Use of genipin-1-β-d gentiobioside in preparation of drug for treating chronic obstructive pulmonary disease

Abstract

Use of genipin-1-β-D gentiobioside in the preparation of a drug for treating chronic obstructive pulmonary disease. Genipin-1-β-D gentiobioside is prepared into a solution for atomized inhalation. Using the administration mode of atomized inhalation can improve lung function in chronic obstructive pulmonary disease, reduce the content of inflammatory factors in lung tissue, and reduce damage to lung tissue.

Description

Use of genipin-1-beta-d-gentiobioside in preparation of drugs for treating chronic obstructive pulmonary disease TECHNICAL FIELD

[0001] The present application relates to the use of genipin-1-beta-d-gentiobioside, in particular the use of genipin-1-beta-d-gentiobioside in preparation of drugs for treating chronic obstructive pulmonary disease. BACKGROUND

[0002] Chronic obstructive pulmonary disease (COPD) is a common chronic airway disease, its main symptoms include persistent cough, expectoration, dyspnea and shortness of breath. The pathogenesis of chronic obstructive pulmonary disease (COPD) is: 1. Airway inflammation: a variety of cells participate in the airway inflammation of COPD, including neutrophils, lymphocytes, eosinophils, etc. Among them, neutrophils are the main effector cells. 2. Oxidative stress: cigarette smoke and other inhaled particles can produce oxygen free radicals, while the endogenous antioxidant production of COPD patients decreases, thereby activating inflammatory genes, inactivating antiproteinases, stimulating mucus hypersecretion, and reducing the anti-inflammatory activity of glucocorticoids. 3. Proteinase / antiproteinase imbalance: elastin is the main component of the lung parenchyma connective tissue, and proteinase-induced elastin destruction is an important cause of emphysema and irreversible damage. 4. Pulmonary emphysema: overinflation and expansion of the lung, decreased lung tissue elasticity, often with the formation of pulmonary bullae, causing respiratory function changes, ventilation and gas exchange dysfunction can cause hypoxia and carbon dioxide retention, and respiratory failure. Chronic obstructive pulmonary disease can be divided into acute exacerbation and stable phase according to the disease process.

[0003] The methods for treating COPD include drug therapy, non-drug therapy (such as pulmonary rehabilitation, smoking cessation and oxygen therapy), and surgical treatment in some cases. Drug therapy mainly includes bronchodilators, inhaled corticosteroids and anticholinergic drugs, aiming to relieve airway spasm and reduce inflammation.

[0004] However, bronchodilators in drug therapy can cause side effects such as palpitations, arrhythmias, muscle spasms, etc.; inhaled corticosteroids can cause oral and throat Candida infections, and long-term use can increase the risk of osteoporosis, cataracts and glaucoma; anticholinergic drugs can cause dry mouth, urinary retention, constipation and other side effects.

[0005] Genipin-1-β-D gentiobioside is a naturally occurring compound belonging to iridoid compounds. It is one of the main effective components in Gardenia jasminoides Ellis, and has various biological activities, including anti-inflammatory, liver protection, choleretic, hypoglycemic effects, etc. The prior art Chinese patent CN102000102A discloses the application of genipin-1-β-D gentiobioside in the preparation of a drug for treating heart failure diseases, and the Chinese patent CN104510747B discloses the application of genipin-1-β-D gentiobioside in the preparation of a drug for protecting against death caused by influenza virus FM1 strain infection. However, the research of genipin-1-β-D gentiobioside in treating chronic obstructive pulmonary disease has not been disclosed.

[0006] SUMMARY

[0007] The application aims to provide an application of genipin-1-β-D gentiobioside in the preparation of a drug for treating chronic obstructive pulmonary disease. The genipin-1-β-D gentiobioside of the application has the characteristics of improving lung function, reducing the content of inflammatory factors in lung tissue, and reducing lung tissue damage in chronic obstructive pulmonary disease.

[0008] The technical solution of the application is: the application of genipin-1-β-D gentiobioside in the preparation of a drug for treating chronic obstructive pulmonary disease.

[0009] In the aforementioned application of genipin-1-β-D gentiobioside, the genipin-1-β-D gentiobioside is administered by the way of atomization inhalation.

[0010] In the aforementioned application of genipin-1-β-D gentiobioside, the concentration of the atomization inhalation solution of the genipin-1-β-D gentiobioside is 75mg / ml or 37.5mg / ml, the atomization flow rate is 7.5L / min, and the atomization time is 0-30min.

[0011] In the aforementioned application of genipin-1-β-D gentiobioside, the atomization inhalation concentration of 75mg / ml of genipin-1-β-D gentiobioside is 2.419mg / L.

[0012] In the aforementioned application of genipin-1-β-D gentiobioside, the atomization inhalation concentration of 37.5mg / ml of genipin-1-β-D gentiobioside is 1.032mg / L.

[0013] In the aforementioned application of genipin-1-β-D gentiobioside, the atomization inhalation solution of the genipin-1-β-D gentiobioside comprises the following raw materials: genipin-1-β-D gentiobioside, a pH adjuster, an osmotic pressure adjuster, and a solvent.

[0014] In the application of the foregoing genipin-1-beta-D-gentobioside, the preparation method of the aerosol inhalation solution of the genipin-1-beta-D-gentobioside comprises the following steps:

[0015] (1) a solvent with a volume of 40% to 90% of the total volume of the prepared solution is measured to obtain a first solution;

[0016] (2) a tonicity adjusting agent is added to the first solution and stirred uniformly to obtain a second solution;

[0017] (3) a pH adjusting agent is added to the second solution to adjust the pH value to 4.5-7.0 to obtain a third solution;

[0018] (4) genipin-1-beta-D-gentobioside is added to the third solution, stirred uniformly, and a pH adjusting agent is added to keep the pH value at 4.5-7.0 to obtain a fourth solution;

[0019] (5) the fourth solution is supplemented with a solvent to a total volume of the prepared solution, stirred uniformly, and the aerosol inhalation solution of the genipin-1-beta-D-gentobioside is obtained.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] The aerosol inhalation solution of the genipin-1-beta-D-gentobioside used in the application has two concentrations of 75mg / ml and 37.5mg / ml, and the median particle size of the aerosol is between 2-3um within 0-30min of atomization, which meets the requirement of the particle size <3um of the inhalation solution for pulmonary administration.

[0022] During the detection of the aerosol inhalation concentration, the filter membrane concentration of the genipin-1-beta-D-gentobioside with a concentration of 75mg / ml is stabilized at 594.133-615.776ug / ml within 5-35min of atomization, and the filter membrane concentration of the genipin-1-beta-D-gentobioside with a concentration of 37.5mg / ml is stabilized at 238.814-271.001ug / ml, which indicates that the content of the genipin-1-beta-D-gentobioside is stable during atomization, and the aerosol inhalation concentration is 2.419mg / L and 1.032mg / L, respectively.

[0023] The application adopts a rat chronic obstructive pulmonary disease (COPD) model, a rat acute exacerbation of chronic obstructive pulmonary disease (AECOPD) model, an ammonia water-induced mouse cough model and a mouse airway phenol red excretion expectorant model to evaluate the effectiveness of 75 mg / ml and 37.5 mg / ml concentrations of genipin-1-beta-D gentiobioside nebulized inhalation solution in treating chronic obstructive pulmonary disease.

[0024] In the rat chronic obstructive pulmonary disease (COPD) model, genipin-1-beta-D gentiobioside is nebulized and inhaled, 20 min each time, once a day, and after 4 weeks of continuous administration, the rat chronic obstructive pulmonary disease (COPD) model has obvious therapeutic effects in the tested dose range, which can increase the peak inspiratory flow rate, peak expiratory flow rate, tidal volume and expiratory volume of the rats, and increase the blood oxygen saturation of the rats.

[0025] In the rat acute exacerbation of chronic obstructive pulmonary disease (AECOPD) model, genipin-1-beta-D gentiobioside is nebulized and inhaled, 20 min each time, once a day, and after 4 weeks of continuous administration, the rat acute exacerbation of chronic obstructive pulmonary disease (AECOPD) model has obvious therapeutic effects in the tested dose range, which can increase the peak inspiratory flow rate, peak expiratory flow rate, tidal volume and expiratory volume of the rats, increase the blood oxygen saturation of the rats, reduce the IL-6, IL-10 and TNF-alpha content in the lung tissue, and significantly reduce the alveolar wall thickening, interstitial inflammatory cell infiltration and bronchial injury.

[0026] In the ammonia water-induced mouse cough model and the mouse airway phenol red excretion expectorant model, genipin-1-beta-D gentiobioside is nebulized and inhaled, 15 min each time, once a day, and after 3 days of continuous administration, the ammonia water-induced mouse cough frequency is reduced, the cough latency is delayed, and the airway phenol red excretion amount is increased in the tested dose range. BRIEF DESCRIPTION OF DRAWINGS

[0027] Fig. 1 is a rat lung tissue pathological photo under a microscope in the COPD model test.

[0028] Fig. 2 is a rat lung tissue pathological photo under a microscope in the AECOPD model test. DETAILED DESCRIPTION

[0029] The application will be further described below in combination with examples, but it is not used as a basis for limiting the application.

[0030] Example:

[0031] The application of genipin-1-beta-D gentiobioside in preparing a drug for treating chronic obstructive pulmonary disease.

[0032] Genipin-1-beta-D gentiobioside is administered in the form of nebulized inhalation.

[0033] Genipin-1-β-D gentiopicroside was nebulized at concentrations of 75 mg / ml and 37.5 mg / ml. After nebulization using a PARI (blue filter) nebulizer at a flow rate of 7.5 L / min for 10 min until the particle size stabilized, the aerosol particle size was measured at the nozzle outlet using a TSI particle size analyzer. Simultaneously, three parallel detection ports were selected, and the particle size values ​​at each outlet were measured at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min.

[0034] A test sample aerosol is considered stable if the particle size difference between consecutive measurements does not exceed ±20% of the mean.

[0035] The test results are shown in Table 1.

[0036] Table 1. Particle size detection results of genipin-1-β-D gentiopicroside nebulized inhalation solution

[0037] Table 1 shows that the concentrations of genipin-1-β-D gentiopicroside in the nebulized inhalation solution were 75 mg / ml and 37.5 mg / ml, respectively. Under the condition of nebulization flow rate of 7.5 L / min, the median aerosol particle size at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min were all between 2 and 3 μm, indicating that the aerosol particle size was stable within 30 min of nebulization.

[0038] Nebulized inhalation concentration detection:

[0039] The nebulizer flow rate was set to 7.5 L / min, and the single-port nebulizer flow rate was set to 1 L / min. Filter membrane samples were collected at seven time points: 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, and 35 min, after nebulization with 75 mg / ml and 37.5 mg / ml genipin-1-β-D gentiopicroside inhalation solution. The filter membranes obtained at different time points were placed in 50 ml centrifuge tubes, and 20 ml of 50% acetonitrile aqueous solution was added quantitatively. The tubes were sealed, shaken up and down, vortexed for 2 min, and then filtered through a 0.45 μm filter membrane. The filtrate was subjected to HPLC analysis under specific chromatographic conditions: an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm), a column temperature of 35℃, a mobile phase of acetonitrile-water (25:75), a flow rate of 0.6 mL / min, water as the solvent, an injection volume of 10 μL, and a run time of 10 min. The peak area obtained from the HPLC analysis was substituted into the standard curve to calculate the drug concentration in the filter membrane. The detection limit was calculated as the drug concentration in the filter membrane multiplied by 20. The detection results are shown in Table 2.

[0040] Table 2: The detection amount of genipin-1-β-D gentiobioside at different nebulization time points

[0041] The results in Table 2 show that, after nebulization for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min and 35 min at a nebulization flow rate of 7.5 L / min and a single-port nebulizer flow rate of 1 L / min, the filter membrane concentration of genipin-1-β-D gentiobioside at a concentration of 75 mg / ml was stabilized at 594.133-615.776 μg / ml, and the filter membrane concentration of genipin-1-β-D gentiobioside at a concentration of 37.5 mg / ml was stabilized at 238.814-271.001 μg / ml, indicating that the content of genipin-1-β-D gentiobioside was stable during nebulization. The average filter membrane detection amount of genipin-1-β-D gentiobioside at a concentration of 75 mg / ml was 12095.041 μg, and the average filter membrane detection amount of genipin-1-β-D gentiobioside at a concentration of 37.5 mg / ml was 5159.191 μg. The calculated nebulization inhalation concentrations of genipin-1-β-D gentiobioside of the two tested drugs were 2.419 mg / L and 1.032 mg / L, respectively.

[0042] Therefore, in the pharmacodynamic test, the administration method was to use genipin-1-β-D gentiobioside nebulization inhalation solution at two concentrations of 75 mg / ml and 37.5 mg / ml, at a nebulization flow rate of 7.5 L / min, for 20 min of nebulization inhalation once a day, for 4 weeks continuously.

[0043] According to the nebulization inhalation concentration, nebulization inhalation time and animal body weight, the actual administration amount was calculated according to the following formula: DD (mg / kg) = C x RMV x D (x IF) / BW

[0044] Note: C is the nebulization inhalation concentration (mg / L); RMV is the inhalation volume per minute (L / min); D is the nebulization time; (x IF) is the proportion of particles in the liquid that can enter the respiratory tract, which is generally not calculated; BW is the animal body weight (kg); RMV (L / min) = 0.608 x BW0.852

[0045] The calculated actual administration amount of genipin-1-β-D gentiobioside of the genipin-1-β-D gentiobioside nebulization inhalation solution at two concentrations of 75 mg / ml and 37.5 mg / ml after 20 min of nebulization was 37.53 mg / kg and 16.01 mg / kg, respectively.

[0046] The pharmacodynamics test of geniposide-1-β-D-gentobioside is as follows:

[0047] Test 1, Effect of Geniposide-1-β-D-Gentobioside Aerosol Inhalation Solution on Chronic Obstructive Pulmonary Disease (COPD) Model in Rats.

[0048] 1.1 Test sample:

[0049] Geniposide-1-β-D-gentobioside aerosol inhalation solution, batch number: 231212; specification: 5ml:375mg; production date: 2023.12.27; expiration date: tentatively 24 months; function and indication: chronic obstructive pulmonary disease (remission or acute exacerbation); providing unit: Yinkoer (Tianjin) Innovative Pharmaceutical Research Co., Ltd.

[0050] 1.2 Positive control drug:

[0051] Inhaled ipratropium bromide solution, batch number: 3982210, production date: 20230719; expiration date: 202506, specification: 2ml:0.5mg; usage and dosage: 1 single dose vial each time. Indications: This product is used as a bronchodilator for chronic obstructive pulmonary disease. Manufacturer: Boehringer Ingelheim (China) Investment Co., Ltd.

[0052] 1.2 Test animals:

[0053] Wistar rats, 80, SPF level, 130-150g, half male and half female, purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd., animal license number: SCXK (Jing) 2021-0006, certificate number: ♀ No. 110011231112531117, ♂ No. 110011231112531048.

[0054] 1.3 Test reagents:

[0055] Table 3 Test reagents

[0056] 1.4 Test instruments:

[0057] Table 4 Test instruments

[0058] 1.5 Test drug dosage:

[0059] Table 5 Test drug

[0060] 1.6 Positive control drug dosage:

[0061] Table 6 Positive control drug

[0062] 1.7 Test grouping and administration method:

[0063] Take 80 Wistar rats, SPF level, body weight 130-150 g, half male and half female. According to the weight level, 10 rats were randomly taken as the normal control group, and the remaining rats were smoked with Huangguoshu cigarettes every day for 2 hours, twice a day, for 6 weeks; During the smoking period, in the first 4 weeks, the rats were anesthetized with isoflurane and then tracheal instillation of tar was performed, 45 μL per rat; In the last 2 weeks, the rats were tracheal instilled with LPS, 100 μg per rat; The normal control group was atomized and inhaled or tracheal instilled with normal saline under the same conditions. In the third week of modeling (15th day of modeling), the model rats were evenly divided into model control group, isopropyl bromide control group, and 75 mg / ml and 37.5 mg / ml two dose groups of genipin-1-β-D gentiopicroside atomized inhalation solution, 11-12 rats in each group. After grouping, the administration groups started to be administered by atomization, once a day, 20 minutes each time, for 4 weeks. The normal control group and the model control group were atomized with water under the same conditions. During the modeling period, some rats died, and finally there were 10 rats in each group. After the administration was completed, the samples were taken for analysis. Test indexes:

[0064] ① During the administration period, the body weight of the rats was measured every week, and the results were statistically analyzed by t test between groups;

[0065] ② After 2 weeks of administration, the lung function was detected every week, including peak inspiratory flow, peak expiratory flow, tidal volume and expiratory volume; The results were statistically analyzed by t test between groups;

[0066] ③ After 2 weeks of administration, the blood oxygen saturation was detected every week; The results were statistically analyzed by t test between groups;

[0067] ④ After the administration was completed, the left lung lobe of the rats was dissected for lung histopathology detection, and the lung lesions were recorded. The statistical analysis was performed by SPSS 20.0 statistical software.

[0068] 1.8 Test results:

[0069] Table 7 Effect of genipin-1-β-D gentiopicroside atomized inhalation solution on chronic obstructive pulmonary disease model in rats Note: Compared with the normal control group # P<0.05, ## P<0.01

[0070] The results of Table 7 show that the body weight of the model control group rats decreased significantly, and there was a significant difference compared with the normal control group (P<0.01); The body weight of the rats in the 75 mg / ml and 37.5 mg / ml dose groups of genipin-1-β-D gentiopicroside showed a growth trend after 4 weeks of administration, but there was no significant difference compared with the model control group.

[0071] Table 8 Effect of neopingon-1-β-D gentiobioside inhalation solution on peak inspiratory flow rate of rats with chronic obstructive pulmonary disease model Note: Compared with the normal control group # P<0.05; compared with the model control group ** P<0.01, * P<0.05

[0072] Table 8 shows that the peak inspiratory flow rate of the model control group was significantly reduced, and there was a significant difference compared with the normal control group (P<0.05); the peak inspiratory flow rate of the 75mg / ml and 37.5mg / ml dose groups of neopingon-1-β-D gentiobioside increased after 4 weeks of administration, and there was a significant difference compared with the model control group (P<0.05).

[0073] Table 9 Effect of neopingon-1-β-D gentiobioside inhalation solution on peak expiratory flow rate of rats with chronic obstructive pulmonary disease model Note: Compared with the normal control group # P<0.05; compared with the model control group ** P<0.01, * P<0.05

[0074] Table 9 shows that the peak expiratory flow rate of the model control group was significantly reduced, and there was a significant difference compared with the normal control group (P<0.05); the peak expiratory flow rate of the 75mg / ml dose group of neopingon-1-β-D gentiobioside increased after 3 weeks of administration, and the peak expiratory flow rate of the 37.5mg / ml dose group increased after 4 weeks of administration, and there was a significant difference compared with the model control group (P<0.05).

[0075] Table 10 Effect of neopingon-1-β-D gentiobioside inhalation solution on tidal volume of rats with chronic obstructive pulmonary disease model Note: Compared with the normal control group # P<0.05; compared with the model control group ** P<0.01, * P<0.05

[0076] Table 10 shows that the tidal volume of the model control group was reduced, and there was a significant difference compared with the normal control group (P<0.05); the tidal volume of the 75mg / ml group of neopingon-1-β-D gentiobioside increased after 4 weeks of administration, and there was a significant difference compared with the model control group (P<0.05).

[0077] Table 11 Effect of neopingon-1-β-D gentiobioside inhalation solution on expiratory volume of rats with chronic obstructive pulmonary disease model Note: Compared with the normal control group # P<0.05; Compared with the model control group ** P<0.01, * P<0.05

[0078] Table 11 shows that the expiratory volume of the model control group rats decreased, and there was a significant difference compared with the normal control group (P<0.05); the expiratory volume of the rats in the 75mg / ml dose group of geniposide-1-β-D gentiobioside increased after 4W of administration, and there was a significant difference compared with the model control group (P<0.05).

[0079] Table 12 Effect of geniposide-1-β-D gentiobioside nebulized inhalation solution on chronic obstructive pulmonary disease model rats Note: Compared with the normal control group ## P<0.01; Compared with the model control group * P<0.05, ** P<0.01

[0080] Table 12 shows that the blood oxygen saturation of the model control group rats decreased significantly, and there was a significant difference compared with the normal control group (P<0.01); the blood oxygen saturation of the rats in the 75mg / ml dose group of geniposide-1-β-D gentiobioside increased significantly after 2W of administration, and there was a significant difference compared with the model control group (P<0.01).

[0081] Effect on pathological changes of lung tissue of rats:

[0082] The lung tissue of rats was fixed in 4% paraformaldehyde fixing solution, and after 48 hours, it was taken out, washed with running water, dehydrated with gradient ethanol, transparentized with xylene, embedded with wax, sliced, HE stained, neutral gum mounted, and observed and photographed under a microscope. The test results are as follows:

[0083] Table 13 Effect of geniposide-1-β-D gentiobioside nebulized inhalation solution on chronic obstructive pulmonary disease model rats Note: Compared with the normal control group##P<0.01

[0084] Table 14 Effect of geniposide-1-β-D gentiobioside nebulized inhalation solution on chronic obstructive pulmonary disease model rats Note: Compared with the normal control group ## P<0.01

[0085] Table 15 Effect of geniposide-1-β-D gentiobioside nebulized inhalation solution on chronic obstructive pulmonary disease model rats Note: Compared with the normal control group ##P<0.01

[0086] Table 16 Effects of nebulized inhalation of geniposide-1-β-D-gentiobioside on the COPD model of rats Note: Compared with the normal control group ## P<0.01

[0087] In Tables 13-16, "-" means normal lung parenchyma structure, no thickening, no or occasional inflammatory cells, and normal bronchial structure. "+" means 5-25% of alveoli damaged, slight thickening, slight inflammatory cell infiltration, bronchial epithelial cell cilia deflection, disorderly arrangement, and individual cell shedding. "++" means 25-50% of alveoli damaged, mild thickening, mild inflammatory cell infiltration, partial bronchial epithelial swelling and degeneration, and mild inflammatory exudate in the lumen. "+++" means 50-75% of alveoli damaged, moderate thickening, moderate inflammatory cell infiltration, massive bronchial epithelial swelling and degeneration, partial epithelial cell shedding, and moderate inflammatory exudate in the lumen. "++++" means 75-100% of alveoli damaged, severe thickening, severe inflammatory cell infiltration, almost all bronchial epithelial necrosis and shedding, and a large amount of inflammatory exudate in the lumen.

[0088] The results of Tables 13-16 show that the normal control group: the lung tissue structure of the rats is complete and clear, the alveolar cavity is not enlarged, the interstitium is not infiltrated with inflammatory cells, the bronchial epithelial cells are arranged in order, and the cilia are not damaged and shed. The model control group: the alveolar exudation of the rats is increased, the alveolar wall is slightly or moderately thickened, the interstitium is congested and edematous, a large amount of inflammatory cells, including lymphocytes, macrophages and neutrophils, are infiltrated, the bronchial epithelium is slightly or moderately swollen and degenerated, the goblet cells are increased, partial epithelial cells are shed, and a large amount of inflammatory exudate is observed. The degree of the lesion is significantly different from that of the normal control group. The ipratropium bromide group and the 75 mg / ml geniposide-1-β-D-gentiobioside group: the lung tissue damage of the rats has no significant difference from that of the model control group. The 37.5 mg / ml geniposide-1-β-D-gentiobioside group: the inflammatory cell infiltration of the lung interstitium is reduced compared with the model control group, and there is no significant difference in alveolitis, alveolar wall thickening and bronchial damage.

[0089] Test Two: Therapeutic effect of nebulized inhalation of geniposide-1-β-D-gentiobioside on the AECOPD model of rats

[0090] 2.1 Test sample:

[0091] Genipin-1-β-D-gentiobioside aerosol inhalation solution, batch number: 240304; specification: 5 ml: 375 mg; production date: 20240304; validity period: tentatively 24 months; indications: chronic obstructive pulmonary disease (remission or acute exacerbation); provided by Yingkerui (Tianjin) Innovative Pharmaceutical Research Co., Ltd.

[0092] 2.2 Positive control drug:

[0093] Ipratropium bromide solution for inhalation, batch number: 3982215, production date: 20230909; validity period: 202508, specification: 2 ml: 0.5 mg; dosage and administration: 1 single-dose vial each time. Indications: This product is used as a bronchodilator for chronic obstructive pulmonary disease. Manufacturer: Boehringer Ingelheim (China) Investment Co., Ltd.

[0094] 2.3 Test animals:

[0095] 70 Wistar rats, SPF grade, 130 - 150 g, half male and half female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal license number: SCXK (Beijing) 2021 - 0006, certificate number: ♀No.110011241102885668, ♂No.110011241102885563.

[0096] 2.4 Bacterial strains: [[ID=十六]] [[ID=十七]]

[0097] Pseudomonas aeruginosa (ATCC: VR - 27853), from the American Type Culture Collection (ATCC), passaged in this laboratory and stored at -80 °C in the refrigerator for standby.

[0098] 2.5 Test reagents:

[0099] Table 17 Test reagents

[0100] 2.6 Test instruments:

[0101] Table 18 Test instruments

[0102] 2.7 Dosage of the test drug:

[0103] Table 19 Test drug

[0104] 2.8 Dosage of the positive control drug:

[0105] Table 20 Positive control drug

[0106] 2.9 Test grouping and drug administration method: [[ID=五十一]]

[0107] Take 70 Wistar rats, SPF level, body weight 130-150 g, half male and half female. According to the weight level, 10 rats were randomly selected as the normal control group, and the rest of the rats were smoked with Huangguoshu cigarettes, 2 times a day, 1 hour each time, for 6 weeks; and respectively on the 1st, 14th, 28th day of smoking, 200 μg per rat (the day of tracheal instillation does not smoke), to prepare a chronic obstructive pulmonary disease model. In the 3rd week of modeling, the model rats were evenly divided into model control group, isopropyl bromide control group, genipin-1-β-D gentiobioside aerosol inhalation solution 75 mg / ml and 37.5 mg / ml two dose groups, 10 rats in each group. After grouping, each drug group started aerosol administration, once a day, 20 minutes each time, for 4 weeks, and the normal control group and the model control group were aerosol injected with water under the same conditions; on the 39th day of modeling (3 days before the end of the test) 6×10 8 CFU / ml of Pseudomonas aeruginosa bacterial solution, 200 μL per rat, to prepare a chronic obstructive pulmonary disease acute exacerbation model. The normal control group was aerosol inhaled or tracheally instilled with normal saline under the same conditions. After the administration was completed, the rats were dissected. The detection indexes were:

[0108] ①During the administration period, the body weight of the rats was weighed every week, and the results were statistically analyzed by intergroup comparison t test;

[0109] ②After 2 weeks and 4 weeks of administration, the lung function was checked, including peak inspiratory flow, peak expiratory flow, tidal volume and expiratory volume, and the results were statistically analyzed by intergroup comparison t test;

[0110] ③After 2 weeks and 4 weeks of administration, the blood oxygen saturation was measured, and the results were statistically analyzed by intergroup comparison t test;

[0111] ④At the end of the test, the right lung lobe of the rat was taken for detection of inflammatory factors IL-6, IL-10 and TNF-α, and the results were statistically analyzed by t test;

[0112] ⑤The left lung lobe of the rat was taken for lung histopathology detection, and the lung lesions were recorded, and the statistical analysis was performed by SPSS20.0 statistical software;

[0113] ⑥The right lung lobe of the rat was taken for proteomics detection.

[0114] 2.10 Test results:

[0115] Table 21 Effect of genipin-1-β-D gentiobioside aerosol inhalation solution on chronic obstructive pulmonary disease acute exacerbation model in rats Note: Compared with the normal control group # P<0.05, ## P<0.01

[0116] Table 21 shows that the weight of the model control group rats was significantly reduced, and there was a significant difference compared with the normal control group (P<0.05, P<0.01); the two dose groups of 75 mg / ml and 37.5 mg / ml of genipin-1-β-D gentiobioside had no significant effect on the weight of rats after administration.

[0117] Table 22 Effect of genipin-1-β-D gentiobioside nebulized inhalation solution on inspiratory peak flow rate of rats with acute exacerbation of chronic obstructive pulmonary disease model Note: Compared with the normal control group # P<0.05, ## P<0.01; compared with the model control group * P<0.05

[0118] Table 22 shows that the inspiratory peak flow rate of the model control group rats was significantly reduced, and there was a significant difference compared with the normal control group (P<0.05, P<0.01); the 37.5 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the inspiratory peak flow rate of rats after 2W of administration, and there was a significant difference compared with the model control group (P<0.05).

[0119] Table 23 Effect of genipin-1-β-D gentiobioside nebulized inhalation solution on expiratory peak flow rate of rats with acute exacerbation of chronic obstructive pulmonary disease model Note: Compared with the normal control group # P<0.05, ## P<0.01; compared with the model control group * P<0.05, ** P<0.01

[0120] Table 23 shows that the expiratory peak flow rate of the model control group rats was significantly reduced, and there was a significant difference compared with the normal control group (P<0.05, P<0.01); the 37.5 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the expiratory peak flow rate of rats after 2W of administration, and there was a significant difference compared with the model control group (P<0.01).

[0121] Table 24 Effect of genipin-1-β-D gentiobioside nebulized inhalation solution on tidal volume of rats with acute exacerbation of chronic obstructive pulmonary disease model Note: Compared with the normal control group ## P<0.01; compared with the model control group * P<0.05, ** P<0.01

[0122] Table 24 shows that the tidal volume of the model control group was significantly reduced, and there was a significant difference compared with the normal control group (P < 0.01); the 75 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W of administration, and the 37.5 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W and 4W of administration, and there were significant differences compared with the model control group (P < 0.05, P < 0.01).

[0123] Table 25 shows that the tidal volume of the model control group was significantly reduced, and there was a significant difference compared with the normal control group (P < 0.01); the 75 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W of administration, and the 37.5 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W and 4W of administration, and there were significant differences compared with the model control group (P < 0.05, P < 0.01). Note: Compared with the normal control group ## P < 0.01; compared with the model control group * P < 0.05, ** P < 0.01

[0124] Table 25 shows that the tidal volume of the model control group was significantly reduced, and there was a significant difference compared with the normal control group (P < 0.01); the 75 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W of administration, and the 37.5 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W and 4W of administration, and there were significant differences compared with the model control group (P < 0.05, P < 0.01).

[0125] Table 26 shows that the tidal volume of the model control group was significantly reduced, and there was a significant difference compared with the normal control group (P < 0.01); the 75 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W of administration, and the 37.5 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W and 4W of administration, and there were significant differences compared with the model control group (P < 0.05, P < 0.01). Note: Compared with the normal control group ## P < 0.01; compared with the model control group * P < 0.05, ** P < 0.01

[0126] Table 26 shows that the tidal volume of the model control group was significantly reduced, and there was a significant difference compared with the normal control group (P < 0.01); the 75 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W of administration, and the 37.5 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W and 4W of administration, and there were significant differences compared with the model control group (P < 0.05, P < 0.01).

[0127] Table 27 shows that the tidal volume of the model control group was significantly reduced, and there was a significant difference compared with the normal control group (P < 0.01); the 75 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W of administration, and the 37.5 mg / ml dose group of genipin-1-β-D gentiobioside could significantly increase the tidal volume of rats after 2W and 4W of administration, and there were significant differences compared with the model control group (P < 0.05, P < 0.01). Note: Compared with the normal control group ## P < 0.01; compared with the model control group ** P < 0.01

[0128] The results of Table 27 show that after modeling, the IL-6, IL-10 and TNF-a contents in the lung tissues of the model control group rats were significantly increased, and had significant differences compared with the normal control group (P < 0.01); the 75 mg / ml and 37.5 mg / ml dose groups of genipin-1-β-D gentiobioside could significantly reduce the IL-6, IL-10 and TNF-a contents in the rats after 4 weeks of administration, and had significant differences compared with the model control group (P < 0.01).

[0129] Effects on the pathological changes of the lung tissues of the rats:

[0130] The lung tissues of the rats were fixed in 4% paraformaldehyde fixing solution, and after 48 hours, they were taken out, washed with flowing water, dehydrated with gradient ethanol, transparentized with xylene, embedded with wax, sliced, HE stained, neutral gum sealed, and observed and photographed under a microscope. The detection results are shown as follows:

[0131] Table 28 Effects of genipin-1-β-D gentiobioside preparation on the acute exacerbation stage model of chronic obstructive pulmonary disease in rats Note: Compared with the normal control group ## P < 0.01

[0132] Table 29 Effects of genipin-1-β-D gentiobioside preparation on the acute exacerbation stage model of chronic obstructive pulmonary disease in rats Note: Compared with the normal control group ## P < 0.01, compared with the model control group ** P < 0.01

[0133] Table 30 Effects of genipin-1-β-D gentiobioside preparation on the acute exacerbation stage model of chronic obstructive pulmonary disease in rats Note: Compared with the normal control group ## P < 0.01, compared with the model control group * P < 0.05 Table 31 Effects of genipin-1-β-D gentiobioside preparation on the acute exacerbation stage model of chronic obstructive pulmonary disease in rats Note: Compared with the normal control group ## P < 0.01, compared with the model control group ** P < 0.01

[0134] Table 28-31, "-" : normal lung parenchyma structure, no thickening, no or occasional inflammatory cells, normal bronchial structure. "+" : 5-25% of alveoli damaged, slight thickening, slight inflammatory cell infiltration, bronchial epithelial cilia deflection, arrangement disorder, individual cell shedding. "++" : 25-50% of alveoli damaged, mild thickening, mild inflammatory cell infiltration, partial bronchial epithelial swelling, degeneration, mild inflammatory exudate in lumen. "+++" : 50-75% of alveoli damaged, moderate thickening, moderate inflammatory cell infiltration, large pieces of bronchial epithelial swelling, degeneration, partial epithelial cell shedding, moderate inflammatory exudate in lumen. "++++" : 75-100% of alveoli damaged, severe thickening, severe inflammatory cell infiltration, almost all bronchial epithelial necrosis and shedding, a large amount of inflammatory exudate in lumen.

[0135] Table 28-31 shows that the normal control group: the lung tissue structure of rats is clear, the alveolar wall is not thickened, the interstitial is not congested, edematous or infiltrated with inflammatory cells, the bronchial epithelial cells are arranged in order, and there is no swelling, degeneration or necrosis. After modeling, the model control group: the alveolar wall of rats is slightly or moderately thickened, the interstitial is congested, edematous and fibroblast proliferative; a large number of neutrophils, lymphocytes and mononuclear macrophages are infiltrated around the blood vessels, bronchi and alveolar septum, and granuloma or abscess formation can be seen in some areas; there is slight inflammatory cell exudation in the alveolar cavity; the bronchial epithelium is slightly to severely swollen and degenerated, and some epithelial cells are shed; there are more inflammatory exudates in the bronchial lumen. The degree of lesion has significant difference compared with the normal control group (P<0.01).

[0136] The isopropyl bromide group: the alveolar wall thickening and lung bronchial injury of rats is slightly reduced compared with the model control group. The 75mg / mL group of geniposide-1-β-D gentiobioside: the alveolar wall thickening, interstitial inflammatory cell infiltration and bronchial injury of rats are slightly reduced compared with the model control group. The 37.5mg / mL group of geniposide-1-β-D gentiobioside: the alveolar wall of rats can be seen slightly or slightly thickened; the interstitial is slightly to moderately infiltrated with inflammatory cells; the bronchial epithelial cells are slightly to moderately swollen and degenerated. The lesion grade is significantly reduced compared with the model control group.

[0137] Test three, the antitussive effect of geniposide-1-β-D gentiobioside aerosol inhalation solution on the cough model of mice induced by ammonium hydroxide.

[0138] 3.1 Test drug:

[0139] Geniposide-1-β-D gentiobioside aerosol inhalation solution, batch number: 230608; production date: 20230630; specification: 5ml:375mg; expiration date: tentatively 24 months; function and indication: chronic obstructive pulmonary disease (remission or acute exacerbation); providing unit: Yike Rui (Tianjin) Innovative Pharmaceutical Research Co., Ltd.

[0140] 3.2 Positive control drug:

[0141] Budesonide Suspension for Inhalation, batch number: 329723, production date: August 2023, expiration date: July 2025. Produced by AstraZeneca Pharmaceuticals Co., Ltd. Composition: The active ingredient of this product is budesonide. Properties: A suspension of fine particles. Indications: Treatment of bronchial asthma. Specification: 1mg:2ml. Dosage and administration: Nebulized inhalation, 1 - 2mg each time, twice a day.

[0142] 3.3 Test animals:

[0143] 50 ICR rats, SPF grade, 18 - 22g, half male and half female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal license number: SCXK(Beijing)2021 - 0006, certificate number: 110324241101008338.

[0144] 3.4 Test instruments:

[0145] Table 32 Test instruments

[0146] 3.5 Dosage of the test drug:

[0147] The dosages for genipin - 1 - β - D - gentiobioside in mice are two dose groups of 75mg / ml / d and 37.5mg / ml / d. Nebulized inhalation administration, once a day, 15 minutes each time.

[0148] 3.6 Dosage of the positive control drug:

[0149] Nebulized inhalation administration of the original 1mg:2ml drug solution, once a day, 15 minutes each time.

[0150] 3.7 Test grouping and administration method:

[0151] (1) Screening of qualified mice: ICR mice, body weight 20 ± 2g, half male and half female. Induce coughing for 15s by ultrasonic atomization spraying of 25% ammonia water, observe the time from the start of spraying to the appearance of abdominal muscle contraction and open mouth in mice. Those that do not cough within 1.5 minutes are discarded.

[0152] (2) Observation of drug effect: The qualified mice were randomly divided into model control group, budesonide positive control group, 75 mg / ml / d and 37.5 mg / ml / d dose groups of genipin-1-β-D gentiopicroside, 10 mice in each group, half male and half female. Each administration group was atomized for 15 min, once a day, for 3 consecutive days. The model control group was given normal saline under the same conditions. One hour after the administration on the 3rd day, each mouse was sprayed with 25% ammonia water ultrasonic atomization for 15 s to induce cough. The mouse abdominal muscle contraction or chest contraction, together with the mouth opening, was the index of cough. The latent period from the start of spraying to the occurrence of cough and the number of coughs within 3 min were observed in each group of mice, and the results were statistically processed.

[0153] 3.8 Test results:

[0154] Table 33 Antitussive effect of genipin-1-β-D gentiopicroside atomized inhalation solution on ammonia water-induced cough model in mice

[0155] Compared with the model control group, **P<0.01, *P<0.05

[0156] Table 33 shows that the 75 mg / ml dose group of genipin-1-β-D gentiopicroside can significantly prolong the latent period of ammonia water-induced cough in mice, and the 37.5 mg / ml dose group can significantly reduce the number of ammonia water-induced cough in mice and prolong the latent period of ammonia water-induced cough in mice, with significant differences compared with the model group (P<0.05, P<0.01).

[0157] Test four, expectorant effect of genipin-1-β-D gentiopicroside atomized inhalation solution on phenol red excretion in mouse airway.

[0158] 4.1 Test drug:

[0159] Genipin-1-β-D gentiopicroside atomized inhalation solution, batch number: 230608; specification: 5 ml: 375 mg; expiration date: tentatively 24 months; functional indication: chronic obstructive pulmonary disease (remission or acute exacerbation); providing unit: Yinkoer (Tianjin) Innovative Pharmaceutical Research Co., Ltd.

[0160] 4.2 Positive control drug:

[0161] Acetylcysteine solution for inhalation: batch number: 28006666, production date: 2023.05, expiration date: 2028.04. Produced by Zambon Pharmaceutical Co., Ltd. Composition: The active ingredient of the product is acetylcysteine. Characteristic: colorless or slightly blue-purple clear liquid with a slight sulfur smell. Indication: treatment of excessive thick and sticky mucus in respiratory diseases. Specification: 3 ml / branch. Method of use: atomization inhalation, 3 ml / time, 1-2 times / day.

[0162] 4.3 Test animals:

[0163] ICR rats, 50, SPF level, 18-22 g, half male and half female, purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd., Animal License No.: SCXK (Jing) 2021-0006, Certificate No.: 110324241101007874.

[0164] 4.4 Test instruments:

[0165] Table 34 Test instruments

[0166] 4.5 Dose of test drug:

[0167] Geniposide-1-β-D gentiobioside for mice was 75 mg / ml / d and 37.5 mg / ml / d in two dose groups. Inhalation administration, once a day, 15 min each time.

[0168] 4.6 Dose of positive control drug:

[0169] Inhalation of acetylcysteine solution, original drug solution was inhaled, once a day, 15 min each time.

[0170] 4.7 Test grouping and administration method:

[0171] ICR mice, 40, body weight 18-22 g, half male and half female, were randomly divided into 4 groups according to body weight, namely model control group, acetylcysteine group, geniposide-1-β-D gentiobioside 75 mg / kg / d and 37.5 mg / kg / d two dose groups, 10 in each group. Each administration group was administered by atomization for 15 min, once a day, for 3 consecutive days, and the model control group was given normal saline under the same conditions. 30 min after administration on the 3rd day, each mouse was injected intraperitoneally with 2.5% phenol red saline solution (250 mg / kg), and 30 min later, the mouse was executed by decapitation, the tissue around the trachea was stripped, a section of trachea from the thyroid cartilage to the tracheal branch was cut off, and then put into a test tube containing 1 ml of normal saline, and then 0.1 ml of 1 mol / L NaOH solution was added. The test tube was stored in the refrigerator, and when measuring the OD value, the solution in all test tubes was diluted to 2 ml with normal saline, centrifuged at 2000 r / min for 8 min, and the supernatant was aspirated and measured by an enzyme marker at 559 nm. According to the standard curve equation, the respiratory tract phenol red excretion of each mouse was calculated, and the phenol red excretion of each mouse was compared, that is, the sputum excretion.

[0172] Preparation of standard curve of phenol red: A certain amount of phenol red was weighed and dissolved in 1 mol / L NaOH solution to make the concentration of phenol red 1.00 mg / ml. The solution was diluted with 1 mol / L NaOH to make the concentration 20.0, 10.0, 5.00, 2.50, 1.25, 0.625, 0.3125, 0.1562 μg / ml, respectively. The absorbance value was determined. The concentration was taken as the horizontal coordinate and the absorbance value was taken as the vertical coordinate to draw the standard curve.

[0173] 4.8 Test results:

[0174] Table 35 The expectorant effect of genipin-1-β-D gentiobioside on the phenol red excretion model of mice

[0175] Compared with the model control group, **P <0.01

[0176] Table 35 shows that the 75 mg / kg / d and 37.5 mg / kg / d dose groups of genipin-1-β-D gentiobioside can significantly increase the tracheal phenol red excretion of mice, and there is a significant difference compared with the model control group (P <0.01).

Claims

1. Application of genipin-1-β-D gentiopicroside in the preparation of drugs for the treatment of chronic obstructive pulmonary disease.

2. The application of genipin-1-β-D gentiopicroside according to claim 1, characterized in that: The genipin-1-β-D gentiopicroside is administered via nebulized inhalation.

3. The application of genipin-1-β-D gentiopicroside according to claim 1, characterized in that: The concentration of the nebulized inhalation solution of genipin-1-β-D gentiopicroside is 75 mg / ml or 37.5 mg / ml, the nebulization flow rate is 7.5 L / min, and the nebulization time is 0–30 min.

4. The application of genipin-1-β-D gentiopicroside according to claim 3, characterized in that: The nebulized inhalation concentration of genipin-1-β-D gentiopicroside at 75 mg / ml is 2.419 mg / L.

5. The application of genipin-1-β-D gentiopicroside according to claim 3, characterized in that: The nebulized inhalation concentration of genipin-1-β-D gentiopicroside (37.5 mg / ml) is 1.032 mg / L.

6. The application of genipin-1-β-D gentiopicroside according to claim 2, characterized in that: The nebulized inhalation solution of genipin-1-β-D gentiopicroside comprises the following raw materials: genipin-1-β-D gentiopicroside, pH adjuster, osmotic pressure adjuster, and solvent.

7. The application of genipin-1-β-D gentiopicroside according to claim 6, characterized in that: The method for preparing the nebulized inhalation solution of genipin-1-β-D gentiopicroside includes the following steps: (1) Measure out 40% to 90% of the total volume of the solvent required to prepare the solution to obtain the first solution; (2) Add an osmotic pressure regulator to the first solution and stir until homogeneous to obtain the second solution; (3) Add a pH adjuster to the second solution to adjust the pH value to 4.5-7.0 to obtain the third solution; (4) Add genipin-1-β-D gentiopicroside to the third solution, stir well, and add a pH adjuster to keep the pH value between 4.5 and 7.0 to obtain the fourth solution; (5) Add solvent to the fourth solution and bring the volume up to the required total volume of the solution to be prepared. Stir well to obtain the nebulized inhalation solution of genipin-1-β-D gentiopicroside.

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