Application of BRD4780 serving as anti-shiga toxin protective agent and related products of BRD4780

Abstract

The invention discloses application of a compound BRD4780 as an anti-shiga toxin protective agent and related products thereof, and provides an anti-shiga toxin pharmaceutical composition and a pharmaceutical preparation, it is found for the first time that the compound BRD4780 can be used as the effective anti-shiga toxin protective agent, the BRD4780 can effectively inhibit the toxicity of shiga toxin I and shiga toxin II, and the compound BRD4780 can effectively inhibit the toxicity of shiga toxin I and shiga toxin II. The BRD4780 can be applied to preparation of anti-shiga toxin drugs, an effective candidate drug is provided for prevention, treatment, alleviation or improvement of shiga toxin poisoning related diseases or symptoms, and reference is provided for new application clinical research of the BRD4780 drug at the same time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to the use of BRD4780 as an anti-Shiga toxin protective agent and related products thereof. BACKGROUND

[0002] Shiga toxin is a strong virulence protein produced by pathogens such as Shigella, mainly including Shiga toxin I and Shiga toxin II, which can cause intestinal infection, hemorrhagic enteritis and other diseases by damaging target cells, and even lead to life-threatening complications such as hemolytic uremic syndrome in severe cases, posing a significant threat to public health. At present, the treatment for diseases related to Shiga toxin poisoning is limited, and the existing treatment plan is mainly symptomatic and supportive treatment, such as fluid replacement to correct electrolyte imbalance, dialysis treatment for kidney failure, etc. There is still a lack of specific protective agents that can directly and effectively inhibit the toxicity of Shiga toxin and protect cells from damage. Therefore, the development of safe and efficient anti-Shiga toxin drugs has become an important demand in the field of anti-Shiga toxin research.

[0003] However, in the prior art, although some related research has explored strategies for anti-Shiga toxin, most of them focus on antibody neutralization and toxin receptor blocking, etc., which have problems such as high preparation cost and limited application range. At the same time, no research has found that the compound BRD4780 has anti-Shiga toxin activity, whether it has anti-Shiga toxin activity, whether it can inhibit the toxicity of Shiga toxin I and Shiga toxin II, and how to be applied to the research and development of anti-Shiga toxin drugs, etc. The core issues have not been explored, i.e. the role of BRD4780 in inhibiting the toxicity of Shiga toxin I and Shiga toxin II and its related applications are still blank, and it is urgent to fill this technical gap in order to provide new candidate effective drugs for the research and development of anti-Shiga toxin drugs.

[0004] At present, there is no related research or report on BRD4780 as an anti-Shiga toxin protective agent. SUMMARY

[0005] Therefore, the purpose of the present application is to provide the use of BRD4780 as an anti-Shiga toxin protective agent and related products thereof.

[0006] The present application achieves the above-mentioned purpose by adopting the following technical solutions:

[0007] The first aspect of the present application provides an anti-Shiga toxin pharmaceutical composition.

[0008] Further, the pharmaceutical composition takes the compound BRD4780 or a pharmaceutically acceptable salt thereof as an active ingredient, and the structural formula of the compound BRD4780 is shown in formula (I):

[0009]

[0010] Formula (I).

[0011] In the present application, the compound BRD4780 corresponds to I1-imidazoline receptor antagonist (I1-imidazoline receptor antagonist), also known as AGN192403, which corresponds to CAS number 175521-95-6, molecular formula is C 10 H 19 N, molecular weight 153.26, structural formula is shown as formula (I) above. The specific source of the compound BRD4780 in the present application is not particularly limited, and those skilled in the art can purchase it through conventional routes according to the above specific information provided in the present application.

[0012] In a specific embodiment of the present application, the compound used is AGN192403 hydrochloride, which corresponds to CAS number 1021868-90-5, molecular formula is C 10 H 20 ClN, molecular weight 189.73, structural formula is shown as formula (II) below:

[0013]

[0014] Formula (II).

[0015] The second aspect of the present application provides a pharmaceutical preparation against Shiga toxin.

[0016] Further, the pharmaceutical preparation is prepared from the pharmaceutical composition of the first aspect of the present application.

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

[0018] The third aspect of the present application provides the use of compound BRD4780 or its pharmaceutically acceptable salt in the preparation of a drug against Shiga toxin.

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

[0020]

[0021] Formula (I).

[0022] Further, the pharmaceutically acceptable salt of the compound BRD4780 is a pharmaceutically acceptable salt of the compound BRD4780 prepared by salt modification of the compound BRD4780.

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

[0024] Further, the Shiga toxin is Shiga toxin I and / or Shiga toxin II.

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

[0026] Further, the drug further comprises a pharmaceutically acceptable excipient.

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

[0028] In some embodiments, the formation of the pharmaceutically acceptable salt of the compound BRD4780 is prepared by a targeted salt modification process taking BRD4780 as the parent compound, with the core purpose of optimizing the physicochemical properties and pharmaceutical properties on the basis of retaining the anti-Shiga toxin activity of BRD4780 (i.e. inhibiting Shiga toxin I toxicity, inhibiting Shiga toxin II toxicity, inducing cell tolerance to toxins), to provide a more suitable active ingredient form for the subsequent research and development of drug compositions and drug preparations.

[0029] In some embodiments, the salts used in the salt modification process all belong to the types recognized by the pharmaceutical field as safe, stable and having good drug properties, including inorganic acid salts and organic acid salts.

[0030] Among them, inorganic acid salts include hydrochloride, sulfate, nitrate, phosphate, hydrobromide, hydroiodide, carbonate and bicarbonate. Hydrochloride, as the salt form of BRD4780 (i.e. AGN192403 hydrochloride) that has been explicitly applied in the present application, has been verified by experiments that it can effectively exert anti-Shiga toxin effect; other inorganic acid salts such as sulfate, phosphate, etc. can be formed by protonation reaction of BRD4780 with corresponding inorganic acids (such as sulfuric acid, phosphoric acid) under suitable reaction conditions (such as controlling reaction temperature, pH value). Such salts usually have high stability, which is beneficial to the long-term storage of drug preparations; carbonate and bicarbonate can be formed by the combination of BRD4780 with carbonate, bicarbonate ion, which can adjust the dissolution rate of the active ingredient in specific drug delivery scenarios (such as oral preparations), and adapt to different dosage form requirements.

[0031] Among them, the organic acid salt includes acetate, citrate, maleate, tartrate and benzoate. Such salts are generated by BRD4780 reacting with corresponding organic acids (such as acetic acid, citric acid, maleic acid), and the advantage is that it can improve the water solubility or fat solubility of BRD4780. For example, citrate and tartrate can improve the solubility of BRD4780 in water, which is convenient for the preparation of oral solution, injection and other liquid preparations; benzoate can enhance the fat solubility of BRD4780, which is suitable for the skin penetration requirement of active ingredients in topical preparations (such as ointment, patch); at the same time, the organic acid salt usually has low irritation, which can reduce the risk of irritation of the drug to the body tissue (such as gastrointestinal mucosa, injection site) during administration.

[0032] It should be noted that no matter which salt is selected for salt modification, the preparation process must follow the conventional salt formation process specification in the pharmaceutical field to ensure complete reaction and product purity up to standard, and the final BRD4780 pharmaceutically acceptable salt needs to be verified for activity to confirm that its anti-Shiga toxin activity is not damaged by salt modification.

[0033] In some embodiments, the pharmaceutically acceptable excipients are combined with the active ingredients to adapt to the preparation requirements of different dosage forms and clinical administration scenarios. These excipients need to meet the safety standards in the pharmaceutical field, do not react with BRD4780 or its pharmaceutically acceptable salt, do not affect its anti-Shiga toxin activity (such as inhibiting Shiga toxin I / II toxicity, inducing cell tolerance), and can play specific functions according to the physical and chemical properties of the drug and the characteristics of the dosage form. For example, in oral preparations (such as tablets, capsules), diluents (such as lactose, microcrystalline cellulose) can adjust the uniformity of drug content to ensure the accuracy of the active ingredient dose in each unit of preparation; binders (such as hydroxypropyl methyl cellulose, povidone) can enhance the flowability and formability of the powder to ensure the hardness of the tablet or the stability of the capsule filling; disintegrants (such as cross-linked povidone, sodium carboxymethyl starch) can promote the rapid disintegration of the preparation after entering the body, accelerating the dissolution and absorption of BRD4780 or its pharmaceutically acceptable salt; lubricants (such as magnesium stearate) can reduce the friction between materials and equipment during preparation, improving production efficiency. In injections (such as subcutaneous injections, intramuscular injections), suspending agents (such as polysorbates) can prevent active ingredient particles from settling to ensure the uniformity of the drug solution; stabilizers (such as edetate disodium) can inhibit drug oxidation or hydrolysis to extend the shelf life of the preparation; and isotonicity adjusting agents (such as sodium chloride, glucose) can maintain the osmotic pressure of the drug solution consistent with the body fluid, avoiding local irritation or hemolysis after administration.

[0034] In some embodiments, the dosage forms of the drug cover various administration routes such as injection, oral administration, inhalation, etc. The selection of various dosage forms is centered on ensuring the bioavailability of BRD4780 or its pharmaceutically acceptable salt and improving the convenience of treatment, and fully covers the different treatment stages and patient population needs of Shiga toxin poisoning related diseases. In actual application, the clinical needs of Shiga toxin poisoning related diseases can be flexibly selected. The present application does not have special restrictions on the specific drug dosage form, and any technical solution using BRD4780 or its pharmaceutically acceptable salt to resist Shiga toxin is within the protection scope of the present application.

[0035] In some embodiments, subcutaneous injection, intramuscular injection, and intradermal injection belong to injection dosage forms, which are suitable for patients with more urgent conditions or poor oral absorption, can make BRD4780 or its pharmaceutically acceptable salt quickly reach the action site through blood circulation, and rapidly exert the effect of inhibiting the toxicity of Shiga toxin, for example, in the acute phase of hemolytic uremic syndrome, intramuscular injection can be used for rapid administration to control toxin damage.

[0036] In some embodiments, oral solution, tablets, capsules, and granules are oral dosage forms, which are convenient to take and have high patient compliance, and are suitable for the prevention or mild treatment of Shiga toxin poisoning related diseases, such as high-risk groups of Shigella infection, which can achieve preventive administration through oral tablets, or mild intestinal infection patients, which can relieve symptoms through oral granules.

[0037] In some embodiments, the suspension has the characteristics of fast absorption of liquid preparations and good stability of solid preparations, and the dosage can be adjusted according to the age of the patient (such as children and elderly patients with difficulty swallowing) to meet the needs of different populations.

[0038] In some embodiments, the aerosol belongs to an inhalation dosage form, which can be used for the prevention or adjuvant treatment of respiratory mucosa damage related complications caused by Shiga toxin. The drug is delivered to the respiratory tract in the form of aerosol, directly acting on the local damaged mucosa, and enhancing the protection effect.

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

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

[0041] The Shiga toxin produced by the Shiga toxin-producing Escherichia coli refers to the Shiga toxin produced by the Shiga toxin-producing Escherichia coli. The Shiga toxin-producing Escherichia coli (STEC) is a new high pathogenic foodborne pathogen carrying one or two Shiga toxin genes encoded by prophages. The Shiga toxin produced by STEC has similar structure and function to the Shiga toxin produced by Shigella, and can also cause severe diseases such as intestinal hemorrhage and hemolytic uremic syndrome.

[0042] In the present application, the Shiga toxin includes but is not limited to Shiga toxin I, Shiga toxin II, Shiga toxin I related variant, and Shiga toxin II related variant.

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

[0044] In some embodiments, the prevention refers to, after the administration of the compound BRD4780 or a pharmaceutically acceptable salt thereof as described above, the pharmaceutical composition as described above, or the pharmaceutical preparation as described above to a subject who has not been infected with Shiga toxin and has not shown symptoms of a related disease, the risk of the subject developing a Shiga toxin poisoning related disease after subsequent exposure to Shiga toxin is reduced, or the occurrence of the disease is delayed, by inducing cells to produce tolerance to Shiga toxin I and / or Shiga toxin II.

[0045] In some embodiments, the treatment refers to, after the administration of the compound BRD4780 or a pharmaceutically acceptable salt thereof as described above, the pharmaceutical composition as described above, or the pharmaceutical preparation as described above to a subject who has been infected with Shiga toxin and has shown symptoms of a Shiga toxin poisoning related disease such as intestinal infection and hemorrhagic enteritis, the toxicity of Shiga toxin I and / or Shiga toxin II is effectively inhibited, and the cell survival state is improved (such as increasing the cell clone amount and improving the cell survival rate), thereby alleviating the disease symptoms, controlling the disease progression, and promoting the recovery of the subject.

[0046] In some embodiments, the alleviation refers to, after the administration of the compound BRD4780 or a pharmaceutically acceptable salt thereof as described above, the pharmaceutical composition as described above, or the pharmaceutical preparation as described above to a subject who has shown symptoms of a Shiga toxin poisoning related disease (such as abdominal pain, diarrhea, and organ damage related manifestations), the damage of the toxin to the cells is reduced, the severity of the existing symptoms is alleviated, such as reducing the frequency of diarrhea, alleviating abdominal pain, or slowing down the deterioration speed of organ function, and the life quality of the subject is improved.

[0047] In some embodiments, the improvement refers to the overall optimization of the body's response to Shiga toxin after administration of the compound BRD4780 or its pharmaceutically acceptable salt as described above, the pharmaceutical composition as described above, or the pharmaceutical preparation as described above to patients with Shiga toxin poisoning-related diseases (including patients in the treatment stage or recovery stage), such as improving the function of damaged cells, improving the body's tolerance to toxins, or promoting the recovery of body functions during the recovery stage, and reducing the possibility of disease sequelae.

[0048] In some embodiments, the effective amount refers to the specific dose of the compound BRD4780 or its pharmaceutically acceptable salt, the pharmaceutical composition containing the compound, or the pharmaceutical preparation administered to a subject in need thereof to achieve the expected effect of preventing, treating, alleviating, or improving Shiga toxin poisoning-related diseases or conditions, which dose should be able to effectively inhibit the toxic effects of Shiga toxin by inducing the tolerance of target cells (such as intestinal-related cells, etc.) in the body of the subject to Shiga toxin I and / or Shiga toxin II, which can be specifically manifested as an increase in the survival rate of target cells under the action of Shiga toxin and an increase in the amount of cell clones, while not causing unacceptable toxic side effects in the subject; the specific amount should be adjusted in combination with the age, body weight, health status, severity of the disease, administration route (such as oral, injection, etc.), and dosage form of the pharmaceutical preparation of the subject to achieve the purpose of safe and effective treatment or prevention.

[0049] In some embodiments, the subject refers to an individual who may be exposed to Shiga toxin, has been exposed to Shiga toxin, or has been diagnosed with Shiga toxin poisoning-related diseases, including but not limited to: human populations, non-human mammals who may be affected by Shiga toxin. In preferred embodiments, the subject is a human. Regardless of human or non-human mammalian subjects, the dose and route of administration of BRD4780 or its pharmaceutically acceptable salt should be adjusted in combination with the species, age, body weight, health status, degree of Shiga toxin exposure, and severity of the disease to ensure safe and effective anti-Shiga toxin effects.

[0050] In some embodiments, the Shiga toxin poisoning-related disease or condition refers to a related disease or condition caused by Shiga toxin (e.g., Shiga toxin I and / or Shiga toxin II), which is centered on the toxic effects of Shiga toxin as the core pathogenic mechanism, and its disease manifestations are closely related to the degree of damage to the body's cells, the site of action, and the body's immune response. It can be specifically divided into the following categories:

[0051] From the perspective of local lesions in the intestinal tract, Shiga toxin (especially Shiga toxin I and Shiga toxin II) can enter cells by binding to specific receptors on the surface of intestinal mucosal cells, inhibit protein synthesis in cells and induce cell apoptosis, and further destroy the integrity of the intestinal mucosal barrier. This can cause pathological changes such as congestion, edema, erosion and ulceration in the intestinal mucosa, leading to acute intestinal infection-related diseases. Patients mainly show typical symptoms such as abdominal pain, diarrhea (initially watery stool, and later may turn into mucous and bloody stool), and tenesmus. Some patients also have fever, fatigue and other systemic inflammatory responses. Such diseases are commonly seen in bacterial dysentery caused by Shigella infection or intestinal infection caused by enterohemorrhagic E. coli carrying Shiga toxin genes.

[0052] From the perspective of systemic serious complications, Shiga toxin (especially the more toxic Shiga toxin II) can break through the intestinal mucosal barrier and enter the blood circulation, flow to multiple organ systems throughout the body with the blood, and cause extensive damage to target organ cells. The most typical and life-threatening complication is hemolytic uremic syndrome (HUS). The pathogenesis of this disease is that Shiga toxin, after entering the blood, specifically acts on vascular endothelial cells (such as renal vascular endothelial cells, red blood cell membranes, etc.), induces vascular endothelial damage, activates the blood coagulation system, and forms microthrombi. Microthrombi not only block renal microvessels, leading to acute renal failure (manifested as oliguria, anuria, elevated blood creatinine and urea nitrogen), but also destroy red blood cells, causing hemolysis (manifested as anemia, jaundice, hemoglobinuria), and consume platelets, leading to thrombocytopenia (manifested as skin and mucosal hemorrhagic spots and ecchymoses), forming a "hemolysis-renal failure-thrombocytopenia" triad, with a high mortality rate. In addition, Shiga toxin can also cause thrombotic thrombocytopenic purpura (TTP), which has a similar pathological mechanism to HUS, but is more likely to involve the central nervous system, causing patients to have neurological symptoms such as disturbance of consciousness, headache, and seizures, further exacerbating the disease.

[0053] In addition, Shiga toxin can also cause other related diseases, such as dehydration, electrolyte imbalance (such as hypokalemia, hyponatremia) and other complications in some patients due to severe intestinal mucosal damage. If not corrected in time, it can induce arrhythmia, shock, etc. A small number of patients may also have organ damage such as myocarditis and pulmonary edema. These diseases, although not specific, are indirectly or directly caused by the toxic effects of Shiga toxin and fall within the scope of "Shiga toxin poisoning-related diseases or conditions" as defined in the present application.

[0054] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0055] The present application first discovers that the compound BRD4780 can be used as an effective anti-Shiga toxin protective agent, 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, and provides an effective candidate drug for the prevention, treatment, alleviation or improvement of Shiga toxin poisoning related diseases or conditions, and also provides a reference for the new application clinical research of the drug BRD4780. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 : The cell survival curve result graph of each group of cells after different doses of BRD4780 pretreatment for 24 h and then using different concentrations of Shiga toxin I to treat the cells for 48 h;

[0057] Figure 2 : The cell survival curve result graph of each group of cells after different doses of BRD4780 pretreatment for 24 h and then using different concentrations of Shiga toxin I to treat the cells for 48 h;

[0058] Figure 3 : The cell survival curve result graph of each group of cells after different doses of BRD4780 pretreatment for 24 h and then using different concentrations of Shiga toxin II to treat the cells for 48 h;

[0059] Figure 4 : The cell survival curve result graph of each group of cells after different doses of BRD4780 pretreatment for 24 h and then using different concentrations of Shiga toxin II to treat the cells for 48 h. DETAILED DESCRIPTION

[0060] The present application will be further described below in conjunction with specific embodiments, and the following specific embodiments are only used to explain the present application and cannot be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents. The experimental consumables, reagents and raw materials used in the present application are easily obtained by those skilled in the art, and if not specifically stated, they can be obtained from commercial channels, and the experimental methods of the present application not specified in the present application are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present application and should not limit the scope of the present application in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present application and should not and will not limit the present application described in detail in the claims.

[0061] Example BRD4780 induces the toxicity effect of cells against Shiga toxin I and Shiga toxin II

[0062] 1. Experimental materials

[0063] In the present embodiment, the small molecule compound BRD4780 used refers to AGN192403 (same as BRD4780) hydrochloride, which is purchased from MedChemExpress (MCE), and its corresponding CAS number is 1021868-90-5, the product number is HY-101374A, and the molecular formula is C 10 H 20 ClN, molecular weight 189.73, and structural formula is shown in the following formula (II):

[0064]

[0065] Formula (II).

[0066] 2. Experimental method

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

[0068] Different doses of BRD4780 (0 μM, 10 μM, 20 μM, 40 μM) were used to pretreat 5637 cells for 24 h. The cells were subcultured into 96-well plates, and after the cells adhered, 1×10 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 of BRD4780-containing serum-free 1640 medium were added, and 3 replicate wells were set for each concentration (n=3). After 48 h of Shiga toxin treatment, 10 μL of CCK-8 reagent was added to each well, and the reaction was carried out at 37°C for 3 h. The absorbance was measured at 450 nm wavelength using an enzyme marker. The cell survival rate of each concentration was calculated according to the formula: cell survival rate=[(experimental well-blank well) / (control well-blank well)]×100%, and the survival curve was fitted and drawn.

[0069] (2) Plate colony formation experiment

[0070] The number of colonies can reflect the survival rate of cells through cell colony staining. Different doses of BRD4780 were used to pretreat 5637 cells for 24 h. The pretreated cells were given 6.4 pg·mL -1 , 32 pg·mL -1 , and 160 pg·mL -1Cells were treated with different concentrations of Shiga toxin I or Shiga toxin II for 48 h. Cells in 96-well plates were fixed with 100 μL of methanol per well for 5 min, and residual methanol was washed with distilled water. 100 μL of 2% ammonium oxalate crystal violet staining solution was added to each well, and the cells were stained for 5-10 min. Residual crystal violet dye was washed off with distilled water, and the 96-well plates were left to dry in the shade. Subsequently, photographs were taken using an optical microscope.

[0071] 3. Experimental results

[0072] (1) BRD4780 induces cell resistance to the toxic effects of Shiga toxin I

[0073] Cells were pretreated with different doses of BRD4780 for 24 h, and then treated with different concentrations of Shiga toxin I for 48 h. The cell survival curves of the different groups are shown in Figure 1 The half maximal inhibitory concentration (IC 50 ) of Shiga toxin I for the solvent control group was 6.89 pg·mL -1 . Compared with the solvent control group, the cells pretreated with 10 μM, 20 μM and 40 μM BRD4780 for 24 h all showed obvious Shiga toxin tolerance effects, and the IC 50 values were increased to 15.25 pg·mL -1 , 32.28 pg·mL -1 and 404.8 pg·mL -1 , respectively, which were 2.2 times, 4.7 times and 58.8 times higher than the IC 50 value of the solvent control group. Pretreatment with BRD4780 for 24 h can induce obvious Shiga toxin I tolerance effects in cells.

[0074] To further confirm the anti-toxic effects of BRD4780, the present application also performed a cell clone staining experiment, and the results are shown in Figure 2 Compared with the solvent control group, the number of cell clones in the group pretreated with 10 μM BRD4780 for 24 h and treated with 32 pg·mL -1 of Shiga toxin I was significantly higher. The number of cell clones in the group pretreated with 20 μM BRD4780 for 24 h and treated with 32 pg·mL -1 and 160 pg·mL -1 of Shiga toxin I was significantly higher. The number of cell clones in the group pretreated with 40 μM BRD4780 for 24 h and treated with different concentrations of Shiga toxin I was significantly higher. The above results show that the Shiga toxin I resistance effect induced by BRD4780 in cells has a certain dose dependence.

[0075] (2) BRD4780 induces cell resistance to the toxic effect of Shiga toxin II

[0076] Different doses of BRD4780 were used to pretreat cells for 24 h, and then different concentrations of Shiga toxin II were used to treat the cells for 48 h, and the cell viability levels of each group were detected. The cell survival curves of each group are shown in Figure 3 The half maximal inhibitory concentration (IC 50 ) of Shiga toxin II for the solvent control group was 4.59 pg·mL -1 . Compared with the solvent control group, the 10 μM, 20 μM and 40 μM BRD4780 pretreated cells for 24 h all showed obvious Shiga toxin II resistance effect, and the toxin effect curve was obviously right-shifted, and the IC 50 was increased to 18.32 pg·mL -1 , 32.26 pg·mL -1 and 227.5 pg·mL -1 , respectively, and the IC 50 values were increased by 4 times, 7 times and 50 times, respectively. Pretreatment of BRD4780 for 24 h can make the cells have obvious Shiga toxin II resistance effect.

[0077] In order to further confirm the anti-toxic effect of BRD4780, the present application also carried out cell clone staining experiment, and the results are shown in Figure 4 Compared with the solvent control group, the cell clone amount of the 32 pg·mL -1 Shiga toxin II treatment group pretreated with 10 μM BRD4780 for 24 h was obviously more than that of the solvent control group. The cell clone amount of the 32 pg·mL -1 and 160 pg·mL -1 Shiga toxin II treatment groups pretreated with 20 μM BRD4780 for 24 h was obviously more than that of the solvent control group. The cell clone amount of the three dose Shiga toxin II treatment groups pretreated with 40 μM BRD4780 for 24 h was obviously more than that of the solvent control group. The above results show that the BRD4780 induced cell resistance to Shiga toxin II has a certain dose dependence.

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

Claims

1. Use of the compound BRD4780 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament against Shiga toxin, characterized in that, The structural formula of the compound BRD4780 is shown as formula (I): Formula (I).

2. Use according to claim 1, characterized in that, The pharmaceutically acceptable salt of the compound BRD4780 is a compound BRD4780 pharmaceutically acceptable salt prepared by salt modification of the compound BRD4780.

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

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

5. The use according to claim 1, characterized in that, The medicine contains an effective amount of the compound BRD4780 or its pharmaceutically acceptable salt.

6. Use according to claim 1, characterized in that, The medicine also contains a pharmaceutically acceptable adjuvant.

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

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