Application of Pladienolide B in the preparation of drugs against Shiga toxin

Abstract

The present invention discloses the use of Pladienolide B in the preparation of drugs against Shiga toxin. The present invention also discloses an anti-Shiga toxin pharmaceutical composition and an anti-Shiga toxin pharmaceutical preparation comprising the compound Pladienolide B. The present invention proves through experiments that the compound Pladienolide B can be used in the preparation of drugs against Shiga toxin, provides a new strategy for the research and development of drugs against Shiga toxin, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Specifically, it relates to a new use of a drug, and more specifically, to the application of Pladienolide B in the preparation of a drug against Shiga toxin. Background Art

[0002] Shiga toxin is produced by Shigella species and some Escherichia coli, and it is a highly toxic protein toxin. STX1 and STX2 are two main types of Shiga toxin. The structures of STX1 and STX2 are similar, both consisting 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 cells. The A subunit has enzymatic activity and can act on ribosomes after entering the cells, inhibiting protein synthesis, thereby causing cell dysfunction and death. Shiga toxin can not only cause severe damage to intestinal mucosal cells, leading to 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 injury, and convulsions, seriously threatening the life and health of patients, especially children, the elderly, and immunocompromised populations.

[0003] Currently, for Shiga toxin (STX1 / STX2) infection, supportive treatment and antibiotic treatment are mainly used. However, antibiotics may induce bacteria to release more Shiga toxin in some cases, aggravating the condition, and they have no neutralizing effect on the already produced toxin. With the widespread use of antibiotics, the drug resistance of Shiga toxin-producing bacteria to various antibiotics has gradually increased, making traditional antibiotic treatment face greater challenges. Although supportive treatment can relieve some symptoms, it has limited effect on severe 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, the transmission routes of Shigella and Shiga toxin-producing Escherichia coli are extensive, and they can be transmitted through contaminated food, water sources, and close contact, and are extremely likely to cause large-scale infections in areas with poor sanitation and dense populations. Therefore, the development of new drugs that can directly combat Shiga toxin is of irreplaceable significance for improving the cure rate of patients, reducing the mortality rate, and controlling the spread of Shiga toxin. Summary of the Invention

[0004] In order to solve the technical problem in the current field that there is an urgent need to develop new drugs against Shiga toxin (STX1 / STX2), the purpose of the present invention is to provide the application of Pladienolide B in the preparation of a drug against Shiga toxin.

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

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

[0008]

[0009] Formula (I).

[0010] Further, the Shiga toxin is STX1 or STX2.

[0011] Further, the drug contains an effective amount of Pladienolide B or a pharmaceutically acceptable salt thereof.

[0012] Further, the drug is prepared by a pharmaceutical method from the Pladienolide B or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients and / or adjuvants.

[0013] Further, the dosage form of the drug is a solution, tablet, capsule, granule, inhalant, gel, emulsion, powder or suspension.

[0014] Further, the Pladienolide B has an anti-Shiga toxin toxic effect.

[0015] In the present invention, the Pladienolide B is the main analogue of the macrolide family isolated from Streptomyces, and its corresponding Chinese name is Pradimicin B. Pladienolide B is a highly efficient inhibitor of hypoxia signaling and cancer cell proliferation. There is currently no research or report on the anti-Shiga toxin toxic effect of Pladienolide B. Its corresponding CAS number is 445493-23-2, the molecular formula is C 30 H 48 O8, the molecular weight is 536.70, and the structural formula is shown in formula (I) above. The present invention has no particular limitation on the specific source of the Pladienolide B, and those skilled in the art can obtain it through conventional channels.

[0016] In a specific embodiment of the present invention, the present invention proves through experiments that the Pladienolide B can effectively counteract the toxicity of Shiga toxin (STX1 / STX2) and has a dose-dependence, that is, it proves that Pladienolide B has an effect of effectively inhibiting the toxicity of Shiga toxin (STX1 / STX2) and can be used in the preparation of drugs against Shiga toxin (STX1 / STX2).

[0017] In the present invention, the Shiga toxin includes the Shiga toxin produced by Shigella and the Shiga toxin produced by Shiga toxin-producing Escherichia coli.

[0018] Among them, the Shiga toxin produced by Shigella refers to the Shiga toxin produced by Shigella dysenteriae type 1 and some type 2 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 enterotoxicity, which has an activity similar to the enterotoxins of Escherichia coli and Vibrio cholerae.

[0019] Among them, the Shiga toxin produced by Shiga toxin-producing Escherichia coli refers to the Shiga toxin produced by Shiga toxin-producing Escherichia coli. Shiga toxin-producing Escherichia coli (STEC) is a newly emerging highly pathogenic foodborne pathogen carrying one or two Shiga toxin genes encoded by a prophage. The Shiga toxin produced by STEC has a similar structure and function to the Shiga toxin produced by Shigella and can also cause severe diseases such as intestinal bleeding and hemolytic uremic syndrome.

[0020] In the present invention, the Shiga toxin is not limited to STX1 or STX2, and STX1 or STX2-related variants are also included within the scope of protection of the present invention. Among them, the related variants mainly refer to various mutants related to STX2, including but not limited to: STX2c, STX2d, STX2f, STX2e.

[0021] In some embodiments, the solvates, hydrates, crystalline forms, enantiomers, diastereomers or derivatives corresponding to Pladienolide B are also within the scope of protection of the present invention, that is, the related applications of the solvates, hydrates, crystalline forms, enantiomers, diastereomers or derivatives corresponding to Pladienolide B in the preparation of anti-Shiga toxin drugs also fall within the scope of protection of the present invention.

[0022] The second aspect of the present invention provides a pharmaceutical composition.

[0023] Furthermore, the pharmaceutical composition contains Pladienolide B or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention.

[0024] In some embodiments, the pharmaceutical composition further contains a pharmaceutically acceptable carrier and / or excipient.

[0025] In some embodiments, the pharmaceutically acceptable carrier and / or excipient include, but are not limited to: any one or a combination of at least two of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, adsorption carriers, lubricants, coating materials, coloring agents, pH regulators, antioxidants, bacteriostatic agents, or buffers, etc.

[0026] In some embodiments, the pharmaceutical composition may further contain other drugs that can be used for treating and / or preventing, or assisting in treating and / or preventing Shiga toxin infection or diseases or symptoms related to Shiga toxin intoxication. Those skilled in the art can make a conventional selection from the drugs disclosed in the prior art according to the actual situation, and use them in combination with Pladienolide B or its pharmaceutically acceptable salt described in the present invention.

[0027] In some embodiments, the pharmaceutical composition is a combination of Pladienolide B or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier and / or excipient, a single compound preparation, or a combination of multiple separate single-agent preparations.

[0028] In some embodiments, the single compound preparation refers to a compound preparation containing Pladienolide B or its pharmaceutically acceptable salt described in the present invention and other drugs that can be used for treating and / or preventing, or assisting in treating and / or preventing Shiga toxin infection or diseases or symptoms related to Shiga toxin intoxication.

[0029] In some embodiments, the combination of multiple separate single-agent preparations refers to a combination of a single-agent preparation containing Pladienolide B or its pharmaceutically acceptable salt described in the present invention and a single-agent preparation containing other drugs that can be used for treating and / or preventing, or assisting in treating and / or preventing Shiga toxin infection or diseases or symptoms related to Shiga toxin intoxication.

[0030] In some embodiments, the administration methods of the multiple single-agent preparations in the combination of multiple separate single-agent preparations include, but are not limited to: simultaneous administration, sequential administration. When the administration methods of the multiple single-agent preparations in the combination of single-agent preparations are sequential administration, the administration methods include: first administering the single-agent preparation containing Pladienolide B or its pharmaceutically acceptable salt described in the present invention, and then administering the single-agent preparation containing other drugs that can be used for treating and / or preventing, or assisting in treating and / or preventing Shiga toxin infection or diseases or symptoms related to Shiga toxin intoxication; first administering the single-agent preparation containing other drugs that can be used for treating and / or preventing, or assisting in treating and / or preventing Shiga toxin infection or diseases or symptoms related to Shiga toxin intoxication, and then administering the single-agent preparation containing Pladienolide B or its pharmaceutically acceptable salt described in the present invention.

[0031] The third aspect of the present invention provides a pharmaceutical preparation.

[0032] Further, the pharmaceutical preparation comprises the pharmaceutical composition described in the second aspect of the present invention.

[0033] Further, the dosage form of the pharmaceutical preparation is a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0034] In some embodiments, the gastrointestinal dosage forms include, but are not limited to: oral solid preparations, oral liquid preparations. In other embodiments, the oral solid preparations include, but are not limited to: tablets, capsules, granules, powders. In other embodiments, the oral liquid preparations include, but are not limited to: solutions, suspensions, emulsions.

[0035] In some embodiments, the non-gastrointestinal dosage forms include, but are not limited to: injection dosage forms, respiratory dosage forms, dermal dosage forms, mucosal dosage forms. In other embodiments, the injection dosage forms include, but are not limited to: injections, sterile powders for injection. In other embodiments, the respiratory dosage forms include, but are not limited to: aerosols, sprays, powder aerosols. In other embodiments, the dermal dosage forms include, but are not limited to: topical solutions, ointments, patches. In other embodiments, the mucosal dosage forms include, but are not limited to: suppositories, eye drops.

[0036] In some embodiments, the pharmaceutical preparation may further contain pharmaceutical excipients, which can be those conventionally used in various preparations, including but not limited to: isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, lubricants, etc.; or those selected for compatibility with the substance, including but not limited to: emulsifiers, solubilizers, bacteriostatic agents, analgesics, 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, etc., thereby improving the metabolism of various active ingredients in the body and further enhancing the administration effect of the composition. In addition, excipients can also be used to achieve specific administration purposes or modes, such as: sustained-release administration, controlled-release administration, pulsed administration, etc., including but not limited to: gelatin, albumin, chitosan, polyethers, and polyester-based polymer materials (such as: polyethylene glycol, polyurethane, polycarbonate, and their copolymers, etc.). The main manifestations beneficial to administration are: improving the therapeutic effect, increasing the bioavailability, reducing the toxicity and side effects, and improving the patient compliance, etc.

[0037] In some embodiments, the suitable dosage of Pladienolide B or a pharmaceutically acceptable salt thereof, the pharmaceutical composition or the pharmaceutical preparation of the present invention can be prescribed in various ways according to factors such as the formulation method, the administration mode, the age, weight, gender, morbidity, diet, administration time, administration route, excretion rate and sensitivity of the patient, and a skilled doctor can usually easily determine the prescription and the desired dosage effective for treatment and / or prevention.

[0038] The fourth aspect of the present invention provides a method for in vitro non-therapeutically inducing cells to resist Shiga toxin.

[0039] Furthermore, the method includes: treating a system in need with Pladienolide B or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention.

[0040] In some embodiments, the present invention places no particular limitation on the system in need. Exemplarily, the system includes a cell system, a subcellular system, a tissue system or an organ system in need.

[0041] The fifth aspect of the present invention provides the use of the pharmaceutical composition described in the second aspect of the present invention in the preparation of a pharmaceutical preparation for resisting Shiga toxin.

[0042] Furthermore, the drug further comprises a pharmaceutically acceptable excipient and / or adjuvant.

[0043] Furthermore, the pharmaceutically acceptable excipient and / or adjuvant is a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler and / or a disintegrant.

[0044] 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 a drop.

[0045] In addition, the present invention also provides a method for treating, preventing, alleviating and / or improving Shiga toxin infection or diseases or symptoms related to Shiga toxin poisoning, the method including: administering an effective amount of Pladienolide B or a pharmaceutically acceptable salt thereof, the pharmaceutical composition or the pharmaceutical preparation as described above to a subject in need.

[0046] In some embodiments, the Shiga toxin infection refers to the process in which Shiga toxin-producing bacteria invade the human body, multiply in vivo, and release Shiga toxin simultaneously. This process emphasizes the entire process of a pathogen (such as Shiga toxin-producing Escherichia coli or Shigella bacteria) infecting the human body and causing diseases, including the colonization, growth of bacteria in the intestine, and the production and release of toxins, resulting in a series of pathophysiological 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, the situation where a person eats food contaminated with Shiga toxin-producing Escherichia coli, leading to the entry of the bacteria into the body and causing diseases, is a Shiga toxin infection.

[0047] 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. It mainly focuses on the damage caused by Shiga toxin to cells, tissues, and organs through mechanisms such as inhibiting protein synthesis after entering the human body, and the resulting acute poisoning manifestations, such as severe vomiting, diarrhea, bloody stools, shock and other symptoms. For example, in some cases, a patient may directly come into contact with a substance containing Shiga toxin, rather than being infected with toxin-producing bacteria, and this situation is Shiga toxin poisoning.

[0048] In the present invention, the use of Pladienolide B or its pharmaceutically acceptable salts 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 will fall within the protection scope of the present invention.

[0049] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0050] The present invention discloses a new use of the compound Pladienolide B, that is, the use of the compound Pladienolide B in the preparation of drugs against Shiga toxin. At the same time, the present invention also discloses an anti-Shiga toxin drug composition and an anti-Shiga toxin drug preparation containing the compound Pladienolide B, solving the technical problem that there is an urgent need to develop new drugs against Shiga toxin (STX1 / STX2) in the current field, providing a new strategy for the research and development of anti-Shiga toxin drugs, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Effect of treating cells with different doses of Pladienolide B on the sensitivity to STX1 toxin;

[0052] Figure 2Effect of cells pretreated with Pladienolide B for different times on the sensitivity to STX1 toxin. Among them, Figure A: pretreatment time is 6 h; Figure B: pretreatment time is 12 h; Figure C: pretreatment time is 24 h;

[0053] Figure 3 Effect of treating cells with different doses of Pladienolide B on the sensitivity to STX2 toxin;

[0054] Figure 4 : Effect of cells pretreated with Pladienolide B for different times on the sensitivity to STX2 toxin. Among them, Figure A: pretreatment time is 6 h; Figure B: pretreatment time is 12 h; Figure C: pretreatment time is 24 h. Detailed implementation manners

[0055] The present invention will be further described below in conjunction with specific embodiments. The following specific embodiments are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention. The scope of the present invention is defined by the claims and their equivalents. In order to further explain the present invention, some terms involved in the present invention are explained as follows:

[0056] 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 obtained by salt-forming modification of Pladienolide B and suitable for use in pharmaceutical preparations and clinical applications. The salts used in the salt-forming modification process include but are not limited to: inorganic acid salts (such as: hydrochloride, sulfate, phosphate), organic acid salts (such as: citrate, maleate, tartrate), and these salts are suitable for contact with patients within the scope of reliable medical judgment and will not cause inappropriate toxicity, irritation, allergic reactions, etc.

[0057] In some embodiments, examples of the pharmaceutically acceptable salt of Pladienolide B include but are not limited to: salts having (as counterions) alkali metal ions such as Na + , Li + or K + , or salts having alkaline earth metal ions such as Ca 2+ or Mg 2+ , or salts having any other pharmaceutically acceptable metal ions such as Zn 2+ or Al 3+a salt; or a pharmaceutically acceptable salt formed with an organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

[0058] In some embodiments, the pharmaceutically acceptable base addition salts are formed with a metal or an amine, such as an alkali metal and an alkaline earth metal hydroxide or an organic amine. 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.

[0059] In some embodiments, the base addition salts of acidic compounds can be prepared by the method described below: 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 and then separating the free acid in a conventional manner.

[0060] In some embodiments, the pharmaceutically acceptable salts can include cations based on alkali metals and alkaline earth metals, 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 covered are salts of amino acids, such as gluconates, argininates, galacturonates, etc.

[0061] As used herein, the term "prevent or treat" means 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. Thus, these terms include improving existing disease symptoms, preventing additional symptoms, improving or preventing the underlying metabolic causes of symptoms, inhibiting a disorder or disease, e.g., preventing the development of a disorder or disease, alleviating a disorder or disease, regressing a disorder or disease, alleviating the conditions caused by a disease or disorder, or stopping the symptoms of a disease or disorder. In a specific embodiment of the present invention, the disease is a related disease or disorder caused by Shiga toxin infection or Shiga toxin intoxication.

[0062] As used herein, the term "effective amount" means the amount of a compound (the active ingredient in the compound Pladienolide B, pharmaceutical composition or pharmaceutical preparation of the present invention) that effectively produces the desired prophylactic, alleviating or therapeutic effect. Depending on factors such as the compound, the symptoms and their severity, the age of the mammal being treated, etc., the amount of the compound Pladienolide B, pharmaceutical composition or pharmaceutical preparation of the present invention that reaches an effective amount will also vary, but the specific dosage can be routinely determined by those of ordinary skill in the art based on the knowledge in the art they possess and the content disclosed in the present invention. As used herein, the term "effective amount" includes "prophylactically effective amount" and "therapeutically effective amount".

[0063] As used herein, 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 recurrence of a disease, disorder or condition. The prophylactically effective amount of a compound refers to the amount of a therapeutic agent, used alone or in combination with other medicaments, that provides a prophylactic benefit in preventing a disease, disorder or condition. The term "prophylactically effective amount" may include an amount that improves overall prophylaxis, or an amount that enhances the prophylactic effect of other prophylactic medicaments.

[0064] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or an amount to delay or minimize one or more symptoms associated with a disease, disorder or condition. The therapeutically effective amount of a compound refers to the amount of a therapeutic agent, used alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder or condition. 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.

[0065] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" includes any substance that is suitable for use in humans and / or mammals without undue adverse side effects (such as 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 drugs, pharmaceutical compositions or pharmaceutical preparations described in 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).

[0066] Exemplarily, 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, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, adsorption carriers, lubricants, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents or buffers, etc.

[0067] In some embodiments, the diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, dextrin, mannitol, powdered sugar, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, water, etc.

[0068] In some embodiments, the fillers include, but are not limited to, mannitol (granular or powdered), glucose, lactose, sucrose, dextrin, xylitol, sorbitol, maltose, erythritol, microcrystalline cellulose, polymeric sugars, coupled sugars, starch, sodium alginate, laminaran powder, agar powder, calcium carbonate, sodium bicarbonate, etc.

[0069] In some embodiments, the binder includes but is not limited to: starch, pregelatinized starch, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid, alginate, xanthan gum, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.

[0070] In some embodiments, the humectant includes but is not limited to: glycerol, starch, etc.

[0071] In some embodiments, the disintegrant includes but is not limited to: crosslinked vinylpyrrolidone, low-substituted hydroxypropylmethyl, croscarmellose sodium, sodium carboxymethyl starch, soy polysaccharide, etc.

[0072] In some embodiments, the surfactant includes but is not limited to: polyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceryl stearate, cetyl alcohol, etc.

[0073] In some embodiments, the adsorption carrier includes but is not limited to: starch, lactose, bentonite, activated carbon, silica gel, kaolin, alumina, diatomaceous earth, saponite, hydroxypropylmethylcellulose, ethylcellulose, chitosan, polystyrene, polylactic acid, polyethylene glycol, etc.

[0074] In some embodiments, the lubricant includes but is not limited to: zinc stearate, monoglyceryl stearate, polyethylene glycol, talc, calcium stearate, magnesium stearate, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, sucrose monolaurate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium dodecyl sulfate, etc.

[0075] As used herein, the term "subject" refers to an animal, preferably a mammal (human and non-human animals), and the mammals include but are not limited to: humans, non-human primates (especially higher primates, such as macaques, cynomolgus monkeys, stump-tailed monkeys, Assamese macaques, flat-faced monkeys, golden monkeys, and tree shrews), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, any domestic animal or pet, etc. In a preferred embodiment, the subject is a human.

[0076] The experimental consumables, reagents, and raw materials used in the present invention are easily obtainable by those of ordinary skill in the art. Unless otherwise specified, they can all be obtained from commercial sources. The experimental methods of the present invention without specific conditions are usually carried out under 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 their 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.

[0077] Example 1: Pladienolide B induces cells to be dose - dependent against STX1

[0078] 1. Experimental method

[0079] In this example, after pretreating 5637 cells with different concentrations of Pladienolide B (PB) for 24 h, different concentrations of STX1 were given for treatment. The IC50 of cells in the blank control DMSO group and the Pladienolide B pretreatment group was compared to judge the cytotoxic effect of Pladienolide B against Shiga toxin STX1 - induced cells. The specific experimental method is as follows:

[0080] Cell viability was detected by CCK - 8 method: 5637 cells were pretreated with different doses of Pladienolide B for 24 h. After sub - culturing into 96 - well plates and waiting for the cells to adhere, 1640 medium without serum with STX1 concentrations 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 was added. Three replicates were set for each concentration (n = 3). After STX1 treatment for 48 h, 10 μL of CCK - 8 reagent was added to each well, and the reaction was carried out at 37℃ for 4 h. The absorbance was measured using a microplate reader at a wavelength of 450 nm. According to the formula: Cell survival rate = [1 - (experimental well - control well) / (control well - blank well)]×100%, the cell survival rate of each concentration experimental well was calculated, and the survival curve was fitted and drawn.

[0081] 2. Experimental results

[0082] 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. The results are as Figure 1 shown. As can be seen from the results shown in Figure 1 , pretreatment with 5 nM Pladienolide B can make cells have a relatively obvious STX1 tolerance effect. With the increase of the Pladienolide B treatment concentration, the ability of cells to resist STX1, especially to tolerate high - concentration STX1, gradually increases, suggesting that the effect of Pladienolide B in inducing cells to resist STX1 is dose - dependent. Among them, when cells were pretreated with 10 nM Pladienolide B, for 1×10 5STX1 at pg / mL exhibited a completely resistant phenotype, while the cell survival rate of the control group was only 10%. The above results indicate that Pladienolide B can effectively induce cells to resist STX1 and shows a dose-dependence. 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.

[0083] Example 2: Pladienolide B induces cells to resist STX1 in a time-dependent manner

[0084] 1. Experimental method

[0085] In this example, it was further explored whether different pretreatment times of Pladienolide B affected the anti-toxic phenotype of the induced cells. The pretreatment time of 10 nM Pladienolide B on cells was set to 6, 12, and 24 h. The specific method for detecting cell viability was as described in the experimental method of Example 1 above. 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 .

[0086] 2. Experimental results

[0087] In this example, the pretreatment time of 10 nM Pladienolide B on cells was set to 6, 12, and 24 h. When the pretreatment time of Pladienolide B was up to 6 or 12 h, there was no significant change in the sensitivity of the experimental group cells to STX1 compared with the DMSO control group cells ( Figure 2 A, 2B); when the pretreatment time of Pladienolide B was up to 24 h, STX1 had almost no killing effect on the cells ( Figure 2 C). The above results indicate that the effect of Pladienolide B in inducing cells to resist STX1 toxicity has a certain time-dependence.

[0088] The experimental results of the above Example 1 and Example 2 indicate that Pladienolide B has the effect of effectively inhibiting STX1, can effectively induce cells to resist the toxicity of STX1, and has a certain dose-dependence and time-dependence.

[0089] Example 3: Pladienolide B induces cells to be resistant to STX2 in a dose-dependent manner

[0090] 1. Experimental method

[0091] 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 cells in the blank control DMSO group and the Pladienolide B pretreatment group was compared to determine the cytotoxic effect of Pladienolide B against STX2-induced cells. The specific experimental method is as follows:

[0092] Cell viability was detected by CCK-8 method: 5637 cells were pretreated with different doses of 1, 5, 10 nM Pladienolide B for 24 h. Passaged into 96-well plates, after the cells adhered, 1640 medium without serum with STX2 concentrations 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 was added. Three replicates were set for each concentration (n = 3). After STX2 treatment for 48 h, 10 μL of CCK-8 reagent was added to each well, reacted at 37°C for 4 h, and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. According to the formula: cell survival rate = [1 - (experimental well - control well) / (control well - blank well)] × 100%, the cell survival rate of each concentration experimental well was calculated, and the survival curve was fitted and plotted.

[0093] 2. Experimental results

[0094] 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 to detect the cell viability level of each group. The results are as Figure 3 shown. As can be seen from the results shown in Figure 3 , pretreatment with 5 nM Pladienolide B can make cells have a relatively obvious STX2 tolerance effect. With the increase of the Pladienolide B treatment concentration, the ability of cells to resist STX2, especially to tolerate high concentrations of STX2, gradually increases, suggesting that the effect of Pladienolide B in inducing cells to resist STX2 is dose-dependent. Among them, when cells were pretreated with 10 nM Pladienolide B, they were resistant to 1×10 5STX2 at pg / mL exhibited a completely tolerant phenotype, while the cell survival rate in the control group was only 8%. The above results indicate that Pladienolide B can effectively induce cells to resist STX2 and shows a dose-dependence. 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.

[0095] Example 4: Pladienolide B induces cells to resist STX2 in a time-dependent manner

[0096] 1. Experimental method

[0097] In this example, it was further explored whether different pretreatment times of Pladienolide B affected the anti-toxic phenotype of induced cells. The pretreatment times of 10 nM Pladienolide B on cells were set to 6, 12, and 24 h, and the specific cell viability detection method was as described in the experimental method of Example 3 above. The pretreatment times of 10 nM Pladienolide B on cells were set to 6, 12, and 24 h, and STX2 with concentrations 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 .

[0098] 2. Experimental results

[0099] In this example, the pretreatment times of 10 nM Pladienolide B on cells were set to 6, 12, and 24 h. When the pretreatment time of Pladienolide B was up to 6 or 12 h, there was no significant change in the sensitivity of the experimental group cells to STX2 compared with the DMSO control group cells ( Figure 4 A, 4B); when the pretreatment time of Pladienolide B was up to 24 h, STX2 had almost no killing effect on the cells ( Figure 4 C). The above results indicate that the effect of Pladienolide B in inducing cells to resist STX2 toxin has a certain time-dependence.

[0100] The experimental results of Example 3 and Example 4 above indicate that Pladienolide B has the effect of effectively inhibiting STX2, can effectively induce cells to resist the toxicity effect 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.

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