Use of sesquiterpenes in the preparation of a medicament for preventing and / or treating pulmonary fibrosis

Abstract

The application relates to application of a sesquiterpenoid compound in preparation of a medicine for preventing and / or treating pulmonary fibrosis. The sesquiterpenoid 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] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of sesquiterpenoid compounds in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease. Fibrotic lung tissue typically presents with a "honeycomb" appearance, bronchiectasis, and thickening of the surrounding alveolar septa. The disease progresses from the base and periphery of the lungs, gradually spreading to the entire lung. Common symptoms include chronic dry cough, dyspnea, and clubbing of the fingers. Pulmonary fibrosis results from excessive accumulation of extracellular matrix and disordered tissue repair after lung tissue damage. It is a difficult-to-treat disease with a high mortality rate; the life expectancy after diagnosis is 2–6 years. In industry, fibrosis accounts for 45% of all deaths. As of July 2020, approximately one-third of hospitalized COVID-19 patients developed pulmonary fibrosis. In 2014, the U.S. Food and Drug Administration (FDA) approved pirfenidone and nintedanib for the treatment of fibrosis; however, their clinical efficacy is limited, primarily slowing disease progression but not fundamentally resolving the disease. Some kinase inhibitors have also been found to be in clinical trials; however, their inhibition of target enzymes in healthy tissues causes systemic toxicity, and they have not been approved. Although lung transplantation remains a last resort, survival rates are still limited and the medical costs are prohibitive. Therefore, the clinical need for novel, highly effective drugs for the prevention and treatment of pulmonary fibrosis with low side effects is increasingly urgent.

[0003] Atractylodes lancea, a traditional Chinese medicine, is believed to have the effects of drying dampness and strengthening the spleen, dispelling cold and wind, and improving eyesight. Clinically, it is mainly used to treat dampness in the middle jiao (middle burner), abdominal distension and diarrhea, and rheumatic pain. In the eighth edition of the COVID-19 diagnosis and treatment guidelines published by the National Health Commission, 14 traditional Chinese medicine formulas were recommended, with Atractylodes lancea appearing frequently, five times. Traditional Chinese medicine literature records that Atractylodes lancea has aromatic, dampness-resolving, and foul-smelling properties, and is widely used in the prevention and treatment of viral pneumonia and pulmonary fibrosis, showing certain advantages in efficacy, prognosis, and recovery. Existing research reports that Atractylodes lancea has effects such as anti-acute lung injury, treatment of allergic pneumonia, treatment of porcine reproductive and respiratory syndrome (PRRS), and promoting wound healing. Existing technology (a patent on the application of a traditional Chinese medicine composition in the preparation of a drug for treating pulmonary fibrosis) provides a drug made from Atractylodes lancea and other traditional Chinese herbs for treating pulmonary fibrosis, but it does not further investigate which specific compounds in Atractylodes lancea are effective in treating pulmonary fibrosis.

[0004] Based on the above, it is of great significance to develop low-toxicity and highly effective drugs for the prevention and treatment of pulmonary fibrosis. Summary of the Invention

[0005] The main objective of this invention is to overcome the lack of low-toxicity and highly effective drugs for treating pulmonary fibrosis in the prior art, and to provide the application of sesquiterpenoid compounds in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis. These sesquiterpenoid compounds exhibit a significant inhibitory effect on collagen deposition in TGF-β1-induced human embryonic lung fibroblasts without showing cytotoxicity, demonstrating good anti-pulmonary fibrosis activity, and can be used to prepare drugs for the prevention and treatment of pulmonary fibrosis.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] The use of sesquiterpenoid compounds in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis, wherein the sesquiterpenoid compounds have a structure as shown in any one of formulas (I) to (XI):

[0008]

[0009] In formula (Ⅰ), R1 is H or OH; in formula (Ⅱ), R2 is α-OH, β-OH or =O; in formula (Ⅲ), R3 is α-OH, β-OH, H or =O, and R4 is H or OH; in formula (Ⅶ), R5 is H or β-OH, R6 is H, α-OH or β-OH, and R7 is H or α-OH.

[0010] The specific sesquiterpenoids of this invention are extracted from Atractylodes lancea (see the inventors' previous research publication, 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). Sesquiterpenoids have 15 carbon atoms and contain two non-aromatic rings linked by either fused rings or spirocyclic rings.

[0011] The inventors of this invention have unexpectedly discovered that certain sesquiterpenoid compounds have a significant inhibitory effect on collagen deposition in TGF-β1-induced human embryonic lung fibroblasts without exhibiting cytotoxicity, and have good anti-pulmonary fibrosis activity, which can be used to prepare drugs for the prevention and treatment of pulmonary fibrosis.

[0012] Preferably, in formula (Ⅲ), R3 is α-OH and R4 is H; or R3 is β-OH and R4 is H; or R3 is H and R4 is OH; or R3 is H and R4 is H; or R3 is α=O and R4 is H.

[0013] Preferably, in formula (VII), R5 is H, R6 is H, and R7 is H; or R5 is β-OH, R6 is H, and R7 is H; or R5 is H, R6 is α-OH, and R7 is H; or R5 is H, R6 is β-OH, and R7 is H; or R5 is H, R6 is H, and R7 is α-OH.

[0014] Preferably, the sesquiterpene compound is any one of the compounds numbered MCZ-1 to 22:

[0015]

[0016] Preferably, the sesquiterpene compound is any one of the compounds numbered MCZ-1 to 14, 16 to 22:

[0017]

[0018] Sesquiterpenoids numbered MCZ-1~14, 16~22 exhibited good anti-pulmonary fibrosis activity and did not show cytotoxicity.

[0019] More preferably, the sesquiterpenoid compound is one of the compounds numbered MCZ-2, MCZ-3, MCZ-8, MCZ-11, MCZ-12, MCZ-13, MCZ14, MCZ-19, MCZ-20, or MCZ-22. These compounds exhibit better anti-pulmonary fibrosis activity, with a collagen inhibition rate of over 70% against HFL1 cells at 10 μM.

[0020] Typically, the drug also includes pharmaceutically acceptable excipients.

[0021] Optionally, the pharmaceutically acceptable excipient is a carrier.

[0022] Optionally, the pharmaceutically acceptable excipient is at least one of a lubricant, filler, binder, disintegrant, surfactant, antioxidant, or pH adjuster.

[0023] Typically, the dosage form of the drug is an injection, tablet, oral liquid, granule, or capsule.

[0024] Preferably, the sesquiterpene compound is used in the preparation of a drug that inhibits collagen deposition in pulmonary fibroblasts.

[0025] More preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] The specific sesquiterpenoid compounds of this invention have a significant inhibitory effect on collagen deposition in TGF-β1-induced human embryonic lung fibroblasts without showing cytotoxicity, and have good anti-pulmonary fibrosis activity, which can be used to prepare drugs for the prevention and treatment of pulmonary fibrosis. Attached Figure Description

[0028] Figure 1 Sesquiterpenoids MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, MCZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ -12. Molecular structure diagrams of MCZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21 and MCZ-22.

[0029] Figure 2 The figure shows the toxicity test results of sesquiterpenoid compounds MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, MCZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ-12, MCZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21 and MCZ-22 on HFL1 cells at a concentration of 10 μM for 48 hours.

[0030] Figure 3 The figure shows the results of the inhibition of collagen deposition in TGF-β1-induced HFL1 cells by sesquiterpenoid compounds MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, MCZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ-12, MCZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21, and MCZ-22 at a concentration of 10 μM. Detailed Implementation

[0031] To more clearly and completely describe the technical solution of the present invention, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Various changes can be made within the scope of the claims of the present invention.

[0032] Example 1

[0033] This embodiment provides sesquiterpenoid compounds MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, MCZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ-12, MCZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21, and MCZ-22. The preparation method of the sesquiterpenoid compounds mainly includes the following steps:

[0034] The dried rhizomes of Atractylodes lancea (Chinese Pharmacopoeia Editorial Committee. Chinese Pharmacopoeia [M]. 2020, 1:107) were pulverized and extracted three times with 80% acetone aqueous solution at room temperature for three days each time. The extract was concentrated under reduced pressure to obtain a crude extract (i.e., crude extract of Atractylodes lancea). The extract was suspended in water and extracted five times with ethyl acetate. The ethyl acetate fraction was concentrated under reduced pressure to obtain the ethyl acetate fraction. The fraction was subjected to silica gel column chromatography at 200-300 mesh, eluted with a gradient of petroleum ether / ethyl acetate (20:1, 10:1, 5:1, 2:1, 1:1, 1:0, v / v). The obtained fraction was then ethanolated to obtain sesquiterpenoid compounds. For more detailed steps and characterization information of each compound, please refer to the literature 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 respectively correspond to MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, M of the present invention. CZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ-12, MCZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21 and MCZ-22.

[0035] Figure 1It is MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, MCZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ-12, Molecular structure diagrams of MCZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21 and MCZ-22.

[0036] Example 2

[0037] This embodiment provides an injection for the prevention and treatment of pulmonary fibrosis, which is prepared by the following process: sesquiterpenoid compounds MCZ-1 to MCZ-22 are respectively mixed with excipients (such as starch paste) and then compressed into tablets.

[0038] Example 3

[0039] This embodiment provides a tablet for preventing and treating pulmonary fibrosis, which is prepared by the following process: sesquiterpenoid compounds MCZ-1 to MCZ-22 are mixed with excipients (such as polyethylene glycol 400) and then made into capsules.

[0040] Example 4: Evaluation of the anti-pulmonary fibrosis activity of sesquiterpenoid compounds MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, MCZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ-12, MCZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21 and MCZ-22.

[0041] 4.1 Cell Culture

[0042] The human embryonic lung fibroblast (HFL1) cell line used in this experiment was purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number CL-0106. HFL1 cells were cultured in Ham's F-12k medium containing 10% FBS and incubated at 37°C with 5% CO2. When the cells reached approximately 90% confluence, they were passaged. The appropriate cell density was adjusted according to experimental needs and seeded into culture plates or dishes. Cells were pre-starved for 12 hours with medium containing 1% FBS before TGF-β1 treatment.

[0043] 4.2 Evaluation of cytotoxicity using the CCK8 assay

[0044] HFL1 cells in logarithmic growth phase were divided into groups of 3 × 10⁻⁶. 3Cells were seeded per well in a 96-well plate. After 24 hours of culture, 10 μM of different sesquiterpenoid compounds were added to each well and incubated for 48 hours. Three replicates were set up, along with a control group and a zeroing well. The old culture medium was discarded and replaced with 100 μL of serum-free medium. The cells were then incubated in the dark with 10 μL of CCK-8 solution in each well for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated based on the absorbance of each well using the following method:

[0045]

[0046] Sesquiterpenoids MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, MCZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ-12, M The toxicity test results of CZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21 and MCZ-22 to HFL1 cells are as follows Figure 2 As shown.

[0047] 4.3 Sirius Red Staining Detection Experiment for Collagen Fiber Deposition

[0048] Human embryonic lung fibroblasts HFL1 were injected at a rate of 1×10 4 Cells were seeded into 96-well plates and cultured for 24 h, then synchronized with serum-free medium for 12 h. Each well was incubated with 5 ng / mTGF-β1 and 10 μM of different sesquiterpenoids for 48 h. The supernatant was removed, and the cells were fixed with 4% paraformaldehyde for 30 min. After washing twice with PBS, the cells were stained with Sirius red for 4 h. The staining solution was removed, and the cells were washed three times with 0.1% acetic acid to remove any remaining stain before imaging. After staining, 0.1M NaOH (100 ml / well) was added to each well, and the cells were shaken for 10 min to dissolve. The absorbance was measured at 540 nm using a microplate reader. The total collagen accumulation inhibition rate was calculated as follows:

[0049]

[0050] The inhibitory effects of sesquiterpenoid compounds MCZ-1, MCZ-2, MCZ-3, MCZ-4, MCZ-5, MCZ-6, MCZ-7, MCZ-8, MCZ-9, MCZ-10, MCZ-11, MCZ-12, MCZ-13, MCZ-14, MCZ-15, MCZ-16, MCZ-17, MCZ-18, MCZ-19, MCZ-20, MCZ-21, and MCZ-22 on TGF-β1-induced collagen deposition in human embryonic lung fibroblasts at a concentration of 10 μM are shown in the figure. Figure 3 As shown.

[0051] 4.4 Statistical Methods

[0052] Data analysis was performed using GraphPad Prism 7.0, and statistical results are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used, and LSD-t test was used for multiple comparisons. Figure 2 and 3 middle, ＊ This indicates that P < 0.05. ＊＊ This indicates that P < 0.01. ＊＊＊ A p < 0.001 is considered statistically significant. ns (not significant) indicates that the difference between groups is not statistically significant.

[0053] Figure 2 The results showed that the sesquiterpenoid compounds MCZ-1 to MCZ-22 did not exhibit cytotoxicity against HFL1 cells at a concentration of 10 μM for 48 hours, indicating that the sesquiterpenoid compounds of the present invention do not exhibit cytotoxicity.

[0054] Figure 3 The results showed that, at 10 μM, all sesquiterpenoids except MCZ-15 significantly inhibited TGF-β1-induced collagen deposition in HFL1 cells, demonstrating their anti-pulmonary fibrosis capabilities. Specifically, sesquiterpenoids MCZ-2, MCZ-3, MCZ-8, MCZ-11–MCZ-14, MCZ-19, MCZ-20, and MCZ-22 exhibited collagen inhibition rates greater than 70% in HFL1 cells at 10 μM.

[0055] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of sesquiterpenoid compounds in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis, characterized in that, The sesquiterpenoid compounds have structures as shown in any of formulas (I) to (III): Equation (I) Formula (II) Formula (Ⅲ); In formula (Ⅰ), R1 is H or OH; in formula (Ⅱ), R2 is α-OH, β-OH or =O. In formula (Ⅲ), R3 is α-OH and R4 is H; or R3 is β-OH and R4 is H; or R3 is H and R4 is OH; or R3 is H and R4 is H; or R3 is α=O and R4 is H.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

3. The application according to claim 1, characterized in that, The dosage form of the drug is injection, tablet, oral liquid, granules or capsule.

4. The application according to claim 1, characterized in that, The sesquiterpenoid compounds inhibit collagen deposition in lung fibroblasts.

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

6. The application according to claim 1, characterized in that, The pulmonary fibrosis mentioned is idiopathic pulmonary fibrosis.

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