Ambroxol and ibuprofen eutectic dry powder inhaler as well as preparation method and application thereof

Abstract

The invention relates to an ambroxol and ibuprofen eutectic dry powder inhaler as well as a preparation method and application thereof, and belongs to the professional field of pharmaceutical preparations. The dry powder inhalant 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 and ibuprofen eutectic is 5-35%. A rat lung tissue distribution experiment result shows that compared with traditional intravenous administration, the delivery efficiency of the medicine in the lung can be remarkably enhanced by adopting a dry powder inhalation administration mode, and the retention time of the medicine in the lung is greatly prolonged. The pharmacodynamic experiment further reveals that the ambroxol and ibuprofen eutectic dry powder inhalant can effectively inhibit the active expression of inflammatory factors in a model animal body, reduce the accumulation of hydroxyproline (HYP) in lung tissues and serum, improve the anti-fibrosis and anti-inflammatory ability and improve the oxidative stress ability, so that the pathological process of pulmonary fibrosis is delayed, and the inhalant can be used for treating pulmonary fibrosis. The compound shows huge development potential as an anti-pulmonary fibrosis drug.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular 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 can cause progressive damage to lung function. It is characterized by progressive dyspnea, excessive degradation of the lung matrix, and damage to the alveolar structure, which in turn destroys the lung structure and eventually leads to pulmonary interstitial fibrosis. Idiopathic pulmonary fibrosis (IPF) is the most common, fatal, and progressive subtype of pulmonary fibrosis. Its clinical course is heterogeneous, including asymptomatic stable state, slow progression with acute exacerbation, or rapid progressive decline in lung function. Clinical symptoms are insidious onset, with no obvious symptoms and little cough in the early stage, progressive hypoxemia and diffuse damage to both lungs in the later stage, inflammation invading the alveolar cavity and alveolar wall, causing pulmonary fibrosis and thickening of the alveolar septum, and eventually causing respiratory failure and death. It is generally believed that inflammatory response and oxidative stress may be important factors involved in the onset and development of IPF. In recent years, the pathogenesis of IPF and how to treat it have become hot topics of research at home and abroad. Idiopathic pulmonary fibrosis can be inhibited by inhaled porous microspheres loaded with relaxin; Ma Xinai, Xia Kexin and other scholars proposed to use inhaled silybin dry powder prepared by nanosuspension spray drying technology to treat idiopathic pulmonary fibrosis, etc.

[0003] IPF continues to develop and is currently incurable. Clinically, appropriate drug therapy, surgery, rehabilitation training and other methods are generally used to slow down the progression of the disease, thereby improving the patient's quality of life and survival time. The existing treatments for IPF include drug therapy and non-drug therapy. In terms of drug therapy, only pirfenidone (an inhibitor of multiple cytokines) and nintedanib (a small molecule inhibitor of receptor tyrosine kinase) are currently approved for marketing to slow down the progression of IPF and prolong survival time. However, both of these therapeutic drugs have side effects, including abdominal pain, diarrhea, skin diseases, and liver toxicity. In terms of the best efficacy of clinical goals, there is no ideal drug for the treatment of IPF so far. Non-drug treatment is mostly the main treatment for respiratory failure in the late clinical stage, mainly oxygen therapy and lung transplantation. The IPF clinical guidelines indicate that lung transplantation is the most direct and effective method for treating IPF, but factors such as the scarcity of donor lungs and high costs objectively limit the implementation of lung transplantation in clinical practice. Therefore, it is urgent to find therapeutic drugs and dosage forms that are effective, safe and effective.

[0004] Ibuprofen (BI) is an aromatic propionic acid nonsteroidal anti-inflammatory drug with antipyretic, analgesic and anti-inflammatory effects. In clinical practice, ibuprofen is widely used to treat arthritis and gout. Its mechanism of action is to inhibit the activity of cyclooxygenase (COX) to reduce the synthesis of prostaglandins, thereby achieving the effect of reducing inflammatory response and analgesia. In the course of research, BI treats pulmonary fibrosis by reducing the inflammatory response of rats with pulmonary fibrosis. When ibuprofen was injected into rats with bleomycin-induced pulmonary fibrosis, it was found that BI could also reduce the levels of pulmonary fibrosis cytokines by downregulating the levels of TGF-β, MyD88 and p-Smad2. The above research results show that BI plays a certain role in the treatment of pulmonary fibrosis and has the potential to be used as an anti-IPF drug. However, due to its poor water solubility, the dissolution rate of general ibuprofen tablets after oral administration is slow, and it is difficult to play a good therapeutic effect in clinical practice. In order to meet clinical requirements, people have been studying new formulations of ibuprofen. 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] As a solid form that improves the physicochemical properties of active pharmaceutical ingredients (API), pharmaceutical cocrystals have attracted great attention from the academic community. Functional groups in APIs can form pharmaceutical cocrystals using intermolecular recognition interactions such as hydrogen bonding and π-π stacking, and cocrystal ligand (CCF) molecules without changing the pharmacological activity of APIs, but their physicochemical properties are improved, such as melting point, stability, solubility, bioavailability, mechanical properties, etc., thereby improving their drugability. However, how to select cocrystal solvents, predict cocrystal structures and effects, and how to mass-produce cocrystals so that cocrystals can be applied to industry are still difficult problems in the research and development of pharmaceutical cocrystals. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides an ambroxol ibuprofen cocrystal dry powder inhaler, a preparation method and application thereof. The dry powder inhaler has low hygroscopicity, good fluidity and high lung deposition rate, thereby greatly promoting the inhalation administration effect of the drug.

[0007] To solve the above problems, the present invention adopts the following technical solutions:

[0008] An ambroxol ibuprofen cocrystal dry powder inhaler, the main components of which include ambroxol ibuprofen cocrystal and lactose, wherein the mass ratio of lactose to ambroxol ibuprofen cocrystal is 5-35%. Within this mass ratio range, the obtained dry powder inhaler can achieve more efficient pulmonary delivery and better therapeutic effect.

[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, the crystal system is monoclinic, the space group is P21 / c, an AM cation and a BI anion form an asymmetric unit, and the cocrystal can improve the solubility of the drug and increase the active ingredients of the drug.

[0012] Further preferably, the preparation method of the ambroxol ibuprofen cocrystal is: ambroxol (AM) and ibuprofen (BI) are mixed at a molar ratio of 1:1, dissolved in a solvent after mixing, heated to a certain temperature, cooled, filtered, and the ambroxol ibuprofen cocrystal product is obtained. The solvent is a C1-C6 solvent, such as methanol, ethanol, propanol, butanol, tetrahydrofuran, etc., preferably ethanol; the reaction temperature is 10-120°C, preferably 80°C.

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

[0014] Preferably, the mass ratio of the ambroxol ibuprofen co-crystal mass to anhydrous ethanol in the ethanol solution of the ambroxol ibuprofen co-crystal 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 the use of the dry powder inhaler in preparing a drug for treating anti-pulmonary fibrosis.

[0019] Beneficial effects of the present invention:

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

[0021] 1. The present invention prepares a eutectic drug by mixing ambroxol (AM) and ibuprofen (BI) at 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 retention 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 ability, 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 currently used clinically for idiopathic pulmonary fibrosis (IPF), the ambroxol-ibuprofen cocrystal drug dry powder inhaler described in the present invention can improve the compliance of alveolar epithelial cells and enhance the compliance of patients, 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 pattern of ambroxol-ibuprofen cocrystal (AB), (a) simulated pattern, (b) measured pattern.

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

[0030] Figure 7 .Fingerprint of compound AB.

[0031] Figure 8 .Contribution of each key.

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

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

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

[0035] Fig.12 .DSC curve graph.

[0036] Fig.13 . Changes in body weight of rats in each group at 1, 7, 14, 21, and 28 days;

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

[0038] Fig.14 .Chart of lung function indexes of rats in different groups;

[0039] Fig.15 .Chart of lung function indexes of rats in different groups;

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

[0041] Fig.17 .The lung coefficient of rats in each group after 28 days;

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

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

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

[0045] Fig.19 .The concentrations of IFN-γ and TGF-β1 in BALF of rats in the eight groups;

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

[0047] Fig. 20 . Expression levels of inflammatory factors IL-1, IL-2, IL-8, and IL-1β in BALF of rats in eight groups;

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

[0049] Fig.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 BM group, *p<0.05, **p<0.01.

[0051] Fig. 22 .Rat alveolar tissue sections.

[0052] Fig.23 .The results of the determination of MPO content in rat serum;

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

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

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

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

[0057] Raw materials used in the embodiments 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, American Hyclone Company); 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: 48 SPF male SD rats weighing 200±20 g were kept at 23-27°C and were allowed to eat and drink freely. This study was approved by the Medical Ethics Committee of Shandong University of Traditional Chinese Medicine and conducted in the Animal Experiment Center. The animals used in the experiment were strictly implemented in 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, and then heated at 80 °C for several minutes until completely dissolved, allowed to stand and cool to room temperature, cooled and crystallized, and colorless block crystals (recorded as AB cocrystal API) were precipitated, with the chemical formula of C 26 H 36 Br2N2O3 (584.39). Yield: 90%.

[0062] AB eutectic structure:

[0063]

[0064] AB eutectic melting point: 147-149°C. Elemental analysis: theoretical value: C, 53.44; H, 6.21; N, 4.79; experimental value: 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 This indicates the presence of a characteristic stretching vibration of NH. -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 vibrations. The infrared band is located at 966-746 cm -1 , indicating that it undergoes in-plane and out-of-plane bending modes of the 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 by using a Bruker CCD diffractometer with Mo Ka (λ = 0.71073) monochromatic radiation. The absorption coefficient was semi-empirically corrected using SADABS software, the data obtained by X-ray diffraction were normalized, and 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, there is a torsion angle (174.8°) between the cyclohexane and benzene rings, and the cyclohexane is in a more stable chair conformation rather than a less stable boat conformation. Both bromine atoms bound to the benzene ring show disordered features, which is completely different from what is observed in the individual ambroxol compounds. This is likely because the proton from the BI part can be easily transferred to the AM molecule. It is worth noting that there are two amino groups in the AM part. At the same time, we found that only one amino group in the aliphatic group can be protonated. The observations suggest that aliphatic amino groups have stronger basicity compared to 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 eutectic, 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 length and bond angle 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 lengths range 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 bonds can generate aggregation between AM cations and BI anions. 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 along the crystal direction to the supramolecular 2-D layer ( Figure 3 ,c). Stacking interaction (C9-H9B···Cg3 b ) and the above hydrogen bonds, through which the supramolecular three-dimensional structure can be generated along the crystallographic a-axis ( Figure 4 ).

[0074] Table 2. Hydrogen bonding related 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 molecular interactions and structure-performance relationships of the compounds. The interactions of compound AB on the Hirshfeld surface were quantified by two-dimensional fingerprints and Hirshfeld surface calculation methods. 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 map ( Figure 7 ) observed that the contribution rate of the peak representing the H···O interaction was 12.8%, second only to H···H. The C···H, which is shaped like two sharp teeth, contributed 11.0%. The contributions of other short contact points (such as C···C, N···C and N···H) were lower and all less than 2.0%, but their intermolecular forces also play a very important role in the formation of crystals. There are also O···O, O···C and N···N with 0% interaction.

[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 aqueous solution using a 0.5-micron microporous filter membrane to remove possible solid particles or impurities 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 needs of pulmonary administration, ensuring that the drug particles can penetrate into the lung tissue and achieve effective therapeutic effects. In addition, the span (GSD) value of the dry powder is 1.5, indicating that its particle size distribution is relatively narrow, which is crucial to 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, and its emptying rate is as high as 98%, which means that there will be almost no drug residue during the inhalation administration process, thereby ensuring the effective use of the drug. In addition, the hygroscopicity of the dry powder is less than 14%, which ensures 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 Fig. 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 dry powder obtained with different proportions of Lactose is shown in Fig.10 As shown, as the amount of Lactose added increases, the moisture content of the cocrystal drug AB shows an overall decreasing trend, indicating that the microparticles combining the cocrystal drug AB and Lactose can reduce the moisture contained in the microparticles to reduce the evaporation of moisture, making it easier to store while also avoiding the weakening of the efficacy.

[0088] The yield of AB dry powder obtained with different ratios of Lactose is shown in Fig.11As shown, the yield of AB-DPI containing 15% Lactose is the highest at about 85%, and the yield of AB-DPI containing 0% Lactose is the lowest at 79.2%, with a difference of 5.8%; whether the content of AB-DPI is greater than or less than 15% Lactose, it will decrease. This shows that adding a proper amount of Lactose to DPI can increase the yield of DPI micropowder to a certain extent. Based on the results of the comprehensive data measurement, the optimal formula is selected when the proportion of Lactose is 15%, which can reduce the loss of the drug and make the morphology more stable.

[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 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 obtained DSC curve is shown in the figure below. Fig.12 . Fig.12 It shows that AM has an endothermic peak at 250℃ and an exothermic peak at 280℃, and BI has a melting exothermic peak at 84.5℃, indicating that the two individual drugs are unstable under heat. At the same time, Mix (physical mixture of the two) can see a melting endothermic peak at 160℃, and there are two adjacent melting endothermic peaks and exothermic peaks at 180℃ and 300℃, which can show that the physical mixture of the two drugs is also extremely unstable. Lactose can see a melting endothermic peak at 315℃, which can be inferred that the melting temperature of AM and BI is reduced due to the influence of excipients. In AB-DPI, there is no characteristic peak in its spectrum, indicating that the crystalline form of AB is transformed into the amorphous form of AB-DPI. The reason for this amorphous form is that AB is encapsulated in Lactose. Compared with the individual drugs and physical mixtures of the two drugs, the cocrystal of the two drugs combines the advantages of the two, which is very advantageous. Moreover, pirfenidone represented by BF also has a melting exothermic peak at 100℃, 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 -1Use a measuring cylinder to measure bleomycin, add physiological saline, and then put it into a 10mL volumetric flask to obtain a concentration of 12.5mg·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 containing 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 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 animal center, without external effects); (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: a certain amount of bleomycin was injected into the rats through endotracheal intubation, and then the rats were shaken upright for 5 minutes; (4) BM+BI-DPI group: on the second day of the construction of the BM model group, endotracheal intubation was performed using the DP-4 rat dry powder pulmonary administration device, BI powder, 3 mg per day, the dose was 9 mg 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 (starting 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 dose was 11.6 mg mL -12mL of pirfenidone, administered for a total of 28 days). Operation of DPI dry powder administration in the lungs: Use a precision balance to weigh the DPI micropowder and put it into the storage room 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 a rat laryngoscope, and immediately insert the nozzle of the drug delivery device from the tracheal opening while the tracheal opening is opened. Place it in the bronchial intubation port, and immediately push air into the drug delivery device to push the DPI micropowder into the rat's lungs. The Sham group inhaled 1.5 to 2 ml of air (without drug) in the same manner as described above. The BM+TI group was injected with AB-DPI micropowder solution, and the dosage was the same as that of the BM+AB-DPI group. At the same time, for the rats in the BM+BF group, the dose given by gavage was 116 mg·kg -1 . Observe the general condition of the experimental rats every day, such as: rat activity, eating, weight, spirit and respiratory status and other physical indexes. The constructed BM model is regarded as the first day. The weight of each group of experimental rats is observed and recorded every 7 days (1 week), a total of five records, and the recorded rat data are statistically analyzed. When the model is established on the 28th day, the EMKA pulmonary function tester is used to detect various respiratory indicators of rats in a free-moving state, including: relaxation time (RT), inspiratory duration (TI), bronchial contraction parameter (Penh), maximum expiratory flow (PEF), maximum inspiratory flow (PIF), respiratory frequency (f), tidal volume (TV), and expiratory duration (TE). After the test, the rats in each group for experimental research were treated, and their abdominal aorta was used for blood sampling. Finally, they were killed, and the alveoli at the bronchus were irrigated with physiological saline to collect the alveolar lavage fluid of the rats, and then the lung tissue was 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 a speed of 4000 r / min -1 The serum in the supernatant was taken and placed in a -80°C refrigerator for testing.

[0099] (3) Rat lung coefficient determination

[0100] Lung coefficient is often used as a measurement index in animal experiments. It refers to the size of the lung volume when the same volume of gas is inhaled per unit time. It is one of the important indicators reflecting the degree of damage to lung tissue, lung edema and lung tissue fibrosis. The higher the number, the more serious the degree of lung tissue lesions. 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 fiberoptic bronchoscope into the patient's airway to the location of inflammation or lesions, injecting a sterile saline solution, and then inhaling it to obtain a liquid. In clinical practice, BALF is mainly used to detect cytokines, oxidative stress-related enzymes and soluble substances, which is very helpful in the diagnosis and treatment of lung-related diseases (such as lung inflammation, IPF, etc.) and improving the prognosis of patients.

[0103] The method of BALF is as follows: blood is drawn from the abdominal aorta of the rat, the rat is killed, the chest cavity is opened, and then the excess fat on the airway is removed to expose the trachea. The retention needle is inserted from the front of the trachea into the right bronchus, and the front and back sides are tightly fixed. 2mL of pre-cooled saline is slowly infused into the lungs, and then aspirated after standing for 2 minutes. This operation is repeated 3 times, and the 3 BALFs are combined into a 10mL EP tube and stored at low temperature. This lavage method is relatively easy to operate, and the recovery rate is >80%.

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

[0105] The left lung of the rat was taken and soaked in 10% formalin solution for 24 hours. After drying in a dryer, the left lung of the rat was taken and wrapped in paraffin to save the slices. A 5-micron thick slice was taken, stained with hematoxylin and eosin, dehydrated until transparent, and sealed with a neutral adhesive. The sliced ​​sample was observed under a microscope to obtain an image.

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

[0107] Determination of total protein content in BALF

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

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

[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 multiple of sample before testing

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

[0115] The 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 response and immune disorder. Our previous studies have found that TGF-β1 can promote the transition of type II epithelial cells to epithelial-mesenchymal cells (EMT) and the transition of primary fibroblasts to muscle fibroblasts (FMT). The content of IFN-γ and transforming growth factor-β1 (TGF-β1) in the supernatant of BALF culture medium was detected by ELISA. The detection was performed according to the method described in the purchased ELISA kit.

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

[0119] Weigh an appropriate amount of rat lung tissue, grind it in a tissue homogenizer using ice-cold physiological saline as the homogenization medium, and then centrifuge the grinding solution at a speed of 3000 r / min. -1 , centrifuged for 10 minutes, and the supernatant was used to prepare 10% tissue homogenate, which was placed in a refrigerator for determination. Enzyme-linked immunosorbent assay was used to determine Hyp, and the operation steps were carried out according to the steps in the kit; the WST-1 method was used to detect the content of superoxide dismutase in serum; malondialdehyde (MDA) and LDH were determined by TBA and LDH methods, 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 use the colorimetric method to detect the MPO level in plasma. The detection method of methamphetamine oxidase should be carried out according to the instructions of 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] Taking 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 brought 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] GraphPadPrism8.0 was used to perform statistical analysis on the experimental data. The experimental results were expressed as mean ± standard deviation (x ± s), and the differences between the two groups were tested by single factor variance analysis and Tukey multiple comparison. p < 0.05 was considered to be different, and p < 0.01 was considered to be significantly different. The statistical significance of the experimental results was explained.

[0126] Result analysis:

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

[0128] Weight changes of rats

[0129] The BM was used to induce pulmonary fibrosis in rats to construct IPF rats. The weight data of the rats obtained were represented by a bar graph (as shown in Figure 2A). Fig.13 The weight changes every 7 days are shown in the figure, which can directly reflect the daily weight changes. At the same time, three control systems were constructed: System 1 used normal control group (Normal) and sham operation group (Sham) rats to discuss the effects 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 were in a depressed mental state, had a long period of inactivity, a high frequency of inactivity, decreased food intake, and more concentrated activities. The 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, which 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, from being more inactive to being active, the excitability increased, and the amount of food and water intake gradually recovered. On days 7-14, the weight gain of rats in the BM+AM-DPI group and the BM+AB-DPI group was more obvious than that in the BM+BI-DPI group, but on days 15-28, the rats in the BM+BI-DPI group and the BM+AB-DPI group showed better recovery effects. Among them, the weight recovery of rats in the BM+AB-DPI group was better than that of the other two groups during the experiment, and it was the optimal group; system three set up BM+TI and BM+BF. After 28 days of tail vein injection and lung administration, the two groups of rats were in basically the same state and both had a certain degree of discomfort. The tail of the BM+TI rats had a certain degree of peeling. After administration, the rats in the BM+BF group had a certain dry cough. It is speculated that pirfenidone may have a certain irritation and damage to the lungs, but the discomfort disappeared after a period of time. The optimal group obtained in system two was compared with the two groups in system three, and it was found that the weight recovery and mental state of rats in the BM+AB-DPI group were better than those in the two groups in system three. Although the rats in this group also had dry cough as the rats in other lung administration groups, the degree was milder and the discomfort disappeared faster. In summary, AB-DPI can significantly improve the weight loss of rats induced by BM.

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

[0131] Generally speaking, lung resistance and compliance are important indicators used to evaluate lung function. Figures 14 to 16It can be seen that compared with the Normal group, the various lung function parameters of the Sham group were almost the same. Among the lung function parameters of the rats in the BM group, the values ​​of AV, EEP, EV, PIF, EF50, PEF, TV, RT, Te, and Ti were significantly decreased (p<0.01), the values ​​of EIP and MVV were slightly decreased (p<0.05), while the values ​​of F and Penh were significantly increased (p<0.01). Compared with the BM group, the lung function parameters of the BM+BI-DPI group 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; among the lung function parameters of the BM+AM-DPI group, 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 the MVV parameter was slightly higher than that 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 shows that pulmonary administration via tail vein injection will cause damage to the lungs, and dry powder inhalation can effectively avoid this situation. In addition, 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 group, BM+BF group and Normal group (p<0.01, p<0.05). This shows that AB-DPI can significantly weaken the changes of various lung function parameters from normal values ​​induced by BM, which is more significant than the effect of pirfenidone.

[0133] (3) Analysis of rat lung coefficient

[0134] The lung coefficient is an important indicator that reflects 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 serious. According to the following formula, the lung coefficient is statistically analyzed:

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

[0136] The lung system values ​​of 6 rats in 8 groups were measured, and the results are shown in Fig.17 The average values ​​of Normal and Sham groups were 4.5±0.5; the lung coefficient of BM group was 9.5±0.5; the lung coefficients of rats in 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 shows that the index meets the requirements of the model and the difference is statistically significant. Fig.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, and there were significant differences (p<0.05 or p<0.01). In summary, AB-DPI has the effect of relieving pulmonary edema and repairing lung function. Compared with simple AM ​​and BI, AB cocrystal drugs have better therapeutic effects on respiratory failure caused by pulmonary fibrosis.

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

[0138] The total protein content in the bronchoalveolar lavage fluid of rats in each group was determined by BCA method. Fig.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 about 17 g·mL -1 Compared with the Sham group, the protein content was significantly increased (p<0.01). Compared with Normal, the total protein content of the BM+BI-DPI group was increased (p<0.05); compared with the BM group, the total protein content of 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; while the total protein content in the BM+TI group decreased and the difference was more obvious. 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 Fig.19As shown in the data, compared with the Normal group, the levels of IFN-γ and TGF-β1 in the Sham group were close to those in the Normal group; the expression level of TGF-β1 in BALF of rats in the BM group was significantly increased (p<0.01), while the concentration of IFN-γ was significantly decreased (p<0.01). Compared with the BM group, the concentration of TGF-β1 in the BM+AB-DPI group and the BM+BF group was significantly decreased (p<0.01), the concentration of IFN-γ was significantly increased (p<0.01), and the concentration of IFN-γ in the BM+TI group was significantly increased (p<0.01). The expression level of TGF-β1 in the BM+AB group was significantly decreased compared with the BM+BI-DPI group and the BM+AM-DPI group (p>0.05). The levels of IFN-γ and TGF-β1 in the BM+AB-DPI group did not change much compared with the BM+BF group and the BM+TI group. 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. Fig. 20 As shown in the figure, the concentrations of inflammatory factors IL-1, IL-2, IL-8, and IL-1β in 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 BALF of rats in the BM group were significantly increased (p<0.01), the concentrations of IL-1β in the BM+AM-DPI group and the BM+TI group were increased (p<0.01, p<0.05), and the concentration of IL-2 in the BM+AM-DPI group was also significantly increased (p<0.05). Compared with the BM group, the IL-1, IL-2, IL-8 and IL-1β in the Sham group, BM+BI-DPI group, BM+AB-DPI group and BM+BF group were significantly reduced (p<0.01 or p<0.05); however, the concentrations of IL-1, IL-2, IL-8 and IL-1β in BALF of rats in the BM+AM-DPI group and BM+BI-DPI group were reduced but not as obvious as those in the BM+AB-DPI group, but the effect of reducing inflammatory factors was almost the same as that in the BM+BF group. The above results show that AB-DPI can more significantly reduce the level of inflammatory factors and has a significant inhibitory effect on the inflammatory response induced by BM in rats.

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

[0145] HE staining was used to classify the degree of lung injury and fibrosis. Fig. 22 ), the lung tissue structure of the normal group was normal, and the alveolar wall structure was clear. The lung tissue of rats in the BM group had extensive lesions, focal infiltration of inflammatory cells, the alveolar cavity was filled with inflammatory exudate, and obvious pulmonary fibrosis occurred. 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 drug dry powder inhalation (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 an important role in the progression of BM-induced IPF. The expression level of MDA and the activity of SOD can reflect whether the body's oxidative and antioxidant effects are balanced. In this study, we measured SOD and MDA together to observe the changes in the content of oxidative-related enzymes in IPF. Since the content of Hyp is the highest in collagen, the level of Hyp in lung tissue homogenate can reflect the collagen metabolism in lung tissue and the degree of pulmonary fibrosis.

[0148] like Fig.21 As shown in the figure, compared with the Normal group, there were significant differences in the levels of LDH, SOD, MDA and Hyp in the Sham group. After BM induction, the SOD content in the lung tissue homogenate of rats decreased significantly (p<0.01), and the MDA, LDH and Hyp contents increased significantly (p<0.01). After 28 days of continuous administration, compared with the BM group, the SOD content in the Sham group, BM+AB-DPI group and BM+BF group increased significantly (p<0.01), and the MDA and LDH contents decreased significantly (p<0.01), but the MDA and Hyp contents in the BM+TI group decreased slightly (p<0.05). The LDH level in the lung tissue of rats in the BM+BI-DPI group and BM+AM-DPI group was slightly lower than that after BM induction (p>0.05), and the SOD level was 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] Fig.21The results of Hyp determination were also shown. There was no difference in the Hyp content in the lung tissue of rats in the Normal group and the Sham group. The Hyp content in the BM group was significantly higher than that in the Normal group and the Sham group (p<0.01), but the Hyp content in the BM+AM-DPI group, the BM+BI-DPI group and the BM group was slightly lower (p>0.05), and the Hyp content in the BM+AM-DPI group and the BM+BI-DPI group was higher than that in the Normal group (p<0.01). Compared with the BM group, the Hyp levels in the lung tissue of rats in the BM+TI, BM+AB-DPI and BM+BF groups were significantly reduced (p<0.05, p<0.01). The above results show that ambroxol ibuprofen cocrystal drug dry powder inhalation (AB-DPI) can increase the content of SOD in lung tissue homogenate and inhibit the expression of MDA, LDH and Hyp under BM induction, enhance its antioxidant capacity, and thus effectively reduce the degree of lung 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. Fig.23 The content of MPO in the serum of each rat is shown in the figure. Compared with the normal group, the MPO content in the serum of rats in the BM group, BM+BI-DPI group and BM+AM-DPI group was significantly increased (p<0.01). Compared with the BM group, the MPO content in the serum of rats in the BM+AB-DPI group and BM+BF group was significantly reduced, indicating that it had a significant inhibitory effect on the activity of MPO (p<0.01); the BM+TI group had an inhibitory effect on the activity of MPO in serum but not significantly (p<0.05), and the BM+BI-DPI group and BM+AM-DPI group had a less obvious inhibitory effect on the activity of MPO in rat serum (p>0.05). In summary, the effect of ambroxol and ibuprofen cocrystal compound in inhibiting MPO activity is more significant than that of single raw materials (ambroxol and ibuprofen); in terms of pulmonary administration, the therapeutic effect of ambroxol and ibuprofen cocrystal drug in DPI mode is significantly better than that of tail vein injection.

[0152] (10) Preliminary pharmacokinetic study in rats (detection 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 Fig.24 and Fig.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, the drug retention time in the body was long, and the drug elimination rate of AM in the pulmonary administration group was large, but the bioavailability was high. 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 is also high. 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 lung 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 prospects for the treatment of lung diseases.

[0154] Mechanism of action: The results of the study showed that ambroxol-ibuprofen cocrystal dry powder inhalation can regulate oxidative stress response and reduce the level of inflammatory factors to alleviate IPF in rats. At the same time, ambroxol-ibuprofen cocrystal dry powder inhalation can significantly inhibit the development of IPF in rats, and reduce IPF in rats by regulating oxidative stress response and reducing the level of inflammatory factors. Its anti-inflammatory effect is through down-regulating the content of IL-1, IL-2, IL-8, IL-1β, down-regulating the expression of LDH (lactate dehydrogenase), and reducing the infiltration of inflammatory cells; its antioxidant effect is considered to be one of its antioxidant mechanisms by increasing the content of interferon (IFN-γ), reducing the content of malondialdehyde (MDA), and causing an abnormal increase in MPO (myeloperoxidase). Its anti-fibrotic effect is mainly achieved by reducing the content of Hyp (hydroxyproline) and the expression of TGF-β1 (transforming growth factor-β1) protein.

[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, lactose and phosphate; the mass ratio of lactose to ambroxol ibuprofen cocrystal is 5%-35%.

2. The dry powder inhaler according to claim 1, characterized in that The preparation method of ambroxol-ibuprofen cocrystal is as follows: ambroxol and ibuprofen are mixed in a molar ratio of 1:1, dissolved in a suitable solvent after mixing, heated to a certain temperature, cooled, filtered, and the ambroxol-ibuprofen cocrystal product is obtained.

3. The dry powder inhaler as claimed in claim 2, characterized in that The solvent is a C1-C6 solvent, preferably one of methanol, ethanol, propanol, butanol, and tetrahydrofuran, preferably ethanol.

4. The dry powder inhaler according to claim 2, characterized in that The reaction temperature is 10-120°C, preferably 80°C.

5. The method for preparing the dry powder inhaler according to any one of claims 1 to 4, characterized in that: The ambroxol ibuprofen cocrystal is dissolved in anhydrous ethanol, lactose is dissolved in water, and the mixture is added dropwise 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, characterized in that: The mass ratio of the ambroxol ibuprofen co-crystal mass to the anhydrous ethanol in the ambroxol ibuprofen co-crystal ethanol solution is 100:0.5-0.

9.

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

8. The preparation method according to claim 5, characterized in that: The filtration is carried out using a 0.2-0.8 μm microporous filter membrane.

9. The preparation method according to claim 5, characterized in that: 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 / s. 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 5 in the preparation of a drug for treating anti-pulmonary fibrosis.

Images