, the molar ratio of ambroxol to ibuprofen is 1:1, and the mass ratio of lactose to ambroxol-ibuprofen cocrystal is 5-35%. The results of the rat lung tissue distribution experiment showed that the administration method of dry powder inhalation can significantly enhance the drug delivery efficiency in the lungs and greatly prolong the drug retention time in the lungs compared with the traditional intravenous administration. Pharmacodynamic experiments further revealed that the ambroxol-ibuprofen cocrystal dry powder inhaler can effectively inhibit the active expression of inflammatory factors in model animals, and reduce the accumulation of hydroxyproline (HYP) in lung tissue and serum, improve anti-fibrosis and anti-inflammatory capabilities, improve oxidative stress capabilities, thereby helping to delay the pathological process of pulmonary fibrosis, showing great development potential as an anti-pulmonary fibrosis drug.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and particularly relates to an ambroxol ibuprofen eutectic dry powder inhaler, a preparation method and application thereof. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible interstitial lung disease that causes progressive lung damage. It is characterized by progressive dyspnea, excessive matrix degradation, and damage to alveolar structure, ultimately leading to pulmonary interstitial fibrosis. IPF is the most common, fatal, and progressive subtype of pulmonary fibrosis. Its clinical course is heterogeneous, ranging from an asymptomatic stable state to a slowly progressive state with acute exacerbations or a rapidly progressive decline in lung function. Clinical symptoms are insidious, with early symptoms being subtle and coughing rare. Later, progressive hypoxemia and diffuse bilateral lung damage develop. Inflammation invades the alveolar spaces and alveolar walls, leading to pulmonary fibrosis and thickening of the alveolar septa, ultimately leading to respiratory failure and death. It is widely believed that inflammation and oxidative stress may be important factors in the initiation and progression of IPF. In recent years, the pathogenesis and treatment of IPF have become hot topics of research both domestically and internationally. Idiopathic pulmonary fibrosis is inhibited by inhaled porous microspheres loaded with relaxin; Ma Xinai, Xia Kexin and other scholars proposed to treat idiopathic pulmonary fibrosis by using inhaled silybin dry powder prepared by nanosuspension spray drying technology, etc.

[0003] IPF is a progressive disease that is currently incurable. Clinically, treatments such as medication, surgery, and rehabilitation are typically used to slow disease progression, thereby improving patients' quality of life and survival. Existing treatments for IPF include both pharmacological and non-pharmacological therapies. Currently, only pirfenidone (a multi-cytokine inhibitor) and nintedanib (a small molecule inhibitor of receptor tyrosine kinases) have been approved for use in IPF to slow progression and prolong survival. However, both drugs are associated with side effects, including abdominal pain, diarrhea, skin disorders, and liver toxicity. Currently, no ideal treatment for IPF exists, aiming for optimal clinical efficacy. Non-pharmacological therapies, primarily oxygen therapy and lung transplantation, are the primary treatment for respiratory failure in the late stages of the disease. Clinical guidelines for IPF indicate that lung transplantation is the most direct and effective treatment for IPF, but the scarcity of donor lungs and the high cost of these treatments objectively limit its clinical implementation. Therefore, the search for effective, safe, and proven therapeutic agents and formulations is urgent.

[0004] Ibuprofen (BI) is an arylpropionic acid nonsteroidal anti-inflammatory drug (NSAID) with antipyretic, analgesic, and anti-inflammatory effects. Clinically, ibuprofen is widely used to treat arthritis and gout. Its mechanism of action is to inhibit cyclooxygenase (COX) activity, thereby reducing prostaglandin synthesis, thereby reducing inflammation and relieving pain. In research, BI has been shown to treat pulmonary fibrosis by reducing the inflammatory response in rats with bleomycin-induced pulmonary fibrosis. Injection of ibuprofen into rats with bleomycin-induced pulmonary fibrosis revealed that BI also downregulates levels of TGF-β, MyD88, and p-Smad2, thereby reducing the levels of pulmonary fibrosis cytokines. These findings suggest that BI plays a role in the treatment of pulmonary fibrosis and has potential as an anti-IPF drug. However, due to its poor water solubility, ibuprofen tablets typically dissolve slowly after oral administration, making them ineffective in clinical practice. To meet clinical needs, new ibuprofen formulations are under investigation. Over the years, people have tried to find new crystal forms, salts, and solvent compounds of ibuprofen to solve its poor water solubility and other problems, but only a few ibuprofen salts have been screened out.

[0005] Pharmaceutical cocrystals, as a solid-state form for improving the physicochemical properties of active pharmaceutical ingredients (APIs), have garnered significant attention in academia. Functional groups in APIs can form drug cocrystals through intermolecular recognition interactions such as hydrogen bonding and π-π stacking, along with cocrystal ligand (CCF) molecules. This process maintains the API's pharmacological activity but improves its physicochemical properties, such as melting point, stability, solubility, bioavailability, and mechanical properties, thereby enhancing its drugability. However, the selection of cocrystal solvents, the prediction of cocrystal structure and efficacy, and the scaled production of cocrystals for industrial application remain challenges in drug cocrystal development. Summary of the Invention

[0006] To address the problems in the prior art, the present invention provides an ambroxol ibuprofen cocrystal dry powder inhaler, a preparation method, and applications thereof. The dry powder inhaler exhibits low hygroscopicity, good fluidity, and a high lung deposition rate, thereby significantly enhancing the inhalation efficacy of the drug.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] An ambroxol ibuprofen cocrystal dry powder inhaler comprises ambroxol ibuprofen cocrystal and lactose as main ingredients, wherein the mass ratio of lactose to ambroxol ibuprofen cocrystal is 5-35%. Within this mass ratio range, the resulting dry powder inhaler can achieve more efficient pulmonary delivery and better therapeutic effects.

[0009] Preferably, lactose accounts for 10-20% by mass of the ambroxol ibuprofen cocrystal, and most preferably 15%.

[0010] Preferably, the particle size of the ambroxol ibuprofen cocrystal dry powder inhaler is 0.5-4.0 μm.

[0011] Preferably, the ambroxol ibuprofen cocrystal is a cocrystal formed by ambroxol (AM) and ibuprofen (BI) in a molar ratio of 1:1, and the cocrystal molecular formula is C 26 H 36 Br2N2O3 has a monoclinic crystal system and a space group of P21 / c. An AM cation and a BI anion form an asymmetric unit. The cocrystal can improve the solubility of drugs and increase the active ingredients of drugs.

[0012] More preferably, the ambroxol-ibuprofen cocrystal preparation method comprises: mixing ambroxol (AM) and ibuprofen (BI) at a molar ratio of 1:1, dissolving the mixture in a solvent, heating to a certain temperature, cooling, and filtering to obtain the ambroxol-ibuprofen cocrystal product. The solvent is a C1-C6 solvent, such as methanol, ethanol, propanol, butanol, tetrahydrofuran, etc., preferably ethanol; and the reaction temperature is 10-120°C, preferably 80°C.

[0013] The present invention also provides a preparation method of the dry powder inhaler, comprising dissolving an ambroxol ibuprofen cocrystal in anhydrous ethanol, dripping a lactose aqueous solution into the ethanol solution containing the ambroxol ibuprofen cocrystal, filtering, and spray drying to obtain the dry powder inhaler.

[0014] Preferably, the mass ratio of the ambroxol ibuprofen cocrystal mass to the anhydrous ethanol in the ethanol solution of the ambroxol ibuprofen cocrystal is 100:0.5-0.9.

[0015] Preferably, the mass ratio of lactose to water in the lactose aqueous solution is 1:1-15.

[0016] Preferably, the filtration is performed using a 0.2-0.8 μm microporous filter membrane.

[0017] Preferably, the spray drying is specifically carried out by using a spray dryer at a spray pressure of 160-240 kPa and a drying wind speed of 0.4-0.8 m 3 The dry powder inhaler is prepared under the conditions of: a liquid supply rate of 4-8 mL / min, a liquid supply rate of 4-8 mL / min, and an inlet temperature of 105-125° C.

[0018] The present invention also provides use of the dry powder inhaler in preparing a drug for treating anti-pulmonary fibrosis.

[0019] Beneficial effects of the present invention:

[0020] The one or more technical solutions provided by the specific embodiments of the present invention have at least the following beneficial effects or advantages:

[0021] 1. The present invention forms a co-crystal drug with ambroxol (AM) and ibuprofen (BI) in a molar ratio of 1:1, thereby improving the solubility, stability and biological activity of the drug.

[0022] 2. The dry powder inhaler provided by the present invention can effectively improve the lung delivery efficiency and significantly prolong the residence time of the drug in the lung. Further pharmacodynamic experiments have also fully confirmed that the dry powder inhaler is excellent in suppressing the expression of inflammatory factors in model animals, and can significantly reduce the concentration of hydroxyproline (HYP) in lung tissue and serum, improve anti-fibrosis and anti-inflammatory abilities, improve oxidative stress ability, thereby effectively slowing down the development process of pulmonary fibrosis. In addition, the dry powder inhaler can also effectively control the inflammatory response of rat pulmonary fibrosis, reduce the production of collagen fibers, and effectively inhibit the remodeling of rat alveolar and bronchial structures, showing an excellent anti-pulmonary fibrosis effect.

[0023] 3. Compared with pirfenidone, which is currently used clinically for idiopathic pulmonary fibrosis (IPF), the ambroxol-ibuprofen cocrystal dry powder inhaler described in the present invention can improve the compliance of alveolar epithelial cells and enhance patient compliance, and has potential application value in treating IPF, relieving cough and reducing phlegm, and improving lung function. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .Infrared spectrum of AB eutectic.

[0025] Figure 2 .Thermogravimetric curve of AB eutectic.

[0026] Figure 3 .(a) Perspective view of compound AB; (b) Supramolecular one-dimensional chain formed along the crystal c axis by hydrogen bonding; (c) Supramolecular two-dimensional layer formed in the crystal ac plane by hydrogen bonding interaction.

[0027] Figure 4 .Diagram of the three-dimensional stacking structure of the compound along the a-axis of the crystal.

[0028] Figure 5 Single crystal X-ray diffraction patterns of ambroxol-ibuprofen cocrystal (AB), (a) simulated pattern, (b) measured pattern.

[0029] Figure 6 .Surface (a), shape index (b), and curvature (c) of the Hirshfeld curve map.

[0030] Figure 7 .Fingerprint of compound AB.

[0031] Figure 8 .Contribution of each key.

[0032] Figure 9.AB-DPI electron microscope scanning image.

[0033] Figure 10 . Moisture content of DPI microparticle powders with different formulations.

[0034] Figure 11 .Comparison of yields of different formulations.

[0035] Figure 12 .DSC curve graph.

[0036] Figure 13 Body weight changes of rats in each group on days 1, 7, 14, 21, and 28;

[0037] Note: Compared with the Normal group, # p<0.05, ## p<0.01; compared with the BM group, *p<0.05, **p<0.01.

[0038] Figure 14 .Graphs of lung function indicators of rats in different groups;

[0039] Figure 15 .Graphs of lung function indicators of rats in different groups;

[0040] Figure 16 .Chart of lung function indicators of rats in different groups; Note: Compared with the Normal group, # p<0.05, ## p<0.01; compared with the BM group, *p<0.05, **p<0.01.

[0041] Figure 17 .Lung coefficient size of rats in each group after 28 days;

[0042] Note: Compared with the Normal group, ## p<0.01; compared with the BM group, *p<0.05, **p<0.01.

[0043] Figure 18 .Total protein concentration result graph;

[0044] Note: Compared with the Normal group, ## p<0.01; compared with the BM group, *p<0.05, **p<0.01.

[0045] Figure 19 .Concentrations of IFN-γ and TGF-β1 in BALF of rats in eight groups;

[0046] Note: Compared with the Normal group, # p<0.05, ##p<0.01; compared with the BM group, *p<0.05, **p<0.01.

[0047] Figure 20 The expression levels of inflammatory factors IL-1, IL-2, IL-8, and IL-1β in BALF of rats in the eight groups;

[0048] Note: Compared with the Normal group, # p<0.05, ## p<0.01; compared with the BM group, *p<0.05, **p<0.01.

[0049] Figure 21 Expression of SOD, MDA, LDH and Hyp in rat lung tissue;

[0050] Note: Compared with the Normal group, # p<0.05, ## p<0.01; compared with the BM group, *p<0.05, **p<0.01.

[0051] Figure 22 .Rat alveolar tissue sections.

[0052] Figure 23 .The results of measuring MPO content in rat serum;

[0053] Note: Compared with the normal group, ## p<0.01; compared with the BM group, *p<0.05, **p<0.01.

[0054] Figure 24 .Concentration curve of BI and AM in plasma (ng·ml -1 ).

[0055] Figure 25 .Concentration curve of BI and AM in lung tissue homogenate (ng·g -1 ). DETAILED DESCRIPTION

[0056] The present invention will be further described in detail below with reference to the examples, but is not limited thereto.

[0057] Raw materials used in the examples and comparative examples: ambroxol (purity>99%, Beijing Bailingwei Technology Co., Ltd.), ibuprofen (BI, purity>99%, Beijing Bailingwei Technology Co., Ltd.); lactose (purity 99%, Shanghai Zeye Biotechnology Co., Ltd.); pirfenidone (PFD, purity 98%, Shanghai Zeye Biotechnology Co., Ltd.); chloral hydrate (Shanghai Zeye Biotechnology Co., Ltd.); PBS×1 (pH 7.4, Hyclone Company, USA); bleomycin (purity 99%, Beijing Solebold Technology Co., Ltd.); rat tumor necrosis factor (TNF-α) ELISA kit (Shanghai Zeye Biotechnology Co., Ltd.); rat transforming growth factor (TGF-β1) ELISA kit (Shanghai Zeye Biotechnology Co., Ltd.); rat interleukin-1 (IL-1) ELISA kit (Shanghai Zeye Biotechnology Co., Ltd.); hydroxyproline kit (Shanghai Zeye Biotechnology Co., Ltd.); Kunming rats (Shandong University of Traditional Chinese Medicine Experimental Animal Co., Ltd.).

[0058] Experimental Animals: Forty-eight SPF male Sprague-Dawley rats weighing 200 ± 20 g were housed at 23–27°C with free access to food and water. This study was approved by the Medical Ethics Committee of Shandong University of Traditional Chinese Medicine and conducted at the Animal Experimentation Center. Animals were used in strict accordance with the approved research protocol.

[0059] Example 1

[0060] Preparation of ambroxol-ibuprofen cocrystal: Ambroxol (AM) (0.189 g, 0.5 mmol) and ibuprofen

[0061] (BI) (0.103 g, 0.5 mmol) was mixed and dissolved in 5 ml of ethanol, then heated at 80 ° C for several minutes until completely dissolved, allowed to cool to room temperature, and cooled to crystallize. Colorless block crystals (recorded as AB cocrystal raw material) were precipitated, with the chemical formula of C 26 H 36 Br2N2O3 (584.3g). Yield: 90%.

[0062] AB eutectic structure:

[0063]

[0064] The melting point of the AB eutectic is 147-149°C. Elemental analysis revealed the following values: theoretical values: C, 53.44; H, 6.21; N, 4.79; experimental values: C, 53.66; H, 6.22; N, 4.78.

[0065] Infrared detection results: IR (KBr, cm -1):3400(s),3308(s),3212(s),2941(s),2864(s),2624(m),2453(m),1906(w),1721(w),1621(s),1546(s),1457(s),1394(s),1365(m),13 18(m),1277(m),1240(m),1188(m),1114(w),1089(s),1007(w),966( w),887(m),865(m),822(w),746(w),727(w),672(m),565(m),415(w). like Figure 1 As shown, at 3400cm -1 A strong peak was observed at 3308 cm-1, which can be attributed to the stretching vibration of the -OH group in compound AB. -1 The strong band at 1546-1457 d cm -1 We can attribute the peaks in the range to the stretching vibrations of aromatic C=C and C=N. -1 The very intense and sharp peak found at is characteristic of carboxylate stretching vibration. The infrared band is located at 966-746 cm -1 , indicating that it undergoes in-plane and out-of-plane bending modes of aromatic -CH.

[0066] Thermogravimetric analysis: Figure 2 It can be seen that below 200°C, the weight of compound AB does not change substantially, indicating that there is no thermal decomposition in this temperature range.

[0067] The corresponding crystallographic data were obtained using a Bruker CCD diffractometer with Mo Ka (λ = 0.71073) monochromatic radiation. The absorption coefficient was semi-empirically corrected using SADABS software, and the X-ray diffraction data were normalized. The structure was refined using SHELXTL software. Single crystal X-ray diffraction pattern of ambroxol ibuprofen cocrystal (AB) Figure 5 Table 1 lists the main crystallographic parameters of compound AB cocrystal.

[0068] Table 1. Crystallographic parameters of compounds AB

[0069]

[0070] a R1=∑||F o |-|F c || / Σ|F o |. b wR2=[Σ[w(Fo 2 -F c 2 ) 2 ] / Σ[w(F o 2 ) 2 ]] 1 / 2 .

[0071] X-ray diffraction analysis results show that the compound AB cocrystal crystallizes in the monoclinic system in the P21 / c space group. An AM cation and a BI anion form an asymmetric unit ( Figure 3 , a). In the AM cation structure, a torsion angle (174.8°) exists between the cyclohexane and benzene rings, and the cyclohexane adopts a more stable chair conformation rather than the less stable boat conformation. Both bromine atoms bound to the benzene ring exhibit disordered features, which is completely different from that observed in the ambroxol compound alone. This is likely due to the ease with which the proton from the BI moiety can transfer to the AM molecule. Notably, the AM moiety contains two amino groups. Meanwhile, we found that only one amino group in the aliphatic group can be protonated. These observations suggest that aliphatic amino groups have stronger basicity than aromatic amino groups.

[0072] In supramolecular structures, hydrogen bonding and stacking interactions are ubiquitous phenomena that play an important role in the assembly and function of supramolecular structures. In the AB cocrystal, OH···O and NH···O occur: (a) Hydrogen bonding O1-H1···O3 a ( a 1+x,y,z) are derived from hydroxyl and carboxyl oxygen atoms, where the bond lengths and bond angles are and 175°. (b) Hydrogen bonds formed by amino nitrogen atoms and carboxyl oxygen atoms, such as N2-H2A···O2, N2-H2B···O2 b ( b 1-x, 1-y, 1-z) and N2-H2B···O3 b The bond length ranges from 2.733(6) to Different from other classical hydrogen bonds, stacking interactions can also regulate the interactions between supramolecular molecules. Compound AB has CH…π stacking interactions C9-H9B···Cg3 b ( b 1-x, 1-y, 1-z), where Cg3 represents a phenyl center, consisting of C1-C2-C3-C4-C5-C6.

[0073] like Figure 3As shown in (b), N2-H2A···O2 hydrogen bond can generate aggregation between AM cation and BI anion. According to O1-H1···O3 a hydrogen bonds, can form supramolecular 1-D chains along the crystallographic a direction, in which adjacent assemblies are arranged in a head-to-tail pattern. Subsequently, it produces O1-H1···O3 a ,N2-H2B···O2 b and N2-H2B···O3 b Through these hydrogen bonds, adjacent supramolecular chains can extend to the supramolecular 2-D layer along the crystal direction ( Figure 3 ,c). Stacking interaction (C9-H9B···Cg3 b ) and the aforementioned hydrogen bonds, through which the supramolecular three-dimensional structure can be generated along the crystallographic a-axis ( Figure 4 ).

[0074] Table 2. Hydrogen bond information of compound AB cocrystal

[0075]

[0076] Symmetry codes: a 1+x,y,z; b 1-x,1-y,1-z for compound AB.

[0077] Table 3. Main bond lengths, bond angles, torsion angles, and dihedral angles in AB eutectic compounds

[0078]

[0079]

[0080]

[0081] Hirshfeld surface analysis: Hirshfeld surface analysis was performed by importing crystal CIF data into CrystalExplorer17 software to further estimate the intermolecular interactions and structure-property relationships of the compounds. The interactions on the Hirshfeld surface of compound AB were quantified by two-dimensional fingerprint mapping and Hirshfeld surface calculation method. Figure 6 As shown, dark red points indicate shorter hydrogen bond interactions, blue areas indicate closer contacts, and white areas indicate nearby van der Waals forces. In the shape index, red indicates concavity and blue indicates convexity. In the curvature, green indicates flat surfaces and blue indicates boundaries. Figure 8It can be clearly seen that H···H close contact accounts for 50%, which is the most significant contribution. At the same time, it can also be seen in the two-dimensional fingerprint ( Figure 7 ) observed in the graph, the peak representing H···O interactions contributes 12.8%, second only to H···H. The C···H, shaped like two sharp teeth, contributes 11.0%. Other short contacts (such as C···C, N···C, and N···H) have lower contributions, all less than 2.0%, but their intermolecular forces also play a significant role in crystal formation. There are also O···O, O···C, and N···N, with interactions of 0%.

[0082] Example 2

[0083] Preparation of AB dry powder inhaler (AB-DPI powder):

[0084] First, dissolve 0.1 grams of AB eutectic API in 0.8 milliliters of anhydrous ethanol to ensure that the API is completely dissolved. Subsequently, under stirring, slowly add the ethanol solution dropwise to the aqueous solution of lactose to ensure that the AB eutectic API and lactose are fully mixed. Next, stir at room temperature for 4 hours to allow the ethanol to fully evaporate and be removed to obtain an aqueous solution containing AB eutectic API and lactose. Then, filter the above aqueous solution using a 0.5-micron microporous filter membrane to remove any solid particles or impurities that may exist to ensure the purity of the final solution. Finally, use a spray dryer to prepare AB dry powder. The optimal spray drying conditions are: spray pressure 220KPa, drying wind speed 0.8m 3 min -1, liquid supply rate 7mL·min, inlet temperature 115℃. The AB dry powder inhaler obtained under these conditions exhibits ideal powder properties. Its fine particle fraction (FPF) is as high as 80.1±1.2%. The increase in this value means that more drug particles can reach the lungs, which is beneficial to the deposition of PTs in the lungs and improves the therapeutic effect of the drug. At the same time, the median particle size (MMAD) is 3.1±0.3μm, which just meets the requirements of pulmonary administration, ensuring that the drug particles can penetrate into the lung tissue and achieve effective therapeutic effects. In addition, the dry powder span (GSD) value is 1.5, indicating that its particle size distribution is relatively narrow, which is crucial for ensuring the consistency and stability of the drug. In terms of powder properties, the dry powder inhaler prepared by the present invention also exhibits excellent performance, with an emptying rate of up to 98%, which means that there will be almost no drug residue during the inhalation administration process, thereby ensuring the effective utilization of the drug. In addition, the hygroscopicity of the dry powder is less than 14%, ensuring its stability in a humid environment. At the same time, the angle of repose is less than 30°, indicating that the dry powder has good fluidity, which is conducive to uniform distribution and inhalation administration of the drug. In summary, the AB dry powder inhaler provided by the present invention performs well in both powder properties and lung deposition efficiency.

[0085] AB dry powder inhalers with different lactose contents were prepared with the ratio of lactose to AB eutectic being 0, 10%, 15%, 20%, 25%, 40% and 50% respectively.

[0086] from Figure 9 It can be seen that the particle diameter of AB-DPI powder ranges from 0.5 to 4.0 μm. Compared with other drugs, it can make the drug have a higher absorption rate and better distribution in the tissue, so that the drug has good stability in the blood and better effect in treating interstitial lung disease.

[0087] The moisture content of AB powder obtained with different ratios of lactose is shown in Figure 10 As shown in the figure, as the amount of lactose added increases, the moisture content of the cocrystal drug AB shows an overall decreasing trend, indicating that the microparticles combined with the cocrystal drug AB and lactose can reduce the moisture contained in the microparticles to reduce water evaporation, making it easier to store while also avoiding the weakening of the drug efficacy.

[0088] The yield of AB dry powder obtained with different ratios of lactose is shown in Figure 11As shown, the yield of AB-DPI containing 15% lactose was the highest, at around 85%, while the yield of AB-DPI containing 0% lactose was the lowest, at 79.2%, a difference of 5.8%. The yield of AB-DPI with both a lactose content of greater than and less than 15% decreased. This suggests that adding a moderate amount of lactose to DPI can improve the yield of DPI micropowder to a certain extent. Based on the comprehensive data, a 15% lactose content is the optimal formulation, reducing drug loss and achieving a more stable form.

[0089] The following comparative experiments and drug activity tests were conducted using AB-DPI powder in which the molar ratio of AM to BI was 1:1 and the mass ratio of lactose to the AB eutectic was 15%.

[0090] Comparative Example:

[0091] AM powder, BI powder, AB-DPI powder, Mix (a mixture of AM and BI in a molar ratio of 1:1) powder, Lactose powder, and BF (pirfenidone) powder were prepared by spray drying. The DSC curves obtained are shown in FIG. Figure 12 . Figure 12 AM shows an endothermic peak at 250°C and an exothermic peak at 280°C, while BI has a melting exothermic peak at 84.5°C, indicating that these two drugs are thermally unstable when isolated. Meanwhile, Mix (a physical mixture of the two drugs) exhibits a melting endothermic peak at 160°C, with adjacent melting endothermic and exothermic peaks at 180°C and 300°C, indicating that the physical mixture of the two drugs is also extremely unstable. Lactose exhibits a melting endothermic peak at 315°C, suggesting that the melting temperatures of AM and BI are reduced due to the influence of the excipients. The absence of these characteristic peaks in the AB-DPI spectrum indicates a transition from the crystalline form of AB to the amorphous form of AB-DPI. This amorphous form is caused by the encapsulation of AB in lactose. Compared to the individual drugs or their physical mixture, the cocrystal of the two drugs combines the advantages of both, offering significant advantages. Furthermore, pirfenidone, represented by BF, also exhibits a melting exothermic peak at 100°C, indicating that pirfenidone is also unstable. In comparison, AB-DPI has great advantages in both efficacy and stability, and the quality process required for production is simple.

[0092] Drug activity test methods for each drug:

[0093] (1) Construction of experimental model and preparation of solution

[0094] Rat IPF model: BM saline solution, administered at a dose of 5 mg kg -1To construct the model, measure bleomycin with a graduated cylinder, add physiological saline, and place it in a 10 mL volumetric flask to obtain a concentration of 12.5 mg mL -1 of BM solution.

[0095] 2% sodium pentobarbital solution (prepared and used immediately): Take 2g of sodium pentobarbital powder and dissolve it completely in 20mL of pure water. Allow it to dissolve completely (shake gently), then inject 0.4mL of the prepared sodium pentobarbital solution into the rat's peritoneal cavity for anesthesia.

[0096] AB-DPI solution (prepared and used immediately): Use a precision balance to weigh 30 mg of AB-DPI powder and completely dissolve it in a volumetric flask with 10 mL of PBS (phosphate buffer solution) to a concentration of 3 mg mL -1 The dosage is 9 mg·kg -1 Drugs were administered by tail vein injection.

[0097] (2) Experimental animal grouping and treatment

[0098] First, 48 SPF-grade male rats weighing 200±20g were randomly divided into 8 groups, with 6 male rats in each group. The treatment methods for the 8 groups were as follows: (1) Normal group (normally raised in an SPF-grade animal center without external influence); (2) Sham group: the same dose of air was injected into the lungs of the rats in this group by direct administration; (3) BM model group: rats were injected with a certain amount of bleomycin through endotracheal intubation and then shaken upright for 5 minutes; (4) BM+BI-DPI group: on the second day of the BM model group, endotracheal intubation was started using the DP-4 rat dry powder pulmonary drug delivery device, BI powder was 3mg per day, and the dose was 9mg·kg -1 , a total of 28 days; (5) BM+AM-DPI group: AM powder, dosage and administration method are the same as (4); (6) BM+AB-DPI group, AB-DPI powder, dosage and administration method are the same as (4); (7) BM+TI group (from the second day of the BM model group, the tail vein injection of the preparation solution was carried out at a concentration of 3 mg mL -1 , 9mg·kg -1 (8) BM+BF group (after the BM model group was successfully constructed, oral gavage was performed from the second day, and the daily dosage was 11.6 mg mL -12mL of pirfenidone, administered for a total of 28 days). Pulmonary DPI dry powder administration operation: Use a precision balance to weigh the DPI powder, place it in the storage chamber of the drug delivery device, install the DP-4 dry powder drug delivery device and connect it to a syringe containing 1.5-2mL of air. After the rat is anesthetized, make the rat lie flat on the endotracheal intubation platform, expose the tracheal opening through the rat laryngoscope, and immediately insert the drug delivery device nozzle from the tracheal opening while the tracheal opening is opened. Place it on the bronchial intubation, and immediately push air into the drug delivery device to propel the DPI powder into the rat's lungs. The Sham group inhaled 1.5 to 2 ml of air (drug-free) using the same method as described above. The BM+TI group was injected with AB-DPI powder solution, and the dosage was the same as the BM+AB-DPI group. At the same time, for the rats in the BM+BF group, the dose administered by gavage was 116 mg·kg -1 The general condition of the experimental rats was observed daily, including activity, food intake, weight, mental state, respiratory status, and other physical indices. The establishment of the BM model was considered the first day, and the weight of each group of experimental rats was observed and recorded every 7 days (1 week), for a total of five records. The recorded rat data were statistically analyzed. On day 28 of modeling, the freely moving rats were tested using an EMKA pulmonary function tester for various respiratory indices, including relaxation time (RT), inspiratory duration (TI), bronchoconstriction parameter (Penh), peak expiratory flow (PEF), peak inspiratory flow (PIF), respiratory rate (f), tidal volume (TV), and expiratory duration (TE). After the tests, the rats in each experimental group were treated, blood was drawn from their abdominal aorta, and they were finally sacrificed. The alveoli in the bronchi were irrigated with physiological saline to collect the alveolar lavage fluid, and the lung tissue was then removed and weighed. The left lung was immersed in 10% neutral formalin fixative for 24 hours, then stained with HE, the remaining tissue was rinsed with saline, the water was absorbed with filter paper, and finally, it was stored at -80℃ for later detection of tissue homogenate. Whole blood was centrifuged for 10 minutes at 4000 rpm. -1 The serum in the supernatant was taken and placed in a -80°C refrigerator for testing.

[0099] (3) Determination of rat lung coefficient

[0100] The lung coefficient is a commonly used measurement in animal experiments. It refers to the lung volume measured when the same volume of gas is inhaled per unit time. It is an important indicator of lung damage, edema, and fibrosis. A higher value indicates more severe lung disease. The lung coefficient is calculated using the following formula: Lung coefficient = lung wet weight (g) / body weight (kg) * 100%.

[0101] (4) Collection of bronchoalveolar lavage fluid (BALF)

[0102] Bronchoalveolar lavage fluid (BALF) is obtained by inserting a bronchoscope into the patient's airway to the site of inflammation or disease, injecting a sterile saline solution, and then inhaling the solution. Clinically, BALF is primarily used to detect cytokines, oxidative stress-related enzymes, and soluble substances, significantly aiding in the diagnosis, treatment, and improvement of patient prognosis for lung diseases such as pneumonitis and IPF.

[0103] The BALF method involves drawing blood from the abdominal aorta of a rat. After killing the rat, the chest cavity is opened, and excess fat over the airway is removed to expose the trachea. A retention needle is inserted from the anterior trachea into the right bronchus, while the anterior and posterior sides are firmly secured. 2 mL of pre-chilled saline is slowly perfused into the lungs, allowed to stand for 2 minutes, and then aspirated. This procedure is repeated three times. The three BALF volumes are combined and placed in a 10 mL EP tube and stored at low temperatures. This lavage method is relatively easy to perform and has a recovery rate of >80%.

[0104] (5) HE staining of rat lung tissue

[0105] The left lung of a rat was infiltrated in 10% formalin for 24 hours. After drying in a desiccator, the left lung was removed, wrapped in paraffin, and sliced. Five-micron-thick slices were stained with hematoxylin and eosin, dehydrated until transparent, and sealed with a neutral adhesive. The slices were observed under a microscope and images were acquired.

[0106] (6) Analysis of bronchoalveolar lavage fluid (BALF)

[0107] Determination of total protein content in BALF

[0108] BALF was heated at 4000 r·min -1 After centrifugation at 4°C for 10 minutes, the BALF was separated to obtain the supernatant. The total protein content was determined by colorimetry according to the method described in the instrument. Table 4 shows:

[0109] Table 4. Total protein assay kit operation instructions

[0110]

[0111] The mixture was shaken and incubated at 37°C for 30 min. The absorbance was detected by colorimetry at a wavelength of 562 nm using an enzyme-labeled instrument.

[0112] The formula is as follows:

[0113] Total protein concentration (μg·mL -1 ) = [(measured OD value - blank OD value) / (standard OD value - blank OD value)] * standard concentration (524 μg mL -1 )*Dilution ratio of sample before testing

[0114] Determination of interleukin-1 (IL-1), IL-2, IL-8, and IL-1β levels in BALF

[0115] The levels of inflammatory factors IL-1, IL-2, IL-8, and IL-1β in the BALF supernatant were determined by ELISA. Detailed experiments were performed according to the instructions of the ELISA kit.

[0116] Determination of interferon-γ (IFN-γ) and transforming growth factor-β1 (TGF-β1) levels in BALF

[0117] Pulmonary interstitial fibrosis is mediated by multiple factors, including inflammatory responses and immune disorders. Our previous studies have shown that TGF-β1 can promote the epithelial-mesenchymal transition (EMT) of type II epithelial cells and the myofibroblast-to-fibroblast transition (FMT) of primary fibroblasts. IFN-γ and transforming growth factor-β1 (TGF-β1) levels in the supernatant of BALF culture medium were measured by ELISA. The assays were performed according to the methods described in the purchased ELISA kit.

[0118] (7) Determination and analysis of hydroxyproline (Hyp) and oxidative stress indicators in lung tissue homogenate

[0119] Weigh an appropriate amount of rat lung tissue, use ice-cold physiological saline as the homogenization medium, grind it on a tissue homogenizer, and then centrifuge the grinding liquid at a speed of 3000 r·min. -1 The cells were centrifuged for 10 minutes, and the supernatant was used to prepare a 10% tissue homogenate, which was then refrigerated for analysis. Hyp was measured using an enzyme-linked immunosorbent assay (ELISA) according to the procedure in the kit. Serum superoxide dismutase (SOD) levels were determined using the WST-1 assay. Malondialdehyde (MDA) and LDH were measured using the TBA and LDH assays, respectively.

[0120] (8) Analysis of serum myeloperoxidase (MPO) content

[0121] Increased MPO activity is closely related to pulmonary interstitial fibrosis and is an important indicator for judging the inflammatory response of lung tissue. -1Centrifuge for 10 minutes and measure plasma MPO levels using a colorimetric assay. Methamphetamine oxidase assays should be performed according to the instructions for the purchased reagent.

[0122] (9) Preliminary pharmacokinetic study in rats

[0123] Detection of BI and AM drug concentrations in plasma and lung tissue homogenate

[0124] Using BI and AM as indicator components, the dynamic changes of drug content in plasma and lung tissue homogenate of the AB-DPI solution tail vein injection group and the AB-DPI pulmonary administration group after administration via intravenous injection and pulmonary administration were investigated at different time periods. The relevant data were entered into WinNonlin6.4 software to calculate pharmacokinetic related parameters, in order to explore the changes in drug content of BI and AM in plasma and lung tissue and the lung targeting effect.

[0125] Statistical analysis of the data was performed using GraphPad Prism 8.0. Results are presented as mean ± standard deviation (x ± s). Differences between the two groups were analyzed using one-way analysis of variance and Tukey's multiple comparison test. A p value < 0.05 was considered a significant difference, and a p value < 0.01 was considered a significant difference. The statistical significance of the results was explained.

[0126] Result analysis:

[0127] (1) In vivo pharmacodynamic study in rats

[0128] Weight changes in rats

[0129] IPF rats were constructed by inducing pulmonary fibrosis in rats using BM. The weight data of rats were expressed as a bar graph starting from the first day of induction (e.g. Figure 13 The weight changes every 7 days are shown in the figure, which can intuitively reflect the daily weight changes. At the same time, three control systems were constructed: System 1 uses normal control group (Normal) and sham operation group (Sham) rats to discuss the effect of surgery on rat weight. As can be seen from the bar graph, the growth conditions of the normal group and the sham operation group are the same ( ##p<0.01); System 2 used IPF rats for comparison under different dosing conditions. The three dosing conditions were BM+BI-DPI, BM+AM-DPI, and BM+AB-DPI. The rats in the BM group had a depressed mental state, a long period of inactivity, a high frequency of inactivity, decreased food intake, and more gathering activities. Their body weight remained basically unchanged within 14 days. After the 14th day, the body weight of the rats in the BM group gradually recovered. The body weight of the rats in the BM+BI-DPI, BM+AM-DPI, and BM+AB-DPI groups changed little in the first 6 days, and was basically equivalent to the body weight of the rats in the BM group. Starting from the 7th day, the body weight of the three groups of rats increased significantly, which was significantly higher than that of the BM group (p<0.01 or p<0.05). The state of the rats improved, and they changed from being mostly inactive to being active, their excitability increased, and their food and water intake gradually recovered. From days 7 to 14, rats in the BM+AM-DPI and BM+AB-DPI groups experienced a more pronounced weight gain than those in the BM+BI-DPI group. However, from days 15 to 28, rats in the BM+BI-DPI and BM+AB-DPI groups showed superior recovery. The BM+AB-DPI group consistently outperformed the other two groups in weight recovery throughout the experiment, making it the optimal group. System three included BM+TI and BM+BF. After 28 days of tail vein and pulmonary administration, the two groups of rats were in essentially identical condition, with both experiencing some discomfort. The BM+TI rats also experienced some tail peeling. Rats in the BM+BF group experienced a dry cough after administration, suggesting that pirfenidone may have caused some irritation and damage to the lungs, but this discomfort resolved after a period of time. Comparison of the optimal group from system two with the two groups from system three revealed that the BM+AB-DPI group exhibited superior weight recovery and mental well-being compared to the two groups from system three. Although this group of rats also experienced dry coughs like the other lung-administered groups, the severity was milder and the discomfort disappeared more quickly. In summary, AB-DPI can significantly improve BM-induced weight loss in rats.

[0130] (2) Comparative analysis of lung function parameters 28 days after modeling

[0131] Generally speaking, lung resistance and compliance are important indicators used to evaluate lung function. Figures 14-16Compared with the Normal group, the various lung function parameters in the Sham group were similar. In the BM group, the lung function parameters of rats, including AV, EEP, EV, PIF, EF50, PEF, TV, RT, Te, and Ti, were significantly decreased (p<0.01), while EIP and MVV values ​​were slightly decreased (p<0.05), while F and Penh values ​​were significantly increased (p<0.01). Compared with the BM group, the lung function parameters of the BM+BI-DPI group, including PEF, PIF, and AV, were significantly increased (p<0.01), while EV, RT, and EF50 were only slightly increased (p<0.05), and EEP and F were slightly decreased. In the BM+AM-DPI group, the lung function parameters, including AV, EV, EF50, Te, and F, were slightly increased (p<0.05), EEP was slightly decreased (p<0.05), Penh was significantly decreased (p<0.01), and PEF, PIF, and RT were significantly increased (p<0.01).

[0132] The Ti, Te, PIF, RT, EV, PEF, and EF50 parameters of the BM+AB-DPI, BM+TI, and BM+BF groups were significantly higher than those of the BM group (p<0.01), and slightly higher than those of the BM group (p<0.01). The Penh parameter was significantly lower than that of the BM group (p<0.01), and the F was slightly lower than that of the BM group in the MVV parameter (p<0.05). The AV and PEF in the BM+TI group were significantly different from those in the EF50 group (p<0.05), but not in the BM+AB-DPI group. This indicates that pulmonary administration via tail vein injection will cause some damage to the lungs, while dry powder inhalation can effectively avoid this. Furthermore, unlike the BM+TI group, the BM+AB-DPI group showed significant differences in Te, Ti, RT, EV, AV, and EEP compared with the BM, BM+BF, and Normal groups (p<0.01, p<0.05). This suggests that AB-DPI can significantly attenuate the changes in various lung function parameters that deviate from normal values ​​induced by BM, a more significant effect than pirfenidone.

[0133] (3) Analysis of rat lung coefficient

[0134] The lung coefficient is an important indicator reflecting the degree of edema, lung tissue damage, and fibrosis, and is often used as a measurement indicator in animal experiments. When pulmonary edema occurs, the lung coefficient will increase significantly, indicating that the degree of lung damage is more severe. The lung coefficient is statistically analyzed according to the following formula:

[0135] Lung coefficient = lung wet weight (g) / body weight (kg) * 100%

[0136] The lung system values ​​of 6 rats in 8 groups were measured. The results are shown in Figure 17 The average values ​​of the Normal and Sham groups were both 4.5±0.5; the lung coefficient of the BM group was 9.5±0.5; the lung coefficients of the BM+BI-DPI group, BM+AM-DPI group, BM+AB-DPI group, BM+TI group, and BM+BF group were all within the range of 6.5±1.5 (p<0.05 or p<0.01). This indicates that this indicator meets the model requirements and the differences are statistically significant. Figure 16 It can be seen that compared with the Normal group, the lung coefficient of rats in the BM group (p<0.01) was significantly increased. Compared with the BM group, the BM+BI-DPI group and the BM+AM-DPI group were able to reduce the lung coefficient of rats, which means that there was no particularly significant difference between them; while the BM+AB-DPI group, the BM+TI group, and the BM+BF group were able to greatly reduce the lung coefficient of rats, all with significant differences (p<0.05 or p<0.01). In summary, AB-DPI has the effect of alleviating pulmonary edema and repairing lung function. Compared with simple AM ​​and BI, AB cocrystal drugs have a better therapeutic effect on respiratory failure caused by pulmonary fibrosis.

[0137] (4) Analysis of the determination results of total protein content in alveolar lavage fluid

[0138] The total protein content in the bronchoalveolar lavage fluid of rats in each group was determined by BCA method. Figure 18 The total protein content of the Normal group and the Sham group was 8 mg·mL -1 There was no significant difference between the two groups; the total protein content of the BM group was approximately 17 g·mL -1 Compared with the sham group, the protein content was significantly increased (p<0.01). Compared with the normal group, the total protein content in the BM+BI-DPI group was increased (p<0.05); however, compared with the BM group, the total protein content in the BM+BI-DPI group was slightly decreased, with a small difference. Compared with the BM group, the total protein content in the bronchoalveolar lavage fluid of rats in the BM+AB-DPI group (p<0.01) and the BM+BF group (p<0.01) was significantly decreased; the total protein content in the BM+TI group was significantly decreased. There was no difference between the BM+AB-DP group and the BM+BF group (p<0.05).

[0139] In summary, it can be seen that the effect of AM and BI cocrystal compound AB in reducing total protein content is better than that of AM and BI alone.

[0140] (5) Analysis of the results of determination of IFN-γ and TGF-β1 levels in BALF

[0141] like Figure 19As shown, compared with the Normal group, the IFN-γ and TGF-β1 levels in the Sham group were similar to those in the Normal group. In the BM group, TGF-β1 expression in the BALF was significantly increased (p < 0.01), while IFN-γ concentration was significantly decreased (p < 0.01). Compared with the BM group, TGF-β1 concentration was significantly decreased (p < 0.01) and IFN-γ concentration was significantly increased (p < 0.01) in the BM+AB-DPI and BM+BF groups. IFN-γ concentration was significantly increased in the BM+TI group (p < 0.01). TGF-β1 expression was significantly decreased in the BM+AB group compared with the BM+BI-DPI and BM+AM-DPI groups (p > 0.05). IFN-γ and TGF-β1 levels in the BM+AB-DPI group remained similar to those in the BM+BF and BM+TI groups. This indicates that ambroxol ibuprofen cocrystal dry powder inhaler has a stronger ability to reduce TGF-β1 levels and increase IFN-γ levels, and has a better anti-inflammatory effect.

[0142] (6) Analysis of the results of determination of inflammatory factors IL-1, IL-2, IL-8, and IL-1β in BALF

[0143] We detected the concentrations of inflammatory factors IL-1, IL-2, IL-8, and IL-1β in BALF to verify whether AB-DPI can regulate the inflammatory process by regulating the secretion of various cytokines involved in the inflammatory response, thereby revealing that AB-DPI plays a significant therapeutic role in the pathogenesis of BM-induced idiopathic pulmonary fibrosis. Figure 20 As shown in the figure, the levels of inflammatory factors IL-1, IL-2, IL-8, and IL-1β in the BALF of rats in the Normal and Sham groups were similar. Compared with the Normal group, the levels of IL-1, IL-2, IL-8, and IL-1β in the BALF of rats in the BM group were significantly increased (p<0.01). The levels of IL-1β were increased in the BM+AM-DPI group and the BM+TI group (p<0.01, p<0.05). The IL-2 level was also significantly increased in the BM+AM-DPI group (p<0.05). Compared with the BM group, IL-1, IL-2, IL-8, and IL-1β levels were significantly decreased in the sham, BM+BI-DPI, BM+AB-DPI, and BM+BF groups (p<0.01 or p<0.05). However, the BALF concentrations of IL-1, IL-2, IL-8, and IL-1β in the BM+AM-DPI and BM+BI-DPI groups were not as pronounced as those in the BM+AB-DPI group, but their effects on reducing inflammatory factors were comparable to those in the BM+BF group. These results indicate that AB-DPI can more significantly reduce inflammatory factor levels and has a significant inhibitory effect on BM-induced inflammatory responses in rats.

[0144] (7) HE staining of rat lung tissue

[0145] HE staining was used to classify the degree of lung injury and fibrosis. Figure 22 ), the lung tissue structure of the normal group was normal, and the alveolar wall structure was clear. The lung tissue of the rats in the BM group developed extensive lesions, with focal infiltration of inflammatory cells, the alveolar cavity filled with inflammatory exudate, and obvious pulmonary fibrosis. The BM+AM-DPI group and the BM+BI-DPI group could alleviate the degree of pulmonary fibrosis in rats, but there was no significant difference. The degree of pulmonary fibrosis and alveolar inflammation in the BM+AB-DPI group and the BM+BF group were significantly lower than those in the BM group (p<0.05), indicating that ambroxol ibuprofen cocrystal dry powder inhaler (AB-DPI) can improve the degree of pulmonary fibrosis and alveolar inflammation in rats with pulmonary fibrosis. Compared with simple AM ​​and BI, AB cocrystal drugs have better therapeutic effects.

[0146] (8) Analysis of the results of determination of LDH, SOD, MDA and Hyp in lung tissue homogenate

[0147] Studies have shown that oxidative stress plays a key role in the progression of BM-induced IPF. MDA expression levels and SOD activity reflect the balance between oxidative and antioxidant functions in the body. In this study, we measured SOD and MDA together to investigate changes in the levels of oxidative enzymes in IPF. Because Hyp is most abundant in collagen, its concentration in lung tissue homogenates can reflect collagen metabolism and the degree of pulmonary fibrosis.

[0148] like Figure 21 As shown, the levels of LDH, SOD, MDA, and Hyp in the sham group were significantly different from those in the normal group. After BM induction, the SOD content in the lung tissue homogenate of rats was significantly decreased (p < 0.01), while the MDA, LDH, and Hyp contents were significantly increased (p < 0.01). After 28 days of continuous administration, compared with the BM group, the Sham group, the BM + AB-DPI group, and the BM + BF group had significantly increased SOD content (p < 0.01), while the MDA and LDH contents were significantly decreased (p < 0.01). However, the MDA and Hyp contents were slightly decreased in the BM + TI group (p < 0.05). LDH levels in the lung tissue of rats in the BM + BI-DPI and BM + AM-DPI groups were slightly lower than those after BM induction (p > 0.05), while SOD levels were slightly higher (p > 0.05). Compared with the Normal group, the MDA level in the lung tissue of rats in the BM+BI-DPI group and the BM+AM-DPI group was higher (p<0.05) and the SOD level was lower (p>0.05).

[0149] Figure 21The results of Hyp analysis are also presented. There was no difference in Hyp levels in lung tissue between the Normal and Sham groups. The Hyp level in the BM group was significantly higher than in the Normal and Sham groups (p < 0.01). However, Hyp levels were slightly lower in the BM+AM-DPI, BM+BI-DPI, and BM groups (p > 0.05). Hyp levels in the BM+AM-DPI and BM+BI-DPI groups were higher than in the Normal group (p < 0.01). Compared with the BM group, Hyp levels in the lung tissue of rats in the BM+TI, BM+AB-DPI, and BM+BF groups were significantly lower (p < 0.05, p < 0.01, respectively). These results suggest that ambroxol / ibuprofen cocrystal dry powder inhaler (AB-DPI), under BM induction, can increase the SOD content in lung tissue homogenates and inhibit the expression of MDA, LDH, and Hyp, thereby enhancing their antioxidant capacity and effectively reducing the degree of pulmonary fibrosis.

[0150] (9) Analysis of MPO content in rat serum

[0151] According to relevant data, the determination of MPO in serum reflects the accumulation of neutrophils in lung tissue, and it is a reliable indicator for judging the infiltration of inflammatory cells in the lungs. Figure 23 Figure 2 shows the serum MPO levels of each rat. Compared with the normal group, serum MPO levels were significantly increased in the BM, BM+BI-DPI, and BM+AM-DPI groups (p < 0.01). Compared with the BM group, serum MPO levels were significantly decreased in the BM+AB-DPI and BM+BF groups, indicating a significant inhibitory effect on MPO activity (p < 0.01). The BM+TI group had a nonsignificant inhibitory effect on serum MPO activity (p < 0.05), while the BM+BI-DPI and BM+AM-DPI groups had less significant inhibitory effects on serum MPO activity (p > 0.05). In summary, the ambroxol / ibuprofen cocrystal exhibits a more potent inhibitory effect on MPO activity than the single APIs (ambroxol and ibuprofen). Regarding pulmonary administration, the therapeutic efficacy of the ambroxol / ibuprofen cocrystal via DPI was significantly superior to that via tail vein injection.

[0152] (10) Preliminary pharmacokinetic study in rats (determination of BI and AM drug concentrations in plasma and lung tissue homogenate)

[0153] The obtained data were analyzed and sorted to obtain drug concentration images in plasma and lung tissue homogenate of the lung administration group and tail vein injection group, as shown in Figure 2. Figure 24 and Figure 25. The results show that, compared with the intravenous injection group, after pulmonary administration of the same dose of AB-DPI micropowder, BI reached the maximum blood concentration in plasma at 30 minutes, and the drug retention time in the body was long. The drug elimination rate of AM in the pulmonary administration group was larger, but the bioavailability was higher. After pulmonary administration, BI reached the maximum blood concentration in plasma at about 32 minutes, and the average retention time in the body was significantly prolonged compared with the tail vein injection. The bioavailability was also higher. It can be seen that pulmonary administration can significantly improve the bioavailability of drugs in plasma and the average retention time in the body. In the lung tissue homogenate, the pulmonary administration group significantly improved the bioavailability of BI in the lung tissue compared with the tail vein injection group. The clearance rate of BI in the lung tissue is low, and the retention time in the body is relatively long. It can be seen that the DPI microparticles prepared by spray drying for pulmonary administration can significantly improve the pulmonary bioavailability of BI and AM, and have a certain lung targeting, which brings potential for the treatment of lung diseases.

[0154] Mechanism of Action: Research results demonstrate that ambroxol-ibuprofen cocrystal powder inhalation can modulate oxidative stress and reduce inflammatory cytokine levels, thereby alleviating IPF in rats. Furthermore, ambroxol-ibuprofen cocrystal powder inhalation significantly inhibits the progression of IPF in rats and alleviates IPF by modulating oxidative stress and reducing inflammatory cytokine levels. Its anti-inflammatory effects are attributed to downregulating IL-1, IL-2, IL-8, and IL-1β levels, downregulating LDH (lactate dehydrogenase) expression, and reducing inflammatory cell infiltration. Its antioxidant effects are believed to be driven by increasing interferon (IFN-γ) levels, decreasing malondialdehyde (MDA) levels, and causing an abnormal increase in MPO (myeloperoxidase). Its anti-fibrotic effects are primarily achieved by reducing Hyp (hydroxyproline) levels and TGF-β1 (transforming growth factor-β1) protein expression.

[0155] In summary, AB-DPI can exert its anti-inflammatory, antioxidant, and anti-fibrotic effects from three aspects, and has outstanding effects in improving and protecting BM-induced IPF. It has the advantages of reducing the dosage, improving the efficacy, and improving patient compliance, thereby avoiding the first-pass effect of the liver.

Claims

1. An ambroxol ibuprofen eutectic dry powder inhaler, characterized in that, The dry powder inhaler comprises ambroxol ibuprofen cocrystal and lactose; the mass ratio of lactose to ambroxol ibuprofen cocrystal is 5%-35%. The preparation method of the ambroxol ibuprofen cocrystal comprises the following steps: mixing ambroxol and ibuprofen at a molar ratio of 1:1, dissolving the mixed mixture in a solvent, heating to a certain temperature, cooling, and filtering to obtain an ambroxol ibuprofen cocrystal product; the solvent is a C1-C6 solvent, and the temperature is 80-120°C.

2. The dry powder inhaler according to claim 1, wherein The solvent is one of methanol, ethanol, propanol, butanol and tetrahydrofuran.

3. The dry powder inhaler according to claim 2, wherein The solvent is ethanol.

4. The dry powder inhaler according to claim 1, wherein The temperature was 80°C.

5. The method for preparing the dry powder inhaler according to any one of claims 1 to 4, wherein: The ambroxol ibuprofen cocrystal is dissolved in anhydrous ethanol to obtain an ethanol solution of the ambroxol ibuprofen cocrystal, lactose is dissolved in water to form a lactose aqueous solution, which is dropwise added to the ethanol solution of the ambroxol ibuprofen cocrystal, stirred at room temperature, filtered, and spray-dried to obtain the dry powder inhaler.

6. The preparation method according to claim 5, wherein The mass ratio of the ambroxol ibuprofen cocrystal mass to the anhydrous ethanol in the ambroxol ibuprofen cocrystal ethanol solution is 100:0.5-0.

9.

7. The preparation method according to claim 5, wherein The mass ratio of lactose to water in the lactose aqueous solution is 1:1-15.

8. The preparation method according to claim 5, wherein The filtration is performed using a 0.2-0.8 μm microporous filter membrane.

9. The preparation method according to claim 5, wherein The spray drying is specifically carried out by using a spray dryer at a spray pressure of 16-40 MPa and a drying wind speed of 0.4-0.8 m 3 min -1 , Liquid supply speed is 4-8mL·min -1 The dry powder inhaler is prepared under the conditions of an inlet temperature of 80-120°C.

10. Use of the dry powder inhaler according to any one of claims 1 to 4 in the preparation of a drug for treating pulmonary fibrosis.

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