Application of MIM2 in preparation of product for treating tissue fibrosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of MIM2 and derivatives thereof in preparation of products for treating tissue fibrosis. It is found that the compound MIM2 can effectively inhibit expression and synthesis of various cancers (extracellular matrix proteins such as collagen and laminin in cells). In-vitro experiments show that after MIM2 treatment, collagen and laminin expression of the cancer cells, skin fibroblasts, hepatic stellate cells and embryonic lung cells is remarkably reduced. Animal experiments further prove that the MIM2 can effectively inhibit collagen deposition in tumor tissues of tumor-bearing mice, can slow down the fibrosis process of liver fibrosis and pulmonary fibrosis mouse models, and can reduce the level of plasma hyaluronic acid at the same time. The results show that the MIM2 has the effects of inhibiting extracellular matrix deposition and delaying tissue fibrosis, and is expected to be developed into small molecular drugs for treating cancer-related fibrosis and other fibrosis diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of MIM2 and its derivatives in the preparation of products for treating tissue fibrosis. Background Art

[0002] Fibrosis is a complex and common pathological process characterized by the abnormal proliferation of connective tissue within a tissue or organ, resulting in the excessive deposition of fibrous connective tissue. This pathological change can be triggered by a variety of factors, including toxins, infectious pathogens, autoimmune reactions, as well as injury and mechanical stress. As fibrosis progresses continuously, it may lead to organ failure and even endanger life in severe cases.

[0003] As a common pathological feature of various diseases, fibrosis widely exists in cancer, complications after radiotherapy, liver diseases, lung diseases, and skin injuries, etc., and has a significant impact on the health and quality of life of patients. For example, cancer-related fibrosis may lead to treatment resistance, while post-radiotherapy fibrosis seriously affects the quality of life and there is currently a lack of effective treatment strategies. The liver-related mortality of patients with advanced liver fibrosis increases significantly, and the mortality rate of patients with stage 4 liver fibrosis reaches about 50%. Idiopathic pulmonary fibrosis is a severe and progressive irreversible interstitial lung fibrosis disease. Currently, there are approximately 1 million idiopathic pulmonary fibrosis patients globally, with a 5-year survival rate of only 30%-50% and an average survival period of less than 3 years. Therefore, there is an urgent need to find new therapeutic targets and develop effective treatment strategies to prevent the further development of fibrosis and improve the prognosis and quality of life of patients.

[0004] Although different types of fibrotic diseases exhibit unique clinical features, they share common pathological mechanisms, including abnormal deposition of the extracellular matrix (ECM), persistent chronic inflammation, increased apoptosis and senescence, and imbalances in growth factors and cytokines. Currently, two drugs have been approved by the US Food and Drug Administration (FDA) for the treatment of idiopathic pulmonary fibrosis, namely pirfenidone and nintedanib. These two drugs can, to a certain extent, slow down the progression of pulmonary fibrosis and play a certain preventive role in cases of sudden symptom exacerbation. However, clinical trial results show that pirfenidone has limited efficacy in patients with poor baseline lung function, while nintedanib can only delay the rate of decline in lung function, cannot completely prevent disease progression, reverse the course of the disease, and its efficacy in patients with severe disease (lung function value FVC%pre < 50%) or acute exacerbation is not yet clear. In addition, these drugs are also accompanied by side effects. For example, nintedanib may cause diarrhea and nausea, while pirfenidone can cause drug-induced liver injury, photosensitive rashes, and severe gastrointestinal reactions. Currently, new drugs and treatment regimens are being developed and tested, but no new treatment methods have been approved by the FDA.

[0005] It is worth noting that targeting the common mechanisms of fibrosis is an important basis for the development of new drugs. Current research has not fully elucidated the mechanism of action and targets of pirfenidone in anti-pulmonary fibrosis. Although some studies suggest that MAPK12 may be its target, the relevant evidence is still insufficient.

[0006] Therefore, further developing drugs targeting fibrosis and their targets has great clinical application value. Summary of the Invention

[0007] An object of the first aspect of the present invention is to provide the use of MIM2 and its derivatives in the preparation of products for treating tissue fibrosis.

[0008] An object of the second aspect of the present invention is to provide a method.

[0009] To achieve the above objects of the present invention, the technical solutions adopted by the present invention are as follows:

[0010] The first aspect of the present invention provides the use of a small molecule compound (MAPK12 inhibitor MIM2) and its derivatives in the preparation of products for treating tissue fibrosis.

[0011] The present invention provides the use of a small molecule compound (MAPK12 inhibitor MIM2) and its derivatives in the preparation of drugs for targeted treatment of tissue fibrosis. The molecular formula of the small molecule compound is C 42 H 50 N 2 O 6, this small molecule is derived from MolPort, with the compound number Molport-044-810-679, and its structural formula is shown in Formula (I):

[0012]

[0013] The small molecule compound (MAPK12 inhibitor MIM2) of this application is screened from the ZINC15 chemical database, named ZINC000824519333. According to the previous research of the inventors, currently this small molecule can play an anti-tumor role in vivo and in vitro, and other functions are unknown, which is referred to as "MIM2" in this specification.

[0014] In some embodiments of the present invention, the MIM2 or its derivatives include at least one of pharmaceutically acceptable salts and pharmaceutically acceptable modifications.

[0015] In some embodiments of the present invention, the pharmaceutically acceptable salts include acid addition salts and base addition salts.

[0016] "Pharmaceutically acceptable acid addition salts" refer to salts that retain the biological effectiveness and properties of the free base and are not undesirable in biological or other aspects, and are formed with inorganic acids and organic acids. Examples of the inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and examples of the organic acids include, but are not limited to, acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, etc.

[0017] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not undesirable biologically or otherwise. These salts are prepared by the addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of the following: primary amines, secondary amines, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0018] In some embodiments of the present invention, the pharmaceutically acceptable modifications include at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phosphatidylation, and halogenation. For example, the lipophilicity can be enhanced by introducing fluorine atoms (fluoroethyl), improving the blood-brain barrier permeability. The corresponding modification method can be selected according to the actual use of the drug.

[0019] In some embodiments of the present invention, the tissue fibrosis includes at least one of tumor and fibrosis after tumor radiotherapy, skin fibrosis, liver fibrosis, and pulmonary fibrosis.

[0020] In some embodiments of the present invention, the tumor includes at least one of colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer.

[0021] In some embodiments of the present invention, the product includes pharmaceuticals.

[0022] In some embodiments of the present invention, the drug includes pharmaceutically acceptable excipients and / or any one or more other active ingredients.

[0023] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, and carriers.

[0024] The present invention provides a drug and a corresponding pharmaceutical dosage form for treating tissue fibrosis, with MIM2 as the active ingredient.

[0025] Among them, the drug exists in the form of an oral preparation, an injection, or a topical administration preparation.

[0026] In particular, the oral preparation includes tablets, capsules, pills, powders, granules, syrups, or solutions; the injection includes an injection dosage form or a freeze-dried powder injection dosage form for injection; the topical administration preparation includes creams, ointments, sprays, aerosols, or patches.

[0027] Among them, the pharmaceutical preparation uses the MIM2 as the effective active ingredient and includes other pharmaceutically acceptable carrier components.

[0028] The carriers in the drug include excipients such as starch, water, etc.; lubricants such as magnesium stearate, etc.; disintegrants such as microcrystalline cellulose, etc.; fillers such as lactose, etc.; binders such as pregelatinized starch, dextrin, etc.; sweeteners; antioxidants; preservatives; flavoring agents; fragrances, etc.

[0029] When preparing the oral preparation, the carriers selected can be conventional pharmaceutical excipients such as starch, dextrin, or cyclodextrin and various chemically modified cyclodextrins, sucrose, stearates, etc. When preparing the freeze-dried powder injection, it can be prepared by methods such as sterile spray drying, low-temperature vacuum drying, freeze drying, etc. The later preparation processes and equipment of each preparation belong to the conventional technologies in the pharmaceutical field, and the present invention does not limit this.

[0030] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and serve as the inactive ingredients of the medicament. Compilations of pharmaceutically acceptable excipients can be found in tools such as "Handbook of Pharmaceutical Excipients" (2nd edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994); "List of Medicinal Excipients in the Pharmacopoeia of the People's Republic of China", etc.

[0031] In some embodiments of the present invention, the dosage form of the product includes a gastrointestinal administration dosage form or a non-gastrointestinal administration dosage form.

[0032] In some embodiments of the present invention, the gastrointestinal administration dosage form includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets.

[0033] In some embodiments of the present invention, the parenteral dosage form includes at least one of an injection dosage form, a respiratory dosage form, a cutaneous dosage form, a mucosal dosage form, and a cavity dosage form.

[0034] In some embodiments of the present invention, the subject to which the product is administered is a mammal.

[0035] In some embodiments of the present invention, the mammal includes humans.

[0036] A second aspect of the present invention provides a method for non-therapeutically inhibiting tissue fibrosis in vitro, including the step of treating cells and tissues with the small molecule compound MIM2.

[0037] In some embodiments of the present invention, the mass concentration of the small molecule compound MIM2 ≥ 1 μM.

[0038] In some embodiments of the present invention, the mass concentration of the small molecule compound MIM2 is 1 - 20 μM.

[0039] The beneficial effects of the present invention are as follows:

[0040] The present invention discloses the use of a small molecule compound and its derivatives in the preparation of a targeted therapeutic for tissue fibrosis. This small molecule compound can be used as a targeted therapeutic fibrosis drug to inhibit the synthesis of extracellular matrix components such as collagen and laminin in cells, and delay the excessive deposition of extracellular matrix proteins.

[0041] The present invention discovers that the compound MIM2 can effectively inhibit the expression and synthesis of extracellular matrix proteins such as collagen and laminin in cells of various cancers (colorectal cancer, gastric cancer, pancreatic cancer, breast cancer). In vitro experiments show that after treatment with MIM2, the expression of collagen and laminin in the above-mentioned cancer cells, as well as skin fibroblasts, hepatic stellate cells, and embryonic lung cells, is significantly reduced. Animal experiments further confirm that MIM2 can effectively inhibit collagen deposition in the tumor tissues of tumor-bearing mice, and can slow down the fibrosis process in mouse models of liver fibrosis and lung fibrosis, while reducing the plasma hyaluronic acid level. These results indicate that MIM2 has the effect of inhibiting extracellular matrix deposition and delaying tissue fibrosis, and is expected to be developed into a small molecule drug for the treatment of cancer-related fibrosis and other fibrosis diseases (such as skin, liver, and lung fibrosis). BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The following further describes the present invention with reference to the drawings and examples, wherein:

[0043] Figure 1Expression of collagen and laminin in colorectal cancer cells (A), gastric cancer cells (B), pancreatic cancer cells (C), and breast cancer cells (D) after treatment with small molecule compound MIM2.

[0044] Figure 2 Drug treatment of small molecule compound MIM2 for mice with spontaneous intestinal cancer.

[0045] Figure 3 Changes in the expression of collagen and laminin in irradiated colorectal cancer cells (A) and embryonic lung cells (B) under the treatment of small molecule compound MIM2.

[0046] Figure 4 Expression of collagen and laminin in skin fibroblasts (A), hepatic stellate cells (B), and embryonic lung cells (C) after treatment with small molecule compound MIM2.

[0047] Figure 5 Therapeutic effect of MIM2 in a mouse model of carbon tetrachloride-induced liver fibrosis; where A is the result of changes in mouse body weight; B is the result of changes in the content of hyaluronic acid in mouse plasma; C is the result of liver tissue size; D is the result of liver nodule changes; E is the result of collagen changes in liver tissue.

[0048] Figure 6 Therapeutic effect of MIM2 in a mouse model of bleomycin-induced pulmonary fibrosis; where A is the result of changes in body weight; B is the result of changes in the content of hyaluronic acid in mouse plasma; C is the result of lung tissue size; D is the result of lung tissue staining; E is the result of collagen changes in lung tissue.

[0049] Figure 7 Comparison of the effects of MIM2 and pirfenidone, where A is the molecular docking results of MIM2 and pirfenidone with MAPK12 respectively, and B is the binding efficiency results of MIM2 and pirfenidone with MAPK12 respectively.

[0050] Figure 8 Comparison results of the therapeutic effects of MIM2 and pirfenidone. Detailed implementation manners

[0051] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0052] The colorectal cancer cells, gastric cancer cells, pancreatic cancer cells, breast cancer cells, skin fibroblasts, hepatic stellate cells, and embryonic lung cells mentioned in this application are derived from the cell lines already available in our laboratory or can be purchased commercially. The culture medium used for culturing the cells was purchased from Gibco, and the fetal bovine serum was purchased from ExCell Bio. The cell lines were cultured in RPMI-1640, DMEM, and McCoy's 5A media containing 10 v / v% fetal bovine serum, and the culture environment was 37 °C and 5% CO 2 incubator, and cells with good growth status were taken for subsequent experiments.

[0053] In some specific embodiments, the term "treatment" refers to a means of obtaining a beneficial or desired result (including but not limited to therapeutic benefit and / or prophylactic benefit) in relation to a disease, disorder, or medical condition. Therapeutic benefit means eliminating or ameliorating the underlying condition being treated. Additionally, a therapeutic benefit is achieved by eliminating or ameliorating one or more of the physiological symptoms associated with the underlying condition, such that an improvement in the individual is observed, even though the individual may still be afflicted with the underlying condition.

[0054] Example 1 MIM2 inhibits the expression of collagen and laminin in tumor cells

[0055] To explore the effect of MIM2 on inhibiting tissue fibrosis, this example detected the expression of the main components of the extracellular matrix (such as collagen and laminin) in tumor cells. 1×10 6 colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cells were seeded in six-well plates and treated with MIM2 drugs at designated concentrations (1, 5, 10 μM) for 24 h. The cells were collected, washed twice with PBS, and RNA was extracted using the TRIzol method. 1 μg of RNA and 4 μL of 4×gDNA wiper Mix (R233-01; Vazyme) were added to a MicroAmp TM reaction tube, and nuclease-free water was added to make the volume up to 20 μL, and then pipetted and mixed well. The above mixture was placed in a PCR instrument and reacted at 42 °C for 2 min, and then placed on ice for later use. 4 μL of 5×HiScript III qRT SuperMix (R233-01; Vazyme) was added, and after pipetting and mixing well, it was placed in a PCR instrument and reacted at 50 °C for 15 min and 85 °C for 5 s to obtain the reverse transcription product. PCR primers were designed using Primer-Blast on the NCBI website, and a PCR experiment was performed using the Cham Q SYBR qPCR Master Mix (Q311-02; Vazyme) kit from Novoprotein to detect the expression of representative fibrosis genes, including the expression of collagen (COL1A1, COL3A1, COL5A1, COL6A1) and laminin (LAMA4, LAMB2). The primer sequences are shown in Table 1.

[0056] Table 1 Primer sequence information

[0057]

[0058] The experimental results are as Figure 1 shown. In colorectal cancer ( Figure 1 A), gastric cancer ( Figure 1 B), pancreatic cancer ( Figure 1 C), and breast cancer cells ( Figure 1 D), after 24 hours of treatment with MIM2 drug, the expression of collagen and laminin was found to be decreased by real-time quantitative fluorescence PCR, especially after treatment with 10 μM MIM2 drug, the expression of related molecules was significantly decreased; it was proved that MIM2 inhibited the expression of extracellular matrix components in tumor cells.

[0059] Example 2 MIM2 inhibits collagen deposition in tumor tissues

[0060] This experiment was conducted in spontaneously tumor-bearing mice (8 - 10 weeks old, male). All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (approval number L025504202109020). Twelve spontaneously tumor-bearing mice were selected. Each mouse was given 50 μl of tamoxifen (1 mg / kg; H7904; Sigma-Aldrich) by rectal enema and continuously given drinking water containing doxycycline (10592 - 13 - 9; Macklin) (1 g / L) to induce intestinal tumors. Approximately 3 weeks later, primary intestinal tumors in mice could be seen under colonoscopy. After all 12 mice developed tumors, the mice were randomly divided into 2 groups: a control group and a treatment group, with 6 mice in each group. Since MIM2 is dissolved in DMSO, the control group was intraperitoneally injected with 10 mg / kg of DMSO daily according to the body weight of the mice, and the treatment group was intraperitoneally injected with 10 mg / kg of MIM2 drug daily according to the body weight of the mice. Since the time sequence of tumor formation in the 12 mice was different, this application started drug administration when all 12 mice had tumors. At the end of the experiment, tumors from 3 mice in each of the control group and the treatment group were fixed and sectioned, and Masson's trichrome staining (S0075; Bioss) and Sirius red staining (S0080; Bioss) were used to analyze the deposition of collagen in the tumors. The operation steps of Masson's trichrome staining were as follows: dewax the paraffin sections to water, stain the nuclei with hematoxylin staining solution for 5 min; differentiate with 1% hydrochloric acid alcohol for 5 - 15 s, and rinse with running water; stain with Ponceau acidic fuchsin solution for 5 - 10 min; wash with 2% glacial acetic acid aqueous solution for 1 min; differentiate with 1% phosphomolybdic acid aqueous solution for 3 - 5 min; without washing with water, directly stain with aniline blue aqueous solution for 1 - 2 min; wash with 0.2% glacial acetic acid aqueous solution for 1 min; dehydrate, clear, and seal with neutral gum. The steps of Sirius red staining were as follows: dewax the paraffin sections to water, drop-stain with Sirius red staining solution for 30 min; slightly rinse with running water to remove the staining solution on the surface of the sections; dehydrate, clear, and seal with neutral gum.

[0061] The experimental results are as Figure 2 shown. Through 14 days of drug administration, it was found that the living state of the mice was not affected and their diet and activities were normal. By calculating the collagen staining of the tumor tissues, it was found that the collagen content in the tumor tissues of the treatment group was significantly less than that of the control group. This shows that MIM2 can inhibit the collagen synthesis of tumor cells and thus inhibit the deposition of extracellular matrix in tumor tissues.

[0062] Example 3 MIM2 inhibits the expression of collagen and laminin in irradiated cells

[0063] In a 10 cm culture dish, 5×10 6Colorectal cancer cells and embryonic lung cells were irradiated with an irradiator (current 15 mA, voltage 160 kV, dose rate 1 Gy / min, total dose 10 Gy), and then the cells were continuously cultured. Subsequently, the cells were treated with 10 μM of MIM2 drug for 24 h. The cells were collected, washed twice with PBS, and RNA was extracted using the TRIzol method. 1 μg of RNA and 4 μL of 4×gDNA wiper Mix (R233-01; Vazyme) were taken into a MicroAmp TM reaction tube, and nuclease-free water was added to make the volume up to 20 μL, and then pipetted and mixed well. The above mixture was placed in a PCR instrument and reacted at 42 °C for 2 min, and then placed on ice for later use. 4 μL of 5×HiScript III qRTSuperMix (R233-01; Vazyme) was added, and after pipetting and mixing well, it was placed in a PCR instrument and reacted at 50 °C for 15 min and at 85 °C for 5 s to obtain the reverse transcription product. PCR primers were designed using Primer-Blast on the NCBI website, and a PCR experiment was carried out using the Cham Q SYBR qPCR Master Mix (Q311-02; Vazyme) kit from Novoprotein to detect the expression of representative fibrosis genes, including the expression of collagen (COL1A1, COL3A1, COL5A1, COL6A1) and laminin (LAMA4, LAMB2). The sequence information is the same as in Table 1.

[0064] The experimental results are as Figure 3 shown. In colorectal cancer ( Figure 3 Group A), embryonic lung cells ( Figure 3 Group B), after irradiation, the expression of collagen and laminin in the cells increased significantly. However, after treatment with 10 μM of MIM2 drug for 24 h, it was detected that the expression of collagen and laminin decreased significantly, proving that MIM2 inhibits the expression of extracellular matrix components after cell radiotherapy.

[0065] Example 4 MIM2 inhibits the expression of collagen and laminin in cells derived from skin / liver / lung

[0066] 1×10 6 skin fibroblasts, hepatic stellate cells, and embryonic lung cells were seeded in six-well plates respectively, and were treated with 5 μM and 10 μM of MIM2 drug for 24 h. The cells were collected, washed twice with PBS, and RNA was extracted using the TRIzol method. 1 μg of RNA and 4 μL of 4×gDNA wiper Mix (R233-01; Vazyme) were taken into a MicroAmp TMThe reaction tube was filled with nuclease-free water to a final volume of 20 μL and pipetted to mix well. The above mixture was placed in a PCR instrument and reacted at 42 °C for 2 min, then placed on ice for later use. 4 μL of 5×HiScript III qRT SuperMix (R233-01; Vazyme) was added, and after pipetting to mix well, it was placed in a PCR instrument and reacted at 50 °C for 15 min and at 85 °C for 5 s to obtain the reverse transcription product. PCR primers were designed using Primer-Blast on the NCBI website, and a PCR experiment was performed using the Cham QSYBR qPCR Master Mix (Q311-02; Vazyme) kit from Novoprotein to detect the expression of representative fibrosis genes, including the expression of collagen (COL1A1, COL3A1, COL5A1, COL6A1) and laminin (LAMA4, LAMB2). The primer sequences were the same as those in Table 1.

[0067] The experimental results are as Figure 4 shown. After 24 h of treatment with MIM2 drug, it was detected that the expression of collagen and laminin decreased in skin fibroblasts ( Figure 4 A in), hepatic stellate cells ( Figure 4 B in), and embryonic lung cells ( Figure 4 C in), especially the expression of related molecules decreased significantly after treatment with 10 μM MIM2 drug; it was proved that MIM2 inhibited the expression of extracellular matrix components in cells derived from skin / liver / lung.

[0068] Example 5 Treatment of MIM2 in liver fibrosis

[0069] The C57BL / 6J mice used in the experiment were all purchased from the Sun Yat-sen University Cancer Center. All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (approval number L025504202109020). Ten female C57 mice at 4-6 weeks of age were selected and intraperitoneally injected with carbon tetrachloride (C805327; Macklin) at a concentration of 0.5 ml / kg, twice a week for 6 consecutive weeks. Subsequently, the C57 mice were randomly divided into 2 groups, namely the control group and the drug treatment group, with 5 C57 mice in each group. Since MIM2 was dissolved in DMSO, the control group was intraperitoneally injected with 10 mg / kg of DMSO daily according to the mouse body weight, and MIM2 was intraperitoneally injected at 10 mg / kg daily according to the C57 mouse body weight for a total of 14 days. The time of the first administration of MIM2 was set as day 0, and the body weight of the C57 mice was weighed every other day with an electronic balance, and the measurement was accumulated 7 times. At the end of the experiment, EDTA anticoagulant tubes were used to collect blood from the orbital venous plexus of the mice, centrifuged at 2000×g for 20 minutes at 4°C, and the upper plasma was collected for plasma enzyme-linked immunosorbent assay (ml500128; Enzyme-linked Biotechnology). The specific operation steps were as follows: Take out the ELISA pre-coated plate, set 1 blank control well and 6 standard wells, and add 50 μl of standard product to each well; directly add 50 μl of the plasma to be tested to each of the remaining test wells. Except for the blank well, add 50 μl of biotinylated antigen to all wells, mix well, stick on the sealing film, and incubate at 37°C for 60 minutes. Discard the liquid in the wells, fill each well with the washing solution, let it stand for 10 seconds and then shake dry, repeat 3 times and then pat dry. Add 50 μl of enzyme-labeled avidin to each well (except the blank control well), mix well, stick on the sealing film, and incubate at 37°C for 30 minutes. Discard the liquid in the wells, fill each well with the washing solution, let it stand for 10 seconds and then shake dry, repeat 3 times and then pat dry. Add 50 μl of chromogenic agent A and 50 μl of chromogenic agent B to each well, mix well by oscillation, and then incubate at 37°C in the dark for 15 minutes. Add 50 μl of stop solution to each well. Read with an enzyme-labeled instrument and measure hyaluronic acid at a wavelength of 450 nm.

[0070] At the same time, the livers of each group of mice were taken for photographing and fixed sectioning, and Masson trichrome staining (S0075; Bioss) was used to analyze the fibrosis progression of the liver. The operation steps of Masson trichrome staining were as follows: Dewax the paraffin sections to water, stain the nuclei with hematoxylin staining solution for 5 min; differentiate with 1% hydrochloric acid alcohol for 5-15 s, and rinse with running water; use Ponceau acid fuchsin solution for 5-10 min; wash with 2% glacial acetic acid aqueous solution for 1 min; differentiate with 1% phosphomolybdic acid aqueous solution for 3-5 min; without washing with water, directly stain with aniline blue aqueous solution for 1-2 min; wash with 0.2% glacial acetic acid aqueous solution for 1 min; dehydrate and clear, and seal with neutral gum.

[0071] The experimental results were as Figure 5 shown. Through 14 days of drug administration, it was found that the diet and activities of the mice gradually returned to normal, and the body weight increased significantly compared with the control group (Figure 5 In A). Plasma enzyme-linked immunosorbent assay detected a decrease in the content of hyaluronic acid in the blood of mice after MIM2 treatment ( Figure 5 In B). Gross and HE pictures of the liver showed that the liver surface tended to be smooth and the nodules decreased after MIM treatment ( Figure 5 In C-D). By calculating the collagen staining of liver tissue, it was found that the content of collagen in the liver tissue of the drug-treated group was significantly less than that of the control group ( Figure 5 In E). It was shown that MIM2 could inhibit the deposition of extracellular matrix in liver tissue and slow down the process of liver fibrosis.

[0072] Example 6 Treatment of MIM2 in pulmonary fibrosis

[0073] The C57BL / 6J mice used in the experiment were all purchased from the Sun Yat-sen University Cancer Center. All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (approval number L025504202109020). Ten female C57 mice at 4-6 weeks of age were selected and instilled with bleomycin solution (0.4 mg / mL, 100 μL per mouse; B802467; Macklin) through the trachea and continued to be fed for 6 weeks. Subsequently, the C57 mice were randomly divided into 2 groups, namely the control group and the drug-treated group, with 5 C57 mice in each group. Since MIM2 was dissolved in DMSO, the control group was intraperitoneally injected with 10 mg / kg of DMSO per day according to the body weight of the mice, and MIM2 was intraperitoneally injected with 10 mg / kg per day according to the body weight of C57 mice for a total of 14 days. The time of the first administration of MIM2 was set as day 0, and the body weight of C57 mice was weighed every other day with an electronic balance, and measured 7 times in total. At the end of the experiment, EDTA anticoagulant tubes were used to collect blood from the orbital venous plexus of mice for plasma enzyme-linked immunosorbent assay (ml500128; Enzyme-linked Biology). At the same time, lung tissues of each group of mice were taken for photography and fixed section, and the fibrosis progress of the lungs was analyzed by Masson trichrome staining (S0075; Bioss).

[0074] The experimental results were as Figure 6 shown. It was found that the diet and activities of mice gradually returned to normal after 14 days of drug administration, and the body weight increased significantly compared with the control group ( Figure 6 In A). Plasma enzyme-linked immunosorbent assay detected a decrease in the content of hyaluronic acid in the blood of mice after MIM2 treatment ( Figure 6 In B). Gross and HE pictures of the lungs showed that the surface was smooth and rosy and no infarction was seen after MIM treatment ( Figure 6 In C-D). By calculating the collagen staining of lung tissue, it was found that the content of collagen in the lung tissue of the drug-treated group was significantly less than that of the control group ( Figure 6 In E). It was shown that MIM2 could inhibit the deposition of extracellular matrix in lung tissue and slow down the process of pulmonary fibrosis.

[0075] Comparison of the Binding Efficacy of MIM2 in Example 7

[0076] Currently, MIM2 has been found to be a member of the p38 MAPK family belonging to MAPK12. The reported functions of MAPK12 mainly include the following aspects: (1) MAPK12 plays an important role in mediating innate immune responses and inflammatory reactions. MAPK12 can promote the elongation and maturation of TNFα by inhibiting the activity of EF2K so that it cannot inhibit the activity of eEF2. (2) MAPK12 was initially discovered during the study of myoblast differentiation. Studies on MAPK12-deficient mice have shown that MAPK12 plays a key role in preventing the premature differentiation of skeletal muscle stem cells. (3) MAPK12 is involved in regulating some processes related to cell malignant transformation. MAPK12 can mediate inflammatory signals to promote the occurrence of colorectal cancer; in triple-negative breast cancer, MAPK12 stimulates the expansion of cancer stem cell-like cells by transactivating Nanog through c-Jun / AP-1, etc. However, there is no direct report in the literature on the regulatory relationship between MAPK12 and fibrosis formation.

[0077] This example further compares the effects of MIM2 with other fibrosis treatment drugs. Pirfenidone is a pleiotropic pyridine compound with anti-inflammatory, anti-fibrotic, and antioxidant properties and has a certain therapeutic effect on idiopathic pulmonary fibrosis. Its mechanism of action mainly inhibits the proliferation of fibroblasts and collagen deposition by inhibiting transforming growth factor-β and other inflammatory mediators.

[0078] This example demonstrates the specific differences between the two drugs through drug affinity detection.

[0079] Based on virtual screening by molecular docking in this example, lead compounds that can bind to the MAPK12 protein are screened from the ZINC15 compound database, and then autonomous docking is performed. The best binding energy Vina score is selected, and the receptor-ligand binding conformation results are output.

[0080] In addition, further in vitro binding experiments were carried out by purifying the MAPK12 protein. The DNA sequence of MAPK12 was synthesized and cloned into the prokaryotic expression vector pET28a. The synthesized pET28a-MAPK12 plasmid was transformed into E. coli BL(Rosetta) competent cells, and positive recombinants were screened on a kanamycin-resistant plate. Colony PCR verification and DNA sequencing verification were performed on them, and the sequencing results were analyzed by NCBI Blast. The strain with correct sequencing was used for subsequent protein purification. In this example, the bacterial solution with correct sequencing was inoculated into LB medium containing ampicillin resistance (100 μg / mL) and cultured overnight at 37 °C with shaking. The next day, the activated bacteria were inoculated into fresh medium for expansion culture at a ratio of 1:100. When the OD600 reached 0.6 at 37 °C with shaking at 180 rpm, the temperature was lowered to 20 °C, and isopropyl β-D-thiogalactopyranoside (IPTG; HY-15921; MedChemExpress) was added. The final concentration of IPTG was 0.5 mM, and the expression was induced for about 18 h under the same conditions. The bacterial cells were collected, centrifuged at 5000 rpm at 4 °C for 30 min, and the bacterial cells were resuspended with 1×PBS buffer and centrifuged again to collect the bacterial cells. The collected bacterial cells were resuspended with 1×PBS buffer again to 10% of the bacterial solution, and then a high-pressure cell disruptor was used to disrupt the cells at a pressure of 900 Pa and a cycle of 3 min at 4 °C. Centrifuge at 10000 rpm at 4 °C for 1 h, discard the precipitate, and filter the supernatant through a 0.22 mm pore size filter membrane. The obtained supernatant was resuspended with Ni-NTA agarose gel (SA005005; Tiandi Renhe) and incubated at 4 °C for 2 h. The color of Ni-NTA agarose gel changed from blue to brown during the incubation. Centrifuge at 1000 g at 4 °C for 1 min, discard most of the supernatant and only retain a little for resuspending Ni-NTA agarose gel, load the column, and wash it 3 times with 1×PBS buffer containing 20 mM imidazole, 2 column volumes each time. Collect 20 μL of the last washing solution. Then, elute with 1×PBS buffer containing 300 mM imidazole. If Ni-NTA agarose gel turns blue again, it indicates that the elution is basically complete. The eluate was centrifuged at 10000 g for 10 min, and the supernatant was concentrated with a 10 kD ultrafiltration tube (UFC900396; Millipore), centrifuged at 4500 g, and ultrafiltered to 500 μL at 4 °C. Then, the concentrate was washed three times with 1×PBS and centrifuged three times repeatedly to obtain the concentrated protein.

[0081] Subsequently, the binding affinity between MIM2 and the purified MAPK12 protein was detected by isothermal titration calorimetry (ITC). In the ITC experiment, 200 μM of MIM2 was titrated into the protein solution of MAPK12 under constant temperature conditions, and the heat released upon binding (ΔH) was recorded in real time. Multiple titrations were carried out continuously. The heat released or absorbed by the binding of molecules is directly proportional to the number of bound molecules. When the system reaches saturation, only the heat of dilution can be observed. By plotting the heat effect generated by each titration against the molar ratio of the titrant and the titrated molecules, a binding curve can be obtained.

[0082] The results are as Figure 7 shown. Molecular docking experiments and target affinity detections found that the binding ability score of MIM2 and MAPK12 was 12.2, which was much greater than that of pirfenidone; and according to the ITC fitting curve, the binding constant (KD) value of MIM2 was at the nM level, while that of pirfenidone was at the μM level, indicating that compared with pirfenidone, MIM2 is a specific inhibitor of MAPK12 ( Figure 7 in A - B).

[0083] Example 8 Comparison of the Efficacy of MIM2

[0084] In this example, the efficacy differences between the two drugs were demonstrated by detecting the collagen content in tumors.

[0085] This experiment was carried out in spontaneously tumor - bearing mice (8 - 10 weeks old, male). All animal experiments were approved by the Animal Ethics Committee of Sun Yat - sen University (approval number L025504202109020). Eighteen spontaneously tumor - bearing mice were selected. Each mouse was given 50 μl of tamoxifen (1 mg / kg; H7904; Sigma - Aldrich) by rectal enema and continuously given drinking water containing doxycycline (10592 - 13 - 9; Macklin) (1 g / L) to induce intestinal tumors. Approximately 3 weeks later, primary tumors in the intestines of the mice could be seen under colonoscopy. After all 18 mice developed tumors, the mice were randomly divided into 3 groups: a control group, a MIM2 group, and a pirfenidone group, with 6 mice in each group. Since MIM2 and pirfenidone were dissolved in DMSO, the control group was given intraperitoneal injection of DMSO daily according to the body weight of the mice, and the treatment groups were given intraperitoneal injection of 10 mg / kg of MIM2 drug and 200 mg / kg of pirfenidone drug daily according to the body weight of the mice. Since the time sequence of tumor formation in the 18 mice was different, this application started drug administration starting from the point when all 18 mice had tumors. At the end of the experiment, tumors from 3 mice in the control group and the treatment groups were taken for fixed - section, and the deposition of collagen in the tumors was analyzed by Masson trichrome staining (S0075; Bioss) and Sirius red staining (S0080; Bioss).

[0086] The results are as Figure 8 shown. In terms of the efficacy of treating fibrosis, by calculating the collagen staining of tumor tissues, it can be found that the content of collagen in the tumor tissues after MIM2 treatment is significantly less than that in the pirfenidone treatment group. This shows that MIM2 has a stronger ability to inhibit tissue fibrosis.

[0087] In summary, the MIM2 drug can be used as a drug for targeted treatment of fibrosis to inhibit the synthesis of extracellular matrix components such as collagen and laminin, delay the process of tissue fibrosis, and is expected to be used as a small molecule compound drug in the treatment of tissue fibrosis such as cancer-related fibrosis, skin fibrosis, liver fibrosis, and pulmonary fibrosis.

Claims

1. The use of MIM2 and its derivatives in the preparation of products for treating tissue fibrosis, characterized in that: The molecular formula of MIM2 is C 42 H 50 N2O6 The structural formula is shown in formula (I); 2. The use according to claim 1, characterized in that: The derivative includes at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable salts include acid addition salts and base addition salts; and / or The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

4. The use according to claim 3, characterized in that: The tissue fibrosis includes at least one of tumor fibrosis and tumor post-radiotherapy fibrosis, skin fibrosis, liver fibrosis, and lung fibrosis.

5. The use according to claim 1, characterized in that: The products include pharmaceutical products.

6. The use according to claim 5, characterized in that: The drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

8. The use according to claim 5, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

9. A method for inhibiting tissue fibrosis in vitro for non-therapeutic purposes, comprising the step of treating cells and tissues with MIM2.

10. The method according to claim 9, characterized in that: The concentration of the MIM2 is 1 to 20 μM.

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