Application of Placienolide B in preparation of anti-shiga toxin medicine

Abstract

The invention discloses application of Pladienolide B in preparation of an anti-shiga toxin medicine, and further discloses an anti-shiga toxin medicine composition containing the compound Pladienolide B and an anti-shiga toxin medicine preparation. Experiments prove that the compound Pladienolide B can be used for preparation of the anti-shiga toxin medicine, and the compound Pladienolide B can be applied to preparation of the anti-shiga toxin medicine. A new strategy is provided for research and development of anti-shiga toxin drugs, and the application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular, relates to a new use of a drug, and more particularly, to the use of Pladienolide B in the preparation of a drug against Shiga toxin. Background Art

[0002] Shiga toxin is produced by Shigella and some Escherichia coli. It is a highly toxic protein toxin. STX1 and STX2 are the two main types of Shiga toxins. STX1 and STX2 have similar structures and are composed of 1 A subunit and 5 B subunits. The B subunit is responsible for binding to specific receptors on the surface of host cells and mediating the entry of the toxin into the cell. The A subunit has enzymatic activity and can act on ribosomes after entering the cell to inhibit protein synthesis, thereby leading to cell dysfunction and death. Shiga toxin can not only cause serious damage to intestinal mucosal cells and cause typical dysentery symptoms such as severe abdominal pain, diarrhea, and bloody stools, but also enter the blood circulation and attack multiple organs throughout the body, such as the kidneys, liver, and nervous system, causing serious complications such as hemolytic uremic syndrome, liver damage, and convulsions, which seriously threaten the life and health of patients, especially children, the elderly, and people with low immunity.

[0003] At present, supportive care and antibiotics are mainly used for Shiga toxin (STX1 / STX2) infection. However, antibiotics may induce bacteria to release more Shiga toxins in some cases, aggravating the condition, and have no neutralizing effect on the toxins that have already been produced. With the widespread use of antibiotics, the resistance of Shiga toxin-producing bacteria to multiple antibiotics has gradually increased, making traditional antibiotic treatments face greater challenges. Although supportive treatment can relieve some symptoms, it has limited effect on serious complications caused by toxins, such as hemolytic uremic syndrome (HUS), and patients still face a high risk of death and long-term health problems. In addition, Shigella and Shiga toxin-producing Escherichia coli are widely transmitted and can be transmitted through contaminated food, water sources and close contact, which can easily cause large-scale infections in areas with poor sanitation and dense populations. Therefore, the development of new drugs that can directly combat Shiga toxins is of irreplaceable importance for improving patient cure rates, reducing mortality rates, and controlling the spread of Shiga toxins. Summary of the invention

[0004] In order to solve the technical problem currently faced in the art of urgently developing new drugs against Shiga toxins (STX1 / STX2), the purpose of the present invention is to provide the use of Pladienolide B in the preparation of drugs against Shiga toxins.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0006] The first aspect of the present invention provides the use of Pladienolide B or a pharmaceutically acceptable salt thereof in the preparation of a drug against Shiga toxin.

[0007] Furthermore, the structural formula of Pladienolide B is shown in formula (I):

[0008] Formula (I).

[0009] Furthermore, the Shiga toxin is STX1 or STX2.

[0010] Furthermore, the drug comprises an effective amount of Pladienolide B or a pharmaceutically acceptable salt thereof.

[0011] Furthermore, the drug is prepared from the Pladienolide B or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable adjuvants and / or excipients via a pharmaceutical method.

[0012] Furthermore, the dosage form of the drug is a solution, tablet, capsule, granule, inhalant, gel, emulsion, powder or suspension.

[0013] Furthermore, the Pladienolide B has an anti-Shiga toxin toxicity effect.

[0014] In the present invention, the Pladienolide B is a major analog of the macrolide family isolated from Streptomyces, and its corresponding Chinese name is pladienolide B. Pladienolide B is a highly effective inhibitor of hypoxia signals and cancer cell proliferation. Currently, there is no research or report on the anti-Shiga toxin toxicity effect of Pladienolide B. Its corresponding CAS number is 445493-23-2, and its molecular formula is C 30 H 48 O8, molecular weight 536.70, structural formula is shown in the above formula (I). The present invention has no particular limitation on the specific source of the Pladienolide B, and those skilled in the art can purchase it through conventional channels.

[0015] In a specific embodiment of the present invention, the present invention has experimentally demonstrated that Pladienolide B can effectively counteract the toxicity of Shiga toxin (STX1 / STX2) and is dose-dependent, that is, it has been demonstrated that Pladienolide B has the effect of effectively inhibiting the toxicity of Shiga toxin (STX1 / STX2) and can be used in the preparation of anti-Shiga toxin (STX1 / STX2) drugs.

[0016] In the present invention, the Shiga toxin includes Shiga toxin produced by Shigella and Shiga toxin produced by Shiga toxin-producing Escherichia coli.

[0017] Among them, the Shiga toxin produced by Shigella refers to the Shiga toxin produced by type I and some type II strains of group A Shigella dysenteriae in the Shigella genus. This toxin has three biological activities, including neurotoxicity, which can act on the central nervous system to cause paralysis of the limbs and death; cytotoxicity, which is toxic to human liver cells, monkey kidney cells, etc.; enterotoxicity, which has activity similar to the enterotoxins of Escherichia coli and Vibrio cholerae.

[0018] Among them, Shiga toxin produced by Shiga toxin-producing Escherichia coli refers to Shiga toxin produced by Shiga toxin-producing Escherichia coli. Shiga toxin-producing Escherichia coli (STEC) is a new type of highly pathogenic foodborne pathogen that carries 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 serious diseases such as intestinal bleeding and hemolytic uremic syndrome.

[0019] In the present invention, the Shiga toxin is not limited to STX1 or STX2, and STX1 or STX2 related variants are also included in the protection scope of the present invention, wherein the related variants mainly refer to various mutants related to STX2, including but not limited to: STX2c, STX2d, STX2f, STX2e.

[0020] In some embodiments, the solvates, hydrates, crystalline forms, enantiomers, diastereomers or derivatives thereof corresponding to the Pladienolide B are also within the protection scope of the present invention, that is, the relevant applications of the solvates, hydrates, crystalline forms, enantiomers, diastereomers or derivatives thereof corresponding to Pladienolide B in the preparation of anti-Shiga toxin drugs also fall within the protection scope of the present invention.

[0021] A second aspect of the present invention provides a pharmaceutical composition.

[0022] Furthermore, the pharmaceutical composition comprises Pladienolide B or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention.

[0023] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0024] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes, but is not limited to, any one or a combination of at least two of diluents, excipients, fillers, binders, humectants, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, adsorption carriers, lubricants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents or buffers.

[0025] In some embodiments, the pharmaceutical composition may also contain other drugs that can be used to treat and / or prevent, or assist in the treatment and / or prevention of Shiga toxin infection or Shiga toxin poisoning-related diseases or symptoms. Those skilled in the art can make routine selections from the drugs disclosed in the prior art according to actual conditions, and use them in combination with the Pladienolide B or a pharmaceutically acceptable salt thereof described in the present invention.

[0026] In some embodiments, the pharmaceutical composition is a combination of Pladienolide B or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient, a single compound preparation, or a combination of multiple separate single preparations.

[0027] In some embodiments, the single compound preparation refers to a compound preparation comprising Pladienolide B or a pharmaceutically acceptable salt thereof of the present invention, and the other drugs mentioned above that can be used for treating and / or preventing, or assisting in treating and / or preventing diseases or symptoms associated with Shiga toxin infection or Shiga toxin poisoning.

[0028] In some embodiments, the combination of multiple separate single preparations refers to a combination of a single preparation comprising Pladienolide B or a pharmaceutically acceptable salt thereof as described herein, and a single preparation comprising the above-mentioned other drugs that can be used for the treatment and / or prevention, or auxiliary treatment and / or prevention of Shiga toxin infection or Shiga toxin poisoning-related diseases or symptoms.

[0029] In some embodiments, the administration methods of the multiple single preparations in the combination of the multiple separate single preparations include but are not limited to: simultaneous administration, sequential administration. When the administration method of the multiple single preparations in the combination of the single preparations is sequential administration, the administration methods include: first administering a single preparation containing Pladienolide B or a pharmaceutically acceptable salt thereof according to the present invention, and then administering a single preparation containing the above-mentioned other drugs that can be used for treating and / or preventing, or assisting in treating and / or preventing diseases or symptoms related to Shiga toxin infection or Shiga toxin poisoning; first administering a single preparation containing the above-mentioned other drugs that can be used for treating and / or preventing, or assisting in treating and / or preventing diseases or symptoms related to Shiga toxin infection or Shiga toxin poisoning, and then administering a single preparation containing Pladienolide B or a pharmaceutically acceptable salt thereof according to the present invention.

[0030] A third aspect of the present invention provides a pharmaceutical preparation.

[0031] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the second aspect of the present invention.

[0032] Furthermore, the dosage form of the pharmaceutical preparation is a dosage form for enteral administration or a dosage form for parenteral administration.

[0033] In some embodiments, the dosage form for administration through the gastrointestinal tract includes, but is not limited to, oral solid preparations and oral liquid preparations. In other embodiments, the oral solid preparations include, but are not limited to, tablets, capsules, granules, and powders. In other embodiments, the oral liquid preparations include, but are not limited to, solutions, suspensions, and emulsions.

[0034] In some embodiments, the non-gastrointestinal dosage forms include, but are not limited to, injection dosage forms, respiratory dosage forms, skin dosage forms, and mucosal dosage forms. In other embodiments, the injection dosage forms include, but are not limited to, injection solutions and sterile powders for injection. In other embodiments, the respiratory dosage forms include, but are not limited to, aerosols, sprays, and powder mists. In other embodiments, the skin dosage forms include, but are not limited to, topical solutions, ointments, and patches. In other embodiments, the mucosal dosage forms include, but are not limited to, suppositories and eye drops.

[0035] In some embodiments, the pharmaceutical preparation may also contain pharmaceutical excipients, which may be conventionally used in various preparations, including but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants and lubricants, etc.; or selected for use in order to adapt to the substance, including but not limited to emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants, etc. Such excipients can effectively improve the stability and solubility of the active ingredients contained in the composition or change the release rate and absorption rate of the active ingredients, thereby improving the metabolism of various active ingredients in the organism, thereby enhancing the administration effect of the composition. In addition, excipients used to achieve specific administration purposes or methods, such as sustained-release administration, controlled-release administration and pulse administration, etc., include but are not limited to gelatin, albumin, chitosan, polyether and polyester polymer materials (such as polyethylene glycol, polyurethane, polycarbonate and its copolymers, etc.). The main manifestations of the advantages of administration are: improving therapeutic effects, improving bioavailability, reducing toxic side effects and improving patient compliance, etc.

[0036] In some embodiments, the suitable dosage of Pladienolide B or its pharmaceutically acceptable salt, pharmaceutical composition or pharmaceutical preparation of the present invention can be prescribed in a variety of ways according to the formulation method, administration mode, patient's age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the desired dosage effective for treatment and / or prevention.

[0037] A fourth aspect of the present invention provides an in vitro method for inducing cell resistance to Shiga toxin for non-therapeutic purposes.

[0038] Furthermore, the method comprises: treating a system in need thereof with Pladienolide B or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention.

[0039] In some embodiments, the present invention has no particular limitation on the system in need thereof. Exemplarily, the system includes a cell system, a subcellular system, a tissue system or an organ system in need thereof.

[0040] The fifth aspect of the present invention provides use of the pharmaceutical composition described in the second aspect of the present invention in the preparation of an anti-Shiga toxin pharmaceutical preparation.

[0041] Furthermore, the medicine also contains pharmaceutically acceptable adjuvants and / or excipients.

[0042] Furthermore, the pharmaceutically acceptable auxiliary materials and / or excipients are diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers and / or disintegrants.

[0043] Furthermore, the dosage form of the drug is a solution, a sustained-release agent, a suspension, a granule, a tablet, a capsule, a powder, an emulsion, a syrup or drops.

[0044] In addition, the present invention also provides a method for treating, preventing, alleviating and / or improving diseases or symptoms related to Shiga toxin infection or Shiga toxin poisoning, the method comprising: administering an effective amount of Pladienolide B or its pharmaceutically acceptable salt, pharmaceutical composition or pharmaceutical preparation as described above to a subject in need.

[0045] In some embodiments, the Shiga toxin infection refers to the process in which Shiga toxin-producing bacteria invade the human body and reproduce in the body, while releasing Shiga toxins. This process emphasizes the entire process of pathogens (such as Shiga toxin-producing Escherichia coli or Shigella bacteria) infecting the human body and causing disease, including bacterial colonization and growth in the intestines, and the production and release of toxins, which leads to a series of pathological and physiological changes and clinical symptoms in the human body, such as fever, diarrhea, abdominal pain, etc., and may also cause serious complications such as hemolytic uremic syndrome. For example, eating food contaminated with Shiga toxin-producing Escherichia coli, causing bacteria to enter the human body and cause disease, is Shiga toxin infection.

[0046] In some embodiments, the Shiga toxin poisoning refers to the toxic effects of Shiga toxin on the human body and the resulting poisoning symptoms, emphasizing the direct damage of Shiga toxin to the body. The main focus is on the damage caused to cells, tissues and organs by Shiga toxin after entering the human body through mechanisms such as inhibition of protein synthesis, as well as the acute poisoning symptoms caused by it, such as severe vomiting, diarrhea, bloody stools, shock and other symptoms. For example, in some cases, the patient may have directly come into contact with substances containing Shiga toxins, rather than being infected with toxin-producing bacteria. This situation is Shiga toxin poisoning.

[0047] In the present invention, the use of Pladienolide B or its pharmaceutically acceptable salt as described above in the preparation of drugs for treating, preventing, alleviating and / or improving diseases or symptoms related to Shiga toxin infection or Shiga toxin poisoning shall fall within the protection scope of the present invention.

[0048] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0049] The present invention discloses a new use of the compound Pladienolide B, namely, the use of the compound Pladienolide B in the preparation of anti-Shiga toxin drugs. At the same time, the present invention also discloses an anti-Shiga toxin drug composition and an anti-Shiga toxin drug preparation comprising the compound Pladienolide B, which solves the current technical problem in the art of urgently needing to develop new drugs against Shiga toxins (STX1 / STX2), provides a new strategy for the research and development of anti-Shiga toxin drugs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The effect of different doses of Pladienolide B on the sensitivity of cells to STX1 toxin;

[0051] Figure 2The effect of different pretreatment time of Pladienolide B on the sensitivity of cells to STX1 toxin, among which, Figure A: pretreatment time 6 h; Figure B: pretreatment time 12 h; Figure C: pretreatment time 24 h;

[0052] Figure 3 The effect of different doses of Pladienolide B on the sensitivity of cells to STX2 toxin;

[0053] Figure 4 : Effects of Pladienolide B pretreatment for different time periods on the sensitivity of cells to STX2 toxin, where A: pretreatment time 6 h; B: pretreatment time 12 h; C: pretreatment time 24 h. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with specific embodiments. The following specific embodiments are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents. In order to further explain the present invention, some of the terms involved in the present invention are explained as follows:

[0055] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of Pladienolide B. Specifically, a pharmaceutically acceptable salt of Pladienolide B refers to a salt form suitable for use in pharmaceutical preparations and clinical applications obtained by salt modification of Pladienolide B. The salts used in the salt modification process include, but are not limited to, inorganic acid salts (e.g., hydrochlorides, sulfates, phosphates), organic acid salts (e.g., citrates, maleates, tartrates), which are suitable for contact with patients within the scope of reliable medical judgment and will not produce inappropriate toxicity, irritation, allergic reactions, etc.

[0056] In some embodiments, examples of pharmaceutically acceptable salts of Pladienolide B include, but are not limited to, those having (as counter ions) alkali metal ions such as Na + , Li + or K + or salts with alkaline earth metal ions such as Ca 2+ or Mg 2+ or any other pharmaceutically acceptable metal ion such as Zn 2+ or Al 3+or a pharmaceutically acceptable salt formed with an organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

[0057] In some embodiments, pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metals and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations are magnesium, potassium, sodium, calcium, etc. Examples of suitable amines are N,N'-dibenzylethylenediamine, diethanolamine, chloroprocaine, choline, ethylenediamine, N-methylglucamine or procaine.

[0058] In some embodiments, base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid in a conventional manner and isolating the free acid.

[0059] In some embodiments, pharmaceutically acceptable salts may include alkali and alkaline earth metal based cations, such as sodium, potassium, calcium, lithium, magnesium, etc., and non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, methylamine, dimethylamine, tetraethylammonium, trimethylamine, triethylamine, ethylamine, etc. Also contemplated are salts of amino acids, such as gluconate, arginate, galacturonate, etc.

[0060] As used herein, the term "prevent or treat" refers to delaying the development of a disease, preventing the development of a disease, and / or reducing the severity of the symptoms that will develop or are expected to develop. Therefore, these terms include improving existing disease symptoms, preventing additional symptoms, improving or preventing potential metabolic causes of symptoms, inhibiting disorders or diseases, for example, preventing the development of disorders or diseases, alleviating disorders or diseases, regressing disorders or diseases, alleviating the symptoms caused by the disease or disorder, or stopping the symptoms of the disease or disorder. In a specific embodiment of the present invention, the disease is a related disease or condition caused by Shiga toxin infection or Shiga toxin poisoning.

[0061] As used herein, the term "effective amount" refers to the amount of a compound (the compound Pladienolide B, the active ingredient in the pharmaceutical composition or the pharmaceutical preparation of the present invention) that effectively produces an ideal preventive, alleviating or therapeutic effect. Depending on factors such as the compound, the symptom and its severity, and the age of the treated mammal, the amount of the compound Pladienolide B, the pharmaceutical composition or the pharmaceutical preparation of the present invention that achieves an effective amount will also vary, but the specific amount can be routinely determined by a person of ordinary skill in the art based on the knowledge in the art mastered by him / her and the contents disclosed in the present invention. As used herein, the term "effective amount" includes "preventive effective amount" and "therapeutic effective amount".

[0062] Wherein, the term "prophylactically effective amount" refers to an amount sufficient to prevent a disease, disorder or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder or condition, or an amount to prevent the recurrence of a disease, disorder or condition. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent, when used alone or in combination with other drugs, that provides a prophylactic benefit in the process of preventing a disease, disorder or condition. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the prophylactic effect of other prophylactic drugs.

[0063] Wherein, the term "therapeutically effective amount" refers to an amount sufficient to provide a therapeutic benefit in the process of treating a disease, disorder or condition, or an amount that delays or minimizes one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent that provides a therapeutic benefit in the process of treating a disease, disorder or condition when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic effect of other therapeutic agents.

[0064] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" includes any substance suitable for human and / or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., having a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers and / or excipients that can be used in the medicaments, pharmaceutical compositions, or pharmaceutical preparations of the present invention are conventional, and suitable pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995).

[0065] Exemplarily, the pharmaceutically acceptable carrier and / or excipient includes, but is not limited to, any one or a combination of at least two of a diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, an adsorption carrier, a lubricant, a coating material, a colorant, a pH adjuster, an antioxidant, an antibacterial agent or a buffer.

[0066] In some embodiments, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, dextrin, mannitol, powdered sugar, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, water, and the like.

[0067] In some embodiments, the filler includes but is not limited to: mannitol (granular or powdered), glucose, lactose, sucrose, dextrin, xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.

[0068] In some embodiments, the binder includes but is not limited to starch, pregelatinized starch, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid, alginate, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like.

[0069] In some embodiments, the humectant includes but is not limited to glycerol, starch, and the like.

[0070] In some embodiments, the disintegrant includes but is not limited to cross-linked vinyl pyrrolidone, low-substituted hydroxypropyl methyl, cross-linked carboxymethyl cellulose sodium, sodium carboxymethyl starch, soybean polysaccharide, and the like.

[0071] In some embodiments, the surfactant includes, but is not limited to, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, cetyl alcohol, and the like.

[0072] In some embodiments, the adsorption carrier includes but is not limited to: starch, lactose, bentonite, activated carbon, silica gel, kaolin, alumina, diatomaceous earth, bentonite, hydroxypropyl methylcellulose, ethyl cellulose, chitosan, polystyrene, polylactic acid, polyethylene glycol, etc.

[0073] In some embodiments, the lubricant includes but is not limited to: zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate, magnesium stearate, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.

[0074] As used herein, the term "subject" refers to an animal, preferably a mammal (human and non-human animal), including but not limited to humans, non-human primates (particularly higher primates, such as macaques, crab-eating macaques, short-tailed macaques, bear monkeys, flat-topped monkeys, golden monkeys and tree shrews), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, any livestock or pets, etc. In a preferred embodiment, the subject is a human.

[0075] The experimental consumables, reagents and raw materials used in the present invention are easily obtained by ordinary technicians in the field. Unless otherwise specified, they can be obtained from commercial channels. The experimental methods for which specific conditions are not specified in the present invention 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 invention and should not limit the scope of the present invention 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 invention, and should not and will not limit the present invention described in detail in the claims.

[0076] Example 1 Pladienolide B induces cells to resist STX1 in a dose-dependent manner

[0077] 1. Experimental methods

[0078] In this example, 5637 cells were pretreated with different concentrations of Pladienolide B (PB) for 24 h, and then treated with different concentrations of STX1. The IC50 of the blank control DMSO group and the Pladienolide B pretreatment group were compared to determine the effect of Pladienolide B on the cytotoxic effect induced by Shiga toxin STX1. The specific experimental method is as follows:

[0079] CCK-8 assay for cell viability: 5637 cells were pretreated with different doses of Pladienolide B for 24 h. Cells were subcultured to 96-well plates and STX1 was added at a concentration of 1×10 5 , 2×10 4 , 4×10 3 ,800,160,32,6.4,1.28,0.256,0.0512,0.01024,0 pg·mL -1 Serum-free 1640 medium was used, and three replicate wells (n=3) were set for each concentration. After 48 h of STX1 treatment, 10 μL of CCK-8 reagent was added to each well, and the reaction was carried out at 37°C for 4 h. The absorbance was measured using a microplate reader at a wavelength of 450 nm. The cell survival rate of each concentration experimental well was calculated according to the formula: cell survival rate = [1-(experimental well-control well) / (control well-blank well)]×100%, and the survival curve was fitted and drawn.

[0080] 2. Experimental results

[0081] In this example, cells were pretreated with different doses of Pladienolide B for 24 h, and then treated with different concentrations of STX1 for 48 h and the cell viability levels of each group were detected. Figure 1 As shown, from Figure 1 The results shown in the figure show that 5 nM Pladienolide B pretreatment can make cells have a more obvious STX1 tolerance effect. With the increase of Pladienolide B treatment concentration, the ability of cells to resist STX1, especially to tolerate high concentrations of STX1, gradually increases, indicating that Pladienolide B induces cells to resist STX1 in a dose-dependent manner. 5pg / mL of STX1 showed a completely tolerant phenotype, while the cell survival rate of the control group was only 10%. The above results show that Pladienolide B can effectively induce cells to resist STX1 and is dose-dependent. That is, this example proves that Pladienolide B has the effect of effectively inhibiting the toxicity of STX1 and can be used in the preparation of anti-STX1 drugs.

[0082] Example 2 Pladienolide B induces cell resistance to STX1 in a time-dependent manner

[0083] 1. Experimental methods

[0084] This example further explored whether different pretreatment times of Pladienolide B affect the induced cell anti-toxic phenotype. The pretreatment time of cells with 10 nM Pladienolide B was set to 6, 12, and 24 h. The specific cell viability detection method was as described in the experimental method of Example 1. Cells were pretreated with 10 nM Pladienolide B for different times, and STX1 was added at a concentration of 1×10 5 , 2×10 4 , 4×10 3 , 800, 160, 32, 6.4, 1.28, 0.256, 0.0512, 0.01024, 2.04×10 -3 , 0 pg·mL -1 .

[0085] 2. Experimental results

[0086] In this example, 10 nM Pladienolide B was used to pretreat cells for 6, 12, and 24 h. When the Pladienolide B pretreatment time was up to 6 or 12 h, the sensitivity of the experimental group cells to STX1 did not change significantly compared with the DMSO control group cells ( Figure 2 A, 2B); When the pretreatment time of Pladienolide B is up to 24 h, STX1 has almost no killing effect on cells ( Figure 2 C). The above results indicate that Pladienolide B-induced cell resistance to STX1 toxicity has a certain time dependence.

[0087] The experimental results of the above Examples 1 and 2 show that Pladienolide B has an effective inhibitory effect on STX1, can effectively induce cells to resist the toxic effects of STX1, and has a certain dose dependence and time dependence.

[0088] Example 3 Pladienolide B induces cells to resist STX2 in a dose-dependent manner

[0089] 1. Experimental methods

[0090] In this example, 5637 cells were pretreated with different concentrations of Pladienolide B (PB) for 24 h, and then treated with different concentrations of STX2. The IC50 of the blank control DMSO group and the Pladienolide B pretreatment group were compared to determine the effect of Pladienolide B on the cytotoxic effect induced by STX2. The specific experimental method is as follows:

[0091] CCK-8 assay for cell viability: 5637 cells were pretreated with different doses of 1, 5, and 10 nM Pladienolide B for 24 h. Cells were cultured in 96-well plates and STX2 was added at a concentration of 1×10 5 , 2×10 4 , 4×10 3 ,800,160,32,6.4,0.256,0.0512,0.01024,0 pg·mL -1 Serum-free 1640 medium was used, and three replicate wells (n=3) were set for each concentration. After 48 h of STX2 treatment, 10 μL of CCK-8 reagent was added to each well, and the reaction was carried out at 37°C for 4 h. The absorbance was measured using an ELISA reader at a wavelength of 450 nm. The cell survival rate of each concentration experimental well was calculated according to the formula: cell survival rate = [1-(experimental well-control well) / (control well-blank well)]×100%, and the survival curve was fitted and drawn.

[0092] 2. Experimental results

[0093] In this example, cells were pretreated with different doses of Pladienolide B for 24 h, and then treated with different concentrations of STX2 for 48 h and the cell viability levels of each group were detected. Figure 3 As shown, from Figure 3 The results shown in the figure show that 5 nM Pladienolide B pretreatment can make cells have a more obvious STX2 tolerance effect. With the increase of Pladienolide B treatment concentration, the ability of cells to resist STX2, especially to tolerate high concentrations of STX2, gradually increases, indicating that Pladienolide B induces cells to resist STX2 in a dose-dependent manner. 5pg / mL of STX2 showed a completely tolerant phenotype, while the cell survival rate of the control group was only 8%. The above results show that Pladienolide B can effectively induce cells to resist STX2 and is dose-dependent. That is, this example proves that Pladienolide B has the effect of effectively inhibiting the toxicity of STX2 and can be used in the preparation of anti-STX2 drugs.

[0094] Example 4 Pladienolide B induces cell resistance to STX2 in a time-dependent manner

[0095] 1. Experimental methods

[0096] This example further explored whether different pretreatment times of Pladienolide B affect the induced cell anti-toxic phenotype. The pretreatment time of cells with 10 nM Pladienolide B was set to 6, 12, and 24 h. The specific cell viability detection method was as described in the experimental method of Example 3 above. The pretreatment time of cells with 10 nM Pladienolide B was set to 6, 12, and 24 h. STX2 was added at a concentration of 1×10 5 , 2×10 4 , 4×10 3 , 800, 160, 32, 6.4, 1.28, 0.256, 0.0512, 0.01024, 2.04×10 -3 , 0 pg·mL -1 .

[0097] 2. Experimental results

[0098] In this example, 10 nM Pladienolide B was used to pretreat cells for 6, 12, and 24 h. When the Pladienolide B pretreatment time was up to 6 or 12 h, the sensitivity of the experimental group cells to STX2 did not change significantly compared with the DMSO control group cells ( Figure 4 A, 4B); When the pretreatment time of Pladienolide B is up to 24 h, STX2 has almost no killing effect on cells ( Figure 4 C). The above results indicate that Pladienolide B-induced cell resistance to STX2 toxin has a certain time dependence.

[0099] The experimental results of the above Examples 3 and 4 show that Pladienolide B has an effective inhibitory effect on STX2, can effectively induce cells to resist the toxic effects of STX2, and has a certain dose dependence and time dependence.

Claims

1. Use of Pladienolide B or a pharmaceutically acceptable salt thereof in the preparation of a drug against Shiga toxin, characterized in that: The structural formula of the Pladienolide B is shown in formula (I): Formula (I).

2. The use according to claim 1, characterized in that: The Shiga toxin is STX1 or STX2.

3. The use according to claim 1, characterized in that: The medicament comprises an effective amount of Pladienolide B or a pharmaceutically acceptable salt thereof.

4. The use according to claim 1, characterized in that: The drug is prepared from the Pladienolide B or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable adjuvants and / or excipients via a pharmaceutical method.

5. The use according to claim 1, characterized in that: The dosage form of the drug is solution, tablet, capsule, granule, inhalant, gel, emulsion, powder or suspension.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Pladienolide B or a pharmaceutically acceptable salt thereof as claimed in claim 1.

7. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 6.

8. The pharmaceutical preparation according to claim 7, characterized in that The dosage form of the pharmaceutical preparation is a dosage form for enteral administration or a dosage form for parenteral administration.

9. A method for inducing cells to resist Shiga toxin in vitro for non-therapeutic purposes, characterized in that: The method comprises: treating a system in need thereof with the Pladienolide B or a pharmaceutically acceptable salt thereof as claimed in claim 1.

10. Use of the pharmaceutical composition according to claim 6 in the preparation of anti-Shiga toxin pharmaceutical preparations.

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