Use of seglitide sodium in the preparation of a drug for treating and / or preventing chronic obstructive pulmonary disease

Abstract

The application discloses an application of seglitide sodium, in particular, an application of seglitide sodium in preparation of a medicine for treating and / or preventing chronic obstructive pulmonary emphysema. It is found that seglitide sodium can effectively improve the lung function, emphysema and airway remodeling and other core pathological characteristics of a COPD model mouse, which indicates that seglitide sodium has the potential to prepare a medicine for treating chronic obstructive pulmonary emphysema and related emphysema lesions.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, specifically to the use of sitagliptin sodium in the preparation of medicaments for the treatment and / or prevention of chronic obstructive pulmonary disease. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease that is both preventable and treatable. It is characterized by persistent airflow limitation and respiratory symptoms, typically progressing over time. This airflow limitation is primarily related to an abnormal chronic inflammatory response in the airways and lung parenchyma triggered by exposure to harmful particles or gases such as cigarette smoke. Pathologically, COPD involves changes in the central airways, peripheral airways, and alveolar spaces, specifically small airway disease and lung parenchymal destruction. These two core pathological changes together lead to expiratory airflow limitation. COPD is a major disease that seriously threatens human health worldwide, carrying an extremely heavy disease burden.

[0003] Currently, treatment strategies for COPD aim to relieve symptoms, reduce the risk of acute exacerbations, improve exercise tolerance, and slow the decline in lung function. Routine treatment during the stable phase primarily involves pharmacological intervention, mainly including inhaled bronchodilators (such as long-acting β2-receptor agonists like LABA and long-acting anticholinergics like LAMA) and inhaled corticosteroids (ICS). While these medications can control symptoms to some extent, they all have their own limitations:

[0004] Bronchodilators primarily act on airway smooth muscle to dilate the airways, but they cannot reverse or repair existing lung parenchymal damage (such as emphysema) and vascular remodeling. Inhaled corticosteroids, while able to suppress airway inflammation, are associated with increased risk of pneumonia and osteoporosis with long-term use, and a significant proportion of patients are insensitive to their treatment. Furthermore, oral phosphodiesterase-4 inhibitors (such as roflumilast), although usable in specific patients, often cause side effects such as nausea, diarrhea, and weight loss, limiting their clinical application. For advanced, severely ill patients, although surgical interventions such as lung volume reduction surgery or lung transplantation can be considered, their widespread use is limited due to their invasiveness, high cost, and scarcity of donors. Therefore, there is an urgent clinical need to develop novel therapeutic drugs that can intervene in the pathological process of COPD at multiple stages and effectively improve emphysema and airway remodeling.

[0005] In recent years, the potential value of peroxisome proliferator-activated receptor γ (PPARγ) in the treatment of COPD has attracted widespread attention. PPARγ is an important nuclear receptor that regulates inflammation, lipid metabolism, and cell differentiation. Studies have shown that intraperitoneal injection of the exogenous PPARγ ligand rosiglitazone can effectively improve emphysema and lung inflammation induced by cigarette smoke exposure in mice. Its mechanism of action may be related to upregulating the transcriptional level of PPARγ in lung tissue, thereby inhibiting the increase of pulmonary inflammatory factors such as interleukin-8 (IL-8), reducing perivascular neutrophil aggregation, and inhibiting pulmonary vascular wall remodeling. However, although rosiglitazone has shown therapeutic potential for COPD in animal models, its clinical translation is constrained by serious safety concerns. Multiple studies have confirmed that rosiglitazone may significantly increase the risk of cardiovascular disease. It can increase the cardiac burden in patients by causing hemodilution, peripheral and pulmonary edema, leading to an increased incidence of serious adverse cardiovascular events such as heart attack, heart failure, and stroke. This significant cardiovascular risk greatly limits its long-term use in COPD patients, especially those at high risk who often have underlying cardiovascular diseases.

[0006] Therefore, finding safer alternative drugs that can avoid the cardiovascular risks of rosiglitazone while retaining or even enhancing the regulatory effect on PPARα / γ targets has become an important direction in the current research and development of COPD drugs. Summary of the Invention

[0007] Based on this, this application provides at least the use of sitagliptin sodium in the preparation of medicaments for the treatment and / or prevention of chronic obstructive pulmonary disease.

[0008] In a first aspect of this application, the use of the preparation of a medicament for treating and / or preventing chronic obstructive pulmonary disease is provided.

[0009] In a second aspect of this application, the use of a medicament in the preparation of a drug that improves emphysema and / or airway remodeling in animal models of chronic obstructive pulmonary disease is provided.

[0010] After treating mice exposed to smoke for six months with nasal drops of sitagliptin for seven days, alveolar fusion, airway wall thickness, and lung function were significantly improved. This indicates that sitagliptin can effectively improve the core pathological features of COPD model mice, such as lung function, emphysema, and airway remodeling, suggesting its potential to be used to develop drugs for the treatment of chronic obstructive pulmonary disease and related emphysematous lesions. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0012] Figure 1 This invention illustrates the effect of different PPARγ agonists on the expression level of interleukin-6 (IL-6) in MH-S cells treated with cigarette extract (CSE) according to one embodiment of the present application.

[0013] Figure 2 This invention demonstrates the effect of different PPARγ agonists on the expression level of tumor necrosis factor-α (TNF-α) in MH-S cells treated with cigarette extract (CSE) according to one embodiment of the present application.

[0014] Figure 3 This illustration shows a schematic diagram of the experimental procedure for administering nasal drops of sitagliptin sodium to a mouse model of chronic obstructive pulmonary disease (COPD) in one embodiment of this application.

[0015] Figure 4 This application demonstrates the effect of nasal drops of sitagliptin sodium in improving lung function indicators (functional residual capacity FRC, FEV20 / FVC, FEV50 / FVC) in COPD model mice according to one embodiment of the present application.

[0016] Figure 5 This invention demonstrates the effect of nasal drops of sitagliptin sodium in improving alveolar fusion in COPD model mice according to one embodiment of the present application (H&E staining).

[0017] Figure 6 This invention presents the quantitative statistical results of the effect of nasal administration of sitagliptin sodium on the alveolar linear intercept in COPD model mice according to one embodiment of this application.

[0018] Figure 7 This invention demonstrates the effect of nasal drops of sitagliptin sodium in improving airway wall thickness in COPD model mice according to one embodiment of the present application (Masson staining).

[0019] Figure 8 This invention presents quantitative statistical results on the effect of nasal administration of sitagliptin sodium on airway wall thickness in COPD model mice according to one embodiment of this application. Detailed Implementation

[0020] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0023] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0024] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0025] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0026] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0027] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0028] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0029] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0030] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0031] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0032] Chronic obstructive pulmonary disease (COPD) is a disease characterized by partially reversible airflow limitation caused by an inflammatory response. Its main symptoms are expectorant cough and progressively worsening dyspnea, and the disease can last for many years. Common physical signs include decreased breath sounds, prolonged expiratory phase, and wheezing. Conventional treatments include the use of bronchodilators, corticosteroids, and antibiotics.

[0033] COPD mainly includes two pathological types: chronic obstructive bronchitis and emphysema, and most patients have both of these characteristics.

[0034] Chronic obstructive bronchitis (COB) is bronchitis characterized by airflow obstruction. It progresses to COB when pulmonary function tests show evidence of airflow limitation. Chronic asthmatic bronchitis is a similar overlapping syndrome characterized by chronic expectorant cough, wheezing, and partially reversible airflow obstruction; it is primarily seen in smokers with a history of asthma. In some cases, the differentiation between COB and chronic asthmatic bronchitis remains unclear.

[0035] Emphysema refers to the destruction of lung parenchyma, leading to loss of lung elastic recoil, alveolar septal damage, and weakened radial airway traction, thereby increasing the risk of airway collapse. Its pathophysiological consequences include lung overinflation, airflow restriction, and gas retention. Air spaces enlarge, potentially developing into bullae.

[0036] Chiglitazar, or sodium 2-(2-(4-fluorobenzoyl)phenylamino)-3-(4-(2-(9H-carbazole-9-yl)ethoxy)phenyl)propionate, is a phenylalanine compound with therapeutic and preventative activity against metabolic diseases and conditions. It selectively activates PPAR-α, PPAR-γ, and PPAR-δ, and its chemical structure is shown below:

[0037] .

[0038] One aspect of this application provides the use of sitagliptin sodium or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of chronic obstructive pulmonary disease.

[0039] While not wishing to be limited by any theory, it has been found that sitagliptin sodium exerts its therapeutic effect on chronic obstructive pulmonary disease (COPD) at least by activating PPARα and / or PPARγ. However, the reverse is not true; that is, not all agents capable of activating PPARα and / or PPARγ are effective in treating and / or preventing COPD.

[0040] For example, the inventors tried the PPARα agonist MRL24 and found that it could not alleviate COPD-related phenotypes.

[0041] In some embodiments, the sitagliptin sodium exerts its therapeutic effect on chronic obstructive pulmonary disease (COPD) through at least one or more mechanisms, including improving lung function, reducing emphysema, and inhibiting airway remodeling.

[0042] For example, the improvement of lung function includes, but is not limited to, reducing functional residual capacity and / or increasing the ratio of forced expiratory volume to forced vital capacity.

[0043] In this application, the form of the drug includes, but is not limited to, sprays, nasal drops, aerosols, powder sprays, tablets, capsules, and granules.

[0044] In some embodiments, the drug is a dosage form administered via the airway. While not wishing to be limited by any particular theory, it is believed that administration via the airway (e.g., nasal administration) minimizes the impact on other organs.

[0045] In some embodiments, the medication is administered via nasal administration. The medication may be in the form of, for example, a nasal spray or nasal drops.

[0046] In some embodiments, the sitagliptin sodium is the sole active ingredient in the drug.

[0047] In some embodiments, the medicament also comprises one or more other pharmaceutically active ingredients used to treat chronic obstructive pulmonary disease.

[0048] For example, other active pharmaceutical ingredients used to treat chronic obstructive pulmonary disease include one or more of bronchodilators, inhaled corticosteroids, and phosphodiesterase-4 inhibitors.

[0049] Another aspect of this application provides a method for treating and / or preventing chronic obstructive pulmonary disease and / or emphysema, comprising administering an effective dose of sitagliptin sodium to a subject in need.

[0050] The subjects can be humans or non-human mammals, such as cats, dogs, rodents, primates, cattle, sheep, or horses.

[0051] In some implementations, the subject is a person, such as a child, woman, or man.

[0052] Unless otherwise specified, the term "treatment" in this application means reversing, alleviating, or inhibiting the progression of a disease or condition to which this term applies, or one or more symptoms of such a disease or condition. "Prevention" means preventing the occurrence of a disease or condition to which this term applies, or one or more symptoms of such a disease or condition.

[0053] The following are some examples.

[0054] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0055] Example 1: Treatment of MHS cells with cigarette extract by different PPARγ agonists

[0056] (I) Preparation of Cigarette Extract

[0057] 1. Take one cigarette, remove the filter, and use sterile scissors to cut off both ends of the cigarette (to remove the sealing paper and make it easier to burn);

[0058] 2. Light a cigarette with a lighter, and connect a dropper and a 50mL sterile syringe containing 10mL of DMEM to the gas outlet of the device.

[0059] 3. Light the cigarette and control the burning speed (avoid burning too fast or too slow, it is recommended to burn 5-8mm per minute) to allow the smoke to completely enter the solvent until the cigarette burns to 1-2cm from the end (avoid burning to the filter or cigarette paper residue to reduce impurities), then extinguish the remaining cigarette;

[0060] 4. If you need to prepare extracts from multiple cigarettes, repeat steps 1-3, passing the smoke from multiple cigarettes into the solvent in the same centrifuge tube (concentration of 1 cigarette / 10mL).

[0061] 5. After the cigarette has finished burning, transfer the contents of the smoke dissolving solution to a sterile 15mL centrifuge tube and vortex for 5-10 minutes to fully dissolve the water-soluble components in the smoke into the solvent.

[0062] 6. Place the centrifuge tubes in a pre-cooled high-speed refrigerated centrifuge and centrifuge at 4°C and 8000 rpm for 10 minutes to remove insoluble impurities (such as cigarette ash, cigarette fragments, etc.) from the solution.

[0063] 7. After centrifugation, carefully aspirate the supernatant and transfer it to a new sterile 15mL centrifuge tube, avoiding aspirating any precipitate at the bottom of the tube.

[0064] 8. Filter the supernatant through a 0.22μm sterile syringe filter. Push the syringe slowly during filtration to avoid excessive pressure that could cause the filter membrane to rupture. Collect the filtered clear liquid (i.e., cigarette extract CSE).

[0065] 9. Using a sterile pipette, dispense the filtered CSE into sterile EP tubes, 100-500 μL per tube (adjust according to the experimental volume), and label them (indicate the preparation date, number of cigarettes, solvent type, and concentration);

[0066] 10. Immediately after repackaging, store CSE in a -80℃ freezer to avoid repeated freezing and thawing (it is recommended to repackage into small portions and use one portion at a time).

[0067] (II) The MH-S cell line was treated with extracted cigarette extract. The control group was treated with 2% CSE (20 μL CSE added to 1 mL of culture medium). After 48 h, cells were harvested and RNA was extracted. The expression of Il6 and Tnfα was detected. The results showed that after 48 h of treatment with 2% CSE, the expression of Il6 and Tnfα increased significantly. The experimental group was treated with 2% CSE for 48 h, and then treated with MRL24, Chiglitazar, and Darglitazone (final concentration 50 µM) for 48 h respectively. After the treatment, cells were harvested and RNA was extracted. The expression of Il6 and Tnfα was detected. The results showed that after 48 h of Chiglitazar treatment, the expression of Il6 decreased by about three times compared with 2% CSE treatment. After 48 h of treatment with MRL24 or Darglitazone, the expression of Il6 did not change significantly. Figure 1 As shown, after 48 hours of Chiglitazar treatment, Tnfa expression decreased approximately three-fold compared to 2% CSE treatment; however, after 48 hours of MRL24 or Darglitazone treatment, Tnfa expression did not change significantly. Figure 2 As shown. Therefore, Chiglitazar has a good effect on reducing the expression of inflammatory factors in CSE-treated MH-S cell lines.

[0068] Example 2: Treatment of COPD model mice with sitagliptin sodium

[0069] Experimental flowchart as follows Figure 3 As shown, the details are as follows:

[0070] (I) Construction of a COPD mouse model:

[0071] 1. Order 20 wild-type C57 male mice (8-10 weeks old, weighing around 25g) from Jiangsu Jicui Yaokang Company, and prepare the necessary items for modeling, including cigarette smoke exposure devices, cigarettes, etc.

[0072] 2. Grouping: Air control group (FA) and smoke group (CS), 10 animals in each group;

[0073] 3. Passive smoking model: Turn on the cigarette smoke exposure device and place the smoke-exposed mice in it for a period of 6 months. The mice were exposed to smoke for about 6 hours a day, 7 days a week, in 4 sessions. Each session used 14 Hongmei cigarettes (tar content 15 mg / cigarette, nicotine content 1.2 mg / cigarette) for 90 minutes.

[0074] (ii) The experimental group received intranasal administration of sitagliptin sodium to mice (CS) that had been exposed to smoke for six months, once a day at a dose of 10 mg / kg of mice. The control groups (two control groups, FA+saline and CS+saline) received intranasal administration of the same volume of physiological saline, with 5 model mice in each experimental group and each control group. The intranasal administration was carried out for a total of 7 days.

[0075] (III) On day 15, five mice from the control group and five mice from the experimental group were taken, and lung function was tested using a small animal lung function testing system (PFT). The specific steps are as follows:

[0076] 1. Turn on the pulmonary function instrument (brand: DSI, model: Buxco PFTcontroller) and calibrate it;

[0077] 2. After weighing the mice, anesthetize them by intraperitoneal injection of 10 mg / ml tribromoethanol according to the standard of 100 μL / 10 g.

[0078] 3. After anesthetizing the mice, fix them to the foam board with adhesive tape and secure the maxillary teeth with rubber bands to fully expose the neck;

[0079] 4. Cut open the skin on the neck and use forceps to bluntly separate the subcutaneous tissue and the soft tissue around the trachea. Place a suture of appropriate length below the trachea, use ophthalmic scissors to make a small horizontal incision in the trachea, insert the endotracheal tube needle for ventilation into the trachea, and fix the endotracheal tube needle to the trachea with sutures;

[0080] 5. Activate the pulmonary function ventilation mode, secure the mouse to the pulmonary function testing board, and connect the endotracheal intubation needle to the instrument. Then, enter the mouse's weight and number, click the testing option, and measure indicators such as FRC (Functional Residual Capacity), FEV50 (50th percentile forced expiratory volume), and FVC (Forced Vital Capacity). Export the pulmonary function data and save it for later use.

[0081] The FRC and FEV50 / FVC indices were evaluated. FRC (Functional Residual Capacity) refers to the amount of gas remaining in the lungs at the end of a quiet expiration, which is the lung volume when the elastic resistance of the lungs and thoracic cage reaches equilibrium at the end of expiration. When airway resistance increases, gas is retained in the lungs during expiration, and FRC will increase. FEV20 / FVC and FEV50 / FVC refer to the exhalation rate of the first 20% and 50% of lung volume during forced expiration. Decreased FEV20 / FVC and FEV50 / FVC indicate airway obstruction and airflow limitation.

[0082] The results are as follows Figure 4 As shown: Compared with FA (fresh air) for six months, CS (cigarette exposure) for six months resulted in increased FRC and decreased FEV20 / FVC and FEV50 / FVC in mice, indicating a significant decline in lung function and successful establishment of the COPD model in mice. After treating mice exposed to smoke for six months with nasal drops of sitagliptin for 7 days, FRC was lower than in the saline-treated group, while FEV20 / FVC and FEV50 / FVC were higher, indicating a significant improvement in lung function in the mice.

[0083] After completing the lung function tests in mice, the left lung was harvested, fixed, embedded, and sectioned for HE staining to detect indicators such as alveolar fusion and airway wall thickness. The specific procedures are as follows:

[0084] I. The specific steps for taking a sample from the left lung are as follows:

[0085] 1. PBS perfusion: Expose the mouse heart with ophthalmic forceps, locate the left ventricle, gently insert the needle into the left ventricle and fix it, while cutting the right atrial appendage with dissecting scissors, and slowly inject pre-cooled PBS buffer (pH=7.4), controlling the perfusion rate at 0.5-1 ml / min, until the lung tissue turns from red to pale, ensuring that the blood in the lungs is completely rinsed out to avoid blood residue affecting the subsequent fixation and staining effect; after perfusion, gently press the chest cavity to expel the residual PBS buffer in the lungs.

[0086] 2. Thoracic cavity exposure: After anesthesia, fix the mouse in a supine position on the dissection table, remove the hair from the abdomen and chest, and disinfect the skin by wiping with 75% alcohol; use dissecting scissors to cut the skin from below the xiphoid process along the midline of the abdomen upwards, separate the subcutaneous tissue, and then cut the thoracic cavity along both sides of the ribs to expose the heart and lung tissue, taking care to avoid damaging the lung tissue.

[0087] 3. Left lung separation: Gently lift the lung tissue with ophthalmic forceps to identify the left lung (the left lung of a mouse is a single lobe, while the right lung is divided into 4 lobes, which makes it easy to distinguish). Use dissecting scissors to cut the connection between the left lung and the main bronchus and hilar vessels, and completely separate the left lung. Try to avoid pulling or compressing the lung tissue to prevent alveolar rupture from affecting subsequent observation.

[0088] 4. Tissue cleaning: Quickly place the separated left lung into a culture dish containing pre-cooled physiological saline, and gently rinse 2-3 times with ophthalmic forceps to remove blood, tissue debris and impurities from the lung surface. After rinsing, gently blot dry the lung surface with filter paper, avoiding pressing hard.

[0089] II. Fixation of left lung tissue

[0090] 1. Fixation: Place the rinsed left lung tissue into a pre-cooled 4% paraformaldehyde fixative solution. The volume of the fixative solution should be 10-15 times the volume of the lung tissue (to ensure complete immersion of the tissue and avoid morphological abnormalities caused by unfixed areas).

[0091] 2. Fixation conditions: Seal the fixation bottle containing lung tissue and place it in a refrigerator at 4°C for 24 hours. During the fixation period, gently shake the fixation bottle once every 6 hours to ensure that the fixative is in full contact with the lung tissue and to avoid uneven fixation.

[0092] 3. Cleaning after fixation: After fixation, the lung tissue is removed with ophthalmic forceps and placed in a culture dish containing PBS buffer (pH=7.4). It is rinsed 3 times for 10 minutes each time to remove residual paraformaldehyde on the tissue surface and avoid affecting the subsequent dehydration and staining effects.

[0093] III. Lung tissue dehydration

[0094] The cleansed lung tissue was placed in a dehydration box and dehydrated sequentially with ethanol in the following gradient. Each dehydration step was carried out at room temperature, and the tissue was kept away from air for extended periods. Specific steps are as follows:

[0095] 1. 70% ethanol: Soak for 1 hour to initially remove moisture from the tissue and prevent tissue shrinkage caused by excessive moisture loss.

[0096] 2. 80% ethanol: Soak for 1 hour to further dehydrate and reduce residual water in the tissue.

[0097] 3. 90% ethanol: Soak for 1 hour, then dehydrate until the tissue is translucent.

[0098] 4. 95% ethanol: Soak for 1 hour to deeply dehydrate and prepare for subsequent xylene clearing.

[0099] 5. 100% ethanol (Ⅰ): Soak for 30 minutes to completely remove water from the tissue.

[0100] 6. 100% ethanol (II): Soak for 30 minutes, then dehydrate a second time to ensure that there is no residual water in the tissue (if the water is not completely removed, the paraffin will not be able to fully wet the tissue during embedding, which will cause the section to break).

[0101] IV. Tissue transparency (removal of ethanol to allow paraffin to infiltrate the tissue).

[0102] 1. Xylene (I): Remove the dehydrated lung tissue from 100% ethanol and immerse it in xylene (I) for 15-20 minutes until the tissue is completely transparent (the tissue is translucent and does not appear white or cloudy to the naked eye).

[0103] 2. Xylene (II): Transfer the tissue into xylene (II) and soak for 15-20 minutes. After a second clearing, the residual ethanol in the tissue is completely removed, ensuring that the paraffin can fully penetrate the tissue.

[0104] 3. Precautions: Xylene is volatile and irritating. It must be handled in a fume hood, with gloves and a mask worn to avoid skin contact and inhalation of vapors. The clearing time should not be too long, otherwise it will cause the tissue to become brittle and affect subsequent sectioning.

[0105] V. Paraffin Embedding

[0106] 1. Paraffin infiltration: After the lung tissue has been cleared, it is removed from xylene and quickly transferred into pre-melted and kept warm (56-58℃) paraffin. The tissue is then placed in a paraffin embedding machine and infiltrated 3 times, 20 minutes each time, to ensure that the paraffin fully infiltrates every part of the tissue and to avoid cavities after embedding.

[0107] 2. Embedding procedure: Place the embedding cassette into the insulated tank of the paraffin embedding machine, pour in an appropriate amount of melted paraffin, and after the paraffin liquid surface is level, use ophthalmic forceps to place the infiltrated lung tissue into the center of the embedding cassette, adjust the tissue orientation (ensure that the cut surface of the lung tissue is parallel to the bottom surface of the embedding cassette, so that subsequent sections can cut a complete cross-section of the lung tissue), and avoid tilting or folding the tissue.

[0108] 3. Paraffin solidification: Remove the embedding cassette containing the tissue from the embedding machine and cool it at room temperature for 30 minutes. After the paraffin has completely solidified (becoming a milky white solid), place it in a 4°C refrigerator for 10 minutes to further solidify and increase the hardness of the paraffin, making it easier to section.

[0109] 4. Post-embedding treatment: After solidification, trim the embedding block with a blade to remove excess paraffin, making the embedding block a regular cuboid to ensure even stress during sectioning and avoid section breakage.

[0110] VI. Paraffin Sectioning (Preparing 5µm thick tissue sections for H&E staining)

[0111] 1. Microtome setup: Turn on the microtome in advance, set the slice thickness to 5μm, adjust the blade angle (usually 15°-20°), and check if the blade is sharp. If the blade is chipped, replace it in time to avoid burrs or breakage in the slices.

[0112] 2. Embedded block fixation: Fix the prepared paraffin embedded block on the sample holder of the microtome, adjust the height and angle of the embedded block so that the cut surface of the embedded block is parallel to the blade, and ensure that the section can cut a complete lung tissue section.

[0113] 3. Sectioning operation: Rotate the microtome handle to slice at a uniform speed, controlling the slicing speed at 1-2 slices / second to avoid excessive speed causing the slices to curl or break; the cut paraffin sections are continuous thin slices. Gently pick up the slices with ophthalmic forceps and place them in a petri dish containing warm water at about 40℃ (warm water can help the slices unfold and remove wrinkles in the slices).

[0114] 4. Slice retrieval: Using a clean glass slide, gently place the slices that have spread out on the water surface close to it and retrieve the slices to the center of the slide (retrieve 1-2 slices per slide to avoid overlapping). Gently absorb the water from the surface of the slide with filter paper, being careful not to touch the slices.

[0115] 5. Drying the sections: Place the glass slides with the sections taken into a constant temperature oven at 60℃ and dry for 30-60 minutes. The purpose is to remove moisture and residual xylene from the sections, so that the sections are tightly bonded to the glass slide and to prevent the sections from falling off during staining.

[0116] VII. H&E staining (hematoxylin-eosin staining, to show histopathological morphology)

[0117] The entire staining process is performed on a staining rack. After each step, rinse with distilled water to avoid stain residue. Specific steps (room temperature operation):

[0118] 1. Dewaxing: Place the dried glass slides into xylene (II) and xylene (I) in sequence, soaking for 10 minutes in each step to completely remove the paraffin (incomplete dewaxing will result in uneven staining and a lighter color).

[0119] 2. Rehydration: Place the dewaxed slides into 100% ethanol (II), 100% ethanol (I), 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol in sequence, soaking for 5 minutes at each step, gradually rehydrating the tissue to restore its moist state and facilitate the penetration of the staining solution.

[0120] 3. Rinse with distilled water: Place the rehydrated slide in distilled water and rinse twice, 3 minutes each time, to remove residual ethanol.

[0121] 4. Hematoxylin staining: Place the slide in the hematoxylin staining solution and stain for 5-10 minutes (the staining time can be adjusted according to the concentration of the staining solution and the room temperature to ensure clear staining of cell nuclei); gently shake the staining rack during staining to ensure that the staining solution is in full contact with the tissue.

[0122] 5. Differentiation: Place the stained slide in a 1% hydrochloric acid ethanol solution and differentiate for 30-60 seconds. The purpose is to remove excess hematoxylin staining solution from the tissue surface, so that the cell nuclei are clearly stained and the background is clean. Immediately after differentiation, rinse with distilled water 3 times for 3 minutes each time to terminate differentiation.

[0123] 6. Blueing: Immerse the slide in 0.5% ammonia (or warm water) for 1-2 minutes to make the cell nuclei turn blue-purple (insufficient blueing will result in a lighter color of the cell nuclei). Then rinse twice with distilled water for 3 minutes each time.

[0124] 7. Eosin staining: Place the slide in eosin staining solution and stain for 30 seconds to 1 minute (eosin is an acidic dye, so the staining time should not be too long to avoid staining the cytoplasm too darkly, which would affect the contrast between the cell nucleus and the cytoplasm).

[0125] 8. Dehydration and clearing: Immerse the stained slides in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol (Ⅰ), and 100% ethanol (Ⅱ) in sequence, soaking for 30 seconds at each step to quickly dehydrate and prevent tissue fading; then transfer them to xylene (Ⅰ) and xylene (Ⅱ), soaking for 5 minutes at each step to clear the slides.

[0126] 9. Mounting: Remove the cleared slide from the xylene, immediately add 1-2 drops of neutral resin to the center of the slide, and gently cover it with a clean coverslip (make sure one end of the coverslip touches the resin first, then slowly lower it to avoid air bubbles); gently blot away any excess resin around the coverslip with filter paper, and let it air dry at room temperature, or bake it in a 60℃ oven for 10 minutes to accelerate resin solidification.

[0127] 8. Index detection (observation and analysis under a microscope after staining)

[0128] 1. Sample observation: After the slide has been sealed and dried, place it under an optical microscope. First, observe the entire lung tissue section under low magnification (10×) to find areas that are intact, undamaged, and without wrinkles. Then, switch to high magnification (40×) to observe the morphology of alveoli and airways.

[0129] 2. Indicator Testing:

[0130] (1) Degree of alveolar fusion: Observe the alveolar structure and count the proportion of fused alveoli to the total number of alveoli (observe at least 5 fields of view in each group, and count no less than 100 alveoli in each field of view). Fusion alveoli are characterized by alveolar cavity enlargement, alveolar wall rupture, and multiple alveoli fusion into one large cavity; compare the alveolar fusion ratio of the experimental group and the control group and analyze the differences between the two groups.

[0131] (2) Airway wall thickness: Select small airways with a diameter of 100-200μm in lung tissue (at least 5 airways in each group are observed), and use the measuring tool on the microscope to measure the thickness of the airway wall (measure the thickest and thinnest parts of the airway wall and take the average value); compare the airway wall thickness of the experimental group and the control group and analyze the differences between the two groups.

[0132] 3. Results Recording: Take typical field-of-view (3-5 images each under low magnification and high magnification) images of HE staining of lung tissue from each group of mice, record the alveolar fusion ratio and airway wall thickness measurement data, and compile them into a table for subsequent statistical analysis.

[0133] like Figure 5 and Figure 6 As shown, compared with FA (fresh air) for six months, CS (cigarette exposure) for six months resulted in significantly more alveolar fusion in mice, indicating significant lung damage after CS treatment, further demonstrating the successful establishment of the COPD model in mice. HE staining of paraffin-embedded lung sections from mice treated with nasal drops of sitagliptin for six months after smoking showed a significant improvement in alveolar fusion in the sitagliptin-treated group, indicating a significant improvement in the emphysema phenotype in the smoking model mice, and the difference was statistically significant.

[0134] like Figure 7 and Figure 8 As shown: Compared with FA (fresh air) for six months, the airway walls of mice were significantly thickened after CS (cigarette exposure) for six months, indicating significant airway obstruction after CS treatment, further demonstrating the successful establishment of the COPD model in mice. Masson staining results of paraffin-embedded lung sections from mice treated with nasal drops of sitagliptin for six months showed that the airway wall thickness was significantly thinner in the sitagliptin-treated group, indicating a significant improvement in the airway obstruction phenotype in the smoke-induced model mice.

[0135] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Use of sitagliptin sodium in the preparation of medicines for the treatment and / or prevention of chronic obstructive pulmonary disease.

2. The application as described in claim 1, characterized in that, Sitagliptin exerts its therapeutic effect on chronic obstructive pulmonary disease by activating PPARα and / or PPARγ.

3. The application as described in claim 1, characterized in that, Sitagliptin exerts its therapeutic effect on chronic obstructive pulmonary disease (COPD) through at least one or more mechanisms, including improving lung function, reducing emphysema, and inhibiting airway remodeling.

4. The application as described in claim 3, characterized in that, The improvement of lung function includes reducing functional residual capacity and / or increasing the ratio of forced expiratory volume to forced vital capacity.

5. The application as described in any one of claims 1 to 3, characterized in that, The form of the drug includes one or more of the following: spray, nasal drops, aerosol, powder, tablet, capsule, and granule.

6. The application as described in any one of claims 1 to 3, characterized in that, The drug is a dosage form administered via the airway.

7. The application as described in any one of claims 1 to 3, characterized in that, Siglitazone sodium is the only active ingredient in the drug.

8. The application as described in any one of claims 1 to 3, characterized in that, The drug also contains one or more other active pharmaceutical ingredients used to treat chronic obstructive pulmonary disease (COPD).

9. The application as described in claim 8, characterized in that, Other active pharmaceutical ingredients used to treat chronic obstructive pulmonary disease include one or more of bronchodilators, inhaled corticosteroids, and phosphodiesterase-4 inhibitors.

10. Use of sitagliptin sodium or its pharmaceutically acceptable salts in the preparation of medicaments for improving pulmonary emphysema and / or airway remodeling in animal models of chronic obstructive pulmonary disease.

Images