Use of small molecule compounds targeting baz2b in the preparation of a medicament for preventing or treating endometrial fibrosis and intrauterine adhesions

Abstract

The application particularly relates to application of a small-molecule compound targeting BAZ2B in preparation of a medicine for preventing or treating endometrial fibrosis and intrauterine adhesion, and finds that five compounds including a small-molecule compound MKI can target BAZ2B protein and combine with a bromodomain of the BAZ2B protein, so that a fibrosis-related process is regulated. Experimental results show that MKI can inhibit endometrial stromal cell activation and myofibroblast formation, down-regulate expression of fibrosis-related molecules, reduce collagen deposition and extracellular matrix abnormal remodeling, so that endometrial fibrosis degree is reduced, damaged endometrium is promoted to repair, endometrial thickness and gland quantity are increased, and normal tissue structure is restored, and pregnancy rate, embryo implantation number and reproductive outcome of an intrauterine adhesion model animal are improved. Therefore, the application provides a new strategy for preventing and treating endometrial fibrosis and intrauterine adhesion by taking BAZ2B as an action target, and provides a new technical approach for developing an anti-fibrosis medicine.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and particularly to the use of small molecule compounds targeting BAZ2B in the preparation of medicaments for the treatment of fibrosis-related diseases, especially in the preparation of medicaments for the prevention or treatment of endometrial fibrosis and intrauterine adhesions. Furthermore, the present invention also relates to pharmaceutical compositions containing the aforementioned small molecule compounds or pharmaceutically acceptable salts thereof. Background Technology

[0002] Intrauterine adhesions (IUA), also known as Asherman's syndrome, are a condition caused by damage to the basal layer of the endometrium, leading to partial or complete closure of the uterine cavity. This can cause reduced menstrual flow, infertility, recurrent miscarriages, and pregnancy complications, severely impacting the reproductive health of women of childbearing age. With the increase in induced abortions, intrauterine procedures, and infections, the incidence of IUA is on the rise. Currently, clinical practice mainly uses hysteroscopic adhesiolysis combined with hormone therapy, biological barrier materials, or intrauterine stents to promote endometrial repair. However, these treatments primarily address existing adhesions and are insufficient to fundamentally inhibit the fibrosis process. Moderate to severe cases still have a high recurrence rate after surgery, and some patients experience limited improvement in fertility outcomes. Therefore, developing novel drugs that can effectively inhibit endometrial fibrosis and promote functional recovery is of great significance.

[0003] Endometrial fibrosis is a crucial pathological basis for the formation and recurrence of intrauterine adhesions, characterized by abnormal fibroblast activation, myofibroblast formation, and excessive extracellular matrix (ECM) deposition. Transforming growth factor β1 (TGF-β1) can induce the transformation of fibroblasts into myofibroblasts and promote the expression of fibrosis-related molecules such as type I collagen, fibronectin, and α-smooth muscle actin, ultimately leading to tissue scarring. Similar pathological processes are widely observed in diseases such as liver fibrosis, pulmonary fibrosis, renal fibrosis, and myocardial fibrosis. Therefore, inhibiting fibroblast activation and abnormal extracellular matrix deposition has become an important direction in antifibrotic therapy.

[0004] Epigenetic regulatory abnormalities are believed to be involved in the development and progression of various tissue fibrosis. BAZ2B (Bromodomain Adjacent to Zinc Finger Domain Protein 2B), a member of the ISWI chromatin remodeling complex, contains a bromodomain (BRD) and can recognize histone acetylation modifications, participating in chromatin remodeling and gene transcription regulation. Previous studies have shown that abnormal chromatin remodeling is closely related to fibroblast activation and the expression of extracellular matrix-related genes. However, further research is needed to understand the mechanism of action of BAZ2B in organ fibrosis, particularly endometrial fibrosis, and its potential as a therapeutic target.

[0005] In recent years, research on small molecule compounds targeting the bromine domains of the BAZ2 protein family has gradually attracted attention. For example, GSK2801 can bind to the bromine domains of BAZ2A and BAZ2B and has been used in related basic research. However, these compounds lack high selectivity for the BAZ2 protein family, and their application in tissue fibrosis, especially endometrial fibrosis, remains limited. Currently, there are no drugs that can effectively inhibit abnormal fibroblast activation, reduce extracellular matrix deposition, and promote endometrial repair by regulating BAZ2B activity.

[0006] In summary, existing treatments are insufficient to effectively halt the progression of endometrial fibrosis, and targeted intervention strategies against BAZ2B remain to be developed. Therefore, providing a novel drug that can target BAZ2B, inhibit fibroblast activation and extracellular matrix deposition, promote endometrial repair, and improve reproductive outcomes is of great significance for the prevention and treatment of endometrial fibrosis and intrauterine adhesions. Summary of the Invention

[0007] This invention addresses the lack of targeted drugs in existing technologies that can effectively intervene in the process of endometrial fibrosis, as well as the high recurrence rate and difficulty in fundamentally improving endometrial function and fertility outcomes of existing treatments. Using BAZ2B as the core target and its bromine domain as the receptor, a compound library was screened using molecular docking. Based on the docking score, the top 200 candidate small molecules were obtained. Furthermore, these 200 candidate compounds were applied to human endometrial stromal cells for 48 hours. The expression levels of type I collagen (COL1A1) and α-smooth muscle actin (α-SMA) were detected by real-time quantitative PCR (qRT-PCR). Five small molecule compounds that could reduce the expression of fibrosis-related genes were obtained: MKI, CRT0066101, Mavacoxib, CB30865, and SP-100030. Among them, MKI showed a strong inhibitory effect on the expression of COL1A1 and α-SMA; therefore, MKI was selected for further research.

[0008] Further analysis was conducted using MK as a representative small molecule compound to examine its binding ability and in vivo and in vitro anti-fibrotic effects. Molecular simulation, in vitro binding experiments, and intracellular target validation experiments showed that MKI can directly bind to the BAZ2B bromine domain in cells, enhancing the stability of the BAZ2B protein. Gene and toxicity assays showed that under different fibrosis induction conditions, MKI treatment reduced the expression levels of fibrosis-related genes and inhibited the transformation of endometrial stromal cells into myofibroblasts, with no significant cytotoxicity observed in the 0-75 μM concentration range. In vitro cell experiments showed that MKI could inhibit the differentiation of endometrial stromal cells into myofibroblasts and suppress the contractile ability of endometrial stromal cells. Animal model experiments showed that MKI can alleviate endometrial fibrosis and promote tissue repair by inhibiting the BAZ2B-mediated fibrosis process, improving embryo implantation ability, fetal development, and pregnancy outcomes, thereby improving fertility in mice with intrauterine adhesions. Therefore, it can be used to prepare drugs for the prevention and / or treatment of endometrial fibrosis and intrauterine adhesions.

[0009] Based on relevant research, the technical solution to be protected by this invention is as follows:

[0010] In a first aspect, this invention provides the use of a small molecule compound targeting BAZ2B or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of fibrosis-related diseases. The small molecule compound is selected from any one of MKI, CRT0066101, Mavacoxib, CB30865, and SP-100030, with MKI being preferred.

[0011] The compound name of MKI is N-(2-hydroxyethyl)-4-(6-(4-(trifluoromethoxy)phenylamino)pyrimidin-4-yl)benzamide; its Chinese name is N-(2-hydroxyethyl)-4-(6-((4-(trifluoromethoxy)phenyl)amino)pyrimidin-4-yl)benzamide; its molecular formula is: C 20 H 17 F3N4O3 has a molecular weight of 418.4. Its CAS number is 778274-97-8, and its chemical structure is shown in Formula I below.

[0012] .

[0013] Formula I

[0014] The chemical name of CRT0066101 is: 2-[4-[[(2R)-2-aminobutyl]amino]pyrimidin-2-yl]

[0015] -4-(1-methyl-1H-pyrazol-4-yl)phenol; Chinese name: 2-[4-[[(2R)-2-aminobutyl]amino]-2-pyrimidinyl]-4-(1-methyl-1H-pyrazol-4-yl)phenol; Molecular formula: C 18 H 22 N6O, molecular weight: 338.41. CAS number: 956123-34-5. Chemical structure is shown in Formula II below:

[0016]

[0017] Formula II

[0018] The chemical name of Mavacoxib is: 4-[5-(3,5-difluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]

[0019] benzenesulfonamide; Chinese name: 4-[5-(3,5-difluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-1-yl]benzenesulfonamide; Molecular formula: C 16 H 10 F5N3O2S, molecular weight: 401.33. CAS number: 170569-88-7. Chemical structure is shown in Formula III below:

[0020]

[0021] Formula III

[0022] The chemical name of CB30865 is: 4-(((7-bromo-2-methyl-4-oxo-1,4-dihydroquinazolin-6-yl)methyl)

[0023] (prop-2-yn-1-yl)amino)-N-(pyridin-3-ylmethyl)benzamide; Chinese name: 4-(7-bromo-2-methyl-4-oxo-1,4-dihydroquinazolin-6-yl)methylamino]-N-(pyridin-3-ylmethyl)benzamide; Molecular formula: C 26 H 22 BrN5O2, molecular weight: 516.39. CAS number: 206275-15-2. Chemical structure is shown in Formula IV below:

[0024]

[0025] Formula IV

[0026] The chemical name of SP-100030 is: N-[3,5-bis(trifluoromethyl)phenyl]-2-chloro-4-(trifluoromethyl)

[0027] pyrimidine-5-carboxamide; Chinese name: N-[3,5-bis(trifluoromethyl)phenyl]-2-chloro-4-(trifluoromethyl)pyrimidine-5-carboxamide; Molecular formula: C 14 H5ClF9N3O, molecular weight: 437.65. CAS number: 154563-54-9. Chemical structure is shown in formula V below:

[0028]

[0029] Formula V

[0030] Preferably, the fibrosis-related diseases are those caused by abnormal activation of myofibroblasts and excessive deposition of extracellular matrix. Further, they include one or more of the following: liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, ovarian fibrosis, skin fibrosis, peritoneal fibrosis, and endometrial fibrosis. These fibroses share similar pathological processes, all resulting from abnormal activation of fibroblasts and excessive deposition of extracellular matrix.

[0031] In a preferred embodiment of the present invention, the primary focus is on exploring and validating the relief of endometrial fibrosis, and subsequently using it to treat the formation and recurrence of intrauterine adhesions caused by endometrial fibrosis. Therefore, the present invention focuses on providing the use of small molecule compounds targeting BAZ2B or pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention or treatment of endometrial fibrosis and intrauterine adhesions.

[0032] Furthermore, the small molecule compound or its pharmaceutically acceptable salt achieves targeted therapy by targeting the BAZ2B gene or protein in fibrotic cells, possessing one or more of the following effects, thereby also conferring corresponding efficacy to the drug:

[0033] 1) It can bind to BAZ2B protein and bind to the bromine domain of BAZ2B protein;

[0034] 2) It can inhibit the activation of endometrial stromal cells and their transformation into myofibroblasts;

[0035] 3) It can reduce the proliferation, migration, and contraction of endometrial stromal cells;

[0036] 4) It can downregulate the expression of α-smooth muscle actin (α-SMA), type I collagen (COL1A1), type III collagen (COL3A1), fibronectin (FN1), and other extracellular matrix-related factors;

[0037] 5) It can reduce collagen deposition and abnormal extracellular matrix remodeling, thus alleviating the degree of endometrial fibrosis;

[0038] 6) It can promote endometrial repair, gland regeneration and recovery of reproductive function.

[0039] Furthermore, the pharmaceutically acceptable salts described in this invention are widely selected and can be salts formed with any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, hydroxyethanesulfonic acid; they can also be selected from halogen salts and salts formed with alkali metals or alkaline earth metals.

[0040] Furthermore, the tumor-targeted therapy drug of the present invention uses a small molecule compound MKI or its salt as the sole active ingredient, or contains MKI or its salt, with a content of 0.1-99 wt%, more preferably 0.1-50 wt%, and can be specifically screened experimentally according to actual conditions.

[0041] In a second aspect, the present invention provides a pharmaceutical composition targeting BAZ2B, comprising an active ingredient and pharmaceutically acceptable excipients, said active ingredient being selected from one of the above five small molecule compounds or a pharmaceutically acceptable salt thereof. The MKI or a pharmaceutically acceptable salt thereof is mixed with a pharmaceutically acceptable carrier, diluent, stabilizer, buffer, preservative, or excipient, and can be prepared into a pharmaceutical composition suitable for different routes of administration using conventional formulation processes.

[0042] Preferably, in actual treatment, the drug composition can be used alone or in combination with other drugs or treatments for endometrial fibrosis. These combined treatments include anti-fibrotic drug therapy, hormone therapy, biomaterial or tissue-engineered scaffold therapy, anti-adhesion material therapy, surgical treatment (such as biological scaffolds), and other adjuvant therapies that promote tissue repair (such as stem cell therapy).

[0043] Furthermore, the dosage form of the pharmaceutical composition is selected from injections, lyophilized powder injections, tablets, capsules, granules, oral liquids, sustained-release preparations, hydrogel preparations, nano-preparations, intrauterine local sustained-release materials, or other pharmaceutically acceptable dosage forms.

[0044] In a third aspect, this application provides a treatment method for fibrotic diseases including endometrial fibrosis and intrauterine adhesions, the method comprising: administering to a subject an effective amount of the aforementioned small molecule compound or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0045] Preferably, the pharmaceutical composition is administered orally, intravenously, intraperitoneally, intrauterinely, or vaginally.

[0046] Preferably, the dosage of the pharmaceutical composition is 1 to 50 mg / kg, more preferably 5 to 20 mg / kg.

[0047] Compared with the prior art, the beneficial effects of this application are as follows:

[0048] Based on previous in vitro and in vivo experiments, this application is the first to discover that the small molecule compound MKI can target the BAZ2B protein and bind to its bromine domain, thereby regulating fibrosis-related processes. Experimental results show that...

[0049] MKI can inhibit the activation of endometrial stromal cells and the formation of myofibroblasts, downregulate the expression of fibrosis-related molecules such as α-SMA, COL1A1, COL3A1, and FN1; reduce collagen deposition and abnormal extracellular matrix remodeling, thereby alleviating the degree of endometrial fibrosis; promote the repair of damaged endometrium, increase endometrial thickness and glandular number, and restore normal tissue structure; and improve pregnancy rate, embryo implantation number, and reproductive outcomes in intrauterine adhesion model animals.

[0050] In summary, this invention provides a novel strategy for the prevention and treatment of endometrial fibrosis and intrauterine adhesions targeting BAZ2B, offering a new technical approach for the development of anti-fibrotic drugs. The small molecule compound MKI provided by this invention has a clear target and good anti-fibrotic activity, and has potential application value in the preparation of drugs for the prevention and / or treatment of fibrosis-related diseases. It is also of great significance in clinical research, clinical treatment, and optimization of treatment safety. Attached Figure Description

[0051] Figure 1 This is a schematic diagram illustrating the screening and anti-fibrotic activity evaluation of small molecule compounds (MKI). Figure 1 A is a schematic diagram of the virtual screening process for small molecule compounds based on the BAZ2B bromine domain; Figure 1 B is a schematic diagram showing the docking scoring results of the candidate compound with the BAZ2B bromine domain molecule; Figure 1 C- Figure 1 G is a schematic diagram illustrating the effects of candidate compounds on the expression of genes related to fibrosis and cell activation in human endometrial stromal cells; Figure 1 H is a schematic diagram illustrating the effect of different doses of MKI on the viability of human endometrial stromal cells.

[0052] Figure 2 This diagram illustrates the interaction between the small molecule compound MKI and the BAZ2B protein, and validates the target. Figure 2 A is a schematic diagram of the molecular dynamics simulation results of the MKI-BAZ2B protein complex; Figure 2 B is a schematic diagram of the molecular docking results between MKI and the bromine domain of BAZ2B protein; Figure 2 C is a schematic diagram showing the results of surface plasmon resonance (SPR) assay to detect the binding ability of MKI to BAZ2B protein; Figure 2 D- Figure 2 E is a schematic diagram of the results of the Cellular Thermal Transfer Assay (CETSA); Figure 2 F~ Figure 2 G is a schematic diagram of the results of the protease protection experiment; Figure 2 H is a schematic diagram of the immunoblotting results.

[0053] Figure 3 This is a schematic diagram illustrating the inhibition of endometrial stromal cell activation by the small molecule compound MKI. Among them, Figure 3 A is a schematic diagram of the cell proliferation experiment results; Figure 3 B~ Figure 3 D is a schematic diagram of the cell migration experiment results; Figure 3 E is a schematic diagram showing the results of immunofluorescence detection of cell transformation into myofibroblasts; Figure 3 F is a schematic diagram of the collagen gel shrinkage experiment results; Figure 3 G~ Figure 3 H is a schematic diagram showing the statistical results of collagen gel shrinkage area.

[0054] Figure 4 This is a schematic diagram illustrating the regulation of extracellular matrix secretion and remodeling by the small molecule compound MKI. Among them, Figure 4 A~ Figure 4 C is a schematic diagram of RNA sequencing analysis results; Figure 4 D~ Figure 4 H is a schematic diagram of the results of gene enrichment analysis and extracellular matrix-related pathway analysis; Figure 4 I is a schematic diagram of the verification results of real-time quantitative PCR.

[0055] Figure 5 This diagram illustrates how the small molecule compound MKI alleviates endometrial fibrosis and promotes endometrial repair in a mouse model of intrauterine adhesions. Figure 5 A is a schematic diagram of the animal experiment procedure and dosing regimen; Figure 5 B is a schematic diagram of the HE staining results; Figure 5 C is a schematic diagram showing the statistical results of the number of uterine glands; Figure 5 D is a schematic diagram of the statistical results of endometrial thickness; Figure 5 E is a schematic diagram of the Masson staining results; Figure 5 F is a schematic diagram showing the statistical results of collagen deposition area; Figure 5 G~ Figure 5 J is a schematic diagram showing the immunohistochemical staining results and quantitative analysis of α-SMA, COL1A1, and FN1.

[0056] Figure 6 This is a schematic diagram illustrating how the small molecule compound MKI improves reproductive outcomes in a mouse model of intrauterine adhesions. Figure 6 A is a schematic diagram of pregnancy and embryo implantation; Figure 6 B is a schematic diagram of the statistical results of embryo absorption rate; Figure 6 C is a schematic diagram showing the statistical results of the number of fetuses; Figure 6 D is a schematic diagram of the placental weight statistics; Figure 6 E is a schematic diagram of the fetal weight statistics. Detailed Implementation

[0057] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0058] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0059] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques from the fields of biomedical engineering, biophysics, pharmaceutics, pharmaceutical analysis, medicinal chemistry, analytical chemistry, molecular biology, biochemistry, and related areas. These techniques have been well described in existing literature.

[0060] The inventors of this application, through extensive research and exploration, discovered the application of five compounds, including the small molecule compound MKI or its pharmaceutically acceptable salts, in the preparation of anti-fibrotic therapeutic drugs, and completed this application based on this discovery.

[0061] In this application, previous studies have found that abnormal chromatin remodeling is closely related to fibroblast activation and the expression of extracellular matrix-related genes, suggesting that BAZ2B could be a novel therapeutic target for anti-fibrosis. Based on this, using the BAZ2B protein as a target, and based on virtual screening of small molecule drugs and the degree of inhibition of fibrosis marker proteins, 200 small molecule inhibitors targeting BAZ2B were further screened and tested. The resulting small molecule compound, MKI, showed strong inhibitory effects on COL1A1 and α-SMA expression.

[0062] In a preferred embodiment of the present invention, the use of a small molecule compound MKI or a pharmaceutically acceptable salt thereof in the preparation of an anti-fibrotic therapeutic medicament is most preferably provided.

[0063] In this invention, small molecule compounds MKI or their pharmaceutically acceptable salts can differentiate into myofibroblasts through targeted differentiation.

[0064] The treatment aims to achieve cell-based anti-fibrotic therapy by regulating the BAZ2B gene or protein in stromal cells, specifically by downregulating the expression level of the BAZ2B gene, and / or reducing the activity of the BAZ2B protein, and / or regulating the function of BAZ2B.

[0065] In a preferred embodiment, the reference to "pharmaceutically acceptable salt" generally refers to any salt that is physiologically tolerable when used in a suitable manner for treatment (particularly when applied or used in humans and / or mammals). This generally means that it is non-toxic, particularly as a result of an anti-ion. These physiologically acceptable salts can be formed with cations or bases, and in the context of this invention, particularly when administered to humans and / or mammals, they should be understood as salts formed from at least one compound provided according to this invention, typically an acid (deprotonated), such as an anion, and at least one physiologically tolerable cation (preferably an inorganic cation). Specifically, in the context of this invention, this may include salts formed with alkali metals and alkaline earth metals, as well as salts formed with ammonium cations (NH4+, ... 4+ Salts formed with (mono) or (di) sodium, (mono) or (di) potassium, magnesium, or calcium can be included, but are not limited to, salts formed with (mono) or (di) sodium, (mono) or (di) potassium, magnesium, or calcium. These physiologically acceptable salts can also be formed with anions or acids, and in the context of this invention, particularly when administered to humans and / or mammals, they should be understood as salts formed by at least one compound provided according to this invention, typically protonated, such as a cation, and at least one physiologically tolerable anion. Salts of the small molecule compound MKI include, but are not limited to, salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or hydroxyethanesulfonic acid. Salts of halides are also applicable. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium).

[0066] In this invention, in addition to the small molecule compound MKI or its pharmaceutically acceptable salt, the related applications of MKI or its hydrates, enantiomers, diastereomers, solvates or crystalline forms in the preparation of antifibrotic drugs also fall within the scope of protection of this invention.

[0067] The present invention also provides a pharmaceutical composition comprising the aforementioned five small molecule compounds (preferably MKIs) or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or excipient.

[0068] In some embodiments, the primary active ingredient of the pharmaceutical composition is the aforementioned small molecule compound MKI or a pharmaceutically acceptable salt thereof. In addition to the small molecule compound MKI or a pharmaceutically acceptable salt thereof described in this invention, the pharmaceutical composition may also contain other drugs that have or may have anti-fibrotic activity.

[0069] In some embodiments, the pharmaceutically acceptable excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used as needed to aid in the stability of the drug or to help improve the activity of the active ingredient (i.e., the small molecule compound MKI or its pharmaceutically acceptable salt as described above in this invention), and include, but are not limited to: diluents, surfactants, humectants, binders, fillers, disintegrants, adsorbents, lubricants, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and the pharmaceutical composition thus formulated can be administered as needed using any suitable route of administration known to those skilled in the art.

[0070] In some embodiments, the diluent includes, but is not limited to, starch, lactose, sucrose, microcrystalline cellulose (MCC), dicalcium phosphate, and calcium sulfate. The surfactant includes, but is not limited to, sodium stearate, sodium lauryl sulfate, benzalkonium chloride, cetrimonium bromide, polysorbates, polyethylene glycol (PEG), fatty acid glycerides, lecithin, and soybean lecithin. The humectant includes, but is not limited to, water, ethanol, polysorbate 80, and sodium lauryl sulfate solution. The binder includes, but is not limited to, starch paste, povidone (PVP), hydroxypropyl methylcellulose (HPMC), and microcrystalline cellulose (MCC). The filler includes, but is not limited to, mannitol, glucose, hydroxypropyl cellulose, calcium carbonate, and sodium carbonate. The disintegrant includes, but is not limited to, dry starch, crospovidone (PVPP), sodium carboxymethyl starch (CMS-Na), and low-substituted hydroxypropyl cellulose (L-HPC). The adsorbent carrier includes, but is not limited to, activated carbon, silica gel, starch, and dextrin. The lubricants include, but are not limited to: magnesium stearate, stearic acid, polyethylene glycol (PEG), sodium dodecyl sulfate (SLS), micronized silica gel, talc, etc. The stabilizers include, but are not limited to: sodium sulfite, sodium bisulfite, vitamin E (α-tocopherol), butylated hydroxyanisole (BHA), ethylenediaminetetraacetic acid (EDTA) and its sodium salt, benzyl alcohol, chlorobutanol, etc. The bactericides include, but are not limited to: ethanol, benzophenol, phenol, cresol, benzalkonium bromide, etc. The buffers include, but are not limited to: phosphate buffer, acetate buffer, borate buffer, etc. The isotonic agents include, but are not limited to: sodium chloride, glucose, glycerol, etc. The chelating agents include, but are not limited to: EDTA and its sodium salt, citric acid, tartaric acid, etc. The pH control agents include, but are not limited to: hydrochloric acid, citric acid, sodium hydroxide, ammonia, amino acids, etc.

[0071] In some embodiments, the drug may additionally contain liquids such as water, saline, glycerin, and ethanol.

[0072] In the pharmaceutical compositions of the present invention, the small molecule compound MKI or its pharmaceutically acceptable salt may be a single active ingredient or may be combined with other active ingredients to form a combined formulation.

[0073] In the pharmaceutical compositions of the present invention, the content of the active ingredient (preferably a small molecule compound MKI or a pharmaceutically acceptable salt thereof) is generally a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the active ingredient generally depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the active ingredient can generally be 1-1000 mg / kg / day, 20-200 mg / kg / day, 1-3 mg / kg / day, or 3-5 mg / kg / day. , 5~10mg / kg / day, 10~20mg / kg / day, 20~30mg / kg / day, 30~40mg / kg / day, 40~60mg / kg / day, 60~80mg / kg / day, 80~100 mg / kg / day, 100~150mg / kg / day, 150~200mg / kg / day, 200~300mg / kg / day, 300~500mg / kg / day, or 500~1000mg / kg / day.

[0074] Those skilled in the art can determine the effective dosage based on the severity of the condition and the recipient's health status and age. The effective dosage typically varies between 0.01 ng / kg body weight and approximately 100 mg / kg body weight.

[0075] The active ingredient or pharmaceutical composition containing the active ingredient provided by this invention can be adapted to any form of administration, including oral or parenteral administration, for example, via nasal, rectal and / or intravenous injection, and more specifically via intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, oral, sublingual, nasal, percutaneous, vaginal, oral or parenteral administration; injection administration includes intravenous injection, intramuscular injection and subcutaneous injection, percutaneous administration, etc.

[0076] As used herein, the dosage form of the pharmaceutical composition is selected from: injections, sterile powders for injection, tablets, pills, capsules, lozenges, tinctures, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories. Those skilled in the art can select appropriate formulations based on the route of administration. For example, formulations suitable for oral administration may include, but are not limited to, pills, tablets, chewable tablets, capsules, granules, solutions, drops, syrups, aerosols, or powder inhalers; formulations suitable for parenteral administration may include, but are not limited to, solutions, suspensions, rehydrated dry preparations, or sprays; suppositories are typically suitable for rectal administration; and injections and sterile powders for injection are suitable for injection administration.

[0077] Tablets, lozenges, pills, and capsules may also contain the following components: binders, such as gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, or alginic acid; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, lactose, or saccharin, or flavorings, such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the above-mentioned substances. Various other substances may exist in coating form or be used to improve the physical form of the unit dosage form. For example, shellac, sugar, or both may be used to coat tablets, pills, or capsules. Syrups or elixirs may contain active compounds, sucrose as a sweetener, methylparaben and propylparaben as preservatives, colorings, and flavorings, such as cherry or orange flavoring. Any substance used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic in the dosage. In addition, active compounds can be incorporated into sustained-release products or formulations.

[0078] Those skilled in the art will understand that although the pharmaceutical compositions mentioned above may further contain pharmaceutically acceptable excipients, when the small molecule compound MKI or its pharmaceutically acceptable salt is used as a drug in humans or animals, it can also be administered in its own form (small molecule compound MKI or its pharmaceutically acceptable salt), that is, the present invention can be achieved without any of the aforementioned pharmaceutically acceptable excipients.

[0079] The present invention further provides a treatment method for fibrotic diseases, the method comprising: administering to a subject an effective amount of the aforementioned small molecule compound or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition. The method may be in vitro or non-therapeutic.

[0080] According to the method of the present invention, the small molecule compound or a pharmaceutically acceptable salt thereof may be co-administered with other therapeutic agents. "Co-administered" means administered simultaneously in the same formulation or in two different formulations via the same or different routes, or administered sequentially via the same or different routes. "Sequentially administered" means a time difference, measured in seconds, minutes, hours, or days, between the administration of two or more different compounds.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0082] The terms "include" and "contain" in this article should be understood as inclusive, without the meaning of exclusivity or exhaustion; that is, "including but not limited to".

[0083] The term "therapeutic effective dose" as used in this article generally refers to a dose that, after an appropriate period of administration, can achieve the therapeutic effect for the diseases listed above.

[0084] In this invention, the object or individual undergoing treatment is preferably a mammal, such as, but not limited to, humans, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), pets (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), or captured wild animals (e.g., foxes, deer). The object is preferably a primate. The most preferred object is a human.

[0085] The present invention is further illustrated below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The processes, conditions, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0086] The compounds of this invention are commercially available and their purity meets pharmaceutical standards.

[0087] Unless otherwise specified, all materials and reagents used in the above embodiments are commercially available. Human endometrial stromal cells were isolated from Shanghai First Maternity and Infant Hospital (Medical Research Ethics Approval No.: KS24243). Cells were cultured in DMEM / F12 (Thermo Fisher Scientific, USA) medium containing 10% fetal bovine serum (Thermo Fisher Scientific, USA), 100 mg / mL streptomycin, and 100 U / mL penicillin at 37°C and 5% CO2. MKI (catalog number: HY-103032), GSK2801 (catalog number: HY-15658), and small molecules for drug screening were purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd.; other reagents: the CCK-8 kit was purchased from Dojin Chemical Co., Ltd., Japan. Female C57BL / 6 mice, 8-10 weeks old, weighing 21-23g, experimental animal ethics approval number: TJBG25924101. Mice were purchased from Shanghai Slack Laboratory Animal Center (Shanghai, China) and were housed in a specific pathogen-free (SPF) environment at Tongji University Laboratory Animal Center for animal experiments. The humidity was 45%–60%, the temperature was constant (23 ± 2) °C, and the daily light exposure was 12 hours. The food and drinking water for the experimental animals were routinely autoclaved.

[0088] Example 1: Obtaining MKI based on structure-based virtual screening

[0089] 1.1 Experimental Methods

[0090] 1.1.1 Virtual Screening of Small Molecule Drugs

[0091] A structure-based virtual screening method was used to screen for potential inhibitors of the BAZ2B bromodomain (BRD). First, the crystal structure of the BAZ2B BRD was obtained from the Protein Data Bank (PDB) database, and the protein structure was preprocessed using Schrödinger Maestro software, including water molecule removal, hydrogen atom addition, hydrogen bond network optimization, and energy minimization. A receptor grid model was generated using the acetylated lysine binding pocket as the active site. Compound structures were obtained from the MedChemExpress (MCE) Bioactive Compound Library (Shanghai Haoyuan Biotechnology Co., Ltd.), and all compounds underwent LigPrep processing, including 3D structure construction, protonation state optimization, and energy minimization. Subsequently, graded molecular docking was performed using the Glide module. Initial screening was conducted using standard precision (SP) mode, followed by re-docking of the top-ranked compounds using high precision (XP) mode. Candidate compounds were ranked according to the Glide docking score, and manual screening was performed using protein-ligand interaction models to finally obtain potential BAZ2B binding molecules for subsequent experimental validation.

[0092] 1.1.2 TGF-β1-induced endometrial fibrosis model

[0093] Human primary endometrial stromal cells were cultured to a density of approximately 60-70%, treated with 10 ng / ml TGF-β1 for 24 hours, and then a small molecule drug was added to a final concentration of 5 μM. After 48 hours, the cells were treated with trizol and then proceeded to subsequent quantitative real-time PCR (qRT-PCR) experiments.

[0094] 1.1.3 qRT-PCR detection of mRNA expression levels

[0095] RNA was extracted from cell and tissue samples and then reverse transcribed into cDNA. qRT-PCR was performed using SYBR Green qPCR premix (Hunan Aike Rui Biotechnology Co., Ltd.), with cDNA and primers added according to the manufacturer's instructions. The qRT-PCR reactions were performed on a QuantStudio 7 real-time PCR system (Thermo Fisher Scientific). All gene expression levels were standardized using β-actin as an internal control gene.

[0096] 1.1.4 Cellular Immunofluorescence Experiment

[0097] Human primary endometrial stromal cells were seeded on coverslips at a density of 40-50%, washed three times with ice-cold PBS for 5 minutes each time, fixed in 4% paraformaldehyde (prepared in 0.1M PBS) solution at pH 7.2 for 10 minutes, washed three times with PBS for 5 minutes each time, permeabilized at room temperature for 15 minutes in permeabilization reagent containing 0.3% Triton X-100, blocked with 5% BSA at room temperature for 1 hour, incubated with primary antibody overnight, washed, incubated with secondary antibody at room temperature for 2 hours, washed, incubated with DAPI for 15-20 minutes, washed, fixed with mounting medium (anti-fluorescence quenching fixative), and photographed and recorded using a confocal microscope.

[0098] 1.1.5 CCK-8 test

[0099] Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Endometrial stromal cells were cultured at 3 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated overnight at 37°C with 5% CO2. After cell attachment, cells were treated accordingly (e.g., TGF-β1, MKI, etc.) and cultured for 24, 48, or 72 hours. 10 μL of CCK-8 working solution was added to each well, and the cells were incubated at 37°C for another 2 hours. The absorbance (OD) was then measured at 450 nm using a microplate reader (BioTek, USA). 450 Cell viability was calculated for each group, using the control group as a baseline, and expressed as a relative percentage.

[0100] All experiments were repeated at least three times, and data are expressed as mean ± SD. Two-tailed Student's t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0101] 1.2 Experimental Results

[0102] During the formation of intrauterine adhesions, the differentiation of endometrial stromal cells into myofibroblasts, accompanied by the deposition of large amounts of extracellular matrix (ECM), is an important pathological feature leading to endometrial fibrosis and scar formation. Previous studies have found that the epigenetic regulator BAZ2B is involved in the activation of endometrial stromal cells and the remodeling of extracellular matrix. Therefore, this invention employs a structure-based virtual drug screening strategy to screen potential BAZ2B-binding small molecules. The specific screening process is as follows: Figure 1As shown in Figure A, using the BAZ2B bromine domain as the acceptor, a compound library was screened via molecular docking. Based on the docking score, the top 200 candidate small molecules were obtained, including the small molecule compound MKI (…). Figure 1 B). The above 200 candidate compounds were treated with human endometrial stromal cells for 48 h, and the expression levels of type I collagen (COL1A1) and α-smooth muscle actin (α-SMA) were detected by real-time quantitative PCR (qRT-PCR). The results showed that 5 candidate compounds were able to reduce the expression of fibrosis-related genes ( Figure 1 C and Figure 1 D). Among them, MKI showed a strong inhibitory effect on the expression of COL1A1 and α-SMA, so MKI was chosen for further research.

[0103] Further treatment with MKI was performed on human endometrial stromal cells (hESCs) derived from patients with intrauterine adhesions (IUA). Results showed that, compared to the control group, MKI treatment decreased the expression levels of COL1A1, α-SMA, and extracellular matrix-related genes, while also reducing cell activation. Figure 1 E and Figure 1 F). Furthermore, under different fibrosis induction conditions, MKI treatment could reduce the expression levels of fibrosis-related genes and inhibit the transformation of endometrial stromal cells into myofibroblasts. Figure 1 G). To evaluate the safety of MKI in normal endometrial stromal cells, its cytotoxicity was detected using a CCK-8 assay. The results showed that no significant cytotoxicity was observed in the MKI concentration range of 0-75 μM, indicating its suitability for subsequent experimental studies. Figure 1 H). The above results suggest that MKI has the effect of regulating the activation of endometrial stromal cells and inhibiting the formation of myofibroblasts, and can be used as a small molecule targeting BAZ2B for further validation studies.

[0104] Example 2: MKI directly binds to BAZ2B protein

[0105] 2.1 Experimental Methods

[0106] 2.1.1 Molecular Dynamics Simulation Experiment (MD)

[0107] To evaluate the binding stability of the small molecule compound MKI with the bromine domain (BRD) of BAZ2B, a 100 ns molecular dynamics simulation was performed on the MKI-BAZ2B complex. The conformational stability of the complex was assessed by analyzing the root mean square shift (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and hydrogen bond formation. The binding free energy was calculated using the MM / PBSA method to evaluate the interaction strength between MKI and BAZ2B.

[0108] 2.1.2 Molecular docking experiment

[0109] Molecular docking analysis of MKI and BAZ2B BRD was performed using the Schrödinger Glide module. The acetylated lysine recognition pocket of the BAZ2B bromine domain was used as the binding site to calculate the ligand binding energy. Hydrogen bonding, hydrophobic interactions, and π-π stacking interactions between MKI and key amino acid residues were analyzed to predict the binding mode.

[0110] 2.1.3 Surface Plasmon Resonance Experiment (SPR)

[0111] Real-time binding analysis was performed using a Biacore T200 surface plasmon resonance (SPR) instrument (GE Healthcare). BAZ2B BRD protein was dissolved at a concentration of 10 μg / mL in 10 mM sodium acetate buffer (pH 5.0) and immobilized on the CM5 chip surface using the standard amino-coupling method, with an immobilization volume of 5362.1 RU. Different concentrations of MKI solution were sequentially injected, using PBS buffer as the mobile phase. A 1:1 Langmuir binding model was used to fit the sensing curves, and the equilibrium dissociation constant (KD) was calculated to evaluate the binding affinity between MKI and BAZ2B protein.

[0112] 2.1.4 Cell heat transfer assay (CETSA)

[0113] Cells were treated with MKI or DMSO for 2 h and then collected. They were resuspended in PBS containing protease inhibitors and lysed by liquid nitrogen freeze-thaw cycles. Equal volumes of protein samples were heated at 42–70 °C for 3 min, cooled, and centrifuged at 12000 g for 20 min at 4 °C. The supernatant was collected, and BAZ2B protein levels were detected by Western blot. ImageJ software was used for grayscale analysis, and protein thermostability curves were plotted to evaluate the effect of MKI on the thermostability of BAZ2B protein.

[0114] 2.1.5 Pronase protection experiment

[0115] Cell lysates were treated with either MKI (20 μM) or DMSO, followed by the addition of Pronase (final concentration 0.1 mg / mL) at total cell protein to Pronase ratios of 1:100, 1:200, 1:300, and 1:600, and incubated at 37 °C for 30 min. The reaction was terminated by adding protein loading buffer. BAZ2B protein degradation was detected by SDS-PAGE electrophoresis and Western blot, and quantitative analysis was performed using ImageJ software to evaluate the protective effect of MKI on BAZ2B protein.

[0116] 2.1.6 Western blot detection

[0117] Total protein was extracted from cells using RIPA lysis buffer containing a protease inhibitor, and protein concentration was determined by the BCA method for quantification. Equal volumes of protein samples were subjected to 10% SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk powder, and incubated overnight at 4°C with primary antibody BAZ2B. After washing the membrane with TBST, HRP-labeled secondary antibody (1:5000) was added, and the membrane was incubated at room temperature for 1 h. Protein expression levels were then detected by ECL colorimetric assay.

[0118] All experiments were repeated at least three times. Data are expressed as mean ± SD. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0119] 2.2 Experimental Results

[0120] To verify whether MKI can directly bind to BAZ2B protein, this invention employed molecular simulation, in vitro binding experiments, and intracellular target verification experiments. Molecular dynamics simulation results showed that the MKI-BAZ2B complex maintained a stable conformation during the simulation, with the RMSD stabilizing after 20 ns. The residue fluctuations in the binding pocket region were minimal, suggesting that MKI can stably bind to the BAZ2B protein. Figure 2 A). Molecular docking results showed that MKI could bind to the acetylated lysine recognition pocket of the BAZ2B bromide domain and form hydrogen bonds and hydrophobic interactions with key amino acid residues, thereby stably binding to the BAZ2B protein. Figure 2 B). Further SPR experiments were used to detect the binding affinity of MKI to the bromodomain (BRD) of BAZ2B protein. The results showed that MKI could directly bind to the BAZ2B BRD, with an equilibrium dissociation constant (KD) of 3.64 μM, indicating that MKI has a good affinity for BAZ2B. Figure 2C). To verify whether MKI can bind to BAZ2B protein intracellularly, a cell heat transfer assay (CETSA) was used. The results showed that, compared with the DMSO group, the stability of BAZ2B protein under different temperature conditions was significantly enhanced after MKI treatment, indicating that MKI can bind to intracellular BAZ2B protein and improve its thermal stability. Figure 2 D、 Figure 2 E). Further verification was performed using a protease protection assay. The results showed that under different concentrations of pronase treatment, the degradation rate of BAZ2B protein in the MKI group was significantly lower than that in the control group, indicating that MKI can bind to BAZ2B protein and exert a protective effect, thereby improving protein stability. Figure 2 F, Figure 2 G). To verify whether MKI regulates myofibroblast activation-related proteins (such as α-SMA, COL1A1, and FN1) by targeting BAZ2B, Western blotting experiments showed that MKI could reverse the promoting effect of BAZ2B on the expression of these proteins. Figure 2 H). The above results indicate that the small molecule compound MKI provided by this invention can directly bind to the bromine domain of BAZ2B, form a stable binding with the BAZ2B protein, and inhibit the expression of its downstream genes.

[0121] Example 3: MKI inhibits the transformation of endometrial stromal cells into myofibroblasts.

[0122] 3.1 Experimental Methods

[0123] 3.1.1 CCK-8 cell viability assay

[0124] Cell proliferation capacity was assessed using the Cell Counting Kit-8 (CCK-8) assay. Human primary endometrial stromal cells were cultured at a concentration of 3 × 10⁶ cells / year. 3 Cells were seeded per well in 96-well plates. After cell adhesion, TGF-β1 (10 ng / mL) was added to induce fibrosis, and MKI (10 μmol / L) was added for treatment. CCK-8 working solution was added at 24 h, 48 h, and 72 h, and the cells were incubated at 37 ℃ for 2 h. The absorbance (OD) was then measured at 450 nm. 450 To evaluate the effect of MKI on cell proliferation.

[0125] 3.1.2 Wound Healing Assay

[0126] Human primary endometrial stromal cells were seeded into six-well plates. When the cell confluence reached approximately 90%, vertical scratches were made using a sterile 200 μL pipette tip. After washing twice with PBS, serum-free culture medium was added, and appropriate treatments were administered. Images of the scratched area were taken at 0 h and 24 h. The scratch area was measured using ImageJ software, and the scratch healing rate was calculated.

[0127] Scratch healing rate (%) = (initial scratch area − remaining scratch area) / initial scratch area × 100%.

[0128] 3.1.3 Transwell migration experiment

[0129] Cell migration ability was assessed using a Transwell chamber (Corning, USA) with an 8 μm pore size. Human primary endometrial stromal cells cultured overnight without serum were digested and collected, then migrated using a 2×10⁻⁶ chamber. 4 Cells were added to the upper chamber, and culture medium containing 10% fetal bovine serum as a chemokine was added to the lower chamber. The cells were incubated at 37 °C in a 5% CO2 incubator for 24 h. After fixation, the cells were stained with 0.1% crystal violet, photographed under a microscope, and the number of migrating cells was counted using ImageJ software.

[0130] 3.1.4 Immunofluorescence detection

[0131] Human primary endometrial stromal cells were seeded on coverslips, induced with TGF-β1, and then treated with MKI. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% BSA. They were then incubated overnight at 4 °C with α-SMA and COL1A1 antibodies, followed by incubation with fluorescently labeled secondary antibody for 2 h. After DAPI staining of the nuclei, confocal microscopy was used to observe the cells and evaluate the effect of MKI on the transformation of endometrial stromal cells into myofibroblasts.

[0132] 3.1.5 Collagen Gel Shrinkage Experiment

[0133] Type I collagen solution (1 mg / mL) and human primary endometrial stromal cells (1.5 × 10⁻⁶) were used. 5 Cells / mL were mixed, with a cell suspension to collagen solution volume ratio of 1:2. After forming a collagen gel at 37 °C, culture medium and TGF-β1 stimulation were added, followed by MKI treatment. Collagen gel shrinkage was observed at 24 h and 48 h, and the area of ​​the collagen gel was measured using ImageJ software after photographing to evaluate cell shrinkage capacity.

[0134] All experiments were repeated at least three times. Data are expressed as mean ± SD. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0135] 3.2 Experimental Results

[0136] During endometrial fibrosis, endometrial stromal cells transform into myofibroblasts, exhibiting enhanced proliferation, migration, and contractile abilities. To evaluate the effect of MKI on endometrial stromal cell activation, this invention employed CCK-8 assay, scratch assay, Transwell assay, immunofluorescence assay, and collagen gel contraction assay. The CCK-8 assay results showed that, compared with the TGF-β1-induced group, MKI treatment significantly reduced cell proliferation (…). Figure 3 A) indicates that MKI can inhibit the abnormal proliferation of endometrial stromal cells in a fibrotic state. Scratch and Transwell assays showed that MKI significantly inhibited TGF-β1-induced migration of endometrial stromal cells, with a significant reduction in both scratch healing rate and the number of migrating cells. Figure 3 B~ Figure 3 D). Further immunofluorescence assays were used to detect the expression of myofibroblast marker proteins. The results showed that TGF-β1 treatment significantly increased the expression of α-SMA, COL1A1, and FN1, while MKI treatment significantly decreased the expression of α-SMA, COL1A1, and FN1, indicating that MKI can inhibit the differentiation of endometrial stromal cells into myofibroblasts. Figure 3 E, Figure 3 F). Since myofibroblasts possess strong contractile ability, this invention further validated this using a collagen gel contraction experiment. The results showed that compared to the TGF-β1-induced group, MKI treatment significantly reduced the degree of collagen gel contraction and increased the collagen gel area, indicating that MKI can inhibit the contractile ability of endometrial stromal cells (F). Figure 3 G, Figure 3 H).

[0137] The test results showed that MKI treatment reduced cell migration and contraction abilities, and decreased the expression level of myofibroblast activation marker proteins.

[0138] 4.1 Experimental Methods

[0139] 4.1.1 RNA sequencing and bioinformatics analysis

[0140] Total RNA was extracted from human primary endometrial stromal cells using TRIzol reagent. RNA integrity and concentration were assessed using an Agilent 2100 bioanalyzer. Library construction and Illumina NovaSeq 6000 PE150 sequencing were performed by Shanghai Ouyi Biomedical Technology Co., Ltd. Raw data underwent quality control using FastQ software and were aligned to the human reference genome (GRCh38) using HISAT2 software. Gene expression quantification was performed using featureCounts. Differential expression analysis was conducted using DESeq2, with adjusted P < 0.05 and |log2FC| > 1 as the screening criteria for differentially expressed genes. Further enrichment analysis using ClusterProfiler software was performed in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Changes in related signaling pathways were analyzed using Gene Set Enrichment Analysis (GSEA).

[0141] 4.2 Experimental Results

[0142] To further clarify the regulatory role of MKI in the extracellular matrix remodeling process of endometrial stromal cells, this invention used RNA sequencing technology to analyze the changes in the cell transcriptome after MKI treatment. RNA sequencing results showed that MKI can regulate the expression of extracellular matrix-related genes and inhibit pathways related to collagen formation and ECM remodeling. Figure 4 A- Figure 4 C). GSEA analysis of differentially expressed genes showed that MKI treatment significantly inhibited signaling pathways related to collagen fiber formation and extracellular matrix tissue formation. Figure 4 D~ Figure 4 H) indicates that MKI can regulate the extracellular matrix remodeling process. Further validation was performed using real-time quantitative PCR to verify the RNA sequencing results. The results showed that, compared with the TGF-β1-induced group, the expression levels of COL1A1, COL3A1, FN1, and other extracellular matrix-related genes were significantly reduced after MKI treatment (H). Figure 4 I). The results showed that the expression levels of multiple extracellular matrix-related genes decreased after MKI treatment.

[0143] Example 5: MKI inhibits endometrial fibrosis in mice with intrauterine adhesions.

[0144] 5.1 Experimental Methods

[0145] 5.1.1 Establishment of a mouse model of intrauterine adhesions

[0146] Female C57BL / 6 mice aged 8–10 weeks were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed at the Laboratory Animal Center of Tongji University, Hubei Campus. All animal experiments were approved by the Tongji University Laboratory Animal Ethics Committee. Mechanical injury modeling was performed during the estrus period of the mice. After anesthesia, the bilateral uterine horns were exposed through a lower abdominal incision. An incision was made approximately 1 mm below the ovary, and the endometrium was repeatedly mechanically scraped using a self-made miniature uterine curette until a noticeably rough feeling appeared in the uterine cavity wall. The incision was then sutured and the abdominal cavity closed. After the model was established, the mice continued to be housed, and uterine tissue was harvested for further analysis after the experiment. HE staining was used to observe changes in the morphology and structure of the uterine tissue and the number of glands; Masson staining was used to detect collagen fiber deposition to evaluate the degree of fibrosis; and immunohistochemistry was used to detect the expression levels of fibrosis-related proteins such as α-SMA, COL1A1, and FN1.

[0147] 5.1.2 Drug preparation and administration regimen

[0148] The vehicle group injection solution was prepared from 2% DMSO, 40% PEG300, 5% Tween-80, and 53% physiological saline. The MKI group injection solution used the same solvent system to dissolve the MKI, with dosages of 5 mg / kg and 10 mg / kg, respectively. The positive control group was treated with the BAZ2A / BAZ2B dual inhibitor GSK2801 (10 mg / kg), and its injection solution was prepared using the same solvent system. Intraperitoneal injections began on day 2 after modeling, administered every 2 days for 14 consecutive days. Mice were sacrificed on day 15, and uterine tissue was collected.

[0149] 5.1.3 Histological and Immunohistochemical Detection

[0150] Uterine tissue was fixed, embedded in paraffin, and sectioned. HE staining was used to observe endometrial morphology, measure endometrial thickness, and count glandular numbers; Masson staining was used to detect collagen fiber deposition area; immunohistochemistry was performed using α-SMA, COL1A1, and FN1 antibodies to detect the expression levels of fibrosis-related proteins. DAB staining was followed by microscopic observation and quantitative analysis using ImageJ software.

[0151] All experiments were repeated at least three times, and data are expressed as mean ± SD. Two-tailed Student's-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0152] 5.2 Experimental Results

[0153] A mechanically induced intrauterine adhesion model was established in 8-10 week old female C57BL / 6 mice after confirming they were in estrus via vaginal smears. Following modeling, mice were treated with Vehicle, MKI (5 mg / kg, 10 mg / kg), and GSK2801 (10 mg / kg), respectively, every two days for a total of 14 days. Figure 5 A). HE staining results showed that, compared with the model group and the GSK2801 treatment group, the MKI treatment group had a more intact endometrial structure, significantly restored intrauterine epithelial continuity, and a significantly increased number of endometrial glands. Figure 5 B and Figure 5 C) suggests that MKI can promote the integrity of damaged endometrial structure. Further statistical analysis revealed that the endometrial thickness in the MKI-treated group was significantly higher than that in the model group and the GSK2801-treated group (C). Figure 5 (D) indicates that MKI promotes endometrial regeneration. Masson staining results showed that a large amount of collagen fiber deposition was present in the uterine tissue of the model group, while the collagen deposition area was significantly reduced after MKI treatment. Figure 5 E and Figure 5 F), suggesting that MKI can effectively alleviate endometrial fibrosis caused by intrauterine adhesions. Further immunohistochemical results showed that, compared with the GSK2801 treatment group, the MKI treatment group had significantly lower levels of α-SMA, COL1A1, and FN1 protein expression. Figure 5 G~ Figure 5 The results indicate that MKI can inhibit the transformation of endometrial stromal cells into myofibroblasts and reduce abnormal extracellular matrix deposition, thereby exerting an anti-fibrotic effect. These results demonstrate that the small molecule compound MKI provided by this invention can alleviate endometrial fibrosis and promote tissue repair by inhibiting the BAZ2B-mediated fibrosis process, and therefore can be used to prepare drugs for the prevention and / or treatment of endometrial fibrosis and intrauterine adhesions.

[0154] In summary, the small molecule compound MKI provided by this invention can significantly improve the endometrial structure of mice with mechanically induced intrauterine adhesions, promote the recovery of endometrial thickness and glandular regeneration, reduce collagen deposition, and downregulate the expression of fibrosis-related proteins. Its anti-fibrotic effect is superior to that of the BAZ2A / BAZ2B dual inhibitor GSK2801, and it can effectively inhibit the occurrence and development of intrauterine adhesion-related endometrial fibrosis.

[0155] The chemical structure of GSK2801 (J Med Chem. 2016 Feb 25;59(4):1410-24.) is as follows:

[0156]

[0157] Example 6: MKI Improves Fertility Outcomes in Mice

[0158] 6.1 Experimental Methods

[0159] 6.1.1 Fertility Experiment

[0160] Fertility is an important indicator for evaluating the recovery of endometrial function. A mouse model of intrauterine adhesions was established using mechanical injury. Drug intervention began on the second day after modeling, with either Vehicle or MKI (10 mg / kg) administered intraperitoneally every two days for 14 consecutive days. The injection solution for the Vehicle group was prepared from 2% DMSO, 40% PEG300, 5% Tween-80, and 53% physiological saline; the injection solution for the MKI group was prepared using the same solvent system. After the drug administration ended, on the 15th day, female mice from each group were paired with normal male mice at a 2:1 ratio. Vaginal plug formation was observed daily. After confirmation of pregnancy, the mice continued to be housed. On day 14.5 of gestation (E14.5), pregnant mice were anesthetized and euthanized. The uterus was dissected, and the embryo resorption rate, number of fetuses, placental weight, and fetal weight were recorded to evaluate the effects of different treatments on the recovery of uterine reproductive function and pregnancy outcomes.

[0161] All experiments were repeated at least three times, and data are expressed as mean ± SD. Two-tailed Student's-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0162] 6.2 Experimental Results

[0163] To further evaluate the effects of MKI on endometrial function recovery and reproductive outcomes, a fertility experiment was conducted in a mouse model of mechanically induced intrauterine adhesions. Figure 6 A). The results showed that, compared with the Vehicle group, the MKI-treated group mice had a significantly higher pregnancy rate, and a significantly increased number of embryo implantation and fetuses. Figure 6 B and Figure 6 C). Meanwhile, the placental weight and fetal weight in the MKI treatment group were significantly higher than those in the Vehicle group ( Figure 6 D and Figure 6 (E) This suggests that MKI can improve adverse pregnancy outcomes caused by intrauterine adhesions. The above results indicate that the small molecule compound MKI provided by this invention can promote the recovery of damaged endometrial function, improve embryo implantation ability, improve fetal development and pregnancy outcomes, thereby improving the fertility of mice with intrauterine adhesions.

[0164] In conclusion, MKI can not only alleviate endometrial fibrosis and promote endometrial regeneration, but also further restore uterine reproductive function, improve pregnancy success rate and fetal development level, indicating that it has good application prospects in the preparation of drugs for the prevention and / or treatment of infertility related to endometrial fibrosis and intrauterine adhesions.

[0165] Example 7 Pharmaceutical Composition

[0166] Small molecule compounds (MKIs) or their pharmaceutically acceptable salts can be mixed with pharmaceutically acceptable carriers, diluents, stabilizers, buffers, preservatives, or excipients, and prepared into pharmaceutical compositions suitable for different routes of administration using conventional formulation processes.

[0167] Preferably, the pharmaceutical composition can be prepared as an injection, lyophilized powder for injection, tablet, capsule, granule, oral liquid, sustained-release preparation, hydrogel preparation, nanopreparation, intrauterine local sustained-release material, or other pharmaceutically acceptable dosage form.

[0168] Preferably, the mass content of the small molecule compound MKI or its pharmaceutically acceptable salt in the pharmaceutical composition is 0.1 to 50 wt%.

[0169] Preferably, the dosage of the pharmaceutical composition is 1 to 50 mg / kg, more preferably 5 to 20 mg / kg.

[0170] Preferably, the pharmaceutical composition can be administered via oral administration, intravenous injection, intraperitoneal injection, intrauterine local administration, vaginal administration, or sustained-release administration.

[0171] Furthermore, the pharmaceutical composition can be used alone or in combination with other treatment methods.

[0172] Preferably, the combined treatment includes antifibrotic drug therapy, hormone therapy, biomaterial or tissue engineering scaffold therapy, anti-adhesion material therapy, surgical treatment, and other adjuvant treatments that promote tissue repair.

[0173] The test results showed that the small molecule compound MKI could reduce the expression level of fibrosis-related molecules, reduce abnormal extracellular matrix deposition and collagen formation, and promote the repair of damaged tissues.

[0174] In some embodiments, the pharmaceutical composition can be used to prevent and / or treat fibrosis-related diseases mediated by abnormal fibroblast activation, myofibroblast formation, and excessive extracellular matrix deposition.

[0175] Preferably, the fibrosis-related diseases include, but are not limited to, endometrial fibrosis, intrauterine adhesions, liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, ovarian fibrosis, skin fibrosis, and peritoneal fibrosis.

[0176] Example 8 Other Implementation Methods

[0177] Those skilled in the art will understand that abnormal activation of fibroblasts, formation of myofibroblasts, and excessive deposition of extracellular matrix are the common pathological basis for the development of fibrosis in various tissues and organs.

[0178] The test results showed that the small molecule compound MKI could reduce the expression level of fibrosis-related molecules, reduce collagen deposition and extracellular matrix remodeling, and promote the repair of damaged tissues.

[0179] Therefore, without departing from the spirit and scope of this invention, the small molecule compound MKI or its pharmaceutically acceptable salt described in this invention can be used not only for the prevention and / or treatment of endometrial fibrosis and intrauterine adhesions, but also for other fibrosis-related diseases.

[0180] In some embodiments, the fibrosis-related diseases include, but are not limited to, liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, ovarian fibrosis, skin fibrosis, and peritoneal fibrosis.

[0181] In some embodiments, the small molecule compound MKI or a pharmaceutically acceptable salt thereof may be used alone or in combination with antifibrotic drugs, hormone therapy, biomaterials, tissue-engineered scaffolds, surgery or other treatments that promote tissue repair.

[0182] Those skilled in the art can make appropriate adjustments to the dosage, administration method, and administration cycle based on the pathological characteristics of fibrosis in different tissues and organs, without departing from the spirit and scope of protection of this invention.

[0183] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. The use of a small molecule compound targeting BAZ2B or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of fibrosis-related diseases, characterized in that, The small molecule compound is selected from any one of MKI, CRT0066101, Mavacoxib, CB30865, and SP-100030, and its structural formulas are shown in formulas (I) to (V) below: Formula I Formula II Formula III Formula IV Formula V.

2. The application as described in claim 1, characterized in that, The fibrosis-related diseases mentioned are diseases caused by abnormal activation of myofibroblasts and excessive deposition of extracellular matrix, selected from one or more diseases among liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, ovarian fibrosis, skin fibrosis, peritoneal fibrosis and endometrial fibrosis.

3. The application as described in claim 1, characterized in that, The small molecule compound or its pharmaceutically acceptable salt achieves antifibrotic treatment by targeting the BAZ2B gene or protein in stromal cells.

4. The application as described in claim 1, characterized in that, The fibrosis-related diseases mentioned are selected from endometrial fibrosis and intrauterine adhesions.

5. The application as described in claim 4, characterized in that, The drug is a drug that has one or more of the following effects: 1) It can bind to BAZ2B protein and bind to the bromine domain of BAZ2B protein; 2) It can inhibit the activation of endometrial stromal cells and their transformation into myofibroblasts; 3) It can reduce the proliferation, migration, and contraction of endometrial stromal cells; 4) It can downregulate the expression of α-smooth muscle actin, type I collagen, type III collagen, fibronectin, and other extracellular matrix-related factors; 5) It can reduce collagen deposition and abnormal remodeling of the extracellular matrix; 6) It can promote endometrial repair, gland regeneration and recovery of reproductive function.

6. The application as described in claim 1, characterized in that, The tumor-targeted therapy drug uses a small molecule compound or its salt represented by any one of formulas (I) to (V) as the sole active ingredient, or contains a small molecule compound or its salt represented by any one of formulas (I) to (V).

7. The application as described in claim 1, characterized in that, In the tumor-targeted therapy drug, the content of any one of the small molecule compounds or their salts represented by formula (I) to (V) is 0.1-99 wt%.

8. A pharmaceutical composition for the treatment of fibrosis, characterized in that, It comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is selected from... The small molecule compound of claim 1 or a pharmaceutically acceptable salt thereof.

9. The pharmaceutical composition for the treatment of fibrotic diseases as described in claim 8, characterized in that, The pharmaceutical composition is used in combination with other antifibrotic treatments. When the fibrotic disease is endometrial fibrosis and intrauterine adhesions, the pharmaceutical composition can be used in combination with hysteroscopic adhesiolysis, hormone therapy, anti-adhesion materials or biological scaffold materials.

10. The pharmaceutical composition for the treatment of fibrotic diseases as described in claim 8, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, granules, injections, lyophilized powder for injection, intrauterine instillation preparations, vaginal administration preparations, sustained-release preparations, hydrogel preparations, or nano-preparations.

Images