WO2023083054A1

WO2023083054A1 - Application of delamanid in preparation of drug for preventing and/or treating lung injury and pulmonary fibrosis

Info

Publication number: WO2023083054A1
Application number: PCT/CN2022/128945
Authority: WO
Inventors: 李平平; 万彦军; 崔冰; 马春晓; 姜茜; 孔丽娟; 刘姗姗; 徐晓军; 侯少聪; 柳星峰; 赵其锦; 邢才轶
Original assignee: 中国医学科学院药物研究所
Priority date: 2021-11-09
Filing date: 2022-11-01
Publication date: 2023-05-19

Abstract

An application of delamanid in preparation of a drug for preventing and/or treating lung injury and pulmonary fibrosis, and use of delamanid in prevention and/or treatment of a silicosis disease. The target of delamanid is Galectin 3. Implementation results of the present invention show that oral administration of delamanid to SiO2-induced silicosis mice can inhibit collagen deposition, delay pulmonary fibrosis, and reduce the number and size of silicotic nodules in lung tissue, and the effect is better than that of pirfenidone. The delamanid can be used in preparation of a drug for preventing and/or treating a silicosis disease.

Description

Application of Dilamanid in the preparation of drugs for preventing and/or treating lung injury and pulmonary fibrosis

technical field

The present invention relates to the technical field of medicine, in particular to the application of Dilamanid in the preparation of drugs for the prevention and/or treatment of lung injury and pulmonary fibrosis, and to a compound of Dilamanid in the preparation of drugs for the treatment of silicosis Applications.

Background technique

Due to its physiological characteristics of opening and exchanging gas with the outside world, the respiratory system is vulnerable to various viruses, bacterial infections and physical and chemical damage, which can cause acute lung injury (Acute lung injury, ALI) and its severe state acute respiratory distress syndrome (Acute respiratory distress syndrome). respiratory distress syndrome, ARDS). The pathological features of ALI and ARDS are injury of alveolar capillaries, endothelial cells and alveolar epithelial cells, manifested as extensive pulmonary edema, microscopic atelectasis, increased intrapulmonary shunt and decreased lung compliance. At present, the treatment of ALI and ARDS mainly adopts glucocorticoid and ventilator-assisted therapy, but many patients have sequelae after recovery, such as diabetes, hypertension, femoral head necrosis, etc., which seriously affect the quality of life. Therefore, there is an urgent need to find safe and effective drugs for the prevention and/or treatment of lung injury.

Pulmonary fibrosis (PF) is the end stage of all interstitial lung diseases (Interstitial lung Disease, ILD). PF is caused by a variety of etiologies, and is mainly characterized by the accumulation of inflammatory cells such as macrophages, neutrophils, and lymphocytes in the alveoli and the accumulation of fibrous connective tissue in the lung tissue, which eventually leads to structural changes in the lung tissue and impaired lung function. damage, respiratory failure. In recent years, affected by factors such as environment and lifestyle, the incidence and mortality of pulmonary fibrosis have been increasing year by year.

Idiopathic pulmonary fibrosis (IPF) is a relatively common type of PF and is a chronic, progressive, interstitial fibrosis lung disease. It is characterized by unexplained recurrent epithelial cell injury, aging of alveolar epithelial cells, accumulation of pro-fibrotic mediators, etc. leading to progressive lung tissue scarring. IPF patients only have a survival period of 2-6 years after diagnosis, generally 3 years. In 2014, the FDA approved pirfenidone and nintedanib for the treatment of IPF, but they can only delay the progression of the disease, and it is unknown whether they can prolong the survival period. Therefore, it is also very important to find safe and effective drugs for preventing and/or treating the occurrence and development of pulmonary fibrosis.

Galectin-3 (Galectin-3) is a β-galactoside-binding lectin, often in the form of homodimers, mainly expressed in tumor cells, macrophages, epithelial cells, fibroblasts and in activated T cells. Important in many biological activities in various organs, including cell proliferation, regulation of apoptosis, inflammation, fibrosis and host defense, etc. Galectin-3 mainly exists in the cytoplasm, and is also expressed in the nucleus and cell surface, and can also be directly secreted by vesicles into biological fluids, such as serum, urine and tissue fluid. Many studies have shown that Galectin-3 can be used as an important biomarker for the diagnosis or prognosis of myocardial fibrosis, renal fibrosis, pulmonary fibrosis, viral infection, autoimmune disease, neurodegenerative disease and tumor formation.

Therefore, inhibiting Galectin 3 may become an effective solution for the prevention and/or treatment of lung injury and pulmonary fibrosis-related diseases.

Silicosis, also known as silicosis, is the most common, fastest-growing, and most harmful type of pneumoconiosis. Its main pathological features are chronic inflammation of lung tissue and continuous formation of silicon nodules. In the advanced stage, severe interstitial fibrosis and respiratory failure are the main symptoms, which can easily lead to death. In addition to getting rid of dust exposure as soon as possible, clinical treatment options for silicosis patients include lung lavage, oxygen therapy and drug therapy. Therapeutic drugs include drugs that delay the process of pulmonary interstitial fibrosis, prevent dust from invading the lung interstitium, promote the discharge of dust in the lungs, improve the body's immunity, and relieve symptoms. However, clinical studies have shown that none of the above treatments can effectively alleviate the progression of the disease, nor can it reverse the damage that has been caused. Lung transplantation is the only option for advanced patients. Therefore, it is particularly urgent to explore new drugs to inhibit the progression of silicosis.

Dilamani, also known as Dramani, is a First-in-Class antibiotic developed by Otsuka Holdings Co Ltd. It is a cell wall inhibitor whose mechanism of action is to interfere with the metabolism of the cell wall of Mycobacterium tuberculosis. The drug is currently approved for marketing for the treatment of multidrug-resistant tuberculosis and pulmonary tuberculosis.

Silicosis injury is caused by long-term inhalation of a large amount of free silicon dioxide (SiO ₂ ) dust. Unlike tuberculosis, SiO ₂ is mainly recognized and phagocytized by macrophages after entering the lungs, and then the macrophages that phagocytose the silica dust undergo a strong inflammatory reaction Afterwards, it ruptures and releases the previously phagocytosed silica dust to continue to attack other macrophages, resulting in continuous destruction of macrophages and damage to other intrinsic cells in the lung, resulting in continuous occurrence of inflammatory damage in the lung; with the continuous inflammatory response, SiO ₂ induces Granulomatous cells aggregate into clusters to form granulomatous nodules (silicon nodules) centered on silica dust, and the nodules will gradually increase, seriously destroying the normal structure of the lungs and impairing ventilation/ventilation function. Patients with advanced silicosis will develop Respiratory failure develops. The clinically existing anti-tuberculosis drugs have no indications for the treatment of silicosis, and the drugs already used for the treatment of silicosis have no indications for the treatment of tuberculosis. Therefore, the etiology, pathogenesis and existing treatments of tuberculosis and silicosis are completely different. At present, there is no literature report on the application of the compound in the preparation of medicines for treating silicosis.

Contents of the invention

The first aspect of the present invention aims at the current lack of safe and effective drugs for the prevention and/or treatment of lung injury and pulmonary fibrosis, and provides a compound dilamanid targeting Galectin 3 signaling pathway and its pharmaceutically acceptable salt in The application of drugs for preventing and/or treating lung injury and pulmonary fibrosis provides a new approach for treating diseases related to lung injury and pulmonary fibrosis.

To achieve the above object, the present invention provides the following scheme:

One of the technical solutions of the present invention is to provide the application of dilamanid and pharmaceutically acceptable salts thereof in the preparation of Galectin 3 inhibitors.

The second technical solution of the present invention is to provide the application of dilamanid and pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and/or treating lung injury diseases.

The "lung injury" mentioned in the present invention may be acute lung injury or acute respiratory distress syndrome. The etiology of lung injury disease includes bacterial and viral infection, gastroesophageal reflux, massive blood transfusion, drowning, shock, pancreatitis, lung contusion, salicylate anesthetic overdose, alveolar hemorrhage, fat and amniotic fluid embolism, smoke and toxic Lung injury due to inhalation of gases, said bacteria and viruses, such as lipopolysaccharide.

The third technical solution of the present invention is to provide the application of dilamanid and pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and/or treating pulmonary fibrosis.

The "pulmonary fibrosis" mentioned in the present invention may be idiopathic pulmonary fibrosis or secondary pulmonary fibrosis. The etiology of the pulmonary fibrosis disease includes pulmonary fibrosis caused by dust, radiation and/or drugs, such as bleomycin.

Further, the dosage forms of the inhibitor include injections, tablets, powders, granules, pills, capsules, oral liquids, ointments, and creams.

Further, the pharmaceutical dosage forms include injections, tablets, powders, granules, pills, capsules, oral liquids, ointments, and creams.

According to the present invention, the compound of the present invention may exist in the form of isomers, and generally speaking "the compound of the present invention" includes the isomers of the compound.

The present invention also relates to a pharmaceutical composition containing the compound in a pharmaceutically effective dose and a pharmaceutically acceptable carrier. When used for this purpose, if necessary, it can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to make an appropriate administration form or dosage form for human medicine.

The pharmaceutical composition of the present invention can be administered in the form of a unit dose, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneal or rectal, etc.

The administration route of the pharmaceutical composition of the present invention can be injection administration. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection. The dosage forms for administration may be liquid dosage forms and solid dosage forms. For example, the liquid dosage forms can be true solutions, colloids, microparticles, emulsions and suspensions. Other dosage forms such as tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories and lyophilized powder injections, etc.

The composition of the present invention can be made into common preparations, and can also be sustained-release preparations, controlled-release preparations, targeted preparations and various particle delivery systems.

Various carriers known in the art can be widely used for tableting unit dosage forms. Examples of carriers such as diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate Wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, arabic mucilage, gelatin slurry, sodium carboxymethylcellulose , shellac, methylcellulose, potassium phosphate and polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbate Sugar alcohol fatty acid esters, sodium lauryl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers , such as quaternary ammonium salts and sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

In order to formulate a dosage unit into a pellet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, Gelucire, kaolin, and talc; binders such as acacia, tragacanth, Gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecylsulfonate, methylcellulose and ethylcellulose, etc.

In order to formulate the administration unit into a suppository, various carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, enzymes of higher alcohols, gelatin, semisynthetic glycerolase and the like.

To form a dosage unit into a capsule, the active ingredient is mixed with the various carriers mentioned above, and the mixture thus obtained is placed in a hard gelatin capsule or a soft capsule. The active ingredients can also be made into microcapsules, suspended in an aqueous medium to form a suspension, and can also be packed into hard capsules or made into injections for application.

For example, the composition of the present invention is made into injection preparations, such as solutions, suspension solutions, emulsions, and freeze-dried powder injections. This preparation can be aqueous or non-aqueous, and can contain one and/or more A pharmaceutically acceptable carrier, diluent, binder, lubricant, preservative, surface active agent or dispersant. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid enzyme, and the like. In addition, in order to prepare isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the preparation for injection, and in addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. These excipients are commonly used in the art.

In addition, coloring agents, preservatives, fragrances, correctives, sweeteners or other materials can also be added to the pharmaceutical preparations as needed.

The dosage of the pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, personality and individual response of the patient or animal, the route of administration, the frequency of administration, etc. , so the therapeutic dosage of the present invention can vary widely. In general, the dosages used for the compounds of the present invention are well known to those skilled in the art. Can be adjusted appropriately according to the actual effective drug quantity contained in the final preparation in the pharmaceutical composition of the present invention, to reach the requirement of its therapeutically effective dose, complete the present invention in preventing and/or treating pulmonary fibrosis and acute pulmonary fibrosis. Drug application in injury-related diseases.

Usually, for patients with a body weight of about 75 kg, the daily dose of the compound of the present invention is 0.001 mg/kg body weight to 200 mg/kg body weight, preferably 1 mg/kg body weight to 100 mg/kg body weight. The above-mentioned dosage may be administered in a single dosage form or divided into several, for example, two, three or four dosage forms, which are limited by the clinical experience of the administering physician and the dosage regimen. The compound or composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs.

The first aspect of the present invention discloses the following technical effects:

The present invention discovers for the first time that the compound dilamanid acts on Galectin 3, which has good application prospects in the preparation of drugs for the prevention and/or treatment of lung injury and pulmonary fibrosis, and has the characteristics of significant effects and low toxic and side effects.

The second aspect of the present invention aims at the current lack of effective drugs for treating silicosis, and provides an application of the compound dilamanid and its pharmaceutically acceptable salts in the treatment of silicosis, so as to fill the gap of drugs for treating silicosis. The invention also discloses a medicine for treating silicosis, the active ingredient of which is dilamani.

The first aspect of the technical solution of the present invention is to provide the application of delamanid represented by formula (I) and pharmaceutically acceptable salts thereof in the preparation of medicaments for treating silicosis.

Preferably, in the application, the dosage of delamanid for human body is 4.40 mg/kg/day.

According to the embodiment of the present invention, according to the mouse body weight 20g and the human body weight 70kg, the drug dose conversion is carried out: the mouse dosage is 40mg/kg/day, and the corresponding human body dosage is: 4.40mg/kg/day, i.e. 308mg/day day. The conversion formula is mouse dose (mg/kg/day)=9.1*human dose (mg/kg/day). Therefore, preferably, the dosage of delamanid for human body is 4.40 mg/kg/day.

The second aspect of the technical solution of the present invention provides an application of a pharmaceutical composition in the preparation of a drug for the treatment of silicosis, characterized in that, the pharmaceutical composition comprises dilamanid as shown in formula (I) and Its pharmaceutically acceptable salt, and pharmaceutically acceptable carrier or excipient;

Further, the pharmaceutical composition includes injections, tablets, powders, granules, pills, capsules, oral liquids, ointments, and creams.

The present invention also relates to a pharmaceutical composition containing the compound in a pharmaceutically effective dose and a pharmaceutically acceptable carrier. When used for this purpose, it can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, if necessary, to make an appropriate administration form or dosage form that can be used as human medicine.

The administration route of the pharmaceutical composition of the present invention can be injection administration. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection. The dosage forms for administration may be liquid dosage forms and solid dosage forms. For example, the liquid dosage forms can be true solutions, colloids, microparticles, emulsions and suspensions. Other dosage forms such as tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories and freeze-dried powder injections, etc.

Various carriers known in the art can be widely used for tableting unit dosage forms. Examples of carriers such as diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate Wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, arabic mucilage, gelatin slurry, sodium carboxymethylcellulose , shellac, methylcellulose, potassium phosphate and polyvinylpyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol Sugar alcohol fatty acid esters, sodium lauryl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers , such as quaternary ammonium salts and sodium lauryl sulfate, etc.; lubricants, such as talc, SiO ₂ , corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. In order to formulate a dosage unit into a pellet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, Gelucire, kaolin, and talc; binders such as acacia, tragacanth, Gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecylsulfonate, methylcellulose and ethylcellulose, etc.

To form a dosage unit into a capsule, the active ingredient is mixed with the above-mentioned various carriers, and the mixture thus obtained is placed in a hard gelatin capsule or a soft capsule. The active ingredients can also be made into microcapsules, suspended in an aqueous medium to form a suspension, and can also be packed into hard capsules or made into injections for application.

Beneficial technical effects of the second aspect of the present invention:

The purpose of the present invention is to overcome the blank of effective medicine for treating silicosis at present, and provide a new application of dilamani in medicine.

The invention discloses a new application of dilamanid in the preparation of medicines for treating silicosis.

Description of drawings

Fig. 1 is the body weight change curve of mice in each group of lung injury model;

Figure 2 shows that Dilamanid regulates the expression changes of inflammation-related genes in the lung injury model;

Figure 3 is the pathological results of each group of lung injury model, Figure 3A is HE staining, Figure 3B is Masson staining;

Fig. 4 is the body weight change curve of mice in each group of pulmonary fibrosis model;

Figure 5 shows that Dilamanid regulates the expression changes of inflammation-related genes in the pulmonary fibrosis model;

Figure 6 is the pathological results of each group of pulmonary fibrosis model, Figure 6A is HE staining, and Figure 6B is Masson staining.

Fig. 7 is the body weight change curve of silicosis model mice;

Figure 8 shows that Dilamani improved the lung tissue morphology of silicosis model mice;

Figure 9 is the pathological results of each group of silicosis model, Figure 3A is HE staining, Figure 3B is Masson staining;

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. The detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present invention.

It should be understood that the terminology described in the present invention is only used to describe specific embodiments, and is not used to limit the present invention. In addition, regarding the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents are described. In case of conflict with any incorporated document, the contents of this specification control.

It will be apparent to those skilled in the art that various modifications and changes can be made in the specific embodiments of the present invention described herein without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the present invention. The description and examples of the invention are illustrative only.

As used herein, "comprising", "comprising", "having", "comprising" and so on are all open terms, meaning including but not limited to.

The "parts" mentioned in the present invention are all in parts by mass unless otherwise specified.

The effect of embodiment 1 Dilamanid on Galectin 3 inhibitor model

1. the experimental material of the present embodiment comprises:

Experimental drug: Dilamani, purchased from MCE Company, its structural formula is:

(2R)-2,3-Dihydro-2-methyl-6-nitro-2-[[4-[4-[4-(trifluoromethoxy)phenoxy]-1-piperidinyl ]phenoxy]methyl]imidazo[2,1-B]oxazole

Test sample solution configuration: Accurately weigh an appropriate amount of Dilamani sample, and prepare a 0.02M storage solution with DMSO for in vitro activity testing.

Experimental reagent: MTT reagent, purchased from Beijing Suo Laibao Technology Co., Ltd., accurately weighed 0.25g of MTT powder, added 50mL PBS to dissolve, filtered through a 0.22μm microporous membrane after dissolution, aliquoted, and stored at -20°C.

Experimental instruments: carbon dioxide incubator, Panasonic Company, Japan; microplate reader, Bio-tek Company, USA; Countstar automatic cell counter, Elite Life Science (Shanghai) Co., Ltd.; inverted microscope, NiKon Company, Japan; ultra-clean bench, China Suzhou Antai Air Technology Co., Ltd.; 96-well culture plate (transparent plate and black plate), American Corning Company.

Experimental cells: RAW264.7 cells were purchased from ATCC; A549 and MRC-5 cells were purchased from Peking Union Medical College Cell Resource Center.

2. Experimental method:

RAW264.7 and MRC-5 cells with logarithmic growth were seeded in 96-well plate (transparent plate) at 5×10 ³ cells/well, and placed in a 37°C incubator containing 5% CO ₂ for 12 hours of adherent culture. Aspirate the medium, wash with PBS once, add different concentrations of culture medium containing Dilamani, continue to culture for 24h, remove the medium, wash twice with PBS, add 0.1mL serum-free medium containing 0.5mg/mL MTT , incubate in an incubator for 3 h, remove the MTT solution, add 0.1 mL DMSO, place on a shaker and shake gently for 5 min, and measure the absorbance with a microplate reader (570/630 nm).

A549 cells in the logarithmic growth phase were seeded in a 96-well plate at 2×10 ⁴ cells/well, and placed in a 37°C incubator containing 5% CO ₂ for 12 hours of adherent culture. Dilamani medium containing different concentrations were added, and the culture was continued for 2 h, 1 μM FITC-Galectin 3 protein was added, incubated in the incubator for 2 h, and the fluorescence intensity was detected by a microplate reader at 485/528 nm.

3. Experimental results:

Dilamanid has low toxicity or no toxicity at a concentration of 5 μM, and significantly inhibits the binding of Galectin3 protein to cells. The results are shown in Table 1.

Table 1 Compound cytotoxicity and its effect on Galectin 3 inhibitor model

Example 2 Effect of Dilamanid on LPS-Induced Lung Injury Model

1. the experimental material of the present embodiment comprises:

Experimental Drugs:

Test sample solution configuration: Accurately weigh an appropriate amount of Dilamani, and prepare a 4 mg/mL solution with water.

Dexamethasone solution configuration: Dexamethasone tablets, 0.75 mg/tablet, produced by Tianjin Lisheng Pharmaceutical Co., Ltd., prepared with water to make a 0.045 mg/mL solution.

Lipopolysaccharide solution configuration: lipopolysaccharide, purchased from Sigma-Aldrich Company, was prepared into a 20 mg/mL stock solution with PBS, and diluted to 0.2 mg/mL immediately before use.

Hydroxyproline detection kit: purchased from Nanjing Jiancheng Institute of Bioengineering.

Experimental animals:

SPF-grade male C57BL/6J mice, 6-8 weeks old, weighing about 20 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., license number SCXK (Beijing) 2019-0010.

Experimental equipment: Flexivent small animal pulmonary function system: FlexiVent, SCIREQ Inc.

2. Experimental method:

Model building:

A mouse model of acute lung injury was established by a single intratracheal injection of lipopolysaccharide (LPS). The specific implementation is as follows: the mice were fasted overnight, after the mice were anesthetized with tribromoethanol (400 mg/kg), LPS (0.5 mg/kg) was injected into the trachea using a non-invasive tracheal intubation technique, with a volume of 50 μL, and then quickly stood upright and rotated Rats, so that LPS evenly into the lung lobes. The mice in the sham operation group were intratracheally injected with the same amount of PBS under the same anesthesia.

Experimental grouping and administration:

The mice were randomly divided into sham operation group (PBS+normal saline), model group (LPS+normal saline), dexamethasone group (LPS+dexamethasone) and dilamanid group (LPS+dilamanid).

On the day of the model, drug intervention was performed after the mice woke up, and the drug was administered once a day. The sham operation group and the model group were given normal saline 0.1mL/10g; the dexamethasone group was given an equal volume of dexamethasone (0.45 mg/kg/d) by intragastric administration; the dilamanid group was given an equal volume of dilamanid ( 40mg/kg/d); continuous administration for 3 days, while observing the general clinical symptoms of mice.

Table 2 Grouping and administration of lung injury model

Animal administration and material collection:

After feeding the model and administration for 48 hours, fast overnight, the next day, tribromoethanol anesthetized the mice, collected the alveolar lavage fluid of the mice, weighed the wet weight of the lung and the right middle lobe of the lung, and calculated the lung index (lung index = lung weight ( g)/body weight (g)×100%); the right middle lobe of the lung was placed in a 60°C constant temperature oven, and after baking for 48 hours, it was weighed as the dry weight of the right middle lobe of the lung, and the ratio of wet to dry weight of the lung was calculated; the left large lobe of the lung was used for hydroxypreserved Amino acid was determined; the right upper lobe of the lung was used for the detection of related cytokines; the right lower lobe of the lung was fixed and stained with HE and Masson. HE staining was used to observe the pathological morphology, and lung tissue fibrosis was scored at the same time. Pulmonary fibrosis grading and scoring criteria: 0, slight thickening of alveolar septa was occasionally seen; no definite fibrosis was seen. 1. Mild fibrous hyperplasia in the alveolar wall or mild fibrous hyperplasia in the bronchiole wall. 2. Moderate fibrous tissue hyperplasia in the alveolar wall or moderate fibrous tissue hyperplasia in the bronchiole wall, without structural damage to the lung tissue. 3. Moderate fibrous tissue hyperplasia in the alveolar wall or bronchiole wall, with a large number of neutrophils and lymphocytes infiltrating, and no structural damage to the lung tissue. 4. Small focal fibrous tissue hyperplasia, accompanied by mild destruction of lung tissue structure. 5. Significant hyperplasia of focal fibrous tissue, obvious destruction of lung tissue structure, accompanied by formation of fiber bundles. 6. Sheet-like fibrous tissue hyperplasia, accompanied by severe destruction of lung tissue structure. 7. Diffuse dimensional tissue hyperplasia, accompanied by severe destruction of lung tissue structure, and hive lung formation. 8. Obvious pulmonary consolidation. Masson staining was selected under a microscope at 200 times to take pictures of 2 areas. The integral absorbance (IOD) of tissue area, fibrous tissue area and fibrosis was measured by Image-Pro Plus 7.2 image analysis software. Calculate the degree of lung tissue fibrosis and the average optical density of fibrous tissue in each picture.

statistical methods:

The data are expressed as mean±standard deviation (Mean±SD), and one-way analysis of variance was used among multiple groups, and p﹤0.05 was considered statistically different. * means p﹤0.05 for the corresponding two groups; ** means p﹤0.01 for the corresponding two groups.

3. Experimental results:

(1) Effects of Dilamanid on the physiological state of lung injury model mice:

Observed beside the cage, it was found that the model group began to have symptoms such as irritability, increased activity, and shortness of breath from the first day, while the symptoms of the dilamanid group and the dexamethasone group were significantly lighter than those of the model group. Body weight was monitored. Compared with the sham operation group, the weight of the mice after the model decreased significantly, but there was no significant difference between the Dilamanid group and the dexamethasone group and the model group. The results are shown in Figure 1.

(2) Effect of Dilamani on respiratory system inflammation in lung injury model mice:

Lung index and lung tissue wet weight/dry weight ratio can reflect the degree of lung tissue inflammation and edema. Protein exudation in alveolar lavage fluid reflects inflammation and permeability of lung tissue. It can be seen from Table 3 that compared with the sham operation group, the lung index, wet weight/dry weight ratio and protein exudation of the mice in the model group were significantly increased; while both dilamanid and dexamethasone could significantly reduce the lung index , Lung wet weight/dry weight ratio and protein exudation.

Table 3 Effects on pulmonary edema in lung injury model

In addition, the determination of key cytokines related to lung tissue inflammation can more clearly describe the body's inflammatory response caused by lipopolysaccharide. The results in Figure 2 show that lipopolysaccharide can significantly increase the mRNA expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in lung tissue; while both dilamanib and dexamethasone can significantly reduce IL-1β, IL-6 and TNF - alpha mRNA expression.

The above results indicated that both dilamanid and dexamethasone could significantly improve LPS-induced lung inflammation.

(3) Effect of Dilamanid on lung tissue fibrosis in lung injury model mice

When pulmonary fibrosis occurs, the main increase in the lung is collagen fibers, and hydroxyproline is unique to collagen fibers. Determination of lung hydroxyproline content can reflect the collagen content of lung tissue, and then reflect the degree of lung tissue fibrosis. The results in Table 5 show that compared with the sham operation group, the hydroxyproline content in the lung tissue of the model group mice was significantly increased; while the administration of Dilamanid group or dexamethasone can significantly reduce the hydroxyproline content in the lung tissue of the mice. content.

In order to visually observe the morphological changes of lung tissue in mice, HE staining was performed on the lung tissue. It can be seen from Figure 3A that there was no significant change in the morphological structure of the lung tissue in the sham operation group; the lung tissue in the model group had extensive fibrosis, local consolidation, and a large number of lymph nodes. Cells and neutrophils were diffusely infiltrated, the remaining alveoli were mildly expanded, the entire lung tissue was honeycombed, the local alveolar epithelium was obviously shrunk and disappeared, and the lung tissue structure was severely damaged; All showed significant improvement, but compared with the sham operation group, there was also a significant difference. The fibrosis score was performed according to the pulmonary fibrosis grading and scoring standard. Compared with the sham operation group, the fibrosis score of the mice in the model group was significantly increased; and compared with the model group, both dilamanid and dexamethasone could significantly reduce the fibrosis score of the lung tissue. The results are shown in Table 5.

Masson staining can measure the area of fibrous tissue and the integrated absorbance of fibrosis, reflecting the degree of fibrosis of lung tissue and the density of fibrous tissue hyperplasia. Figure 3B is a set of typical Masson staining pictures taken. It can be seen from Figure 3B that the lung tissue of the mice in the sham operation group contains a small amount of collagen fibers, which are the main components of the extracellular matrix; the collagen fibers in the mice in the model group are significantly increased, and the density of fibrous tissue hyperplasia is significantly increased, and typical lung tissue appears. Interstitial fibrosis; the mice in the Dilamanid group and the Dexamethasone group also showed pulmonary interstitial fibrosis, but compared with the model group, the collagen fibers in the lung tissue of the mice in the Dilamanid group and the Dexamethasone group Both the area and the density of fibrous tissue hyperplasia were reduced. From the quantitative results in Table 4, compared with the model group, both dilamanid and dexamethasone significantly reduced the fibrosis area and fibrosis density.

Table 4 Effects on lung tissue fibrosis in lung injury model

The above results indicated that both dilamanid and dexamethasone could significantly improve LPS-induced pulmonary fibrosis.

Example 3 Effect of Dilamanid on Bleomycin-Induced Pulmonary Fibrosis Model

1. the experimental material of the present embodiment comprises:

Experimental Drugs:

Test sample solution configuration: Accurately weigh an appropriate amount of Dilamani, and prepare a 2mg/mL solution with water.

Dexamethasone solution configuration: Dexamethasone tablets, 0.75 mg/tablet, produced by Tianjin Lisheng Pharmaceutical Co., Ltd., prepared with normal saline to make a 0.045 mg/mL solution.

Bleomycin solution configuration: Bleomycin hydrochloride for injection, 15U/cartridge, produced by Hanhui Pharmaceutical Co., Ltd., made into 100U/mL stock solution with PBS, and diluted before use.

Experimental animals:

laboratory apparatus:

Flexivent Small Animal Pulmonary Function System: flexiVent, produced by SCIREQ Inc.

2. Experimental method:

Model building:

A mouse model of pulmonary fibrosis was established by a single intratracheal injection of bleomycin (BLM). The specific implementation is as follows: the mice were fasted overnight, after tribromoethanol (400mg/kg) anesthetized the mice, bleomycin (3U/kg) was injected into the trachea using a noninvasive tracheal intubation technique, with a volume of 50 μL, and then quickly stood upright and moved Rotate the mouse so that the BLM enters the lung lobes evenly. The mice in the sham operation group were intratracheally injected with the same amount of PBS under the same anesthesia.

Experimental grouping and administration:

The mice were randomly divided into sham operation group (PBS+normal saline), model group (BLM+normal saline), dexamethasone group (BLM+dexamethasone) and delamanid group (BLM+delamanid).

On the day of the model, drug intervention was performed after the mice woke up, and the drug was administered once a day. The sham operation group and the model group were given normal saline 0.1mL/10g; the dexamethasone group was given an equal volume of dexamethasone (0.45 mg/kg/d) by intragastric administration; the dilamanid group was given an equal volume of dilamanid ( 20mg/kg/d); continuous administration for 21 days, while observing the general clinical symptoms of the mice.

Table 5 Grouping and administration of pulmonary fibrosis model

Animal material:

After feeding the model and administration for 21 days, fast overnight, anesthetize the mice with tribromoethanol, detect the lung function of the mice, weigh the lung weight, and calculate the lung index (lung index=lung weight (g)/body weight (g)×100% ); the left lobe of the lung was used for the determination of hydroxyproline; the right upper lobe of the lung was used for the detection of related cytokines; the right lower lobe of the lung was fixed and stained with HE and Masson. HE staining was used to observe the pathological morphology, and lung tissue fibrosis was scored at the same time. Pulmonary fibrosis grading and scoring criteria: 0, slight thickening of alveolar septa was occasionally seen; no definite fibrosis was seen. 1. Mild fibrous hyperplasia in the alveolar wall or mild fibrous hyperplasia in the bronchiole wall. 2. Moderate fibrous tissue hyperplasia in the alveolar wall or moderate fibrous tissue hyperplasia in the bronchiole wall, without structural damage to the lung tissue. 3. Moderate fibrous tissue hyperplasia in the alveolar wall or bronchiole wall, with a large number of neutrophils and lymphocytes infiltrating, and no structural damage to the lung tissue. 4. Small focal fibrous tissue hyperplasia, accompanied by mild destruction of lung tissue structure. 5. Significant hyperplasia of focal fibrous tissue, obvious destruction of lung tissue structure, accompanied by formation of fiber bundles. 6. Sheet-like fibrous tissue hyperplasia, accompanied by severe destruction of lung tissue structure. 7. Diffuse dimensional tissue hyperplasia, accompanied by severe destruction of lung tissue structure, and hive lung formation. 8. Obvious pulmonary consolidation. Masson staining was selected under a microscope at 200 times to take pictures of 2 areas. The integrated absorbance (IOD) of tissue area, fibrous tissue area and fibrosis was measured by Image-Pro Plus 7.2 image analysis software. The fibrosis degree of lung tissue and the average optical density of fibrous tissue in each picture were calculated.

statistical methods:

3. Experimental results:

(1) Effects of Dilamanid on the physiological state of pulmonary fibrosis model mice

Observed beside the cage, it was found that the mice in the model group began to have symptoms such as hair color difference, fried hair, irritability, increased activity, and shortness of breath from the fifth day; the symptoms of the dexamethasone group and the dilamani group were significantly lighter than those of the model group. The weight of the mice was monitored, and it was found that the weight of the mice decreased after modeling, and began to increase after 6 days; the weight loss of the dexamethasone group was more obvious; there was no significant difference between the Dilamanid group and the model group, the results are shown in Figure 4 . Statistical survival rate, as shown in Table 7, at 21 days, the survival rate of the sham operation group was 100%, the survival rate of the model group was 75%, the survival rate of the Dilamanid group was 91.67%, and the survival rate of the dexamethasone group was 66.66%.

Table 6 Impact on survival rate of pulmonary fibrosis model mice

(2) Effect of Dilamanid on the respiratory function of pulmonary fibrosis model mice

Using the Flexivent Small Animal Pulmonary Function System to detect the respiratory system function of mice. The impaired respiratory function of mice was judged by respiratory system resistance (Rrs) and respiratory system elasticity (Ers).

It can be seen from Table 7 that bleomycin can induce significant impairment of respiratory function in mice, manifested as a significant increase in respiratory system resistance and respiratory system elasticity; dexamethasone can significantly reduce the above indicators, and the delamanid group also decreased trend.

Table 7 Effects on Pulmonary Function of Pulmonary Fibrosis Model

The above results indicated that both dilamanid and dexamethasone could improve bleomycin-induced respiratory function damage in mice.

(3) Effect of Dilamanid on respiratory system inflammation in pulmonary fibrosis model mice

Determination of key cytokines in the inflammatory response of lung tissue can describe the inflammatory response of the body caused by bleomycin. The results in Figure 5 showed that bleomycin significantly increased the mRNA expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in lung tissue, while both dilamanib and dexamethasone could significantly reduce IL-1β, IL- 6 and TNF-αmRNA expression, close to the level of the sham operation group. The results showed that both delamanid and dexamethasone could significantly improve bleomycin-induced lung tissue inflammation.

(4) Effect of Dilamanid on pulmonary fibrosis in pulmonary fibrosis model mice

Lung index can reflect the degree of lung tissue fibrosis in pulmonary fibrosis model mice. It can be seen from Table 8 that compared with the sham operation group, the lung index of the mice in the model group was significantly increased; Dilamanid can significantly reduce the lung index, and the dexamethasone group only had a downward trend.

When pulmonary fibrosis occurs, the main increase in the lung is collagen fibers, and hydroxyproline is unique to collagen fibers. Determination of lung hydroxyproline content can reflect the collagen content of lung tissue, and then reflect the degree of lung tissue fibrosis. It can be seen from Table 8 that compared with the sham operation group, the content of hydroxyproline in the model group was significantly increased; both dilamanid and dexamethasone could significantly reduce the content of hydroxyproline.

In order to visually observe the changes in the lung tissue morphology of mice, HE staining was performed on the lung tissue. It can be seen from Figure 6A that the morphological structure of the lung tissue in the sham operation group did not change significantly; Shrinking alveolar space, local patchy pulmonary fibrosis, severe destruction of lung tissue structure, and diffuse infiltration of a large number of lymphocytes, plasma cells, and neutrophils in the lesion site; the above pathological changes were alleviated in the dexamethasone group and the delamanid group , but compared with the sham operation group, there was also a significant difference.

According to the pulmonary fibrosis grading and scoring standard, the lung tissue scores of the mice were scored. Compared with the sham operation group, the fibrosis score of the mice in the model group was significantly increased; Dilamanib could significantly reduce the fibrosis score, but the decrease was not obvious in the dexamethasone group. The results are shown in Table 8.

Masson staining can measure the area of fibrous tissue and the integrated absorbance of fibrosis, reflecting the degree of fibrosis of lung tissue and the density of fibrous tissue hyperplasia. Figure 6B is a set of typical Masson staining pictures taken. It can be seen from Figure 6B that the lung tissue of mice in the sham operation group contains a small amount of collagen fibers, which are the main components of the extracellular matrix; the collagen fibers in the model group are significantly increased, and the density of fibrous tissue hyperplasia is significantly increased, and a typical lung interstitium appears Fibrosis: Pulmonary interstitial fibrosis also appeared in the dexamethasone group and the delamanid group, but compared with the model group, the degree of fibrosis was reduced. From the quantitative results in Table 8, compared with the model group, Dilamanid significantly reduced the fibrosis area and fibrosis density, and the dexamethasone group also had a decreasing trend, but the effect was slightly worse.

Table 8 Effects on pulmonary fibrosis in pulmonary fibrosis model

The above results showed that both delamanid and dexamethasone could significantly improve bleomycin-induced pulmonary fibrosis, and the effect was slightly better than that of dexamethasone.

From the above experimental results, it can be seen that Dilamanid can significantly reduce alveolar inflammation, pulmonary interstitial inflammation and collagen deposition in lung tissue in lipopolysaccharide-induced lung injury model mice; interstitial inflammation and pulmonary fibrosis. Thereby reducing the degree of lung injury, preventing pulmonary fibrosis, improving lung function and increasing survival rate.

Dilamani has a good effect on preventing and treating lung injury and pulmonary fibrosis.

The above-mentioned embodiments are only to describe the preferred mode of the present invention, and are not intended to limit the scope of the present invention. Variations and improvements should fall within the scope of protection defined by the claims of the present invention.

Example 4 Dilamanid on SiO ₂ The effect of inducing the mouse silicosis model

1. the experimental material of the present embodiment comprises:

Experimental Drugs:

Dilamani, purchased from MCE company, its structural formula is:

Test sample solution configuration: Accurately weigh an appropriate amount of Dilamani, and prepare a 4mg/mL suspension with water.

Pirfenidone solution configuration: pirfenidone powder, prepared into 30mg/mL suspension with water.

Experimental reagents:

SiO ₂ solution configuration: SiO ₂ powder, purchased from Sigma-Aldrich Company, was prepared into a 100 mg/mL solution with PBS.

Experimental animals:

laboratory apparatus:

Flexivent Small Animal Pulmonary System: FlexiVent, SCIREQ Inc.

Other materials involved are also commercial sources, and used according to the instructions provided by the manufacturer.

2. Experimental method:

Model building:

A mouse silicosis model was established using a single intratracheal injection of _SiO2 . The specific implementation is as follows: mice were fasted overnight, tribromoethanol (400 mg/kg) anesthetized the mice, and injected SiO ₂ (100 mg/kg) into the trachea using non-invasive tracheal intubation technology, with a volume of 50 μL, and then quickly stood upright and rotated Rats, so that SiO ₂ evenly into the lung lobes. The mice in the sham operation group were intratracheally injected with the same amount of PBS under the same anesthesia.

On the 30th day of model construction, the mice were fasted overnight, and after the mice were anesthetized with tribromoethanol (400mg/kg), the lung function of the mice was detected by non-invasive tracheal intubation technology, and the timing of treatment was determined according to the results of the lung function.

Experimental grouping and administration:

According to the results of pulmonary function test, drug treatment was started on the 32nd day of model construction. The mice in the model group were randomly divided into model group (SiO ₂ + normal saline), pirfenidone group (SiO ₂ + pirfenidone) and delamanid group (SiO ₂ + delamanid). The sham operation group and the model group were given normal saline 0.1mL/10g; the dilamanid group was given an equal volume of dilamanid (40 mg/kg/d) by intragastric administration; the pirfenidone group was given an equal volume of pirfenidone by intragastric administration (300mg/kg/d) (Table 1), once a day, administered continuously for 10 weeks, while observing the general clinical symptoms of mice.

Table 1 Grouping and administration

Animal material:

After 10 weeks of continuous administration, the mice were fasted overnight, and after the mice were anesthetized with tribromoethanol (400 mg/kg), the lung function of the mice was detected by non-invasive tracheal intubation technology. The weight of the lung tissue was weighed, and the left lobe of the lung was used for the determination of hydroxyproline; the right lower lobe of the lung was fixed and stained with HE and Masson.

The pathological morphology was observed by HE staining, and the lung tissue fibrosis score and silicon nodules were graded at the same time. Pulmonary fibrosis grading and scoring criteria: 0, slight thickening of alveolar septa was occasionally seen; no definite fibrosis was seen. 1. Mild fibrous hyperplasia in the alveolar wall or mild fibrous hyperplasia in the bronchiole wall. 2. Moderate fibrous tissue hyperplasia in the alveolar wall or moderate fibrous tissue hyperplasia in the bronchiole wall, without structural damage to the lung tissue. 3. Moderate fibrous tissue hyperplasia in the alveolar wall or bronchiole wall, with a large number of neutrophils and lymphocytes infiltrating, and no structural damage to the lung tissue. 4. Small focal fibrous tissue hyperplasia, accompanied by mild destruction of lung tissue structure. 5. Significant hyperplasia of focal fibrous tissue, obvious destruction of lung tissue structure, accompanied by formation of fiber bundles. 6. Sheet-like fibrous tissue hyperplasia, accompanied by severe destruction of lung tissue structure. 7. Diffuse dimensional tissue hyperplasia, accompanied by severe destruction of lung tissue structure, and hive lung formation. 8. Obvious pulmonary consolidation.

Pathological grading criteria for silicon nodules: 1. Cellular nodules (grade I fibrosis): Dust cells are the main components of nodules in lung tissue lesions, and argyrophilic fiber staining shows individual fine argyrophilic nodules in the nodules. fibers, no collagen fibers were seen. 2. Fibrous and cellular nodules (grade II fibrosis): Dust cells are the main components of the nodules in lung tissue lesions, and a small amount of fine collagen fibers can be seen in collagen fiber staining. 3. Cellular and fibrous nodules (grade III fibrosis): the main component of nodules in lung tissue lesions is collagen fibers, but a certain amount of cellular components can still be seen. 4. Fibrous nodules (grade IV fibrosis): the main component of nodules in lung tissue lesions is mainly collagen fibers, and generally there are only a small amount or no cellular components.

Masson staining was selected under a microscope at 200 times to take pictures of 2 areas. The integral absorbance (IOD) of tissue area, fibrous tissue area and fibrosis was measured by Image-Pro Plus 7.2 image analysis software. Calculate the degree of lung tissue fibrosis and the average optical density of fibrous tissue in each picture.

statistical methods:

3. Experimental results:

(1) Intratracheal injection of SiO ₂ induced the formation of mouse silicosis model:

After intratracheal injection of SiO ₂ , the general physiological state of the mice was observed, and the body weight of the mice was monitored at the same time. The mice in the sham operation group had normal diet, shiny hair, normal respiration and activity levels. The mice in the model group had a small diet, poor mental state, and reduced activity, and some mice also experienced shortness of breath. The body weight of the mice decreased significantly on the second day after modeling, and then the weight change trend was consistent with that of the sham-operated group, gradually increasing, but the body weight of the model group was still lower than that of the sham-operated group (Figure 1A).

After SiO ₂ intratracheal injection for 30 days, the mouse respiratory system function was detected by Flexivent small animal lung function system. It can be seen from Table 2 that intratracheal injection of SiO ₂ can induce significant impairment of respiratory function in mice, manifested as a significant increase in respiratory system resistance (Rrs) and respiratory system elasticity (Ers), and a significant decrease in respiratory system compliance (Crs).

Table 2 Effects on lung function of silicosis model

The above results indicate that intratracheal injection of SiO ₂ can reproduce the animal model similar to the pathological process of human silicosis patients. After 30 days of modeling, the respiratory system function of the mice was significantly impaired, which was consistent with the clinical symptoms of silicosis patients, such as dyspnea, chest tightness, and chest pain, indicating that the silicosis model was formed and it was time to start drug treatment.

(2) Effects of dilamanid on the pathological process of lung tissue in the mouse silicosis model:

The body weight of the mice in the model group began to drop at about 40 days and reached the lowest point at about 60 days, and then rose slowly; the body weight change curves of the dilamanid group and the pirfenidone group were different from those of the model group (Fig. 1B). It shows that dilamanid and pirfenidone may affect the physiological state and pathological process of mouse silicosis model.

When SiO ₂ particles continue to enter the lung tissue of the body and then deposit in the bronchioles of the distal lung, inflammation and emphysema will appear in the lung tissue. The results in Figure 2 showed that compared with the sham operation group, the lung tissue of the mice in the model group was hypertrophic with abnormal shape; the lung tissue of the mice in the Dilamanid group and the pirfenidone group was still hypertrophic, but the shape tended to be slightly normal.

In the mouse silicosis model, lung weight and lung index can reflect lung tissue inflammation and emphysema to a certain extent. It can be seen from Table 3 that compared with the sham operation group, the lung weight and lung index of the mice in the model group were significantly increased; the lung weight and lung index of the Dilamanid group and the pirfenidone group had a downward trend, but there was no significant difference. sexual difference.

Table 3 Effects on lung tissue inflammation and emphysema in silicosis model

The above results showed that both dilamanid and pirfenidone had a tendency to reduce lung tissue inflammation and emphysema in SiO ₂ -induced mouse silicosis model, but the effect was not significant.

(3) Effect of Dilamani on pulmonary fibrosis in silicosis model mice

SiO ₂ dust can be phagocytized by macrophages in the lung, forming silicon nodules and involving the alveoli, releasing pro-inflammatory mediators, oxygen free radicals, and pro-fibrotic factors, and inducing lung fibroblasts to proliferate, differentiate, and secrete collagen. Eventually leading to fibrosis formation. When lung tissue fibrosis occurs, the main increase in the lung is collagen fibers, and hydroxyproline is unique to collagen fibers. Determination of the content of lung hydroxyproline can reflect the content of collagen in lung tissue, and then reflect the degree of lung tissue fibrosis. . The results in Table 4 show that compared with the sham operation group, the content of hydroxyproline in the lung tissue of the model group mice was significantly increased; and the administration of Dilamanid or pirfenidone can significantly reduce the content of hydroxyproline in the lung tissue of mice. content.

In order to visually observe the morphological changes of lung tissue in mice, HE staining was performed on the lung tissue. It can be seen from Figure 3A that there was no significant change in the morphological structure of the lung tissue in the sham operation group; the lung tissue in the model group had extensive fibrosis, local consolidation, and a large number of lymph nodes. Cells and neutrophils were diffusely infiltrated, the remaining alveoli were mildly expanded, the entire lung tissue was honeycombed, the local alveolar epithelium was obviously shrunk and disappeared, and the lung tissue structure was severely damaged; The changes were significantly improved, but compared with the sham operation group, there were also significant differences. The fibrosis score was carried out according to the pulmonary fibrosis grading and scoring standard. Compared with the sham operation group, the fibrosis degree of the mice in the model group was significantly increased; and compared with the model group, both dilamanid and pirfenidone could significantly reduce the level of lung tissue fibrosis. The fibrosis level, the results are shown in Table 4.

Masson staining can measure the area of fibrous tissue and the integrated absorbance of fibrosis, reflecting the degree of fibrosis of lung tissue and the density of fibrous tissue hyperplasia. Figure 3B is a set of typical Masson staining pictures taken. It can be seen from Figure 3B that the lung tissue of the mice in the sham operation group contains a small amount of collagen fibers, which are the main components of the extracellular matrix; the collagen fibers in the mice in the model group are significantly increased, and the density of fibrous tissue hyperplasia is significantly increased, and typical lung tissue appears. Interstitial fibrosis; the mice in Dilamanid group and Pirfenidone group also showed changes in pulmonary interstitial fibrosis, but compared with the model group, the collagen in the lung tissue of mice in Dilamanid group and Pirfenidone group Both the fiber area and the density of fibrous tissue hyperplasia were reduced. From the quantitative results in Table 4, compared with the model group, both the dilamanid group and pirfenidone significantly reduced the fibrosis area and fibrosis density.

Table 4 Effects on lung tissue fibrosis in silicosis model

The above results showed that Dilamanid could significantly improve _SiO2- induced lung tissue fibrosis in silicosis model mice, and the effect was slightly better than that of pirfenidone.

(4) Effect of Dilamani on lung tissue silicosis in silicosis model mice

SiO ₂ dust can be phagocytosed by macrophages in the lungs, forming extensive nodular fibrosis in the lungs, which is the main feature of silicosis. It can be seen from Figure 3A that the model group has obvious fibrous silicon nodules, which are more in number and larger in size; the dilamanid group and the pirfenidone group have fewer silicon nodules, and most of them are cellular silicon nodules. Nodules, small in size.

Table 5 Effects on silicon nodules in lung tissue of silicosis model

Silicon nodules were graded and counted according to King's grading standard. There were no silicon nodules in the sham operation group; the number of grade I fibrosis, grade II fibrosis, grade III fibrosis and grade IV fibrosis silicon nodules in the lung tissue of the model group were more, and the silicon nodules were larger; Dilama Neither the niger group nor the pirfenidone group had grade IV fibrotic silicon nodules, and compared with the model group, silicon nodules at all levels were significantly reduced, and the ratio of nodules to lung lobe area was significantly reduced.

The above results showed that Dilamanid could significantly inhibit the formation of _SiO2- induced silicon nodules in the lung tissue of silicosis model mice, and the effect was slightly better than that of pirfenidone.

Claims

Application of Dilamanid and pharmaceutically acceptable salts thereof as shown in formula (I) in the preparation of Galectin 3 inhibitors;
Application of dilamanid and pharmaceutically acceptable salts thereof as shown in formula (I) in the preparation of medicines for preventing and/or treating lung injury diseases;
The use according to claim 2, wherein the lung injury is acute lung injury or acute respiratory distress syndrome.
Application of dilamanid and pharmaceutically acceptable salts thereof as shown in formula (I) in the preparation of medicines for preventing and/or treating pulmonary fibrosis;
The use according to claim 4, characterized in that the pulmonary fibrosis is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.
Application of delamanid and pharmaceutically acceptable salts thereof shown in formula (I) in the preparation of medicaments for treating silicosis;
The application according to claim 6, characterized in that, the dosage of dilamani to human body is 0.2-20mg/kg/day;
A kind of application of pharmaceutical composition in the preparation of medicine for treating silicosis disease, it is characterized in that, described pharmaceutical composition comprises delamanid and its pharmaceutically acceptable salt thereof as shown in formula (I), and pharmaceutically acceptable carrier or excipient;
The application according to claim 8, characterized in that the pharmaceutical composition comprises tablets, capsules, granules, oral liquids or injections.