Use of polyeneyne compounds for the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis

Abstract

The application relates to application of a polyene alkyne compound in preparation of a medicine for preventing and / or treating pulmonary fibrosis. The specific polyene alkyne compound has obvious inhibiting collagen deposition effect on TGF-beta 1 induced human embryo lung fibroblasts, does not show cytotoxicity, has good anti-pulmonary fibrosis activity, and can be used for preparing the medicine for preventing and / or treating pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to the use of polyene-yne ​​compounds in preparing drugs for preventing and / or treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is an interstitial lung disease characterized by airway damage, excessive secretion of inflammatory factors, fibroblast proliferation, and abnormal deposition of extracellular matrix (ECM). Common triggers of PF include environmental pollutants (cigarette smoke, silica, and PM2.5), connective tissue diseases (scleroderma and rheumatoid arthritis), and the side effects of certain medications (amiodarone and methotrexate). PF typically develops between the ages of 40 and 50, and is more common in men than in women. Dyspnea is the most common symptom of PF. In mild PF, dyspnea often occurs during strenuous activity and is therefore often overlooked or misdiagnosed as other conditions. As PF progresses, dyspnea may also occur at rest, and patients with severe PF may experience progressive dyspnea. Other symptoms include dry cough and fatigue. Some patients experience digital clubbing and cyanosis. The serious consequences of PF lead to structural changes and functional loss of normal lung tissue. When large amounts of fibrotic tissue, which lacks gas exchange function, replace the alveoli, oxygen cannot enter the blood. Patients suffer from respiratory distress, hypoxia, acidosis, and loss of productivity. In severe cases, death can occur. The incidence and mortality of idiopathic pulmonary fibrosis are increasing annually, with an average survival of only 2.8 years after diagnosis. This mortality rate is higher than that of most cancers, earning it the nickname "tumor-like disease."

[0003] Traditional treatments such as glucocorticoids, immunosuppressants, and antioxidants not only fail to improve patient outcomes but may even accelerate disease progression, and are therefore rarely used clinically. Pirfenidone (PFD) and nintedanib (BIBF1120) are the only recommended treatments. With increasing air pollution, the incidence of pulmonary fibrosis is steadily increasing. Therefore, the search for new, highly effective, and minimally toxic drugs for the prevention and treatment of pulmonary fibrosis remains urgent.

[0004] Atractylodes lancea is a traditional Chinese medicine. Chinese herbal compound containing Atractylodes lancea is widely used in the prevention and treatment of viral pneumonia and pulmonary fibrosis. For example, a Chinese patent entitled "Use of a Chinese herbal composition in the preparation of a drug for treating pulmonary fibrosis" provides a drug made from various Chinese herbs such as Atractylodes lancea for the treatment of pulmonary fibrosis, but no further research was conducted on the specific active ingredient(s) in Atractylodes lancea for the treatment of pulmonary fibrosis.

[0005] Therefore, further research and development of drugs for the treatment of pulmonary fibrosis is needed. Summary of the Invention

[0006] The primary purpose of the present invention is to overcome the significant side effects and limited availability of recommended therapeutic agents for the treatment of pulmonary fibrosis in the prior art. The present invention provides the use of polyene-yne ​​compounds in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis. The specific polyene-yne ​​compounds of the present invention exhibit significant inhibition of collagen deposition in TGF-β1-induced human embryonic lung fibroblasts, exhibit no cytotoxicity, and possess excellent anti-pulmonary fibrosis activity. These compounds can be used to prepare medicaments for the prevention and / or treatment of pulmonary fibrosis.

[0007] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0008] The present invention provides a method for preparing a drug for preventing and / or treating pulmonary fibrosis. The polyene-acetylene compound has a structure as shown in any one of formulas (I) to (V):

[0009]

[0010] In formula (I), R1 is OAc, OB or OH, R2 is OAc, OA, OB or OH, and R3 is OH or H; wherein A in OA is The B in OB is

[0011] The inventors of the present invention extracted specific polyene-yne ​​compounds from Atractylodes lancea (for details, see the document Diverse Sesquiterpenoids and Polyacetylenes from Atractylodes lancea and Their Anti-Osteoclastogenesis Activity, Zhejun Sun, Yuting Zhang, Xing Peng, Shijie Huang, Huihao Zhou, Jun Xu, Qiong Gu, J. Nat. Prod. 2022, 85, 866-877).

[0012] Through further research, the inventors of the present invention found that specific polyene-yne ​​compounds have a significant inhibitory effect on collagen deposition in human embryonic lung fibroblasts induced by TGF-β1, do not show cytotoxicity, have good anti-pulmonary fibrosis activity, and can be used to prepare drugs for preventing and / or treating pulmonary fibrosis.

[0013] Preferably, in formula (I), R1 is OAc, R2 is OA, and R3 is OH; or R1 is OB, R2 is OH, and R3 is H; or R1 is OAc, R2 is OAc, and R3 is OH; or R1 is OB, R2 is OH, and R3 is OH; or R1 is β-OH, R2 is OB, and R3 is OH; or R1 is OAc, R2 is OAc, and R3 is H; or R1 is OH, R2 is OB, and R3 is OH.

[0014] Preferably, the polyene-yne ​​compound is any one of the compounds numbered BCZ-1 to BCZ-11:

[0015]

[0016] Preferably, the polyene-yne ​​compound is any one of the compounds numbered BCZ-2 to 3, BCZ-5 to 7, and BCZ-9 to 11:

[0017]

[0018] Preferably, the polyene-yne ​​compound is any one of the compounds numbered BCZ-1 to 3 and BCZ-5 to 11:

[0019]

[0020] Preferably, the polyene-yne ​​compound is used in the preparation of a drug for inhibiting collagen deposition in lung fibroblasts.

[0021] Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

[0022] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0023] More preferably, the pharmaceutically acceptable excipient is a carrier.

[0024] More preferably, the pharmaceutically acceptable excipient is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH regulator.

[0025] Preferably, the dosage form of the drug is injection, tablet, oral solution, granule or capsule.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The specific polyene-yne ​​compounds of the present invention have a significant effect of inhibiting collagen deposition in human embryonic lung fibroblasts induced by TGF-β1, do not show cytotoxicity, have good anti-pulmonary fibrosis activity, and can be used to prepare drugs for preventing and / or treating pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The molecular structures of polyene-yne ​​compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10 and BCZ-11.

[0029] Figure 2 The crude extract of Atractylodes lancea was 1, 3, and 10 μg ml -1 The results of the inhibition of α-SMA and Fibronectin protein expression in human embryonic lung fibroblasts induced by TGF-β1 at different concentrations.

[0030] Figure 3 This is a graph showing the toxicity test results of polyene-yne ​​compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10 and BCZ-11 on HFL1 cells at a concentration of 10 μM for 48 hours.

[0031] Figure 4 This is a graph showing the results of an inhibitory assay of polyeneyne compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10 and BCZ-11 on TGF-β1-induced collagen deposition in HFL1 cells at a concentration of 10 μM. DETAILED DESCRIPTION

[0032] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0033] Example 1

[0034] This embodiment provides polyene-acetylene compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10 and BCZ-11. The preparation method of the polyene-acetylene compounds mainly includes the following steps:

[0035] The dried rhizome of Atractylodes lancea is crushed, extracted with an 80% acetone aqueous solution at room temperature for three times, each time for three days; the extract is concentrated under reduced pressure to obtain a crude extract (i.e., Atractylodes lancea crude extract); the extract is suspended in water, extracted with ethyl acetate for five times, concentrated under reduced pressure to obtain an ethyl acetate fraction, and subjected to 200-300 mesh silica gel column chromatography, eluted with a petroleum ether / ethyl acetate (20:1, 10:1, 5:1, 2:1, 1:1, 1:0, v / v) mixed solution according to a gradient elution; and the obtained fraction is alcoholized to obtain a polyene-yne ​​compound. For more detailed steps and characterization information, please refer to the literature previously published by the inventors of the present invention, "Diverse Sesquiterpenoids and Polyacetylenes from Atractylodes lancea and Their Anti-Osteoclastogenesis Activity," Zhejun Sun, Yuting Zhang, Xing Peng, Shijie Huang, Huihao Zhou, Jun Xu, Qiong Gu, J. Nat. Prod. 2022, 85, 866-877. In the literature, compounds 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33 correspond to the polyene-yne ​​compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10, and BCZ-11 of the present invention, respectively.

[0036] Figure 1 This is the molecular structure diagram of the polyene-yne ​​compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10 and BCZ-11.

[0037] Example 2

[0038] This embodiment provides an injection for preventing and treating pulmonary fibrosis, which is prepared by the following process: respectively mixing polyene-yne ​​compounds BCZ-1 to BCZ-11 with water for injection, fine filtering, encapsulating, and sterilizing to prepare the injection.

[0039] Example 3

[0040] This embodiment provides a tablet for preventing and treating pulmonary fibrosis, which is prepared by the following process: polyene-yne ​​compounds BCZ-1 to BCZ-11 are separately mixed with excipients (such as starch slurry) and then pressed into tablets.

[0041] Example 4 Evaluation of the Anti-Pulmonary Fibrosis Activity of Compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10, and BCZ-11

[0042] 4.1 Cell Culture

[0043] The cell line used in this experiment was human embryonic lung fibroblast (HFL1) cells purchased from Wuhan Pronocell Life Science Co., Ltd., catalog number CL-0106. HFL1 cells were cultured in Ham's F-12k medium supplemented with 10% FBS in a 37°C incubator with 5% CO2. Cells were passaged when they reached approximately 90% confluency. The appropriate cell density was adjusted according to experimental needs and plated onto culture plates or dishes. Before treatment with TGF-β1, cells were starved for 12 hours using medium supplemented with 1% FBS.

[0044] 4.2. Western blot

[0045] HFL1 cells in the logarithmic growth phase were seeded in six-well plates and attached to the wall for 24 hours. The cells were divided into a blank group, a model group, and a group treated with crude extract of Atractylodes lancea. The blank group was treated with culture medium, and the model group was treated with TGF-β1 (5 ng·ml -1 ) induced the formation of lung myofibroblast model, and the Atractylodes lancea crude extract group was also treated with TGF-β1 (5 ng·ml -1 ) and crude extracts of Atractylodes lancea (1, 3, 10 μg ml -1 ) After 48 hours of co-culture, RIPA lysis buffer was added for lysis, centrifugation was performed, and the supernatant was collected. The protein concentration was determined by BCA method, and loading buffer was added. The protein was denatured by boiling in a 100°C metal bath for 10 minutes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein components, and the protein was transferred to a polyvinylidene fluoride (PVDF) membrane by wet transfer, blocked with 5% skim milk powder, and washed with TBST. Primary antibody (1:1000) was added and incubated overnight at 4°C. Washed with TBST. Goat anti-rabbit secondary antibody (1:5000) was added (incubated at room temperature for 1 hour, exposed and photographed using a Tanon multifunctional imaging system.

[0046] The crude extract of Atractylodes lancea was 1, 3, and 10 μg ml -1 The results of the inhibition of α-SMA and Fibronectin protein expression in human embryonic lung fibroblasts induced by TGF-β1 were as follows: Figure 2 shown.

[0047] 4.3. Evaluation of cytotoxicity by CCK8 assay

[0048] HFL1 cells in the logarithmic growth phase were cultured at a rate of 3×10 3 Cells were inoculated into 96-well plates at 10 μM per well. After 24 hours of cell culture, 10 μM of different polyene-based compounds were added to each well and incubated for 48 hours. Three replicate wells were set up, along with a control group and a zero well. The old culture medium was discarded and replaced with 100 μL of serum-free culture medium. The cells were protected from light and 10 μL of CCK-8 solution was added to each well. The cells were incubated in an incubator for 2 hours. The absorbance at 450 nm (i.e., OD value) was measured using a microplate reader. Cell viability was calculated based on the absorbance of each well as follows:

[0049]

[0050] The results of cytotoxicity assays of polyene-yne ​​compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10 and BCZ-11 on HFL1 cells are shown in Figure 2. Figure 3 shown.

[0051] 4.4. Sirius red staining to detect collagen fiber deposition

[0052] Human embryonic lung fibroblasts HFL1 were cultured at a rate of 1×10 4 Cells / well were seeded into 96-well plates. After 24 hours of cell culture, serum-free medium was changed for synchronization for 12 hours. 5 ng / mL TGF-β1 and 10 μM of different polyene-yne ​​compounds were added to each well and incubated for 48 hours. The supernatant was removed and fixed with 4% paraformaldehyde for 30 minutes. After washing twice with PBS, the cells were stained with picrosirius red stain for 4 hours. The stain was removed and 0.1% acetic acid was added to wash away the remaining stain. After washing three times, the cells were photographed. After staining, 0.1 M NaOH (100 ml / well) was added to the stained cells and shaken to dissolve for 10 minutes. The absorbance was detected at 540 nm by a microplate reader. The total collagen accumulation inhibition rate was calculated as follows:

[0053]

[0054] The results of the inhibition of TGF-β1-induced collagen deposition in human embryonic lung fibroblasts by polyene-yne ​​compounds BCZ-1, BCZ-2, BCZ-3, BCZ-4, BCZ-5, BCZ-6, BCZ-7, BCZ-8, BCZ-9, BCZ-10 and BCZ-11 at a concentration of 10 μM are shown in Figure 2. Figure 4 shown.

[0055] 4.5 Statistical Methods

[0056] Data were analyzed using GraphPad Prism 7.0, and statistical results are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used, and multiple comparisons were performed using the LSD-t test. Figure 2 and 3 middle, ＊ indicates P < 0.05, ＊＊ Indicates P < 0.01, ＊＊＊ P < 0.001 was considered statistically significant. ns (not significant) indicates that there was no statistically significant difference between the groups.

[0057] Figure 2 The results showed that the crude extract of Atractylodes lancea had the following concentrations: 1, 3, 10 μg·ml -1 It has a significant and dose-dependent inhibitory effect on the expression of α-SMA and Fibronectin proteins in human embryonic lung fibroblasts induced by TGF-β1.

[0058] Figure 3 Results showed that the polyene-yne ​​compounds BCZ-1, BCZ-4, and BCZ-8 reduced the survival rate of HFL1 cells to less than 80% at a concentration of 10 μM, indicating that these compounds had an effect on the survival or proliferation of HFL1 cells at a concentration of 10 μM. The remaining polyene-yne ​​compounds BCZ-2, BCZ-3, BCZ-5 to BCZ-7, BCZ-9 to BCZ-11 did not produce significant toxicity to HFL1 cells at a concentration of 10 μM for 48 hours.

[0059] Figure 4 The results showed that, at 10 μM, all polyenyne compounds, except BCZ-4, significantly inhibited TGF-β1-induced collagen deposition in HFL1 cells, demonstrating their anti-pulmonary fibrosis potential. Compounds BCZ-1, BCZ-6 to BCZ-8, and BCZ-10 exhibited greater than 80% inhibition of collagen deposition in HFL1 cells at 10 μM.

[0060] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of a polyene-yne ​​compound in the preparation of a medicament for preventing and / or treating pulmonary fibrosis, characterized in that: The polyene-acetylene compound has a structure as shown in formula (II):

2. Use of a polyene-yne ​​compound in the preparation of a drug for inhibiting collagen deposition in lung fibroblasts, characterized in that: The polyene-acetylene compound has a structure as shown in formula (II):

3. The application according to claim 1, characterized in that The pulmonary fibrosis is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

4. The use according to claim 1 or 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that: The pharmaceutically acceptable excipient is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH regulator.

6. The use according to claim 1 or 2, characterized in that: The dosage form of the medicine is injection, tablet, oral solution, granule or capsule.

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