Pharmaceutical composition for treating chronic obstructive pulmonary disease and application

Abstract

The invention relates to the technical field of lung disease treatment, in particular to a medicine for treating chronic obstructive pulmonary disease and application. The pharmaceutical composition can obviously inhibit lung inflammatory cell infiltration and inflammatory factor generation of patients with chronic obstructive pulmonary diseases and induce malignant cell apoptosis, effectively regulate TNF-alpha, IL-6, calcium and phosphorus levels of the patients with chronic obstructive pulmonary diseases, increase the walking distance of the patients with chronic obstructive pulmonary diseases and improve the curative effect of the patients with chronic obstructive pulmonary diseases. The pharmaceutical composition can be used for effectively treating chronic obstructive pulmonary disease patients and improving prognosis conditions of the chronic obstructive pulmonary disease patients, and is safe to use, easy to control and convenient for clinical application and popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of the treatment of lung diseases, and particularly relates to a pharmaceutical composition for treating chronic obstructive pulmonary disease and its application. Background Art

[0002] Chronic obstructive pulmonary disease is mainly characterized by chronic inflammation of the lung parenchyma, airways and pulmonary blood vessels. In patients, the number of inflammatory cells such as eosinophils, lymphocytes, alveolar macrophages and neutrophils in the lungs increases. After being activated, these inflammatory cells will release a large number of inflammatory mediators, thereby destroying the airway structure and leading to airway remodeling.

[0003] Pterostilbene is one of the polyphenols mainly present in blueberries and grapes. Pterostilbene has anti-tumor, anti-dyslipidemia, cardiovascular and neuroprotective effects, and its mechanism is related to anti-inflammatory and antioxidant functions. Research shows that a decrease in vitamin D levels is one of the risk factors for lung function damage in patients with chronic obstructive pulmonary disease. Its deficiency can induce respiratory muscle weakness and rib osteoporosis in COPD patients, resulting in poor sputum excretion and decreased respiratory movement in patients, aggravating the deterioration of lung function in patients and increasing the incidence of pulmonary infections. 25-hydroxyvitamin D is the main form of vitamin D in the body, with a large content and stable properties. However, there is still no reliable solution regarding the treatment of chronic obstructive pulmonary disease patients with pterostilbene and 25-hydroxyvitamin D.

[0004] In view of the above problems, the present invention provides a solution. Summary of the Invention

[0005] The object of the present invention is to provide a pharmaceutical composition for treating chronic obstructive pulmonary disease and its application. The present invention can effectively treat chronic obstructive pulmonary disease and improve the prognosis of patients with chronic obstructive pulmonary disease.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A pharmaceutical composition for treating chronic obstructive pulmonary disease uses pterostilbene and 25-hydroxyvitamin D as active ingredients;

[0008] Furthermore, the pharmaceutical composition can be administered via the gastrointestinal tract or by inhalation. Preferably, the pharmaceutical dosage forms for gastrointestinal administration are selected from: tablets, capsules, granules, solutions, infusion powders, pills, and powders; the pharmaceutical dosage forms for inhalation administration are selected from: aerosols, sprays or powder inhalants.

[0009] Furthermore, in the pharmaceutical composition, pterostilbene and 25-hydroxyvitamin D account for 2 - 50% of the total weight of the drug, preferably 5 - 25%, and most preferably 10%. The molar ratio of pterostilbene to 25-hydroxyvitamin D is 3:7;

[0010] The pterostilbene described above is Figure 1 the compound shown or the hydrate, solvate, metabolite and pharmaceutically acceptable salt of the compound shown;

[0011] Pterostilbene belongs to the class of polyhydroxylated stilbene compounds and is a chemical component contained in red sandalwood, and is a white or off-white powder crystal; the molecular formula is C 16 H 16 O3, with a molecular weight of 256.3; the melting point is 89 - 92 °C, soluble in hot methanol and dimethyl sulfoxide, insoluble in water; it is easily decomposed in light and air and is not resistant to storage, and its dimethyl sulfoxide solution can be stored for a long time at -20 °C.

[0012] The 25-hydroxyvitamin D described above is Figure 2 the compound shown or the hydrate, solvate, metabolite, pharmaceutically acceptable salt of the compound shown;

[0013] 25-Hydroxyvitamin D is the main existing form of vitamin D in the body and is a white crystalline powder; the molecular formula is C 28 H 44 O2, with a molecular weight of 412.65; the melting point is 45 - 51 °C, soluble in ethanol and dimethyl sulfoxide, slightly soluble in water; it is easily decomposed in light and at room temperature and is not resistant to storage, and its dimethyl sulfoxide solution can be stored for a long time at -20 °C.

[0014] Furthermore, a pharmaceutical composition comprises the pterostilbene and 25-hydroxyvitamin D and a pharmaceutically acceptable carrier, excipient, adjuvant or their pharmaceutical composition;

[0015] Furthermore, an application of a pharmaceutical composition for treating chronic obstructive pulmonary disease, wherein the pharmaceutical composition uses pterostilbene and vitamin D as active ingredients, and the pharmaceutical composition is used for preventing, treating or alleviating chronic obstructive pulmonary disease.

[0016] In the present invention, the pterostilbene and 25-hydroxyvitamin D can significantly improve the lung function of chronic obstructive pulmonary disease.

[0017] In the present invention, the pterostilbene can significantly inhibit the viability and proliferation of lung cancer cells, and at the same time stimulate the apoptosis of lung cancer cells. The up-regulation of the expression of cyclooxygenase-2 has the ability of tumorigenic induction and has an indispensable carcinogenic effect. Pterostilbene can regulate the proliferation and apoptosis of lung cancer cells by targeting cyclooxygenase-2.

[0018] In the present invention, the 25-hydroxyvitamin D can significantly inhibit the infiltration of inflammatory cells in the lungs of chronic obstructive pulmonary disease and can significantly inhibit the production of inflammatory factors of chronic obstructive pulmonary disease. The inflammatory factors of chronic obstructive pulmonary disease include but are not limited to TNF-α, IL-1β and IL-17A.

[0019] In the present invention, the 25-hydroxyvitamin D can significantly reduce the proportion of neutrophils in the lungs of patients with chronic obstructive pulmonary disease. Therefore, 25-hydroxyvitamin D can be used in the preparation of drugs for preventing and treating neutrophil inflammation-related chronic obstructive pulmonary disease, especially bronchial inflammatory chronic obstructive pulmonary disease related to neutrophil inflammation.

[0020] Furthermore, the chronic obstructive pulmonary disease described in the present invention includes, but is not limited to, airway inflammation, emphysema, lung ventilation injury, and the resulting respiratory and critical illnesses. In the present invention, the chronic obstructive pulmonary disease refers to a disease characterized by airflow limitation, and its clinical features include reduced expiratory airflow, slow forced emptying of the lungs, wheezing, coughing, and expectoration, accompanied by chronic airway obstruction and pulmonary hyperinflation. Chronic obstructive bronchitis and obstructive emphysema often occur simultaneously during the onset.

[0021] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows: the pharmaceutical composition of the present invention can significantly inhibit the infiltration of inflammatory cells, the production of inflammatory factors, and induce the apoptosis of malignant cells in the lungs of patients with chronic obstructive pulmonary disease, effectively regulate the levels of TNF-α, IL-6, calcium, and phosphorus in patients with chronic obstructive pulmonary disease, increase the walking distance of patients with chronic obstructive pulmonary disease, effectively treat patients with chronic obstructive pulmonary disease, improve the prognosis of patients with chronic obstructive pulmonary disease, and the pharmaceutical composition of the present invention is safe to use, easy to control, and convenient for clinical application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the molecular structure diagram of pterostilbene.

[0023] Figure 2 It is the molecular structure diagram of 25-hydroxyvitamin D.

[0024] Figure 3 It is the histogram of cell viability of A549 cells measured based on CCK-8.

[0025] Figure 4 It is the histogram of cell viability of H1299 cells measured based on CCK-8. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] In order to make the technical means, creative features, achieved purposes, and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments and diagrams.

[0027] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0028] Example 1

[0029] In vitro intervention of a pharmaceutical composition for treating chronic obstructive pulmonary disease

[0030] 1: Two NSCLC cell lines, A549 (CCL-185-LUC2, ATCC, Manassas, VA, USA) and H1299 (CRL-5803, ATCC), were purchased from the American Type Culture Collection (ATCC);

[0031] 2: The cells were divided into 4 groups: a control group, a radiation group, a drug treatment group, and a radiation + drug group;

[0032] The control group was treated as follows:

[0033] The cells were placed in an incubator (51030286, ThermoFisher Scientific) at 37°C with 5% CO2, and cultured for 24 h using Dulbecco's modified Eagle's medium (DMEM) (31331093, Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS) (12664025, Gibco, USA), 2 mM L-glutamine (G7513, Sigma-Aldrich, St Louis, MO, USA), 100 U / ml penicillin, and 100 μg / ml streptomycin (15070063, ThermoFisher Scientific, Waltham, MA, USA);

[0034] The treatment method for the drug group was: adding 15 μM pterostilbene and 35 μM 25-hydroxyvitamin D to the medium, with other conditions unchanged;

[0035] The treatment method for the radiation group was: irradiating with a cobalt-60 gamma ray irradiator (Gammacell 220, MDS Nordion, Ottawa, Canada), with a total radiation dose of 2 Gy and a power of 2 Gy / min, and other conditions unchanged;

[0036] The treatment method for the radiation + drug treatment group was as follows: 15 μM pterostilbene and 35 μM 25-hydroxyvitamin D were added to the culture medium, and a cobalt-60 gamma-ray irradiator (Gammacell 220, MDS Nordion, Ottawa, Canada) was used with a total radiation dose of 2 Gy and a power of 2 Gy / min, with other conditions remaining unchanged;

[0037] 3: CCK-8 (C0037, Beyotime, Shanghai, China) was used to measure cell viability. The cells were seeded into a 96-well plate at an inoculation amount of 1000 cells per well and cultured for 24 hours. After culturing, 10 μl of CCK-8 solution was added to each well until the cells adhered completely to the wall. After incubation for 2 hours, the absorbance at 450 nm was measured using a microplate reader (Varioskan LUX, Thermo, USA);

[0038] 4: The cells were seeded into the culture medium at a density of 500 cells / 60 mm and cultured in a 37 °C incubator with 5% CO2. The fresh culture medium was changed every 24 hours. After incubation for 14 days, the cells were washed and suspended twice with PBS, then fixed with 4% paraformaldehyde solution (158127, Sigma-Aldrich) for 30 minutes and stained with 0.2% crystal violet staining solution (V5265, Sigma-Aldrich) for 15 minutes. Finally, the images were captured using a camera and the number of colonies (≥50 cells / colony) was counted under a microscope;

[0039] 5: The TUNEL assay was used to evaluate apoptosis in A549 and H1299 cells. The cells were fixed with 4% paraformaldehyde and washed with PBS, and then permeabilized with 0.1% Triton X-100 (X100, Sigma-Aldrich) and 0.1% ice-cold sodium citrate solution (1613859, Sigma-Aldrich) for 2 minutes. Then, the TUNEL reaction was carried out for 1 h, and the TUNEL-positive cells were labeled with fluorescein isothiocyanate. After that, the nuclei of all cells were stained with 4',6-diamidino-2-phenylindole. Finally, the results of apoptosis were observed under a fluorescence microscope;

[0040] Result analysis: By analyzing Figure 3 and Figure 4It can be seen that compared with the control group, the cell viability of the radiation group and the drug group decreased, and the cell viability of the radiation + drug treatment group decreased more. In addition, the results of the colony formation assay showed that radiation significantly inhibited the proliferation of A549 and H1299 cells, and the same was true for drug treatment alone. In addition, compared with single radiation or drug treatment, the combination of radiation + drug treatment further inhibited the proliferation of A549 and H1299 cells, indicating that the drug composition provided by the present invention can further combine with radiation to inhibit the proliferation of malignant cells; TUNEL staining showed that radiotherapy significantly promoted the apoptosis of malignant cells, and drug treatment also showed the same effect. In addition, the combination of radiotherapy and drug treatment was more prominent in the apoptosis rate of malignant cells.

[0041] Example 2

[0042] In vivo intervention of a drug composition for treating chronic obstructive pulmonary disease

[0043] A total of 120 patients with stable chronic obstructive pulmonary disease diagnosed and treated in Taizhou Central Hospital from January to December 2019 were selected, including 85 males and 35 females; the age was 28 - 64 (45.83 ± 10.69) years. Inclusion criteria:

[0044] 1: According to the diagnostic criteria of the Global Initiative for Chronic Obstructive Lung Disease Revised Edition (GOLD 2018 Edition) 3: Stable COPD refers to patients with relatively mild or stable clinical symptoms such as shortness of breath, expectoration, and cough;

[0045] 2: The stable phase lasts for more than 1 month, without symptoms such as cough, expectoration, and dyspnea after activity;

[0046] 3: No short-acting bronchodilators were used within the previous 24 hours, no long-acting bronchodilators were used within 48 hours, and no local or systemic glucocorticoids were used in the recent 1 month;

[0047] 4: Clearly diagnosed as vitamin D deficiency patients: fasting blood 25-hydroxyvitamin D < 20 nmol / L in the early morning. Exclusion criteria: (1) Patients with vitamin D allergy; (2) Patients with bronchial asthma, bronchiectasis, lung abscess or other severe lung diseases; (3) Immunosuppressed patients; (4) History of taking drugs that may affect vitamin D levels within half a year; (5) Patients with acute exacerbation within 1 month or history of using antibacterial drugs and systemic glucocorticoids; (6) Patients who require mechanical ventilation; (7) Pregnant and lactating patients; (8) Patients with kidney stones, hypercalcemia (fasting blood calcium > 2.75 mmol / L in the early morning), and intestinal malabsorption; (9) Patients with end-stage diseases (expected survival < 12 months); (10) Patients with severe complications such as congestive heart failure and severe liver and kidney insufficiency. The patients were divided into a study group and a control group by the random number table method, with 60 cases in each group.

[0048] Table 1. Comparison of General Data of Two Groups of Patients

[0049]

[0050] Administration method: Both groups of patients received conventional treatment, including home oxygen therapy, active smoking cessation, nutritional support, inhalation preparations, etc. The following 4 situations were allowed for inhalation preparations:

[0051] (1) Use tiotropium bromide spray (National Medicine Approval No.: J20120047) alone, 1 puff / time, once a day;

[0052] (2) Use salmeterol fluticasone powder for inhalation 50 / 500 (National Medicine Approval No.: H20140164) alone, 1 puff / time, twice a day;

[0053] (3) Use tiotropium bromide spray in combination, 1 puff / time, once a day; salmeterol fluticasone powder for inhalation 50 / 500, 1 puff / time, twice a day;

[0054] (4) Do not use inhalation preparations. The patients in the study group were simultaneously given a self-made soft capsule pharmaceutical composition (ingredients: 15 mM pterostilbene and 35 mM 25-hydroxyvitamin D / g, specification: 0.25 μg / tablet), 0.25 μg / time, once a day, orally.

[0055] (5) The patients in the control group were simultaneously given a placebo (self-made preparation: soft capsule, ingredients: starch, specification: 0.25 μg / tablet), 0.25 μg / time, once a day, orally.

[0056] Both groups of patients took the medicine continuously for 8 weeks. During the treatment process, follow-up was strengthened. Inpatients were observed at any time. After the course of treatment, the curative effect was evaluated, and relevant laboratory indexes were observed.

[0057] The pulmonary function indexes, blood 25-hydroxyvitamin D, TNF-α, IL-6, calcium, and phosphorus levels, 6-minute walking distance, and CAT score of the two groups of patients before and after treatment were compared. The data after treatment were collected on the 2nd day after the end of the course of treatment;

[0058] Lung function indices were detected using a MasterScreen pulmonary function tester (manufactured by JAEGER, Germany) to measure lung function indices. All patients were uniformly measured in the morning. Sitting upright, the nose was clipped with a nose clip. The patient first took a deep breath and then exhaled quickly and forcefully. After exhaling completely, an ideal forced vital capacity (FVC) curve could be obtained. The total number of times was <8 times. It was required to obtain 3 qualified curves measured repeatedly, and the highest value was taken. The measured indices included FVC, forced expiratory volume in one second (FEV₁), percentage of FEV₁ in predicted value (FEV₁ / predicted value, FEV₁ / pred), and FEV₁ / FVC was calculated.

[0059] For the levels of blood 25-hydroxyvitamin D, TNF-α, IL-6, calcium, and phosphorus, 5 ml of fasting venous blood was collected from the patients, centrifuged at 3000 r / min for 10 min, and the supernatant was taken and stored in a -80°C refrigerator for later use; ELISA was used to measure blood 25-hydroxyvitamin D, TNF-α, and IL-6, spectrophotometry was used to measure blood calcium levels, and phosphomolybdic acid method was used to measure blood phosphorus levels.

[0060] 6-minute walk distance measurement: A straight, flat, hard-surfaced, appropriately temperature-controlled, and well-ventilated corridor in the hospital was selected, and marks were made at both the starting point and the ending point, with a length of approximately 30 m. Before the experiment, the patients were instructed to wear comfortable clothes and not to do excessive exercise. During the test, the patients walked at their normal walking speed, and no obviously suggestive body language or encouraging words were used for the patients. At the same time, the operator could not accompany the patients while walking. If the patients showed intolerable limb twitching, dyspnea, pale complexion, cold sweating, chest pain, dizziness, fatigue, etc. during this period, the experiment should be stopped immediately and the walking distance should be determined.

[0061] Table 2. Comparison of lung function indices between the two groups of patients before and after treatment

[0062]

[0063] Table 3. Comparison of the levels of blood 25-hydroxyvitamin D, TNF-α, IL-6, calcium, and phosphorus between the two groups of patients before and after treatment

[0064]

[0065] Table 4. Comparison of 6-minute walk distance and CAT score between the two groups of patients after treatment

[0066]

[0067] It can be seen from the analysis of Table 2-4 that after drug treatment, the serum calcium level of the patients in the study group was higher than that in the control group, indicating that the pharmaceutical composition of the present invention has a good effect on treating the serum calcium level of patients with chronic obstructive pulmonary disease; after treatment, the levels of FVC, FEV, FEV / FVC and FEV / pred of the patients in the study group were all higher than those in the control group, and the levels of TNF-α and IL-6 of the patients in the study group were lower than those in the control group, indicating that the pharmaceutical composition of the present invention can relieve the inflammatory state of patients with chronic obstructive pulmonary disease; after treatment, the 6-minute walking distance of the patients in the study group was higher than that in the control group and the CAT score was lower than that in the control group, indicating that the pharmaceutical composition of the present invention can improve the prognosis of patients with chronic obstructive pulmonary disease.

[0068] The foregoing shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and all these changes and improvements fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for treating chronic obstructive pulmonary disease, characterized in that: The pharmaceutical composition uses pterostilbene and 25-hydroxyvitamin D as active ingredients; in the pharmaceutical composition, pterostilbene and 25-hydroxyvitamin D account for 2-50% of the total weight of the drug, preferably 5-25%, and most preferably 10%, wherein the molar ratio of pterostilbene to 25-hydroxyvitamin D is 3:

7.

2. A pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 1, characterized in that: The pterostilbene is the compound shown in FIG. 1 or a hydrate, solvate, metabolite or pharmaceutically acceptable salt of the compound shown.

3. A pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 1, characterized in that: The 25-hydroxyvitamin D is the compound shown in Figure 2 or a hydrate, solvate, metabolite, or pharmaceutically acceptable salt of the compound shown.

4. A pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 1, characterized in that: The pharmaceutical composition comprises the pterostilbene and 25-hydroxyvitamin D and a pharmaceutically acceptable carrier, excipient, adjuvant or a pharmaceutical composition thereof.

5. A pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 1, characterized in that: The pharmaceutical composition can be administered via the gastrointestinal tract or by inhalation. Preferably, the pharmaceutical dosage form for administration via the gastrointestinal tract is selected from: tablets, capsules, granules, solutions, granules, pills, powders; the pharmaceutical dosage form for administration by inhalation is selected from: aerosols, sprays or powder inhalers.

6. Use of a pharmaceutical composition for treating chronic obstructive pulmonary disease, characterized in that: The pharmaceutical composition uses pterostilbene and vitamin D as active ingredients, and the pharmaceutical composition is used for preventing, treating or alleviating chronic obstructive pulmonary disease.

7. Use of a pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 6, characterized in that: The pterostilbene and 25-hydroxyvitamin D can significantly improve the lung function of chronic obstructive pulmonary disease.

8. The use of a pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 6, characterized in that: The pterostilbene can significantly inhibit the activity and proliferation of lung cancer cells and stimulate the apoptosis of lung cancer cells.

9. The use of a pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 6, characterized in that: The 25-hydroxyvitamin D can significantly inhibit the infiltration of inflammatory cells in the lungs of patients with chronic obstructive pulmonary disease, significantly inhibit the production of inflammatory factors in patients with chronic obstructive pulmonary disease, and significantly reduce the proportion of neutrophils in the lungs of patients with chronic obstructive pulmonary disease.

10. The use of a pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 6, characterized in that: The chronic obstructive pulmonary disease includes, but is not limited to, airway inflammation, emphysema, lung ventilation damage, and respiratory and critical illnesses caused thereby.

Images