Use of brd4780 as a protective agent against shiga toxin and related products

Abstract

The application discloses use of a compound BRD4780 as an anti-Shiga toxin protective agent and related products, and provides an anti-Shiga toxin pharmaceutical composition and a pharmaceutical preparation. The application first discovers that the compound BRD4780 can be used as an effective anti-Shiga toxin protective agent, and BRD4780 can effectively inhibit Shiga toxin I and Shiga toxin II toxicity, and can be used in preparation of an anti-Shiga toxin drug. The application provides an effective candidate drug for prevention, treatment, alleviation or improvement of Shiga toxin poisoning related diseases or conditions, and also provides a reference for new application clinical research of the drug BRD4780.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of BRD4780 as an anti-Shiga toxin protectant and related products. Background Technology

[0002] Shiga toxins are highly toxic proteins produced by pathogens such as Shigella, mainly including Shiga toxin I and Shiga toxin II. They can damage target cells, causing intestinal infections, hemorrhagic enteritis, and in severe cases, even life-threatening complications such as hemolytic uremic syndrome, posing a significant threat to public health. Currently, treatment options for Shiga toxin poisoning-related diseases are limited. Existing treatments are mostly symptomatic and supportive, such as fluid replacement to correct electrolyte imbalances and dialysis to treat renal failure. There is a lack of specific protective agents that can directly and effectively inhibit Shiga toxin toxicity and protect cells from damage. Therefore, developing safe and effective anti-shiga toxin drugs has become an important need in the field of anti-shiga toxin research.

[0003] However, while existing research has explored strategies for combating Shiga toxin, it primarily focuses on antibody neutralization and toxin receptor blockade, which suffers from high preparation costs and limited applicability. Furthermore, no studies have yet discovered that compound BRD4780 possesses anti-shiga toxin activity. Key questions remain unanswered, including whether it exhibits anti-shiga toxin activity, its ability to inhibit the toxicity of Shiga toxin I and Shiga toxin II, and its potential applications in anti-shiga toxin drug development. In other words, its role in inhibiting the toxicity of Shiga toxin I and Shiga toxin II and its related applications remain unexplored. This technological gap urgently needs to be filled to provide new and effective drug candidates for the development of anti-shiga toxin drugs.

[0004] Currently, there are no studies or reports on BRD4780 as an anti-Shiga toxin protectant. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide the use of BRD4780 as an anti-shiga toxin protectant and related products.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0007] A first aspect of the present invention provides a pharmaceutical composition for treating Shiga toxin.

[0008] Furthermore, the pharmaceutical composition uses compound BRD4780 or its pharmaceutically acceptable salt as the active ingredient, the structural formula of which is shown in formula (I):

[0009]

[0010] Formula (I).

[0011] In this invention, the compound BRD4780 is known in Chinese as I1-imidazoline receptor antagonist, also called AGN192403, with the corresponding CAS number 175521-95-6 and the molecular formula C. 10 H 19 N, with a molecular weight of 153.26, has the structural formula shown in formula (I) above. This invention does not impose any particular limitation on the specific source of the compound BRD4780; those skilled in the art can obtain it through conventional means based on the specific information provided above.

[0012] In a specific embodiment of the present invention, the compound used is AGN192403 hydrochloride, with the corresponding CAS number 1021868-90-5 and the molecular formula C. 10 H 20 ClN, with a molecular weight of 189.73, has the structural formula shown in formula (II) below:

[0013]

[0014] Equation (II).

[0015] A second aspect of the present invention provides a pharmaceutical preparation for treating Shiga toxin.

[0016] Furthermore, the pharmaceutical preparation is prepared from the pharmaceutical composition described in the first aspect of the present invention.

[0017] Furthermore, the dosage form of the pharmaceutical preparation is selected from one of the following: subcutaneous injection, intramuscular injection, intradermal injection, oral solution, tablet, capsule, suspension, granule, and aerosol.

[0018] A third aspect of the invention provides the use of compound BRD4780 or a pharmaceutically acceptable salt thereof in the preparation of an anti-Shiga toxin medicament.

[0019] Furthermore, the structural formula of the compound BRD4780 is shown in formula (I):

[0020]

[0021] Formula (I).

[0022] Furthermore, the pharmaceutically acceptable salt of compound BRD4780 is prepared by modifying compound BRD4780 into a salt.

[0023] Furthermore, the salts used in the salt formation modification process are hydrochloride, sulfate, nitrate, phosphate, acetate, citrate, maleate, hydrobromide, tartrate, hydroiodide, carbonate, bicarbonate and / or benzoate.

[0024] Furthermore, the shiga toxin is shiga toxin I and / or shiga toxin II.

[0025] Furthermore, the drug contains an effective amount of the compound BRD4780 or a pharmaceutically acceptable salt thereof.

[0026] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0027] Furthermore, the dosage form of the drug is selected from one of the following: subcutaneous injection, intramuscular injection, intradermal injection, oral solution, tablet, capsule, suspension, granule, and aerosol.

[0028] In some embodiments, the pharmaceutically acceptable salt of the compound BRD4780 is prepared by using BRD4780 as the parent compound through a targeted salt-forming modification process. The core objective is to optimize its physicochemical properties and pharmaceutical performance while retaining the anti-shiga toxin activity of BRD4780 (i.e., inhibiting Shiga toxin I toxicity, inhibiting Shiga toxin II toxicity, and inducing cell tolerance to toxins), thereby providing a more suitable active ingredient form for the subsequent development of pharmaceutical compositions and formulations.

[0029] In some implementations, the salts selected in the salt-forming modification process are all of the types that are recognized in the pharmaceutical field as safe, stable and with good drug-forming properties, including two major categories: inorganic acid salts and organic acid salts.

[0030] The inorganic acid salts include hydrochloride, sulfate, nitrate, phosphate, hydrobromide, hydroiodide, carbonate, and bicarbonate. Hydrochloride, as the BRD4780 salt form explicitly used in this invention (i.e., AGN192403 hydrochloride), has been experimentally verified to effectively exert its anti-Shiga toxin effect. Other inorganic acid salts, such as sulfate and phosphate, can be formed by protonation reactions between BRD4780 and corresponding inorganic acids (such as sulfuric acid and phosphoric acid) under suitable reaction conditions (such as controlling reaction temperature and pH). These salts typically exhibit high stability, which is beneficial for the long-term storage of pharmaceutical preparations. Carbonates and bicarbonates can be formed by the combination of BRD4780 with carbonate and bicarbonate ions, which can regulate the dissolution rate of the active ingredient in specific drug delivery scenarios (such as oral preparations) to adapt to different dosage form requirements.

[0031] Organic acid salts include acetates, citrates, maleates, tartrates, and benzoates. These salts are formed by the reaction of BRD4780 with corresponding organic acids (such as acetic acid, citric acid, and maleic acid). Their advantage lies in improving the water or lipid solubility of BRD4780. For example, citrates and tartrates can increase the solubility of BRD4780 in water, facilitating the preparation of oral solutions, injections, and other liquid formulations. Benzoates may enhance the lipid solubility of BRD4780, meeting the skin penetration requirements of active ingredients in topical formulations (such as ointments and patches). At the same time, organic acid salts generally have lower irritation, reducing the risk of drug irritation to body tissues (such as the gastrointestinal mucosa and injection sites) during administration.

[0032] It should be noted that, regardless of which type of salt is used for salt formation modification, the preparation process must follow the conventional salt formation process specifications in the pharmaceutical field to ensure complete reaction, product purity meets the standards, and the final BRD4780 pharmaceutically usable salt must pass activity verification to confirm that its anti-Shiga toxin activity is not impaired by salt formation modification.

[0033] In some implementations, the pharmaceutically acceptable combinations of excipients and active ingredients are tailored to the preparation requirements of different dosage forms and clinical administration scenarios. These excipients must meet pharmaceutical safety standards, not react with BRD4780 or its pharmaceutically acceptable salts, not affect its anti-shiga toxin activity (such as inhibiting Shiga toxin I / II toxicity or inducing cell tolerance), and perform specific functions based on the drug's physicochemical properties and dosage form characteristics. For example, in oral formulations (such as tablets and capsules), diluents (such as lactose and microcrystalline cellulose) can adjust the uniformity of drug content, ensuring accurate dosage of the active ingredient per unit of formulation; binders (such as hydroxypropyl methylcellulose and povidone) can enhance powder flowability and formability, ensuring tablet hardness or capsule filler stability; disintegrants (such as crospovidone and sodium carboxymethyl starch) can promote rapid disintegration of the formulation after it enters the body, accelerating the dissolution and absorption of BRD4780 or its pharmaceutically acceptable salts; and lubricants (such as magnesium stearate) can reduce friction between materials and equipment during formulation preparation, improving production efficiency. In injectable preparations (such as subcutaneous and intramuscular injections), suspending agents (such as polysorbates) can prevent the active ingredient particles from settling and ensure the uniformity of the drug solution; stabilizers (such as disodium edetate) can inhibit drug oxidation or hydrolysis and prolong the shelf life of the preparation; while isotonic regulators (such as sodium chloride and glucose) can maintain the consistency of the osmotic pressure of the drug solution with that of the human body fluids, avoiding local irritation or hemolytic reactions after administration.

[0034] In some embodiments, the dosage forms of the drugs described in this invention cover multiple routes of administration, including injection, oral administration, and inhalation. The selection of each dosage form focuses on ensuring the bioavailability of BRD4780 or its pharmaceutically acceptable salts and improving treatment convenience, comprehensively covering different treatment stages and patient groups' needs related to Shiga toxin poisoning. In practical applications, the dosage form can be flexibly selected according to the clinical needs of Shiga toxin poisoning-related diseases. This invention does not impose any particular limitation on specific drug dosage forms; any technical solution using BRD4780 or its pharmaceutically acceptable salts for Shiga toxin treatment is within the scope of protection of this invention.

[0035] In some implementation schemes, subcutaneous injections, intramuscular injections, and intradermal injections are injectable dosage forms, suitable for patients with urgent conditions or poor oral absorption. They enable BRD4780 or its pharmaceutically acceptable saline to quickly reach the site of action through blood circulation and rapidly exert their inhibitory effect on Shiga toxin toxicity. For example, in the acute phase of hemolytic uremic syndrome, it can be rapidly administered via intramuscular injection to control toxin damage.

[0036] In some implementation schemes, oral solutions, tablets, capsules, and granules are oral dosage forms, which are convenient to take and have high patient compliance. They are suitable for the prevention or mild treatment of Shiga toxin poisoning-related diseases. For example, oral tablets can be used for prophylactic administration in high-risk populations of Shigella infection, or oral granules can be used to relieve symptoms in patients with mild intestinal infections.

[0037] In some implementation schemes, suspensions combine the advantages of rapid absorption of liquid formulations with the good stability of solid formulations, and the dosage can be adjusted according to the patient's age (such as children or elderly patients with swallowing difficulties) to meet the needs of different populations.

[0038] In some implementations, the aerosol is an inhaled formulation that can be used to prevent or assist in the treatment of complications related to respiratory mucosal damage caused by Shiga toxin. By delivering the drug to the respiratory tract in the form of an aerosol, it acts directly on the locally damaged mucosa, enhancing the protective effect.

[0039] In some embodiments, the Shiga toxin includes Shiga toxin produced by Shigella or Shiga toxin produced by Shiga-producing Escherichia coli.

[0040] Among them, Shiga toxin produced by Shigella refers to the Shiga toxin produced by Shigella dysenteriae group A type I and some type II strains. This toxin has three biological activities, including neurotoxicity, which can act on the central nervous system and cause paralysis of the limbs and death; cytotoxicity, which is toxic to human liver cells, monkey kidney cells, etc.; and enterotoxicity, which has similar activities to enterotoxins of Escherichia coli and Vibrio cholerae.

[0041] Shiga toxins produced by Shiga-toxin-producing Escherichia coli (STEC) refer to the toxins produced by Shiga-toxin-producing Escherichia coli. Shiga-toxin-producing Escherichia coli (STEC) is a newly emerging group of highly pathogenic foodborne pathogens carrying prophage genes encoding one or two Shiga toxins. Shiga toxins produced by STEC have similar structure and function to those produced by Shigella bacteria and can also cause serious diseases such as intestinal bleeding and hemolytic uremic syndrome.

[0042] In this invention, the Shiga toxin includes, but is not limited to: Shiga toxin I, Shiga toxin II, Shiga toxin I-related variants, and Shiga toxin II-related variants.

[0043] The present invention also provides a method for preventing, treating, alleviating or improving diseases or symptoms related to Shiga toxin poisoning, the method comprising: administering to a subject in need an effective amount of the compound BRD4780 of the present invention as described above, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition as described above, or a pharmaceutical preparation as described above.

[0044] In some implementations, the prevention refers to administering the compound BRD4780 or its pharmaceutically acceptable salt, the pharmaceutical composition, or the pharmaceutical preparation described above to subjects who have not yet been infected with Shiga toxin and have not shown related disease symptoms, thereby inducing cell tolerance to Shiga toxin I and / or Shiga toxin II, reducing the risk of subsequent exposure to Shiga toxin-related diseases or delaying the onset of disease.

[0045] In some implementations, the treatment refers to administering the aforementioned compound BRD4780 or its pharmaceutically acceptable salts, the aforementioned pharmaceutical composition, or the aforementioned pharmaceutical preparation to subjects infected with Shiga toxin and exhibiting symptoms of Shiga toxin poisoning such as intestinal infection or hemorrhagic enteritis, thereby effectively inhibiting the toxicity of Shiga toxin I and / or Shiga toxin II, improving cell survival status (such as increasing cell clone count and improving cell survival rate), thereby alleviating disease symptoms, controlling disease progression, and promoting the recovery of the subjects.

[0046] In some implementations, the relief refers to the reduction of cell damage caused by the toxin after administering the compound BRD4780 or its pharmaceutically acceptable salt, the pharmaceutical composition, or the pharmaceutical preparation described above to subjects who have developed symptoms of Shiga toxin poisoning (such as abdominal pain, diarrhea, or organ damage-related manifestations), thereby alleviating the severity of existing symptoms, such as reducing the frequency of diarrhea, relieving abdominal pain, or slowing the rate of organ function deterioration, and improving the subject's quality of life.

[0047] In some implementations, the improvement refers to the overall optimization of the body's response to Shiga toxin after administration of the previously described compound BRD4780 or its pharmaceutically acceptable salts, the previously described pharmaceutical compositions, or the previously described pharmaceutical preparations to patients with Shiga toxin poisoning-related diseases (including those in the treatment phase or recovery phase). This improvement may include improving the function of damaged cells, increasing the body's tolerance to the toxin, or promoting the recovery of bodily functions during the recovery phase and reducing the likelihood of sequelae.

[0048] In some implementations, the effective dose refers to the specific dosage of compound BRD4780 or its pharmaceutically acceptable salts, pharmaceutical compositions or formulations containing the compound, administered to a subject in need to achieve the expected effect of preventing, treating, alleviating or improving diseases or symptoms related to Shiga toxin poisoning. This dosage needs to effectively inhibit the toxic effects of Shiga toxin by inducing tolerance of target cells (such as intestinal-associated cells) to Shiga toxin I and / or Shiga toxin II in the subject. Specifically, this can be manifested in improving the survival rate of target cells under the action of Shiga toxin and increasing the cell clonal number, while not causing unacceptable toxic side effects in the subject. The specific dosage needs to be adjusted comprehensively based on factors such as the subject's age, weight, health status, disease severity, route of administration (such as oral, injection, etc.) and dosage form of the pharmaceutical formulation to achieve a safe and effective therapeutic or preventive purpose.

[0049] In some implementations, the subject refers to an individual who may be exposed to Shiga toxin, has been exposed to Shiga toxin, or has been diagnosed with a Shiga toxin poisoning-related disease, including but not limited to: human populations and non-human mammals that may be affected by Shiga toxin. In a preferred implementation, the subject is a human. Regardless of whether the subject is a human or a non-human mammal, the dosage and route of administration of BRD4780 or its pharmaceutically acceptable salts must be adjusted based on the species, age, weight, health status, level of Shiga toxin exposure, and severity of disease to ensure safe and effective anti-Shiga toxin action.

[0050] In some implementations, the Shiga toxin poisoning-related diseases or symptoms refer to diseases or symptoms caused by Shiga toxins (e.g., Shiga toxin I and / or Shiga toxin II), all of which have the toxic effect of Shiga toxin as the core pathogenic mechanism. The manifestations of these diseases are closely related to the degree of damage to the body's cells by the toxin, the site of action, and the body's immune response. Specifically, they can be categorized as follows:

[0051] From the perspective of local intestinal lesions, Shiga toxins (especially Shiga toxin I and Shiga toxin II) can enter intestinal cells by binding to specific receptors on the surface of intestinal mucosal cells, inhibiting cellular protein synthesis and inducing apoptosis, thereby disrupting the integrity of the intestinal mucosal barrier. This leads to pathological changes in the intestinal mucosa such as congestion, edema, erosion, and ulceration, triggering acute intestinal infection-related symptoms. Patients mainly present with typical symptoms such as abdominal pain, diarrhea (initially watery stools, which may later turn into mucus and bloody stools), and tenesmus. Some patients also experience systemic inflammatory responses such as fever and fatigue. This type of disease is commonly seen in bacterial dysentery caused by Shigella infection, or intestinal infection caused by enterohemorrhagic Escherichia coli carrying the Shiga toxin gene.

[0052] From the perspective of severe systemic complications, Shiga toxins (especially the more potent Shiga toxin II) can cross the intestinal mucosal barrier and enter the bloodstream, traveling to multiple organ systems throughout the body and causing widespread damage to target organ cells. The most typical and life-threatening complication is hemolytic uremic syndrome (HUS). The pathogenesis of this disease lies in the fact that after Shiga toxins enter the bloodstream, they specifically act on vascular endothelial cells (such as renal vascular endothelial cells and erythrocyte membranes), inducing vascular endothelial damage, activating the coagulation system, and forming microthrombi. Microthrombi not only obstruct renal microvessels, leading to acute renal failure (manifested as oliguria, anuria, elevated serum creatinine and blood urea nitrogen), but also destroy erythrocytes, causing hemolysis (manifested as anemia, jaundice, and hemoglobinuria), while simultaneously consuming platelets, leading to thrombocytopenia (manifested as petechiae and ecchymosis on the skin and mucous membranes), forming a triad of "hemolysis-renal failure-thrombocytopenia," with a high mortality rate. In addition, Shiga toxin may also cause thrombotic thrombocytopenic purpura (TTP), whose pathological mechanism is similar to that of HUS, but it is more likely to affect the central nervous system, causing patients to experience neurological symptoms such as altered consciousness, headache, and seizures, further aggravating the harm of the disease.

[0053] In addition, Shiga toxin may also cause other related symptoms. For example, some patients may experience dehydration and electrolyte imbalance (such as hypokalemia and hyponatremia) due to severe damage to the intestinal mucosa. If not corrected in time, these symptoms may induce arrhythmia and shock. A few patients may also experience organ damage such as myocarditis and pulmonary edema. Although these symptoms are not specific, they are all indirectly or directly caused by the toxic effects of Shiga toxin and fall under the category of "Shiga toxin poisoning-related diseases or symptoms" as defined in this invention.

[0054] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0055] This invention is the first to discover that compound BRD4780 can serve as an effective anti-shiga toxin protectant. BRD4780 can effectively inhibit the toxicity of Shiga toxin I and Shiga toxin II and can be used in the preparation of anti-shiga toxin drugs. This invention provides an effective candidate drug for the prevention, treatment, relief or improvement of diseases or symptoms related to Shiga toxin poisoning, and also provides a reference for clinical research on the new application of BRD4780. Attached Figure Description

[0056] Figure 1 Cell survival curves for each group were obtained by pretreating cells with different doses of BRD4780 for 24 h and then treating cells with different concentrations of Shiga toxin I for 48 h.

[0057] Figure 2 Cell clone staining results: Cells were pretreated with different doses of BRD4780 for 24 h and then treated with different concentrations of Shiga toxin I for 48 h.

[0058] Figure 3 Cell survival curves for each group were obtained by pretreating cells with different doses of BRD4780 for 24 h and then treating them with different concentrations of Shiga toxin II for 48 h.

[0059] Figure 4 Cell clone staining results: Cells were pretreated with different doses of BRD4780 for 24 h and then treated with different concentrations of Shiga toxin II for 48 h. Detailed Implementation

[0060] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0061] Example: BRD4780 induces cells to resist the toxic effects of Shiga toxin I and Shiga toxin II.

[0062] 1. Experimental materials

[0063] In this embodiment, the small molecule compound BRD4780 used refers to AGN192403 (same as BRD4780) hydrochloride, purchased from MedChemExpress (MCE), with the corresponding CAS number 1021868-90-5, catalog number HY-101374A, and molecular formula C. 10 H 20 ClN, with a molecular weight of 189.73, has the structural formula shown in formula (II) below:

[0064]

[0065] Equation (II).

[0066] 2. Experimental Methods

[0067] (1) CCK-8 assay for cell viability

[0068] 5637 cells were pretreated for 24 h with different doses of BRD4780 (0 μM, 10 μM, 20 μM, and 40 μM). Cells were then passaged into 96-well plates, and after cell attachment, 1×10⁻⁶ Shiga toxin I and 1×10⁻⁶ Shiga toxin II were added. 5 2×10 4 4×10 3 800, 160, 32, 6.4, 1.28, 0.256, 0.0512, 0.01024, 0.002048, 4.096×10 -4 pg·mL -1 Serum-free 1640 medium containing BRD4780 compound was used, with three replicates (n=3) for each concentration. After treatment with Shiga toxin for 48 h, 10 μL of CCK-8 reagent was added to each well, and the reaction was carried out at 37℃ for 3 h. The absorbance was measured at 450 nm using a microplate reader. The cell viability of each concentration well was calculated according to the formula: Cell viability = [(Experimental wells - Blank wells) / (Control wells - Blank wells)] × 100%, and survival curves were plotted.

[0069] (2) Plate colony formation experiment

[0070] Cell clonal staining can reflect cell viability by counting the number of clones. 5637 cells were pretreated with different doses of BRD4780 for 24 h. The pretreated cells were then administered 6.4 pg / mL of the solution. -1 32 pg·mL -1 and 160 pg·mL -1Cells were treated with Shiga toxin I or Shiga toxin II at a concentration of 100 μL for 48 hours. In a 96-well plate, cells were fixed with 100 μL of methanol per well for 5 min. Residual methanol was washed away with distilled water, and 100 μL of 2% ammonium oxalate crystal violet staining solution was added to each well. The cells were stained by standing for 5-10 min. Residual crystal violet dye was washed away with distilled water, and the 96-well plate was allowed to air dry in a cool place before photographing using an optical microscope.

[0071] 3. Experimental Results

[0072] (1) BRD4780 induces cells to counteract the toxic effects of Shiga toxin I.

[0073] Cells were pretreated with different doses of BRD4780 for 24 h, followed by treatment with different concentrations of Shiga toxin I for 48 h, and cell viability was measured in each group. Cell survival curves for each group are shown below. Figure 1 As shown, the half-maximal effective dose (IC50) of Shiga toxin I in the solvent control group inhibited cell proliferation. 50 The value was 6.89 pg·mL. -1 Compared with the solvent control group, cells pretreated with 10 μM, 20 μM, and 40 μM BRD4780 for 24 h all showed significant Shiga toxin tolerance effects, with the toxin effect curves shifting significantly to the right and the IC50 value decreasing. 50 The values ​​increased to 15.25 pg·mL. -1 32.28 pg·mL -1 and 404.8 pg·mL -1 Its IC 50 The values ​​increased by 2.2 times, 4.7 times, and 58.8 times, respectively. Pretreatment with BRD4780 for 24 h induced a significant Shiga toxin I tolerance in the cells.

[0074] To further confirm the antitoxic effect of BRD4780, this invention also conducted a cell clonal staining experiment. The results of the cell clonal staining experiment are as follows: Figure 2 As shown, compared with the solvent control group, pretreatment of cells with 10 μM BRD4780 for 24 h resulted in a decrease in cell count to 32 pg·mL⁻¹. -1 The cell clone count in the Shiga toxin I treatment group was significantly higher than that in the solvent control group. Cells were pretreated with 20 μM BRD4780 for 24 h at 32 pg / mL. -1 and 160 pg·mL -1 The cell clone count in the Shiga toxin I treatment group was significantly higher than that in the solvent control group. Pretreatment of cells with 40 μM BRD4780 for 24 h resulted in significantly higher cell clone counts in all three Shiga toxin I treatment groups compared to the solvent control group. These results indicate that the BRD4780-induced anti-Shiga toxin I effect in cells is dose-dependent.

[0075] (2) BRD4780 induces the toxic effects of Shiga toxin II on cells.

[0076] Cells were pretreated with different doses of BRD4780 for 24 h, followed by treatment with different concentrations of Shiga toxin II for 48 h, and cell viability was measured in each group. Cell survival curves for each group are shown below. Figure 3 As shown, the half-maximal effective dose (IC50) of Shiga toxin II inhibited cell proliferation in the solvent control group. 50 The value was 4.59 pg·mL. -1 Compared with the solvent control group, cells pretreated with 10 μM, 20 μM, and 40 μM BRD4780 for 24 h all showed significant Shiga toxin II tolerance, with the toxin effect curve shifting significantly to the right and the IC50 value decreasing. 50 Increased to 18.32 pg·mL -1 32.26 pg·mL -1 and 227.5 pg·mL -1 Its IC 50 The values ​​increased by 4-fold, 7-fold, and 50-fold, respectively. Pretreatment with BRD4780 for 24 h induced a significant Shiga toxin II tolerance effect in the cells.

[0077] To further confirm the antitoxic effect of BRD4780, this invention also conducted a cell clonal staining experiment. The results of the cell clonal staining experiment are as follows: Figure 4 As shown, compared with the solvent control group, pretreatment of cells with 10 μM BRD4780 for 24 h resulted in a decrease in cell count to 32 pg·mL⁻¹. -1 The cell clone count in the Shiga toxin II treatment group was significantly higher than that in the solvent control group. Cells were pretreated with 20 μM BRD4780 for 24 h at 32 pg / mL. -1 and 160 pg·mL -1 The cell clone count in the Shiga toxin II treatment group was significantly higher than that in the solvent control group. Pretreatment of cells with 40 μM BRD4780 for 24 h resulted in significantly higher cell clone counts in all three Shiga toxin II treatment groups compared to the solvent control group. These results indicate that the BRD4780-induced anti-Shiga toxin II effect in cells is dose-dependent.

[0078] The above experimental results demonstrate that the small molecule compound BRD4780 has an effective inhibitory effect on the toxicity of Shiga toxin I and Shiga toxin II, and can be used in the preparation of anti-Shiga toxin drugs.

Claims

1. The use of compound BRD4780 or its pharmaceutically acceptable salt in the preparation of antishiga toxin drugs, characterized in that, The structural formula of the compound BRD4780 is shown in formula (I): Formula (I).

2. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt of compound BRD4780 is prepared by modifying compound BRD4780 into a salt.

3. The application according to claim 2, characterized in that, The salts used in the salt formation modification process are hydrochloride, sulfate, nitrate, phosphate, acetate, citrate, maleate, hydrobromide, tartrate, hydroiodide, carbonate, bicarbonate and / or benzoate.

4. The application according to claim 1, characterized in that, The Shiga toxin is Shiga toxin I and / or Shiga toxin II.

5. The application according to claim 1, characterized in that, The drug contains an effective amount of the compound BRD4780 or a pharmaceutically acceptable salt thereof.

6. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

7. The application according to claim 1, characterized in that, The dosage form of the drug is selected from one of the following: subcutaneous injection, intramuscular injection, intradermal injection, oral solution, tablet, capsule, suspension, granule, and aerosol.

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