Application of matrix metalloproteinase MMP-8 inhibitor in preparation of medicine for treating chronic obstructive pulmonary disease

Abstract

The invention discloses an application of a matrix metalloproteinase MMP-8 inhibitor in preparation of a medicine for treating chronic obstructive pulmonary disease. Clinical chronic obstructive pulmonary disease lung tissue in-situ neutrophil sample transcriptome sequencing finds that differential genes are concentrated in a neutrophil chemotactic pathway, enrichment expression of MMP-8 in neutrophil is further analyzed, and expression enhancement in the neutrophil of chronic obstructive pulmonary disease is achieved. Cigarette smoke (CS) and cigarette smoke extract (CSE) are used for mouse chronic obstructive pulmonary disease modeling experiments to confirm that the MMP-8 inhibitor can effectively protect mouse body weight loss; a whole body plethysmography detection system finds that the MMP-8 inhibitor can effectively protect the lung function; further analysis finds that the MMP-8 inhibitor can relieve lung injury and reduce lung tissue collagen deposition of mice with chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of matrix metalloproteinase MMP-8 inhibitors in the preparation of drugs for treating chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD), also known as chronic obstructive lung disease, is a common chronic inflammatory lung disease. COPD is a highly heterogeneous disease, but it commonly has pathological features such as persistent inflammation, progressive airflow limitation, and irreversible airway remodeling. Exposure to inhalable particulate matter (such as cigarette smoke or other harmful external particles) is one of the most important risk factors. The pathogenesis of COPD involves the interaction of multiple factors such as chronic inflammation, protease-antiprotease imbalance, excessive oxidative stress, and abnormal repair. Clinical features include airway chronic obstructive bronchiolitis (fibrosis), chronic bronchitis (excessive mucus secretion), and emphysema (destruction of alveolar walls).

[0003] Exposure to cigarette smoke (CS) is the primary risk faced by COPD. CS can induce abnormal inflammatory responses, which are characterized by the recruitment of a large number of white blood cells from the circulation. Activated immune cells, together with damaged airway epithelium, lung epithelium, and endothelial cells, secrete a variety of pro-inflammatory cytokines (such as TNF-α, IL-6, IL-8, etc.), leading to further destruction of lung tissue structure, causing a positive feedback loop of white blood cell recruitment, exacerbating airway inflammation, and ultimately worsening COPD.

[0004] Chronic obstructive pulmonary disease can be controlled and treated to a certain extent, but it cannot be cured at present. Traditional drugs such as bronchodilators, glucocorticoids, and antibiotics all show limited therapeutic effects and are accompanied by obvious side effects. Bronchodilators mainly include long-acting muscarinic antagonists (LAMA) and long-acting beta-agonists (LABA). Although they have long been used as first-line drugs for treating COPD, they have limitations such as limited efficacy and inconvenient use; inhaled corticosteroids (ICS) are often used as adjuvant drugs for bronchodilators in clinical practice, but inhaled corticosteroids have the side effect of causing pneumonia. Therefore, it is crucial to find new drugs for COPD.

[0005] Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. Their abnormal release in COPD mediates protease-antiprotease imbalance, excessive degradation of extracellular matrix, amplification of inflammatory response, and activation of other corresponding MMPs, which together promote the development and continued aggravation of COPD. MMP-2, MMP-9, and MMP-12 play a bridging role in COPD. For example, they cooperate with inflammatory factors such as TNF-α, IL-6, and IL-8 to promote the progression of COPD patients, and MMP genetic polymorphisms can also promote the disease progression of COPD patients. MMP-8 is a member of the matrix metalloproteinase family. It is mainly secreted by neutrophils and can efficiently cut type I collagen. In clinical studies, abnormal increases in MMP-8 levels in serum and sputum of COPD patients were detected, suggesting that MMP-8 is closely related to the occurrence and development of COPD, but the biological function and targeted intervention of MMP8 in COPD have not been reported.

[0006] MMPs have three different domains: a propeptide domain (~80AS) that is cleaved during activation, a catalytic domain (~180AS) (including a conserved HEXXHXXGXXH zinc-binding group) and a hematoporphyrin-like domain (~250AS). Matrix metalloproteinase-8 inhibitor (English name MMP8-I, CAS No.: 236403-25-1) is a hydroxamic acid derivative that contains a hydroxylamine group (-NHOH) that can act as a strong zinc-binding group and bind to Zn through its oxygen and nitrogen atoms. 2+ Form a coordination bond, directly occupy the active center of the enzyme, and block Zn 2+ The catalytic effect on the substrate prevents the binding of natural substrates (such as collagen) to MMP8, forming competitive inhibition.

[0007] Although studies have pointed out the various uses of MMP inhibitors, no relevant reports on the use of MMP-8 inhibitors (including MMP8-I) in the treatment of COPD have been found so far. Summary of the invention

[0008] The purpose of the present invention is to provide a use of a matrix metalloproteinase MMP-8 inhibitor in the preparation of a drug for treating chronic obstructive pulmonary disease in view of the above problems.

[0009] In order to achieve its purpose, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention provides the use of a matrix metalloproteinase MMP-8 inhibitor in the preparation of a medicament for treating chronic obstructive pulmonary disease.

[0011] The matrix metalloproteinase MMP-8 inhibitor is MMP-8 inhibitor-1.

[0012] The MMP-8 inhibitor-1 has the following chemical structural formula:

[0013]

[0014] In the above application technical solution, the MMP-8 inhibitor alleviates weight loss in the subject.

[0015] In the above application technical solution, the MMP-8 inhibitor improves the inspiratory muscle fatigue of the subject, reduces airway obstruction and resistance, and protects lung function.

[0016] In the above application technical solution, the MMP-8 inhibitor reduces the abnormal accumulation of neutrophils in the lungs and alleviates lung inflammation in the subject mice.

[0017] In the above application technical solution, the MMP-8 inhibitor can improve lung injury and lung structure. The MMP-8 inhibitor improves the degree of pulmonary bullae and lung tissue structure in the subject, alleviates lung injury, and protects lung tissue.

[0018] In the above application technical solution, the MMP-8 inhibitor reduces the deposition of collagen fibers in the lungs and improves pulmonary fibrosis.

[0019] In the above application technical solution, the drug includes an MMP-8 inhibitor as an active ingredient and a pharmaceutically acceptable carrier.

[0020] In the above application technical solution, the drug is an oral preparation, an injection, or an inhalant.

[0021] The beneficial effects of the present invention are:

[0022] Through transcriptome sequencing of in-situ neutrophil samples of lung tissues of clinical patients with chronic obstructive pulmonary disease (COPD), the present invention found that the differentially expressed genes were concentrated in the neutrophil chemotaxis pathway. Further analysis showed that MMP-8 was highly expressed in neutrophils and its expression was enhanced in neutrophils of COPD patients. Mouse models of COPD were established using cigarette smoke (CS) and cigarette smoke extract (CSE), and it was confirmed that the MMP-8 inhibitor could effectively protect mice from weight loss; the whole-body plethysmography detection system was used to find that the MMP-8 inhibitor could effectively protect lung function; further analysis found that the MMP-8 inhibitor could alleviate lung injury and reduce collagen deposition in the lung tissues of COPD mice.

[0023] The present invention discovers the clinical application value of MMP-8 inhibitors in the preparation of preventing and treating chronic obstructive pulmonary disease (COPD) caused by cigarettes, and proposes that MMP-8 inhibitors have a good protective effect on lung injury caused by COPD and can improve pulmonary pathological damage, thus having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It shows the pathway enrichment results of differential genes (A) obtained by transcriptome sequencing of neutrophils derived from lung tissues of clinical healthy control group (HVT) and COPD patients; the expression levels of MMP8 in neutrophils of lung tissues of normal control group and COPD patients were compared and analyzed (B).

[0025] Figure 2 It shows that the MMP8 inhibitor (MMP8-I) can effectively protect the body weight (B, C) and lung function (D-I) of COPD mice: A. Schematic diagram of the establishment of the COPD mouse model and drug administration; B. Results of mouse body weight monitoring; C. Quantification diagram of mouse body weight loss; D. Detection results of airway stenosis index (Penh); E. Detection results of peak expiratory time ratio (Rpef); F. Detection results of inspiratory time (Ti); G. Detection results of expiratory time (Te); H. Detection results of mid-expiratory flow rate (EF50); I. Detection results of apnea (PAU).

[0026] Figure 3 It shows that the MMP8 inhibitor (MMP8-I) can reduce the abnormal accumulation of neutrophils in the lungs of COPD mice: A, B. Detection and results of bone marrow neutrophils in COPD mice; C, D. Detection and results of peripheral blood neutrophils in COPD mice; E, F. Detection and results of pulmonary neutrophils in COPD mice; In the figure: BM, bone marrow; Neu, neutrophil, neutrophil.

[0027] Figure 4 It shows that the MMP8 inhibitor (MMP8-I) can effectively reduce lung injury in COPD mice: A. H&E staining of lung tissue; B. Quantification results of the mean linear intercept of alveoli in the lung tissue of COPD mice; C. Quantification results of the mean alveolar septum in the lung tissue of COPD mice; D. Quantification results of the destruction index of the lung tissue of COPD mice; E. Quantification results of the mean alveolar number in the lung tissue of COPD mice.

[0028] Figure 5 It shows that the MMP8 inhibitor (MMP8-I) can effectively improve pulmonary fibrosis in COPD mice: A. Masson staining of lung tissue; B. Quantification results of peribronchial collagen fibers in the lung tissue of COPD mice. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereby.

[0030] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0031] The matrix metalloproteinase-8 inhibitor used in the implementation of the present invention is MMP-8 inhibitor-1 (MMP8-I), purchased from MCE Company, CAS No.: 236403-25-1, molecular weight is 362.40, and chemical formula is C 17 H 18 N2O5S, and its chemical structural formula is as follows:

[0032]

[0033] Example 1: Extract neutrophil samples from the lung tissues of clinical patients with chronic obstructive pulmonary disease (COPD) and healthy control subjects for transcriptome sequencing and analyze the related pathways of differentially expressed genes and the expression levels of neutrophils.

[0034] 1. Determine the samples

[0035] A total of 15 COPD lung tissue samples and 18 normal human (HVT) lung tissue samples were included according to the "China Guidelines for Primary Diagnosis, Treatment and Management of Chronic Obstructive Pulmonary Disease (2024)". The inclusion criteria included: (1) age ≥ 55 years old; (2) no immunosuppressive therapy and hormone therapy; (3) no acute or chronic infectious diseases, such as pulmonary tuberculosis, etc.; (4) no other immune-related diseases, such as bronchial asthma, etc. Clinical lung tissue neutrophils were isolated, extracted and subjected to transcriptomic sequencing by density gradient centrifugation (percoll).

[0036] 2. Metascape analysis

[0037] The obtained transcriptomic sequencing data was applied to the Metascape database (http: / / metascape.org / ) to analyze the enrichment entry clustering analysis (Reactome, KEGG, Hallmark and GO) of differentially expressed genes in COPD and HVT neutrophils.

[0038] The enrichment entry clustering analysis of differentially expressed genes is as Figure 1 shown in A. The main biological processes of these differentially expressed genes are biological pathways such as neutrophil chemotaxis and activation of matrix metalloproteinases, and there is an interaction in the expression of these differentially expressed genes. It is suggested that the COPD inflammatory microenvironment has a significant impact on the function of neutrophils.

[0039] 3. Immgen database analysis

[0040] Given the above results, which suggest a key role of neutrophils in regulating the course of COPD, we then used the Immgen database (http: / / immgen.org / ) to analyze the expression of the differentially expressed genes measured by transcriptomics in immune cells (B cells, T cells, NK cells, neutrophils, etc.), and sorted them according to the expression level of neutrophils. MMP-8 was relatively specifically expressed in neutrophils.

[0041] 4. MMP8 Expression Analysis

[0042] Subsequently, the expression of MMP8 in pulmonary neutrophils of the COPD group and the HVT group was further analyzed.

[0043] Statistics and analysis: Graphpad prism 9 was used to plot and analyze the experimental results, and the two-group normal distribution unpaired T-test method was used for the analysis method. The figures in this specification are expressed as mean ± SD. If p ≤ 0.0001: ****, p ≤ 0.001: ***, p ≤ 0.05: *, the difference is considered significant.

[0044] The results are as Figure 1 shown in Figure B. The expression of MMP8 in pulmonary neutrophils of COPD patients increased. This suggests that high expression of MMP-8 plays an important role in the process of human chronic obstructive pulmonary disease, and inhibiting the activity of MMP-8 may be used for the treatment of human COPD.

[0045] Example 2: Study on the therapeutic effect of MMP8-I on COPD

[0046] I. MMP-8 inhibitor (MMP8-I) can effectively protect the body weight of COPD mice and improve the abnormal accumulation of pulmonary neutrophils

[0047] To verify whether inhibiting MMP-8 can effectively treat COPD, we used a whole-body smoke exposure system to establish a mouse model of COPD. Ten wild C57BL / 6 mice were randomly divided into a drug administration group and a control group, and the drug administration group was treated with an MMP-8 inhibitor (MMP8-I).

[0048] The modeling method is as Figure 2 shown in Figure A: The establishment of the COPD mouse model was for 6 weeks. The mice were exposed to smoke for 2 hours, twice a day, for 5 consecutive days per week for 6 weeks, and were intraperitoneally injected with cigarette smoke extract (CSE) (150 μl / 20 g, 150 μl of CSE was injected per 20 g of mouse body weight) on the sixth day of each week.

[0049] The method of CS exposure is as follows: Use the Shanghai Yuyan whole-body cigarette smoke exposure system, which is equipped with a fully automatic cigarette smoke generator, an exposure chamber, and a laminar flow biosafety cabinet. Place the modeled mice in the exposure chamber. Each time, 7 cigarettes (brand: Longfeng Chengxiang; tar content 10 mg, carbon monoxide content in cigarette smoke 12 mg, nicotine content in cigarette smoke 1.0 mg, Chongqing China Tobacco Industry Co., Ltd.) are placed on the cigarette track. The cigarettes are generated by the automatic cigarette smoke generator and pumped into the exposure chamber (7 min / cigarette). Set the aerosol flow rate (1.5 L / min), dilution flow rate (6.5 L / min), and extraction flow rate (8 L / min). During the exposure, the oxygen concentration is controlled at (20.5 ± 0.5)%, and the air humidity is (80 ± 5)%.

[0050] The administration method is as Figure 2 shown in A: MMP8-I group: MMP8-I stock solution is dissolved in DMSO (20 mg / ml), and intraperitoneal injection (1 mg / kg) is performed after dilution with normal saline 1 hour before cigarette exposure on the 1st, 3rd, and 5th days of each week; Vehicle group: Perform intragastric administration with the same volume and time of DMSO normal saline as the MMP8-I group for 6 weeks.

[0051] During the modeling and administration period, body weight monitoring is carried out, as Figure 2 shown in B and C: After drug treatment, the body weight of the mice decreased less during the COPD modeling process (P < 0.01), indicating a protective effect on the body weight of COPD mice.

[0052] After 6 weeks of modeling, lung function is detected using a whole-body plethysmography detection system (DSI Buxco, USA). Before use, the airtightness of the instrument is detected and calibrated. After the mice breathe smoothly in the measurement chamber for 5 minutes, start the measurement. Detect lung function indexes such as the airway narrowing index (Penh), peak expiratory time ratio (Rpef), and inspiratory time (Ti) of the mice. The data is automatically exported by the software for analysis and processing.

[0053] As Figure 2 shown in D-I: Drug treatment can reduce the lung function indexes Penh (P < 0.0001), Ti (P < 0.0001), Te (P < 0.05), PAU (P < 0.0001) of the mice; improve the lung function indexes Rpef (P < 0.0001), EF50 (P < 0.05), indicating that after administering the MMP8 inhibitor, it can improve the inspiratory muscle fatigue of COPD mice, reduce airway obstruction and resistance, and effectively protect the lung function of mice.

[0054] II. MMP-8 inhibitor (MMP8-I) can reduce the abnormal accumulation of pulmonary neutrophils

[0055] After the pulmonary function test was completed, the mice were sacrificed, and peripheral blood, lung tissue, and bone marrow (BM) samples were collected from the mice for further analysis of the effect of the MMP8 inhibitor on neutrophils in COPD.

[0056] Peripheral blood: Mouse peripheral blood was collected into a purple-top anticoagulant tube (containing EDTA-K2). Then, 200 μl of the sample was taken and lysed twice with 5 ml of erythrocyte lysate in a 37°C water bath for 15 minutes each time, washed twice with PBS, and finally resuspended in 100 μl of PBS to prepare a cell suspension. Flow antibodies (FITC anti-mouse CD45 Antibody, APC anti-mouse / human CD11bAntibody, BD Pharmingen TM PE RatAnti-Mouse Ly-6G, PerCP anti-mouse Ly-6CAntibody) were used for staining at room temperature in the dark for 15 minutes. After washing, the sample was subjected to flow cytometry analysis.

[0057] Lung tissue: After collecting the blood samples, the mouse thorax was exposed, the right upper lobe of the mouse was ligated, and cardiac perfusion was performed. The mouse lung tissue (except the right upper lobe) was collected and washed twice in PBS. The lung tissue was minced with sterile ophthalmic forceps. 100 mg of lung tissue samples from each mouse were weighed into an EP tube and digested in 1.5 ml of digestion solution (1.0 mg of collagenase per 1.5 ml of RPMI 1640) in a 37°C water bath for 1 hour. Then, it was taken out and pipetted until there were no tissue clumps, and the cell suspension was filtered through a 70-μm filter. Then, it was lysed with 3 ml of erythrocyte lysate for 5 minutes, washed twice with PBS, and finally resuspended in 3 ml of PBS. 100 μL of the cell suspension was taken and stained with flow antibodies (FITC anti-mouseCD45 Antibody, APC anti-mouse / human CD11bAntibody, BD Pharmingen TM PE RatAnti-Mouse Ly-6G) at room temperature in the dark for 15 minutes. After washing, the sample was subjected to flow cytometry analysis.

[0058] Bone marrow (BM): Mouse bone marrow was flushed into an EP tube with a 1-ml syringe filled with PBS, lysed at room temperature with 100 μL of erythrocyte lysate for 5 minutes, washed twice with PBS, and finally resuspended in 300 μl of PBS. 100 μL of the cell suspension was taken into another EP tube and stained with flow antibodies (FITC anti-mouse CD45 Antibody, APC anti-mouse / human CD11bAntibody, BDPharmingen TMStain with PE Rat Anti-Mouse Ly-6G at room temperature in the dark for 15 min, wash, and then perform flow cytometry analysis.

[0059] The results were exported by the flow cytometer and analyzed using FlowJo software. By analyzing and detecting the proportion of neutrophils (CD11b Figure 3 Ly6G Figure 3 in bone marrow cells (such as Figure 3 A, B), peripheral blood (such as + C, D), and lungs (such as + E, F)). It was found that administration of the MMP-8 inhibitor could effectively reduce neutrophil mobilization from the bone marrow (P < 0.05), reduce the proportion of peripheral blood neutrophils (P < 0.001), thereby reducing the abnormal accumulation of neutrophils in the lungs (P < 0.05), and effectively alleviating lung inflammation in COPD mice.

[0060] III. MMP-8 inhibitor (MMP8-I) can effectively reduce lung injury in COPD mice

[0061] Collect the ligated right upper lobe lung tissue of mice, fix it with 4% paraformaldehyde, dehydrate it routinely, embed it in paraffin, stain it with HE, make sections, and observe the degree of lung tissue injury under an ordinary optical microscope at 400x (such as Figure 4 A). Analyze four indicators of the tissue sections: mean linear intercept (MLI), mean alveolar septal thickness (MAST), destructive index (DI), and mean alveolar number (MAN).

[0062] Statistics of MLI and MAST: Analyze using ImageJ-pro Plus 17.0 software. Draw a crosshair at the center of each field of view, calculate the number of alveolar septa (Ns) intersecting the crosshair, and measure the total length of the crosshair (L) and the area of each field of view (S) at the same time. Calculate the MLI of the lung tissue according to the formula MLI = L / Ns, and automatically calculate the mean alveolar septal thickness (MAST) of the lung tissue sections.

[0063] Determination of DI and MAN: Analysis was performed using ImageJ software. The destruction of alveolar structure was recorded as normal (N) or damaged (D). When the alveolar wall was intact or had only one break, the alveolus was considered normal and labeled as N. The criteria for judging damaged alveoli were as follows: The alveolar wall had two or more breaks; the alveolar wall was thickened and there was hyperplasia of cuboidal epithelial cells; typical emphysema destruction was present. Any of these changes was considered a damaged alveolus and labeled as D. Ten different sections needed to be analyzed for each mouse. DI = D / (D + N)×100% was calculated. At the same time, the total area of the field of view (S) was calculated, and MAN = S / (N + D).

[0064] Results showed that after administration of MMP8-I, the infiltration of inflammatory cells in lung tissue sections decreased (as shown in Figure 4 A), and it could effectively reduce COPD MLI (as shown in Figure 4 B, P < 0.0001), MAST (as shown in Figure 4 C, P < 0.05), DI (as shown in Figure 4 D, P < 0.0001), and increase MAN (as shown in Figure 4 E, P < 0.0001), indicating that after administration, it could improve the degree of bullae and the alveolar tissue structure in COPD mice and effectively protect the lung tissue.

[0065] IV. MMP-8 inhibitor (MMP8-I) can effectively reduce the deposition of lung collagen fibers in COPD mice

[0066] Mouse lung tissue was paraffin-embedded and sectioned, and then Masson staining was performed. The changes in airway fibrosis were observed using an ordinary optical microscope at 400x magnification (as shown in Figure 5 A), where collagen fibers were blue. Analysis was performed using ImageJ software. The relative collagen area = (collagen fiber area / total tissue area)×100%. Ten bronchi were selected for measurement in each sample.

[0067] Results showed that after administration of MMP8-I, the collagen fibers around the airways in lung tissue decreased (as shown in Figure 5 A - B, P < 0.0001), indicating that after administration, it could reduce the deposition of collagen fibers in the lungs of mice and improve pulmonary fibrosis.

Claims

1. Application of matrix metalloproteinase (MMP-8) inhibitors in the preparation of drugs for the treatment of chronic obstructive pulmonary disease.

2. The use according to claim 1, characterized in that: The matrix metalloproteinase MMP-8 inhibitor is MMP-8inhibitor-1.

3. The use according to claim 2, characterized in that: The MMP-8inhibitor-1 has the following chemical structure:

4. The use according to claim 1, characterized in that: The MMP-8 inhibitor alleviates weight loss in the subject.

5. The use according to claim 1, characterized in that: The MMP-8 inhibitor improves the inspiratory muscle fatigue of the subject, reduces airway obstruction and resistance, and protects lung function.

6. The use according to claim 1, characterized in that: The MMP-8 inhibitor reduces the abnormal accumulation of neutrophils in the lungs and reduces lung inflammation in the subject mice.

7. The use according to claim 1, characterized in that: The MMP-8 inhibitor can improve lung damage and lung structure. The MMP-8 inhibitor improves the degree of lung bullae and lung tissue structure of the subject, alleviates lung damage, and protects lung tissue.

8. The use according to claim 1, characterized in that: The MMP-8 inhibitor reduces lung collagen fiber deposition and improves lung fibrosis.

9. The use according to claim 1, characterized in that: The medicament comprises a MMP-8 inhibitor as an active ingredient and a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that: The medicine is an oral preparation, an injection or an inhalation preparation.

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