Active component for preventing and / or treating pulmonary fibrosis and pharmaceutical application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to an application of an active component in preparation of a medicine for preventing and / or treating pulmonary fibrosis, the active component is a compound # imgabs0 # with a structure shown in a formula 1, and the invention also comprises the medicine for treating pulmonary fibrosis containing the active component and a combined medicine of a medicine combination capable of inducing pulmonary fibrosis. Researches show that the compound with the structure in the formula 1 can realize synergy based on the combination of molecular structures of the compound, can show excellent activity for treating and / or preventing pulmonary fibrosis, and can be used for preparing medicines for preventing and / or treating pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of drug preparation, and in particular to the field of drug preparation for pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a terminal change of a large class of lung diseases characterized by fibroblast proliferation and accumulation of large amounts of extracellular matrix, accompanied by inflammatory damage and destruction of tissue structure. It is one of the four major respiratory diseases and the most serious pathological condition of the lungs. Its pathological changes are mostly manifested as initial inflammation of the lower respiratory tract and damage to alveolar epithelial cells and vascular endothelial cells, accompanied by proliferation of fibroblasts and type II alveolar cells, release of cytokines, and deposition of extracellular matrix proteins and collagen, which ultimately cause lung changes. In patients with pulmonary fibrosis, the alveoli of the lungs are gradually replaced by fibrous substances, causing the lung tissue to harden and thicken, and the lungs to gradually lose their gas exchange capacity, resulting in patients experiencing dyspnea due to varying degrees of hypoxia, and ultimately death from respiratory failure.

[0003] There are some essential differences between pulmonary fibrosis and other fibrosis, such as liver fibrosis. Liver fibrosis is a chronic liver disease caused by multiple causes. Its main feature is the diffuse and excessive deposition of extracellular matrix (especially collagen) in the liver. Pulmonary fibrosis, liver fibrosis and fibrosis of different organs are all organ fibrosis lesions, but different cell types in different organs are involved in the occurrence and development of fibrosis, such as hepatic stellate cells, pancreatic stellate cells, fibroblasts and myofibroblasts. The causes and pathogenesis of fibrosis in different organs are also different. Therefore, the clinical treatment methods for fibrosis of different organs are also different.

[0004] It can be seen that for fibrosis of different organs, the causes and phenotypes of the diseases are different, the treatment methods are difficult to switch, and the treatment effects of the switches are difficult to predict. For pulmonary fibrosis, its etiology is complex and the pathogenesis is unclear. The current drugs and methods for treating pulmonary fibrosis are very limited, mainly including some gene and small molecule modification schemes. For example, the Chinese patent document with publication number CN118286250A discloses an application of miR-15b in the preparation of drugs for treating pulmonary fibrosis. The patent document with publication number WO2024139978A1 discloses a method for treating pulmonary fibrosis by using Vofopitant to improve pulmonary inflammatory lesions and reduce collagen deposition. The Chinese patent document with publication number CN114984026A discloses an application of glycyrrhetinic acid monosaccharide in the preparation of drugs for preventing and treating pulmonary fibrosis.

[0005] In summary, although there are some methods for treating pulmonary fibrosis in existing technologies, it is still difficult for existing methods to take into account multiple advantages such as efficacy, low side effects and cost. The industry needs to further expand the treatment methods for pulmonary fibrosis. Summary of the Invention

[0006] In view of the problems in the prior art, the present invention aims to provide an active ingredient for use in preparing a drug for preventing and / or treating pulmonary fibrosis.

[0007] The present invention also provides a pharmaceutical composition comprising the active ingredient.

[0008] Use of an active ingredient in the preparation of a drug for preventing and / or treating pulmonary fibrosis, wherein the active ingredient is a compound having the structure of Formula 1 and a pharmaceutically acceptable crystal, solvate or salt thereof;

[0009]

[0010] The present invention studies have shown that the compounds of formula 1 can achieve synergy based on the combination of their molecular structures, can exhibit excellent activity in treating and / or preventing pulmonary fibrosis, and can be used to prepare drugs for preventing and / or treating pulmonary fibrosis.

[0011] In the present invention, the pulmonary fibrosis is at least one of primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, immune pulmonary fibrosis, and drug-induced pulmonary fibrosis.

[0012] In the present invention, the secondary pulmonary fibrosis is drug-induced pulmonary fibrosis.

[0013] In the present invention, the drug that induces drug-induced pulmonary fibrosis includes at least one of rituximab, propranolol, bleomycin and nitrofurantoin.

[0014] In the present invention, the active ingredient is combined with a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable dosage form; for example, it can be an oral preparation and / or an injectable preparation.

[0015] The present invention also provides a drug for preventing and / or treating pulmonary fibrosis, comprising a pharmaceutically effective amount of the active ingredient of formula 1.

[0016] The drug for preventing and / or treating pulmonary fibrosis of the present invention further comprises pharmaceutically acceptable excipients.

[0017] The drug for preventing and / or treating pulmonary fibrosis of the present invention has a pharmaceutically acceptable dosage form.

[0018] The drug for preventing and / or treating pulmonary fibrosis of the present invention is in the form of oral or injection administration.

[0019] The present invention also provides an anticancer pharmaceutical composition capable of preventing and / or treating pulmonary fibrosis, comprising an anticancer active ingredient that causes pulmonary fibrosis and an active ingredient of formula 1;

[0020] The anticancer active ingredient comprises at least one of toximab, propranolol, bleomycin and nitrofurantoin.

[0021] The present invention has shown that the formula 1 of the present invention can be combined with known anticancer active ingredients capable of inducing pulmonary fibrosis to obtain excellent anticancer activity while also preventing and treating the side effects of pulmonary fibrosis caused by the anticancer drugs.

[0022] In the present invention, the formula 1 has high biological safety and will not cause any side effects or dependence to the human body. In addition, it can also enhance the body's response to immunotherapy. Moreover, formula 1 can inhibit Th17 cell differentiation and IL17 signaling pathway both in vivo and in vitro, thereby weakening lung fibroblast proliferation, reducing collagen deposition, and alleviating pulmonary fibrosis.

[0023] Beneficial effects

[0024] This invention applies Formula 1 to research related to pulmonary fibrosis for the first time, demonstrating for the first time that Formula 1 can significantly improve bleomycin-induced pulmonary fibrosis and revealing its mechanism of action as inhibition of the IL-17 signaling pathway. Compared to conventional symptomatic treatments, Formula 1 does not cause any side effects or dependency, and can enhance the body's response to immunotherapy, making it safe to use.

[0025] In addition, the present invention has also found that the formula 1 can be used to prevent and / or treat drug-induced pulmonary fibrosis caused by drugs such as bleomycin. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are the Masson-stained pathological images of each group.

[0027] Figure 2 The results show the hydroxyproline content in the lung tissue of bleomycin mice treated with normal saline or different drugs.

[0028] Figure 3 The results are shown in Table 1. The results show the expression of fibrosis-related genes in the lung tissue of mice treated with saline or Formula 1.

[0029] Figure 4 The expression of IL17A, RORγT, IL23R, and CCR6 genes in Example 3;

[0030] Figure 5 This is a graph showing the results of formula 1 inhibiting Th17 cell differentiation. DETAILED DESCRIPTION

[0031] In the present invention, conventional pharmaceutical methods can be used to prepare drugs with Formula 1 as the active ingredient or with other pharmaceutical carriers, and can be prepared into clinically acceptable conventional oral or injectable dosage forms in pharmacy, such as tablets, capsules, powders and injections.

[0032] In the present invention, the dosage of Formula 1 is 50 mg / kg; preferably, the continuous use period of Formula 1 is 30 days. Furthermore, the administration method includes subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, and oral gavage; preferably, the administration method is oral gavage.

[0033] In the present invention, the pulmonary fibrosis may be pulmonary fibrosis of any form and stage, for example, it may be at least one of primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and drug-induced pulmonary fibrosis.

[0034] In the present invention, the drug-induced pulmonary fibrosis can be drug-induced pulmonary fibrosis caused by the treatment phase of a pharmacodynamic molecule with a side effect of inducing pulmonary fibrosis. For example, the pharmacodynamic molecule can be bleomycin. Formula 1 described in the present invention can be used to prevent and / or treat pulmonary fibrosis caused by bleomycin treatment, and can be administered before, during, and after bleomycin treatment to prevent and / or alleviate pulmonary fibrosis that may be caused by bleomycin. "Treatment" described in the present invention refers to reducing the degree of pulmonary fibrosis, or curing pulmonary fibrosis to normalize it, or slowing down the progression of pulmonary fibrosis.

[0035] The "treatment" mentioned in the present invention refers to reducing the degree of pulmonary fibrosis, or curing pulmonary fibrosis to normalize it, or slowing down the progression of pulmonary fibrosis.

[0036] The reagents and raw materials used in the present invention are commercially available.

[0037] Example 1 Preparation of Pulmonary Fibrosis Animal Model

[0038] 1.1 Main reagents and experimental animals

[0039] The bleomycin used in the experiment was purchased from Nippon Kayaku, batch number X81040.

[0040] Unless otherwise specified, all compounds used in the experiments were purchased from Sigma.

[0041] The SPF-grade C57BL / 6 mice used in the experiment (male, 6-8 weeks old, 16-18 g) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.

[0042] 1.2 Preparation of animal model of pulmonary fibrosis

[0043] Male C57BL / 6 mice (aged 6-8 weeks) were fasted overnight, anesthetized with sodium pentobarbital (45 mg / kg, ip), and injected intratracheally with bleomycin (1 U / kg) for a total of 6 doses, each time with an interval of 14 days.

[0044] The specific protocol is as follows: After anesthesia, place the mouse in the prone position. Illuminate the neck with a cold light source. Using forceps in the right hand, pull the tongue outward while using forceps in the left hand, open the mouth as much as possible until the glottis is exposed. A 20G trocar is then inserted into the mouse's trachea over a guidewire. Approximately 50 μL of bleomycin is then injected into the trachea using a microinjection needle. The mouse is then rapidly rotated and held upright for 5 minutes to ensure even distribution of the bleomycin to both lung lobes. The entire procedure is performed on an operating table at approximately 60°C.

[0045] Example 2 Treatment and efficacy analysis of mice with pulmonary fibrosis

[0046] 2.1. Formula 1 for the treatment of mice with bleomycin-induced pulmonary fibrosis

[0047] The specific scheme is: after the animal model prepared in Example 1 is successfully established, the animal model is divided into groups and dosed. The grouping and dosing conditions are shown in Table 1:

[0048] Table 1 Grouping and drug administration of pulmonary fibrosis animal model after modeling

[0049]

[0050] Note: The structure of comparative formula a is:

[0051] Contrast b

[0052] 2.2 Masson staining pathological imaging analysis of mice with pulmonary fibrosis

[0053] Masson staining is a classic and authoritative method for staining collagen fibers. After staining, muscle fibers appear red, while collagen fibers appear blue, which mainly distinguishes muscle fibers from collagen fibers.

[0054] The right lower lobe lung tissue of the animal was obtained, fixed with 4% paraformaldehyde, and then embedded in paraffin. Sections were made on the largest surface of the paraffin block of the embedded lung tissue and stained with Masson's stain to observe the fibrosis. High-definition color pathology image analysis system SpotAdvanced3.0 was used to obtain high-definition Masson's special staining pathology images (200 times). The results are shown in Figure 1 The results showed that the area of ​​blue collagen fibers in the lung tissue of mice increased significantly after bleomycin administration, while the area of ​​blue collagen fibers in the lung tissue of mice decreased significantly after treatment with Formula 1, while the other control groups did not reduce the collagen deposition in the lung tissue.

[0055] 2.3 Determination of Hydroxyproline Content in Mice with Pulmonary Fibrosis

[0056] Hydroxyproline accounts for 13.4% of collagen, a very small amount of elastin, and does not exist in other proteins. Therefore, the content of collagen is detected by hydroxyproline. The hydroxyproline content of the entire left lung lobe of the animal was detected to evaluate the condition of pulmonary fibrosis. The specific method is as follows: take all the left lung lobes of the model animal prepared in Example 1, record the wet weight, prepare a 10% tissue homogenate with normal saline ultrasonic homogenization, take about 150 μL of the homogenate supernatant, add 500 μL of alkaline hydrolyzate, vortex mix, and treat with alkaline hydrolysis at 120 degrees and 0.1Kpa for 40 minutes (the method refers to the kit instructions of Nanjing Jiancheng Bioengineering Technology Co., Ltd., with slight changes), adjust the pH value and make the volume constant, and take the supernatant after activated carbon treatment. Hydroxyproline determination was carried out according to the instructions (chloramine T method). The results are shown in Table 2 and Figure 2 As can be seen from Table 2, the hydroxyproline content in the bleomycin group was significant, indicating that the pathological changes of fibrosis were serious. Formula 1 treatment could significantly reduce the hydroxyproline content in the lungs of mice with pulmonary fibrosis, while the other three drugs did not reduce the hydroxyproline content in the lungs of mice with pulmonary fibrosis.

[0057] Table 2 Formula 1 reduces the hydroxyproline content in lung tissue of mice with pulmonary fibrosis

[0058]

[0059]

[0060] 2.4 Determination of pulmonary fibrosis-related gene expression in pulmonary fibrosis mice

[0061] Acta2, FN1, Col1a1, and Col3a1 genes are commonly used to assess mouse pulmonary fibrosis. Increased expression levels of these genes in lung tissue indicate the severity of lung fibrosis.

[0062] Real-time PCR was used to detect the expression of Acta2, FN1, Col1a1, and Col3a1 genes: Lung tissue was ground and total RNA was extracted using the Trizol method. Chloroform, isopropanol, and 75% anhydrous ethanol were sequentially added to extract RNA. A gel-like precipitate of RNA was dissolved in RNase-free water. Once completely dissolved, a small amount was sampled for RNA quality testing. The spectrophotometer was zeroed with RNase-free water, and the absorbance at 260 nm and 280 nm was read to determine the RNA concentration. The A260 / A280 ratio of the RNA solution was used to determine RNA purity, ensuring a range of 1.8 to 2.1. Reverse transcription was performed according to established laboratory procedures to generate cDNA. The PCR reaction mixture was prepared according to the Real-time PCR reagent manual. A predetermined amount of cDNA was added to the primers, and the RNA was plated on a 384-well plate for analysis of Acta2, FN1, Col1a1, and Col3a1 expression levels.

[0063] Table 3 Formula 1 reduces the expression levels of pulmonary fibrosis-related genes in lung tissue of mice with pulmonary fibrosis

[0064]

[0065] In this experiment, pathological examination, pathological imaging, and other analysis methods revealed that Formula 1 significantly inhibited bleomycin-induced pulmonary fibrosis and significantly reduced hydroxyproline and collagen content in the lung tissue of mice with pulmonary fibrosis, as well as the expression levels of genes associated with pulmonary fibrosis. These experimental results demonstrate that Formula 1 has excellent therapeutic potential for pulmonary fibrosis.

[0066] The results of the above examples show that Formula 1 of the present invention has a significant effect in resisting pulmonary fibrosis and can be used as an active ingredient in the preparation of anti-pulmonary fibrosis drugs.

[0067] Example 3: Mechanism of Formula 1 in Alleviating Pulmonary Fibrosis

[0068] 3.1 Determination of IL17 signaling pathway-related gene expression in lung tissue of mice with pulmonary fibrosis

[0069] IL17A, RORγT, IL23R, and CCR6 are typical genes of the IL17 signaling pathway and are often used to evaluate the expression strength of this pathway.

[0070] Real-time PCR was used to detect the expression of IL17A, RORγT, IL23R, and CCR6 genes; the specific operation steps were the same as above. The results are shown in Table 4 and Figure 4 .

[0071] Table 4: Formula 1 inhibits the expression of genes related to the IL17 signaling pathway in mice with pulmonary fibrosis

[0072]

[0073] 3.2 Formula 1 inhibits Th17 cell differentiation

[0074] The specific plan is: 96-well plates were coated with CD3 and CD28 one night in advance, mouse spleens were separated, red blood cells were lysed, and CD4 + Mouse T cells were isolated using a T cell negative selection kit and differentiated into Th17 cells under the stimulation of IL6, TGFβ, IL23, and IL1β. Formula 1 solution was then added. Real-time PCR was used to detect the expression of IL17A and RORγT to verify the effect of Formula 1 on Th17 differentiation. Group F served as the control group, Group G served as the Th17 induced differentiation group, and Group H served as the Th17 + Formula 1 group. The results are shown in Tables 5 and Figure 5 .

[0075] Table 5: Formula 1 inhibits the differentiation of Th17 cells in vitro

[0076]

[0077] In this experiment, Formula 1 can inhibit the IL17 signaling pathway both in vivo and in vitro, thereby alleviating pulmonary fibrosis.

[0078] The results of the above examples show that Formula 1 alleviates pulmonary fibrosis by inhibiting the IL17 signaling pathway, and its active ingredients have good pharmaceutical prospects.

[0079] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of an active ingredient in the preparation of a drug for preventing and / or treating pulmonary fibrosis, characterized in that: The active ingredient is a compound having a structure of Formula 1 and a pharmaceutically acceptable crystal, solvate or salt thereof; 2. The use according to claim 1, characterized in that: The pulmonary fibrosis is at least one of primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, immune pulmonary fibrosis, and drug-induced pulmonary fibrosis.

3. The use according to claim 2, characterized in that The secondary pulmonary fibrosis is drug-induced pulmonary fibrosis.

4. The use according to claim 3, characterized in that Drugs that induce drug-induced pulmonary fibrosis include at least one of rituximab, propranolol, bleomycin, and nitrofurantoin.

5. The use according to any one of claims 1 to 4, characterized in that: The active ingredients are combined with pharmaceutically acceptable excipients to prepare pharmaceutically acceptable dosage forms.

6. The use according to claim 5, characterized in that The dosage form is an oral preparation or an injection preparation.

7. A drug for preventing and / or treating pulmonary fibrosis, characterized in that: The invention comprises a pharmaceutically effective amount of the active ingredient of formula 1 described in any one of claims 1 to 6.

8. The drug for preventing and / or treating pulmonary fibrosis according to claim 7, characterized in that: Also contains pharmaceutically acceptable excipients.

9. The drug for preventing and / or treating pulmonary fibrosis according to claim 8, characterized in that: It has a pharmaceutically acceptable dosage form; preferably an oral or injectable dosage form.

10. An anticancer pharmaceutical composition capable of preventing and / or treating pulmonary fibrosis, characterized in that: Containing an anticancer active ingredient that causes pulmonary fibrosis and the active ingredient of formula 1 described in any one of claims 1 to 6; The anticancer active ingredient comprises at least one of toximab, propranolol, bleomycin and nitrofurantoin.

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