Application of BT2 or salt thereof in preparation of medicine for preventing and treating pulmonary fibrosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of BT2 or a salt thereof in preparation of a medicine for preventing and treating pulmonary fibrosis. The invention discloses that BT2 or the salt thereof can inhibit the production of collagen and relieve collagen deposition of patients with pulmonary fibrosis for the first time, so that the effect of preventing and treating pulmonary fibrosis is achieved, and BT2 is low in price.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of BT2 or its salts in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is characterized by fibroblast proliferation and extracellular matrix deposition, causing damage to lung tissue structure and ultimately leading to respiratory failure. Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease. Currently, there is no specific drug for pulmonary fibrosis, and it cannot be completely cured if it progresses to a certain stage. Clinically, measures are often taken to stabilize the condition and slow its progression. In most patients, due to the unknown cause, it is classified as idiopathic pulmonary fibrosis (IPF), and the incidence and prevalence of IPF are showing an increasing trend year by year. Idiopathic pulmonary fibrosis (IPF) is an irreversible interstitial pulmonary fibrosis disease. Due to the abnormal increase in interstitial cells and extracellular matrix, the alveolar walls of patients thicken, leading to irreversible decline in lung function and even respiratory failure. Currently, there are very limited drugs for treating IPF; pyrfenidone and nintedanib are commonly used drugs for IPF treatment. While both types of drugs can slow disease progression, they do not improve lung function or patients' quality of life, and both therapies suffer from drug tolerance issues. For example, oral pirfenidone can cause photosensitivity, liver damage, dizziness, and drowsiness, while oral nintedanib can cause liver and kidney damage, bleeding, nausea, and diarrhea. Furthermore, pirfenidone and nintedanib are expensive, placing a heavy burden on patients. Therefore, it is necessary to develop drugs to prevent and treat pulmonary fibrosis.

[0003] The small molecule compound BT2, chemically named 3,6-dichloro-benzo[b]thiophene-2-carboxylic acid, is a small molecule inhibitor targeting the protein kinase BCKDK. It has been shown to effectively alleviate heart failure, insulin resistance, and diabetes, but its role in pulmonary fibrosis has not been reported. Summary of the Invention

[0004] The first aspect of the present invention is to provide the application of BT2 or its salts.

[0005] A second aspect of the present invention is to provide a method for preventing and treating pulmonary fibrosis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides the use of BT2 or a salt thereof in any one of (1) to (3):

[0008] (1) Prepare drugs for the prevention and / or treatment of pulmonary fibrosis;

[0009] (2) Preparation of reagents to inhibit collagen production;

[0010] (3) In vitro non-therapeutic target inhibition of collagen production.

[0011] Preferably, the pulmonary fibrosis mentioned in (1) includes at least one of primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia; further, it is idiopathic pulmonary fibrosis.

[0012] Preferably, the collagen mentioned in (2)-(3) is type I collagen (COL1A1).

[0013] Preferably, the reagent for inhibiting collagen production in (2) is a reagent for inhibiting collagen production in vitro without therapeutic purpose.

[0014] Preferably, the inhibition of collagen production in (2)-(3) is to inhibit collagen production in lung fibroblasts; more preferably, it is to inhibit collagen production in mouse primary lung fibroblasts.

[0015] Preferably, the salt comprises at least one of a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0016] Preferably, the metal salt comprises at least one of alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.), and aluminum salts.

[0017] Preferably, the salt formed with the organic base comprises a salt formed with one or more of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0018] Preferably, the salt formed with the inorganic acid comprises a salt formed with one or more of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0019] Preferably, the salt formed with the organic acid comprises a salt formed with one or more of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0020] Preferably, the salt formed with the basic amino acid comprises a salt formed with one or more of the following basic amino acids: arginine, lysine, ornithine.

[0021] Preferably, the salt formed with the acidic amino acid comprises a salt formed with one or more of the following acidic amino acids: aspartic acid, glutamic acid.

[0022] Preferably, the drug described in (1) also contains other active ingredients for the prevention and / or treatment of pulmonary fibrosis, such as: arbutin (CN117731656A), sodium mannitol (CN115887472A), bufotalin (CN117379441A), kavapiperine A (CN114931566A), Paris polyphylla saponin VI (CN113827655A), kynurenine (CN115381806A), desloratadine (CN115040515A), microcystin-RR (CN114470153A), low molecular weight heparin (CN108117613A), plasminogen (CN110121358A), Chk1 inhibitor (CN113631179A), pirfenidone, nintedanib, etc.

[0023] Preferably, the reagent described in (2) further comprises other active ingredients that inhibit collagen production, such as: bufotalin (CN117379441A), kavapiperine A (CN114931566A), Paris polyphylla saponin VI (CN113827655A), kynurenine (CN115381806A), desloratadine (CN115040515A), arbutin (CN117731656A), sodium mannitol (CN115887472A), kynurenine (CN115381806A), microcystin- RR (CN114470153A), low molecular weight heparin (CN108117613A), plasminogen (CN110121358A), Chk1 inhibitor (CN113631179A), pirfenidone, nintedanib, etc.; further, bufotalin (CN117379441A), kavapiperidine A (CN114931566A), Paris polyphylla saponin VI (CN113827655A), kynurenine (CN115381806A), desloratadine (CN115040515A), etc.

[0024] Preferably, the drug described in (1) further comprises pharmaceutically acceptable excipients.

[0025] Preferably, the pharmaceutically acceptable excipients include at least one of diluents, excipients, binders, humectants, surfactants, lubricants, and disintegrants.

[0026] Preferably, the dosage form of the drug described in (1) is a dosage form suitable for children or an adult.

[0027] Preferably, the dosage form is selected from gastrointestinal dosage forms or non-gastrointestinal dosage forms.

[0028] Preferably, the gastrointestinal dosage form includes at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0029] Preferably, the non-gastrointestinal drug delivery dosage form includes at least one of the following: injectable dosage forms (e.g., injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory dosage forms (e.g., sprays, aerosols, powder inhalers, etc.); skin dosage forms (e.g., topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (e.g., eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, films, etc.); and cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).

[0030] Preferably, the drug in (1) is a tablet, capsule or injection.

[0031] Preferably, the drug described in (1) is administered to animals.

[0032] Preferably, the animal is a mammal; further selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; and even further selected from humans, including minors, adults, and the elderly.

[0033] Preferably, in (1), the active ingredient of the prepared drug, BT2 or its salt has a mass content of 0.1%-40%, more preferably 0.1%-20%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0034] Preferably, the application described in (3) includes the following steps: treating lung fibroblasts with a reagent that inhibits collagen production, wherein the reagent that inhibits collagen production includes BT2 or a salt thereof.

[0035] Preferably, the final concentration of the reagent for inhibiting collagen production when treating lung fibroblasts is 5-200 μM; further, 10-120 μM; even further, 80-120 μM; and still further, 90-110 μM.

[0036] Preferably, the processing time is 1-12 days; more preferably 1-3 days.

[0037] Preferably, the reagent for inhibiting collagen production further comprises other active ingredients that inhibit collagen production, such as: bufotalin (CN117379441A), kavapiperine A (CN114931566A), Paris polyphylla saponin VI (CN113827655A), kynurenine (CN115381806A), desloratadine (CN115040515A), arbutin (CN117731656A), sodium mannitol (CN115887472A), kynurenine (CN115381806A), and microcystin. Heparin-RR (CN114470153A), low molecular weight heparin (CN108117613A), plasminogen (CN110121358A), Chk1 inhibitor (CN113631179A), pirfenidone, nintedanib, etc.; further, bufotoxin (CN117379441A), kavapiperidine A (CN114931566A), Paris polyphylla saponin VI (CN113827655A), kynurenine (CN115381806A), desloratadine (CN115040515A), etc.

[0038] A second aspect of the present invention provides a method for preventing (and / or treating) pulmonary fibrosis by administering a drug to a subject, said drug being the drug described in (1) of the first aspect of the present invention.

[0039] Preferably, the pulmonary fibrosis includes at least one of primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia; more preferably, it is idiopathic pulmonary fibrosis.

[0040] Preferably, the subject is an animal; further, a mammal; even further, selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; and even further, humans, including minors, adults, and the elderly.

[0041] Preferably, the dosage of the drug is 5-30 mg / kg per dose for mice (further, 10-30 mg / kg per dose; even further, 15-25 mg / kg per dose); the dose per unit body weight for different subjects can be calculated using equivalent dose conversion relationships. For example, based on the equivalent dose conversion relationships between experimental animals and humans known to those skilled in the art (usually refer to the guidance of drug regulatory agencies such as the FDA and SFDA, or "Huang Jihan et al. Equivalent dose conversion between animals and between animals and humans in pharmacological experiments. Chinese Journal of Clinical Pharmacology and Therapeutics, 2004 Sep; 9(9): 1069-1072"), the effective dose for humans can be derived from the dose for experimental animals.

[0042] The beneficial effects of this invention are:

[0043] Currently, FDA-approved drugs for treating pulmonary fibrosis (pirfenidone and nintedanib) can slow disease progression, but neither drug can improve lung function or patients' quality of life. Both therapies also have issues with drug tolerance and systemic side effects: for example, oral pirfenidone can cause photosensitivity, liver damage, dizziness, and drowsiness; oral nintedanib can cause liver and kidney damage, bleeding, nausea, and diarrhea. Furthermore, pirfenidone and nintedanib are expensive, placing a heavy burden on patients. This invention discloses for the first time that BT2 or its salts can inhibit collagen production and alleviate collagen deposition in patients with pulmonary fibrosis, thereby achieving the effect of preventing and treating pulmonary fibrosis. Moreover, BT2 is inexpensive. Attached Figure Description

[0044] Figure 1 The results of BT2 inhibiting collagen production in MLF cells in Example 1 are shown in Figure A: WB results of BT2 inhibiting collagen production in MLF cells in Example 1 (N=2); and RT-PCR results of BT2 inhibiting collagen production in MLF cells in Example 1 (N=3).

[0045] Figure 2 The following are the results of BT2 inhibiting BLM-induced pulmonary fibrosis in mice in Example 2: A is a schematic diagram of the experiment in Example 2 on BT2 inhibiting BLM-induced pulmonary fibrosis in mice; B is a statistical graph of mouse body weight data for each treatment in the experiment on BT2 inhibiting BLM-induced pulmonary fibrosis in mice in Example 2 (** indicates p<0.01); C is a micro-CT image of mice for each treatment in the experiment on BT2 inhibiting BLM-induced pulmonary fibrosis in mice in Example 2; D is a statistical graph of fibrosis gene expression in lung tissue of mice for each treatment in the experiment on BT2 inhibiting BLM-induced pulmonary fibrosis in mice in Example 2; E is an H&E staining image (bar = 500 μm; bar = 100 μm) of lung tissue sections from mice for each treatment in the experiment on BT2 inhibiting BLM-induced pulmonary fibrosis in mice in Example 2; F is a Masson staining image (bar = 500 μm; bar = 100 μm) of lung tissue sections from mice for each treatment in the experiment on BT2 inhibiting BLM-induced pulmonary fibrosis in mice in Example 2. Detailed Implementation

[0046] The present invention will be further described in detail below through specific embodiments.

[0047] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0048] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0049] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in commonly used reference books in the field, such as "Molecular Cloning: A Laboratory Manual" (3rd edition, Science Press, 2005), or according to the conditions recommended by the reagent manufacturers.

[0050] In this invention, BT2, whose chemical name is 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid, English name is 3,6-dichloro-benzo[b]thiophene-2-Carboxylic Acid, chemical formula is C9H4Cl2O2S, CAS number is 34576-94-8, has the structural formula shown in formula (I):

[0051]

[0052] Using BT2 as the active ingredient and adding pharmaceutically acceptable excipients, it can be formulated into different dosage forms, such as injections and tablets, as detailed below:

[0053] Preparation of the injection: Mix 200mg BT2, 700mg mannitol, 10mg PEG3000 and 100mL distilled water, adjust the pH of the mixture to 7.0, filter to make the concentration of filtrate 3mg / mL, dispense 2mL per ampoule, freeze dry to obtain the injection.

[0054] Tablet preparation: Take 10mg BT2, 35mg microcrystalline cellulose, 45mg starch, 4mg polyvinylpyrrolidone, 4.5mg sodium carboxymethyl starch, 0.5mg magnesium stearate, and 1mg talc. First, BT2, starch, and microcrystalline cellulose are sieved through a 100-mesh sieve and thoroughly mixed to obtain a mixture. Mix polyvinylpyrrolidone with the above mixture, sieve through a 100-mesh sieve, and obtain wet granules. Dry the wet granules at 50℃. First, sieve sodium carboxymethyl starch, magnesium stearate, and talc through a 24-mesh sieve, and then add them to the above wet granules and compress them into tablets to obtain tablets.

[0055] The experimental procedures related to the following examples are as follows:

[0056] 1. Cells and reagents

[0057] Primary mouse lung fibroblasts (MLFs) were isolated from the lung tissue of 5-6 week old male C57BL / 6 mice; the small molecule drug BT2 (T14834) was purchased from TargetMol; dimethyl sulfoxide (DMSO, D8418) was from Sigma; premium fetal bovine serum (S711-001s) was purchased from Lonsera; DMEM / F12 medium (C11330500BT) was purchased from Gibco; antibody COL1A1 (72026T) was purchased from CST; ACTA2 (Ab7817) was purchased from Abcam; FN1 (A16678) and GAPHD (AC002) were purchased from Abclonal.

[0058] 2. Isolation of MLF from primary mouse lung fibroblasts

[0059] Immediately after euthanasia, 5-6 week old male C57BL / 6 mice were dissected, and their lungs were harvested and washed twice with PBS containing PS. The lung tissue fragments were then minced using scissors or a scalpel in a 10cm dish. The lung tissue fragments were transferred to a 50mL sterile centrifuge tube, and 1mL of Collagenase I was added. The mixture was digested at 37°C, 400rpm, for 1 hour. The digested lung tissue and Collagenase I mixture was filtered through a 70μm nylon filter and washed with 10-20mL of PBS. The filtrate was collected and centrifuged at 4°C, 400g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 10mL of pre-warmed medium and transferred to a 10cm dish. The cells were incubated at 37°C. The medium was changed after 30 minutes, and the cells were returned to the incubator for further incubation, changing the medium every 2-3 days. When the cell density reached 100%, the cells were passaged and cryopreserved.

[0060] 3. Lung tissue staining

[0061] After fixing the upper lobe of the left lung of the mouse with fixative, the tissue sections were prepared by embedding in dehydrated paraffin and then stained with hematoxylin and eosin (H&E) and Masson staining, and observed under a light microscope.

[0062] 4. RNA extraction

[0063] Add 1 mL of Trizol to the cells or tissue, vortex or sonicate to fully lyse the tissue or cells. Add 200 μL of chloroform, vortex for 15 s, incubate on ice for 15 min, centrifuge at 12000 rpm for 15 min at 4 °C, and collect the supernatant. Add an equal volume of isopropanol to the supernatant, mix well, incubate at 4 °C for 10 min or at -20 °C overnight to precipitate; centrifuge at 12000 rcf for 30 min at 4 °C, and discard the supernatant. Add 1 mL of 75% ethanol to the RNA precipitate, gently tap the tube wall to wash the precipitate; centrifuge at 12000 rcf for 5 min at 4 °C, and discard the supernatant. Air dry (about 10 min), add an appropriate amount of ddH2O to dissolve the RNA precipitate. Determine the RNA concentration; the RNA sample can be used for subsequent experiments or stored at -80 °C.

[0064] 5. Real-time quantitative PCR

[0065] First, reverse transcription: RNA samples were reverse transcribed using the Novizan HiScript III RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit. ① Removal of genomic DNA from RNA samples: 4 μL of 4×gDNAwiper Mix + 1 μg of template RNA, and finally, RNase-free ddH2O to bring the total volume to 16 μL. Gently pipette to mix, incubate at 42°C for 2 min. ② Reverse transcription of RNA into cDNA: Add 4 μL of 5×HiScript III qRT SuperMix to the reaction mixture from step 1, and gently pipette to mix. Incubate at 37°C for 15 min; then at 85°C for 15 s. The product can be used immediately for qPCR or stored at -20°C. Finally, qPCR was performed using the Novizan ChamQ Universal SYBR qPCR Master Mix kit (Q711-03). The qPCR reaction mixture consisted of: 5 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.2 μL of Primer 1 (10 μM), 0.2 μL of Primer 2 (10 μM), 2 μL of Template DNA / cDNA, and ddH2O to a final volume of 10 μL. The relative gene expression levels were calculated using the 2-ΔΔCt method based on the comparison of cycle thresholds. Primer sequences are shown in Table 1.

[0066] Table 1 Primer sequences

[0067]

[0068] 6. Western blot (WB)

[0069] First, protein sample preparation: Prepare protein lysis buffer: Mix PBS, 2×SDS, and β-mercaptoethanol in a volume ratio of 5:4:1. Lyse cells: Collect cells by trypsin digestion, wash once with PBS, and add an appropriate amount of protein lysis buffer according to the cell volume. Denature proteins: Heat at 98℃ for 15 min to fully denature the proteins. Load the sample directly for electrophoresis or store at -20℃. Then, perform electrophoresis: Select an appropriate loading volume and spot the sample; set a constant voltage program at 80V and electrophoresis for 30 min until the protein markers reach the lower gel layer. Then, change the voltage to 120V and continue electrophoresis for about 60 min, adjusting the electrophoresis time as needed based on protein size. Next, transfer the membrane: Activate the cut PVDF membrane in methanol for 2-5 min. After electrophoresis, transfer the protein gel to transfer filter paper according to the marker bands, and simultaneously cover the protein gel with the activated PVDF membrane, gently rolling it with a roller to remove air bubbles (following the principle of black gel, white membrane). Constant current, 120V, 280mA, for 12 min. After transfer, immerse the PVDF membrane in 5% skim milk powder (prepared with PBST) and incubate for 30 min; wash three times with PBST, 10 min each time. After blocking, immerse the PVDF membrane in primary antibody dilution buffer and incubate at room temperature for 2 h or overnight at 4°C. After primary antibody application, wash the PVDF membrane three times with PBST, 10 min each time, and incubate at room temperature for 2 h with the corresponding secondary antibody (prepared with 5% skim milk powder). Finally, prepare developing solution and evenly drop it onto the PVDF membrane or incubate with developing solution for 2 min for development.

[0070] Example 1: BT2 significantly inhibited the expression of COL1A1.

[0071] The effect of BT2 on COL1A1 expression was evaluated as follows:

[0072] First, primary mouse lung fibroblasts (MLFs) were seeded at a density of 2 × 10^5 cells / mL into 12-well plates, with 1 mL of cell culture solution added to each well. After culturing at 37°C for 24 h, the cells were divided into a blank control group and a model group. The blank control group was given 20 μL of PBS, while the model group was given 20 μL of TGF-β1 (initial concentration 1 μg / mL, solvent: PBS) to achieve a final concentration of 20 ng / mL. Simultaneously, the model group was divided into a DMSO control group and a drug treatment group. The DMSO control group was given 1 μL of DMSO, while the drug treatment groups were given 1 μL of BT2 (initial concentrations of 10, 20, 50, and 100 mM, solvent: DMSO) to achieve final concentrations of 10, 20, 50, and 100 μM, respectively. After incubation at 37℃ for 48 hours, Western blotting was performed on cells from the drug treatment groups (final BT2 concentrations of 10, 20, 50, and 100 μM), the DMSO control group, and the blank control group (N=2). The results of the Western blotting are as follows: Figure 1As shown in Figure A: BT2 can inhibit MLF cells from producing collagen.

[0073] First, primary mouse lung fibroblasts (MLFs) were seeded at a density of 2 × 10^5 cells / mL into 12-well plates, with 1 mL of cell culture solution added to each well. After incubation at 37°C for 24 h, the cells were divided into a control group and a model group. The control group was given 20 μL of PBS, while the model group was given 20 μL of TGF-β1 (initial concentration 1 μg / mL, PBS solvent) to achieve a final concentration of 20 ng / mL. Simultaneously, the control group was further divided into a blank control group and a drug control group. The blank control group was given 1 μL of DMSO, while the drug control group was given 1 μL of BT2 (initial concentration 100 mM, DMSO solvent) to achieve a final concentration of 100 μM. The model group was divided into a DMSO control group and a drug treatment group. The DMSO control group was given 1 μL of DMSO, while the drug treatment group was given 1 μL of BT2 (initial concentration 100 mM, DMSO solvent) to achieve a final concentration of 100 μM. After culturing at 37℃ for 48 h, RT-PCR was performed on cells from the blank control group, drug control group, DMSO control group, and drug treatment group (N=3). The RT-PCR results are as follows: Figure 1 As shown in Figure B: BT2 can inhibit the expression of collagen genes in MLF cells.

[0074] Example 2: BT2 significantly inhibited BLM-induced pulmonary fibrosis

[0075] A schematic diagram of the experiment on BT2 treatment of bleomycin (BLM)-induced lung fibrosis in mice is shown below. Figure 2 As shown in Figure A: 10-12 week old male C57BL / 6 mice, weighing 25-30g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were SPF-grade. For the BT2 animal experiment: Mice were randomly divided into three groups: normal control group (PBS group, N=7), model group (BLM group, N=8), and treatment group (BLM+BT2 group, N=7). During modeling, the model and treatment groups received intratracheal injections of 3.5 mg / kg BLM (solvent: PBS). The control group received an equal volume of PBS via intratracheal injection. Three days after modeling, drug treatment was initiated. BT2 (solvent: DMSO solution) was administered intraperitoneally at a dose of 20 mg / kg. After two consecutive days of injection, a one-day interval was observed before resuming administration. The control group received an intraperitoneal injection of the same concentration and volume of DMSO solution. Body weight was measured and recorded midway through the treatment. After 14 days of treatment as described above, the mice were sacrificed, and lung tissue was collected. Animal experiments were conducted at the Animal Center of Guangzhou National Laboratory, with ethics code GZLAB-AUCP-2023-02-A02.

[0076] The results are as follows Figure 2As shown: In mice with BLM-induced pulmonary fibrosis, body weight was significantly lower than in the control group, while after BT2 treatment, body weight was significantly higher than in the BLM group. Figure 2 (B); Micro-CT results showed that BT2 treatment (14 days after administration) significantly improved lung lesions in mice. Figure 2 RT-PCR experiments showed that the fibrosis genes Col1a1 and Fn1 in lung tissue were significantly increased in the BLM group, while they were significantly downregulated after BT2 treatment (14 days of administration). Figure 2 (D); H&E and Masson staining revealed a large amount of collagen deposition in the alveoli of mice in the BLM group, while collagen deposition was significantly improved after BT2 administration. Figure 2 (E, F); It can be seen that BT2 treatment can significantly alleviate collagen deposition in BLM-induced pulmonary fibrosis mice, thus achieving the effect of preventing and treating pulmonary fibrosis.

[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of BT2 or its salts in any of (1) to (3): (1) Prepare drugs for the prevention and / or treatment of pulmonary fibrosis; (2) Preparation of reagents to inhibit collagen production; (3) In vitro non-therapeutic target inhibition of collagen production.

2. The application according to claim 1, characterized in that: The pulmonary fibrosis described in (1) includes at least one of primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia; further, it is idiopathic pulmonary fibrosis. Preferably, the collagen mentioned in (2)-(3) is type I collagen.

3. The application according to any one of claims 1-2, characterized in that: The drug described in (1) also contains other active ingredients for the prevention and / or treatment of pulmonary fibrosis; Preferably, the reagent in (2) further contains other active ingredients that inhibit collagen production.

4. The application according to any one of claims 1-3, characterized in that: The drug described in (1) also contains pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include at least one of diluents, excipients, binders, humectants, surfactants, lubricants, and disintegrants.

5. The application according to any one of claims 1-4, characterized in that: The dosage form of the drug mentioned in (1) is a dosage form suitable for children or an adult; Preferably, the dosage form is selected from gastrointestinal dosage forms or non-gastrointestinal dosage forms; Preferably, the gastrointestinal dosage form includes at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-gastrointestinal dosage form includes at least one of the following: injectable dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

6. The application according to any one of claims 1-5, characterized in that: The drug mentioned in (1) is a tablet, capsule or injection.

7. The application according to any one of claims 1-6, characterized in that: (1) The drug is administered to animals; Preferably, the animal is a mammal; further selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; and even further selected from humans.

8. The application according to any one of claims 1-7, characterized in that: The BT2 or its salt described in (1) contains 0.1%-40% by mass in the drug; more specifically, 0.1%-20%.

9. The application according to any one of claims 1-8, characterized in that: The application described in (3) includes the following steps: treating lung fibroblasts with a reagent that inhibits collagen production, wherein the reagent that inhibits collagen production includes BT2 or a salt thereof; Preferably, the final concentration of the reagent for inhibiting collagen production when treating lung fibroblasts is 5-200 μM; more preferably, it is 10-120 μM. Preferably, the reagent for inhibiting collagen production further comprises other active ingredients that inhibit collagen production.

10. The application according to any one of claims 1-9, characterized in that: The salt comprises at least one of the following: metal salt, ammonium salt, salt formed with an organic base, salt formed with an inorganic acid, salt formed with an organic acid, salt formed with a basic amino acid, and salt formed with an acidic amino acid. Preferably, the metal salt comprises at least one of an alkali metal salt, an alkaline earth metal salt, and an aluminum salt; Preferably, the salt formed with the organic base comprises a salt formed with one or more of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine. Preferably, the salt formed with the inorganic acid comprises a salt formed with one or more of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid; Preferably, the salt formed with the organic acid comprises a salt formed with one or more of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Preferably, the salt formed with the basic amino acid comprises a salt formed with one or more of the following basic amino acids: arginine, lysine, ornithine; Preferably, the salt formed with the acidic amino acid comprises a salt formed with one or more of the following acidic amino acids: aspartic acid, glutamic acid.

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