Use of GSDME inhibitor for manufacturing medicament for preventing and treating peripheral nerve injury during chemotherapy

Abstract

The invention provides an application of a GSDME inhibitor for manufacturing a medicine for preventing and treating peripheral nerve injury during chemotherapy. The invention discloses the effect of the GSDME in the peripheral nerve injury induced by chemotherapy, and proves that the GSDME inhibitor can be used as an adjuvant of the chemotherapeutic drug for cancer treatment and has an effect of relieving neurotoxicity caused by chemotherapy, so that a treatment thought and a theoretical basis are provided for the peripheral nerve injury induced by various chemotherapeutic drugs; and molecular targets and preparation ideas can be provided for screening and preparing drugs for treating peripheral nerve injury induced by chemotherapy drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to use of a GSDME inhibitor in manufacturing a medicine for preventing and treating peripheral nerve damage during chemotherapy. Background Art

[0002] The methods of comprehensive tumor treatment are changing with each passing day, but for most tumors, especially those in advanced stages, chemotherapy is still one of the main methods for treating malignant tumors. Repeated chemotherapy can aggravate the adverse reactions of chemotherapy drugs and even cause irreversible tissue damage. CIPN (chemotherapy-induced peripheral neuropathy, CIPN) is a common dose-limiting adverse reaction caused by motor, sensory and autonomic nerve damage caused by anti-tumor drugs. Among them, sensory dysfunction is the most prominent. Typical symptoms include symmetrical numbness of the limbs, loss of proprioception, obvious pins and needles, hyperalgesia or ectopic pain in the hands and feet [1]. In 2014, the American Society of Clinical Oncology (ASCO) specifically issued guidelines for the prevention and treatment of chemotherapy-induced peripheral neuropathy CIPN [2,3], pointing out that "CIPN is a very important adverse reaction event that needs our attention, but there is no effective treatment for CIPN at present" [4]. CIPN-related anti-tumor drugs include platinum drugs (such as carboplatin, cisplatin and oxaliplatin), taxane drugs (such as paclitaxel and docetaxel), eboxilone drugs (such as isabendone), vincristine alkaloids (such as vincristine and vinblastine), bortezomib and thalidomide. CIPN limits the improvement of chemotherapy drug dosage and efficacy to a certain extent and is the main dose-limiting toxicity of chemotherapy drugs [5-8].

[0003] In recent years, how to prevent and alleviate chemotherapy-induced neurotoxicity has become a consensus problem in the field of cancer drug treatment. Based on the pathogenesis of CIPN, a large number of compounds have been developed to prevent or treat CIPN by blocking ion channels, targeting inflammatory cytokines and anti-oxidative stress. Glutathione and mangafodipir are promising for preventing CIPN, and duloxetine is expected to be effective for CIPN intervention, but the mechanisms of these drugs in preventing and intervening in CIPN have not yet been elucidated [9-12]; while drugs such as calcium magnesium injection, venlafaxine, minocycline hydrochloride and gabapentin have limited efficacy in preventing and alleviating CIPN [13-15]; erythropoietin, menthol and aminofastin are accompanied by a variety of adverse reactions during use

[16] . Since the mechanism of peripheral neurotoxicity caused by chemotherapy drugs has not been clarified, there is no targeted target for drug treatment, and drugs that have been proven to have application prospects in animal experiments have not achieved the expected efficacy in large-scale clinical trials

[17] . Therefore, no specific drug for CIPN has been found so far, and clinical prevention and treatment are the main methods, and drug treatment is still controversial.

[0004] Pyroptosis is a pro-inflammatory cell death characterized by cell swelling, cell membrane lysis, and the release of inflammatory factors such as HMGB1, IL-1β and IL-18, and LDH. It is also an important natural immune defense mechanism of the host against intracellular pathogen infection. When cells are stimulated by exogenous pathogen-related molecular patterns such as bacteria and viruses, the cytoplasmic sensor molecules (pattern recognition receptors such as NLRP1, NLRP2, NLRP3, NLRC4, AIM2), adapter proteins (apoptosis associated speck-like protein containing a CARD, ASC) and effector molecules (Caspase-1) form inflammasomes, activate Caspase-1 / GSDME, mediate cell pyroptosis, and release HMGB1, IL-1β and IL-18 inflammatory factors and LDH. This technical field urgently needs to solve whether polypeptide inhibitors targeting GSDME can be used as potential drugs for the prevention and treatment of chemotherapeutic neurotoxicity. The difference in the expression of GSDME between tumor cells and normal cells is the key to the adverse reaction of chemotherapeutic drugs causing tissue damage. This may also serve as a therapeutic target to improve side effects of chemotherapy such as peripheral nerve damage and gastrointestinal damage without affecting the anti-tumor effect of chemotherapy.

[0005] In summary, pyroptosis plays an important role in peripheral nerve damage induced by tumor chemotherapy, and GSDME is a key molecule that mediates chemotherapeutic drug-induced pyroptosis. Inhibitors targeting GSDME can be used as potential drugs for the prevention and treatment of peripheral nerve damage induced by tumor chemotherapy, which has become an issue that technicians in this field urgently need to study. Summary of the invention

[0006] The purpose of the present invention is to provide a use of a GSDME inhibitor for manufacturing a drug for preventing and treating peripheral nerve damage, so as to solve the above technical problem, namely preventing or treating peripheral nerve damage caused by chemotherapy drugs during chemotherapy.

[0007] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention discloses the use of a GSDME inhibitor for manufacturing a medicament for preventing or treating peripheral nerve damage during chemotherapy.

[0008] Preferably, the peripheral nerve damage is damage to peripheral nerve cells induced by chemotherapy drugs.

[0009] Furthermore, the chemotherapy drug is a cancer chemotherapy drug.

[0010] More preferably, the cancer chemotherapy drug is selected from one or more of platinum chemotherapy drugs, taxane chemotherapy drugs, vincristine chemotherapy drugs, bortezomib and its derivatives.

[0011] Furthermore, the platinum chemotherapy drug is one or more of cisplatin, carboplatin, cyclothioplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0012] Furthermore, the taxane chemotherapy drug is paclitaxel.

[0013] Furthermore, the vincristine chemotherapy drug is one or more of vinblastine and vincristine.

[0014] Preferably, the peripheral nerve cells are ND7 / 23 cells.

[0015] Preferably, the symptoms of peripheral nerve injury include one or more of the following: numbness and pain in the skin of the extremities, glove or stocking-like sensation, cold skin, paresthesia, a sense of ants, weakness in holding objects, a feeling of walking on cotton, burning pain, shooting pain, and knife-like pain.

[0016] Preferably, the GSDME inhibitor is dimethyl fumarate (DMF).

[0017] In a second aspect, the present invention provides a medicament for preventing or treating peripheral nerve injury, comprising a GSDME inhibitor and a pharmaceutically acceptable carrier.

[0018] In a third aspect, the present invention further provides a method for preparing a drug for preventing or treating peripheral nerve damage during chemotherapy, the method comprising mixing a GSDME inhibitor with a pharmaceutically acceptable carrier in proportion.

[0019] Compared with the prior art, the present invention has the following beneficial effects: GSDME is the target of chemotherapy-induced cell pyroptosis. The inventors have determined the role of GSDME in chemotherapy-induced peripheral nerve injury, thereby providing a treatment idea and theoretical basis for peripheral nerve injury induced by various chemotherapy drugs, and can also provide molecular targets and preparation ideas for screening and preparing drugs for treating peripheral nerve injury induced by chemotherapy drugs.

[0020] The present invention screens dimethyl fumarate, an inhibitor for GSDME, and further discloses the application of the inhibitor in the preparation of drugs for preventing and treating peripheral nerve damage caused by chemotherapy drugs. Further experiments have found that dimethyl fumarate has a protective effect on the neurotoxicity of ND7 / 23 cells induced by oxaliplatin, paclitaxel, and bortezomib; in animal models, dimethyl fumarate has a protective effect on the peripheral neurotoxicity of mice induced by oxaliplatin, paclitaxel, and bortezomib. The drug dimethyl fumarate of the present invention can be used as an adjuvant of chemotherapy drugs for cancer treatment, and has a mitigating effect on neurotoxicity caused by chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of Western detection of GSDME protein activation in mice in the oxaliplatin-induced peripheral nerve injury model; Figure 2 The knockdown of GSDME has a protective effect on oxaliplatin-induced peripheral nerve injury in mice. Figure 3 The chemical structure diagram of dimethyl fumarate, a GSDME inhibitor; Figure 4 The effects of dimethyl fumarate, oxaliplatin, paclitaxel, and bortezomib on neuronal cell viability; Figure 5 The effect of GSDME inhibitor dimethyl fumarate on the activation level of GSDME in DRG of mice with oxaliplatin-induced peripheral nerve injury model; Figure 6 The effect of GSDME inhibitor dimethyl fumarate on oxaliplatin-induced peripheral nerve injury model mice; Figure 7 This is the effect of GSDME inhibitor dimethyl fumarate on the mechanical pain threshold of mice with peripheral nerve injury model induced by paclitaxel and bortezomib.

[0022] Note: Data are expressed as mean ± standard deviation (n = 3). Figure 1-7 Two-way ANOVA was performed, followed by Tukey's post hoc comparison. *P<0.05, **P<0.01 for comparison between treatment group and control group. DETAILED DESCRIPTION

[0023] The present invention provides a use of a GSDME inhibitor to prevent peripheral nerve damage during chemotherapy, belonging to the field of biomedicine technology. The present invention proposes that a GSDME inhibitor can reduce GSDME oligomerization and inhibit its formation of pores on the plasma membrane, thereby inhibiting GSDME-mediated cell pyroptosis in vivo and being used to prevent and treat peripheral nerve damage.

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like indicating the orientation or positional relationship are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0026] In this specification, the reference to "an implementation method" or "an implementation method" means that specific features, structures and attributes are incorporated into the implementation method. Among them, these characteristics and structures are included in at least one implementation method. Therefore, the expression "in an implementation method" or "in an implementation method" does not necessarily mean that it is equivalent to the same implementation method. In addition, in one or more implementation methods, specific characteristics, structures and attributes can be combined and used in any manner as required. It should be pointed out that the singular forms "one", "a kind" and "the one" used in this specification and the attached claims actually include the plural meaning, unless the specific content expressly indicates otherwise. Finally, the word "or" usually has its literal meaning, that is, "and / or", unless the context has other special provisions.

[0027] As used herein, the term "peripheral nerves" generally refers to those nerves that branch out from the spinal cord and extend to various parts of the body. Nerve repair is more common in the peripheral nervous system than in the central nervous system.

[0028] As used in this article, chemotherapy-induced peripheral nerve damage (CIPN) is a type of nerve damage caused by chemotherapy drugs, usually manifested as paresthesia, pain, numbness, tingling and other symptoms. Chemotherapy drugs affect the normal function of nerve cells, resulting in impaired nerve conduction, which in turn causes peripheral nerve damage. Common chemotherapy drugs include taxanes (such as paclitaxel), platinum drugs (such as oxaliplatin), enzyme inhibitors (such as bortezomib), etc. Peripheral nerve damage may affect the patient's quality of life, so the occurrence and development of CIPN needs to be closely monitored and managed during chemotherapy.

[0029] The terms "therapeutic", "treatment" or "relief", "relief" in the present invention not only refer to medical methods used to treat diseases (such as slowing down or curing symptoms), stop (or reduce) the progression of diagnosed conditions, and prevent peripheral nerve damage, but also cover measures to prevent the slow regeneration process of peripheral nerves. Therefore, the patients treated include people with peripheral nerve damage symptoms; and those who are susceptible to peripheral nerve damage. The ideal medical effect may include but is not limited to symptom relief, reduced degree of nerve tissue damage, maintaining a stable (i.e., not deteriorating) state of nerve damage, slowing down or preventing the development of damage, recovery of nerve tissue (whether partial or complete) or even regeneration. The people treated include people who have suffered from peripheral nerve damage or suspected of having such a disease, as well as people who may be susceptible to such damage. These people may need to prevent this from happening or avoid greater damage for various reasons. In addition, the population that needs special treatment also includes animals whose nerve regeneration activity ability degenerates with age.

[0030] In the present invention, the term "therapeutically effective amount" refers to the amount of small organic molecules or other drugs that are effective for "treating" a disease or condition in a subject or mammal. In the case of peripheral nerve injury or trauma (e.g., sciatic nerve trauma), a therapeutically effective amount of a drug can promote axon regeneration of sensory neurons, epidermal innervation, and functional recovery of neurons after nerve injury in subjects with nerve regeneration decline, by increasing, for example, the proliferation, differentiation, migration and / or survival of neural stem cells / precursor cells; reducing, delaying or stopping the reduction of nerve cells; inhibiting, for example, suppressing, delaying, preventing, stopping or reversing the reduction of nerve cells; increasing the number, density and / or concentration of nerve cells; changes in the morphology or function of nerve cells; or changes in the interaction between nerve cells; alleviating one or more symptoms associated with nerve injury to a certain extent, such as pain; improving the quality of life; or a combination of these effects.

[0031] The treatment subjects of the present invention include individuals, animals and mammals. These animals include but are not limited to humans, household pets, livestock, animals in wild animals, competitive animals and pets, such as dogs, cats, hamsters, rabbits, rats, mice, horses, cattle, cows and bears.

[0032] The administration of the compounds of the present invention can be oral, but, as an alternative to oral drugs, in certain situations, other routes of administration can be applied to the technology and application scenarios described herein, such as intravenous administration. There are many routes of intravenous administration, such as subcutaneous, intravenous, muscle, joint, synovial membrane, sternum, sheath, liver, lesions, and intracranial sites. For example, the compound can also be administered by intravenous, intramuscular, sheath or subcutaneous injection. In some applications, in order to achieve the purpose of oral drugs, the dosage of the compound can be reduced to a level suitable for intravenous administration, and then achieved by injection.

[0033] According to the usage and function described in this article, there are other suitable scenarios for applying the compound for administration, such as: sublingual, buccal (such as applying a film or other mixture that can be dissolved in the mouth under the tongue or in the cheek), eyes (such as as eye drops), ears (such as through ear drops), oral inhalation (such as by blowing or atomization), skin or local area (such as by applying emulsions or lotions) or directly on the skin (such as through patches). In addition to oral administration, it can also be delivered through other intestinal administration methods such as vaginal and rectal administration, such as through ointments, suppositories or enemas.

[0034] In practical applications, the operations and purposes involved in this study are usually achieved by adding effective compounds (such as oral administration) to achieve the goal of alleviating symptoms. The specific effective dose range used is 0.05 mg / kg to 20 mg / kg, including 0.05 mg / kg to 10 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.05 mg / kg to 5 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.05 mg / kg to 2 mg / kg, 0.1 mg / kg to 2 mg / kg, and any dose range with any interval set between 0.05 mg / kg and 20 mg / kg or other intermediate values ​​as endpoints. In some cases, this strategy may require the use of a compound with a dose range of 0.1-1 mg / kg, or a compound with a dose range of 0.2-0.5 mg / kg. Likewise, the methods or uses may involve administering a dose of the compound in some cases in the range of 0.05-20 mg / kg, or in some cases in the range of 1-10 mg / kg.

[0035] In some embodiments, the methods and uses described herein, such as methods or uses for treating pain in a subject (e.g., a human) in need thereof, are achieved by administering (e.g., orally) a therapeutically effective amount of a compound per day, such as 5 to 1000 mg of a compound per day, such as 5 to 500 mg or 5 to 250 mg of a compound per day. For example, the method or use may include administering (e.g., orally) about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135mg, about 140mg, about 145mg, about 150mg, about 155mg, about 160mg, about 165mg, about 170mg, about 175mg, about 180mg, about 185mg, about 190mg, about 195mg, about 200mg, about 205mg, about 210mg, about 215mg, about 220mg, about 225mg, about 230mg, about 235mg, about 240mg, about 245mg, about 250mg, about 255mg, about 260mg, about 265mg, about 270mg, about 275mg, about 280mg, about 285mg, about 290mg, about 295mg, about 300mg, about 305mg, about 310mg, about 315mg, about 320mg, about 325mg, about 330mg, about 335mg, about 340, about 345mg, about 350mg, about 355mg, about 360mg, about 365mg, about 370mg, about 375mg, about 380mg, about 385mg, about 390mg, about 395mg, about 400mg, About 405mg, about 410mg, about 415mg, about 420mg, about 425mg, about 430mg, about 435mg, about 440mg, about 445mg, about 450mg, about 455mg, about 460mg, about 465mg, about 470mg, about 475mg, about 480mg, about 485mg, about 490mg, about 495mg, about 500mg or about 1000mg of a compound, or daily administration (e.g., oral) by using two of the above quantities as any number range created by endpoints. In some aspects, the method or uses include oral administration of 10 to 200mg of a compound to a subject (e.g., a person) every day, for example, 10, 15, 20, 25, 30, 35 or 40mg to 75, 100, 125, 150, 175 or 200mg of a compound per day, including 20 to 150mg per day.In some aspects, the subject (e.g., human) is orally administered a compound comprising 50, 75, 100 or 125 mg per day, for example, 100 mg per day. In some cases, the daily dose of the compound may need to be achieved by multiple doses (e.g., oral), such as 5 times of 20 mg, 4 times of 25 mg, 3 times of 33.3 mg or 2 times of 50 mg, etc. For example, a daily dose of 100 mg can be allocated to different dosing regimens such as 5 times of 20 mg, 4 times of 25 mg, 3 times of 33.3 mg or 2 times of 50 mg per day. In some methods, the daily dose of the compound can also be regarded as a single dose for use (e.g., oral). For example, the daily dose of a compound can be allocated to a single dose of 5 mg, 10 mg, 15 mg, 20 mg, 25 mg or 30 mg, or a dose of 5 mg, 10 mg, 15 mg, 20 mg, 25 mg or 30 mg, such as taking 5 mg once a day or taking 10 mg once a day. Also, any dose of the compound discussed in the previous section may be distributed in a single dosage unit or multiple dosage unit dosage form, such as two, three or four unit dosage forms.

[0036] In some embodiments, the methods and uses described herein for treatment by administering (e.g., orally) a compound include administering the compound at the same time as, before, or after chemotherapy, for example, 0.5h, 1h, 1.5h, 2h before chemotherapy, or 0.5h, 1h, 1.5h, 2h after chemotherapy; it can also be administered 1 day, 2 days, 3 days before chemotherapy, 1 day, 2 days, 3 days after chemotherapy, etc.

[0037] The drug complex of the present invention covers various forms (not limited): injection, tablets, capsules, gas release agents, patches, films, pills, external coatings, controlled release or slow release or nanoparticles. This drug complex contains a primary compound or a pharmacologically acceptable salt thereof that is safe and pharmacologically acceptable within its effective dose range, and a pharmacologically acceptable carrier. The so-called "effective dose" means that the content of the drug can significantly improve the symptoms without causing serious side effects. Generally speaking, the drug complex contains 1-2000 mg of the primary compound / dose, and even better, it contains 10-1000 mg of the primary compound / dose. In the best case, the so-called "one unit" is a capsule or tablet. "Acceptable carrier" refers to one or more compatible solid or liquid filling materials suitable for the human body, which must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the various components in the drug complex can be mixed with the primary compound and between them, and will not significantly reduce the efficacy of the compound. For example, some raw materials of acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween 80) and colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0038] There are many types of solid preparations for oral administration, such as capsules, tablets, pills, powders and granules. The active ingredients in these preparations are mixed with some common inactive adjuvants, such as sodium citrate or dicalcium phosphate, or with other specific ingredients: (i) fillers or thickeners, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (ii) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (iii) humectants, such as glycerol; (iv) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (v) sustained-release agents, such as paraffin; (vi) absorption promoters, such as quaternary ammonium compounds; (vii) wetting agents, such as cetyl alcohol and glyceryl monostearate; (viii) adsorbents, such as kaolin; (ix) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. Buffers may also be added to the three preparations of capsules, tablets and pills. Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be obtained by coating and outer shelling methods, such as enteric coatings and other common materials. These preparations may also contain opaque fillers, and in the preparations, the active compound or its release mode can be released in a specific part of the digestive tract. Buffers may also be added to capsules, tablets and pills. Oral liquid preparations include emulsions, solutions, suspensions, syrups or tinctures accepted in the medical field. In addition, liquid preparations may also contain inactive diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition, in addition to these inactive diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices. The suspension may also contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methylate and agar or mixtures of these substances. In the composition for parenteral injection, a physiologically acceptable sterilized water or anhydrous solution, dispersion, suspension or emulsion and a sterile powder for re-dissolving into a sterile injectable solution or dispersion may be contained. Suitable as water, ethanol, polyols and their appropriate mixtures. In the pharmaceutical formulation for local use, ointments, powders, patches, sprays and inhalants are included. Under sterile conditions, the active ingredient is mixed with a physiologically acceptable carrier, any preservative, buffer, or, if necessary, a propellant. This treatment method can be used independently or in combination with other treatment methods or drugs.

[0039] Example 1: Tail vein injection of AAV-U6-GSDME shRNA adeno-associated virus into mice can knock down the expression level of GSDME in the dorsal root ganglia of mice.

[0040] 1. Experimental materials: AAV-U6-GSDME shRNA adeno-associated virus was purchased from Nanjing Yike Biotechnology Co., Ltd., and oxaliplatin (CAS No.: HY-17371) was purchased from MCE. Oxaliplatin was prepared with 5% glucose solution at a dosage of 3 mg / kg.

[0041] Experimental animals: Male C57BL / 6 mice, weighing 20-24g, clean grade, purchased from Shanghai Slake Experimental Animal Center, and kept in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine. The experimental temperature was controlled at 25±1℃, and free access to drinking water and animal feed was provided. The experiment began after one week of feeding.

[0042] 2. Experimental methods: The experimental animals were randomly divided into two groups: a control group and an AAV-GSDME shRNA group.

[0043] Control group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, 5 days on and 5 days off, for a total of two cycles of 20 days; the solvent was injected into the tail vein; AAV-GSDME shRNA group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, with 5 days of administration and 5 days of rest, for a total of two cycles of 20 days; AAV-GSDME shRNA virus was injected into the tail vein two weeks before oxaliplatin administration.

[0044] On the 21st day, the mice were killed, and the dorsal root ganglia of the mice were collected and weighed. Protein lysis buffer was added and proteins were collected for Western detection.

[0045] 3. Experimental results: The protein lysates of the two parts were tested for GSDME activation using anti-mouse GSDME antibody and anti-mouse GAPDH antibody. The Western results showed that after tail vein injection of AAV-GSDME shRNA virus, the expression and activation of GSDME were significantly reduced. Figure 1 (Lane 1-3 is the control group, and lane 4-6 is the AAV-GSDME shRNA virus group).

[0046] Example 2: GSDME knockdown has a protective effect on oxaliplatin-induced peripheral nerve injury in mice.

[0047] 1. Experimental materials: Experimental drugs: Oxaliplatin (CAS No.: HY-17371), AAV-U6-GSDME shRNA adeno-associated virus were purchased from Nanjing Yike Biotechnology Co., Ltd. Oxaliplatin was prepared with 5% glucose solution, and the dosage was 3 mg / kg.

[0048] Instruments: Electronic von Frey analgesia meter, Plantar Test (Hargreaves method) Glass stands, and Hot Cold Plate Analgesia Meter for Mice and Rats were all purchased from IITC (IITC Inc. Life Science, Woodland Hills, CA, USA).

[0049] Experimental animals: Male C57BL / 6 mice, weighing 20-24g, clean grade, purchased from Shanghai Slake Experimental Animal Center, and kept in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine. The experimental temperature was controlled at 25±1℃, and free access to drinking water and animal feed was provided. The experiment began after one week of feeding.

[0050] 2. Experimental methods: (1) The experimental animals were randomly divided into two groups: a control group and an AAV-GSDME shRNA group.

[0051] Control group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, 5 days on and 5 days off, for a total of two cycles of 20 days; the solvent was injected into the tail vein; AAV-GSDME shRNA group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, 5 days of administration and 5 days of rest, for a total of two cycles of 20 days; AAV-GSDME shRNA virus was injected into the tail vein; (2) The first day of drug treatment was designated as Day 1, and the weight and behavioral tests (mechanical pain test and cold pain test) of mice were performed on Days 0, 6, 11, 16, and 21.

[0052] (A) Body weight: The body weight of mice was measured at the same time point every day.

[0053] (B) Experimental measurement of mechanical pain sensation in mice: Referring to the method of Meng Zhao et al. (KIMeng Zhao, Saki Nakamura, Takayu K1 nakagawa, Shuji Kaneko, Acute cold hypersensitivity characteristically induced by oxaliplatin is caused by the enhanced responsiveness of TRPA1 in mice, Molecular Pain. 8 (2012) 55), the mechanical withdrawal threshold (von Frey test) was used to evaluate the mechanical allodynia of mice. The mice were placed in a (20*17*13cm) plexiglass box with a metal mesh at the bottom of the mouse toes. After the mice adapted to the measurement box environment for 15 minutes and were at rest, the soles of the feet of the mice were vertically stimulated with von Frey fiber filaments. After the mice showed a withdrawal reaction, the electronic display stress value was recorded. Each mouse was measured 10 times.

[0054] (C) Mouse cold pain test: Before the measurement, the mice were placed in the measurement environment to adapt for 30 minutes. During the test, the mice were placed on a cold plate at 5°C and covered with a transparent plexiglass box. The measurement time was 60 seconds. During the measurement time, the escape behavior of the mice was recorded and scored: 0 points = no reaction; 1 point = mild cold escape reaction, such as lifting the hind legs or walking backwards; 2 points = strong cold escape reaction, such as jumping up, etc. The total score within the measurement time of 60 seconds was recorded. Each mouse was measured 3 times and the average was taken.

[0055] 3. Experimental results: The results are as follows Figure 2 As shown, according to the diagram: (1) Effects on mouse body weight: Figure 2 As shown in (A), the mice in the control group showed a significant weight loss due to gastrointestinal adverse reactions caused by oxaliplatin stimulation. During the withdrawal of oxaliplatin, the weight of the mice showed a slow increase trend; the weight of the mice in the AAV-GSDME shRNA group also showed a decreasing trend during the administration of oxaliplatin. After the withdrawal of oxaliplatin, the weight of the mice increased significantly, and the overall weight loss was slower than that of the control group. The weight of the mice in the AAV-GSDME shRNA group was significantly different from that of the mice in the control group.

[0056] (2) Effects on mechanical pain perception in mice: Figure 2As shown in (B), during the measurement period, the mechanical paw withdrawal threshold of the control group mice decreased significantly; compared with the control group, the mechanical pain threshold of the AAV-GSDME shRNA group mice increased significantly, indicating that knockdown of GSDME can reduce the mechanical pain sensitivity of mice caused by oxaliplatin.

[0057] (3) Effects on cold pain perception in mice: Before the experiment, mice in each group were insensitive to cold pain perception. Figure 2 As shown in (C), after 6 days of the experiment, the cold pain scores of the mice in the control group increased significantly, showing obvious cold pain sensitivity. From 11 days to 21 days, the cold pain scores of the mice in the AAV-GSDME shRNA group were lower than those in the control group, and there was a significant difference. This shows that knocking down GSDME can reduce the cold pain sensitivity of mice caused by oxaliplatin.

[0058] In summary, in the peripheral nerve injury model caused by oxaliplatin, mice were sensitive to mechanical pain and cold pain, and GSDME knockdown reduced the sensitivity of mechanical pain and cold pain in mice. GSDME knockdown had a protective effect on peripheral nerve injury caused by oxaliplatin in mice. Two-way ANOVA was used, followed by Tukey's post hoc comparison. *P<0.05, **P<0.01 were compared between the AAV-GSDME shRNA group and the control group.

[0059] Example 3: Chemical structure of the GSDME inhibitor dimethyl fumarate.

[0060] Example 4: The GSDME inhibitor dimethyl fumarate has a protective effect on ND7 / 23 cell injury induced by oxaliplatin, paclitaxel and bortezomib.

[0061] 1. Experimental materials: Oxaliplatin (CAS No.: HY-17371) and dimethyl fumarate (CAS No.: HY-17363) were purchased from MCE, paclitaxel (CAS No.: HWG53914) was purchased from Beijing Huawei Ruike Chemical Co., Ltd., and bortezomib (CAS No.: 179324-69-7) was purchased from Taoshu Biological. The concentration of the storage solution was 10 mM (dissolved in DMSO) and was directly diluted in the culture medium when used.

[0062] ND7 / 23 cells are rat / mouse neuronal fusion cells purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) in a constant temperature incubator at 37°C and 5°C. The cells can be used in experiments when they grow to 70%-80% confluence.

[0063] 2. Experimental methods: ND7 / 23 cells were digested and inoculated on a 96-well plate, 100uL of cell suspension was added to each well, and the cells were cultured overnight in an incubator. The next day, ND7 / 23 cells were treated with dimethyl fumarate at different concentrations (1μM, 10μM) for 2 hours, followed by the addition of 2.5μM oxaliplatin, 0.1μM paclitaxel, and 100nM bortezomib. After the cells were co-treated with dimethyl fumarate and chemotherapy drugs for 24 hours, 10μL of CCK-8 (Cat. No.: HY-K0301, purchased from MCE, China) was added to each well, and the absorbance was measured using a full-wavelength multifunctional microplate reader (model: Varioskan flash, purchased from Thermo Scientific, USA) after incubation in an incubator for 2 hours.

[0064] 3. Experimental results: The results are as follows Figure 4 As shown in the figure, it can be seen that the single administration of oxaliplatin, paclitaxel and bortezomib resulted in a significant decrease in cell viability. When oxaliplatin, paclitaxel and bortezomib were co-treated with 10μM dimethyl fumarate, the cell viability increased, indicating that dimethyl fumarate had a protective effect on the decrease in cell viability caused by oxaliplatin, paclitaxel and bortezomib. One-way ANOVA was used, followed by Tukey's post hoc comparison. Compared with the control group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0065] Example 4: The GSDME inhibitor dimethyl fumarate can reduce the level of GSDME activation in mouse dorsal root ganglia.

[0066] 1. Experimental materials: Oxaliplatin (CAS No.: HY-17371) and dimethyl fumarate (CAS No.: HY-17363) were purchased from MCE. Oxaliplatin was prepared with 5% glucose solution, and dimethyl fumarate was prepared with 50% PEG300 plus 50% saline.

[0067] Experimental animals: Male C57BL / 6 mice, weighing 20-24g, clean grade, purchased from Shanghai Slake Experimental Animal Center, and kept in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine. The experimental temperature was controlled at 25±1℃, and free access to drinking water and animal feed was provided. The experiment began after one week of feeding.

[0068] 2. Experimental methods: The experimental animals were randomly divided into two groups: a normal group, a model group and a dimethyl fumarate group.

[0069] Normal group: intraperitoneal injection of solvent; Model group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, 5 days of administration and 5 days of rest, for a total of two cycles of 20 days; the same dose of corn oil solvent was intragastrically administered every day.

[0070] Dimethyl fumarate group: 50 mg / kg dimethyl fumarate solution was administered intragastrically for 2 hours, and then 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg. Dimethyl fumarate was administered every day, and oxaliplatin was administered for 5 days and stopped for 5 days, for a total of two cycles of 20 days.

[0071] On the 21st day, the mice were killed, and the dorsal root ganglia of the mice were collected and weighed. Protein lysis buffer was added and proteins were collected for Western detection.

[0072] 3. Experimental results: The protein lysates of the five parts were subjected to Western detection of GSDME activation using anti-mouse GSDME antibody and anti-mouse GAPDH antibody. The results showed that compared with the control group, GSDME in the dorsal root ganglia of mice in the oxaliplatin group was significantly activated; and compared with the oxaliplatin group, the GSDME inhibitor dimethyl fumarate significantly reduced the GSDME activation level in the dorsal root ganglia. This indicates that the GSDME inhibitor dimethyl fumarate can significantly reduce the GSDME activation level in the dorsal root ganglia of mice with oxaliplatin-induced peripheral nerve injury model. Figure 5 (Lane 1-3 is the model group, and lane 4-6 is the dimethyl fumarate group).

[0073] Example 5: The GSDME inhibitor dimethyl fumarate has a protective effect on oxaliplatin-induced peripheral nerve injury in mice.

[0074] 1. Experimental materials: Experimental drugs: Oxaliplatin (CAS No.: HY-17371) and dimethyl fumarate (CAS No.: HY-17363) were purchased from MCE. Oxaliplatin was prepared with 5% glucose solution, and dimethyl fumarate was prepared with 50% PEG300 plus 50% saline.

[0075] Instruments: Electronic von Frey analgesia meter, Plantar Test (Hargreaves method) Glass stands, and Hot Cold Plate Analgesia Meter for Mice and Rats were all purchased from IITC (IITC Inc. Life Science, Woodland Hills, CA, USA).

[0076] Experimental animals: Male C57BL / 6 mice, weighing 20-24g, clean grade, purchased from Shanghai Slake Experimental Animal Center, and kept in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine. The experimental temperature was controlled at 25±1℃, and free access to drinking water and animal feed was provided. The experiment began after one week of feeding.

[0077] 2. Experimental methods: (1) The experimental animals were randomly divided into three groups: normal group, model group and dimethyl fumarate group.

[0078] Normal group: intraperitoneal injection of solvent; Model group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, 5 days of administration and 5 days of rest, for a total of two cycles of 20 days; the same dose of corn oil solvent was intragastrically administered every day.

[0079] Dimethyl fumarate group: 50 mg / kg dimethyl fumarate solution was administered intragastrically for 2 hours, and then 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg. Dimethyl fumarate was administered every day, and oxaliplatin was administered for 5 days and stopped for 5 days, for a total of two cycles of 20 days.

[0080] (2) The first day of drug treatment was designated as Day 1. The weight and behavioral characteristics of mice were measured on Days 0, 6, 11, 16, and 21 (mechanical pain test and cold pain test, see Example 2 for specific experimental methods).

[0081] 3. Experimental results: The results are as follows Figure 6 As shown, according to the diagram: (1) Effects on mouse body weight: Figure 6 As shown in (A), the weight of mice in the normal group increased over time. The mice in the model group showed a significant weight loss due to gastrointestinal adverse reactions caused by oxaliplatin stimulation. During the withdrawal of oxaliplatin, the weight of mice showed a slow increase trend. The weight of mice in the dimethyl fumarate group also showed a decreasing trend during the administration of oxaliplatin. After the withdrawal of oxaliplatin, the weight of mice increased significantly, and the overall weight loss was slower than that of the control group. The weight of mice in the dimethyl fumarate group changed significantly compared with that of mice in the control group.

[0082] (2) Effects on mechanical pain perception in mice: Figure 6 As shown in (B), during the measurement period, the mechanical paw withdrawal threshold of mice in the normal group tended to be stable. The mechanical paw withdrawal threshold of mice in the model group decreased significantly; compared with the model group, the mechanical pain threshold of mice in the dimethyl fumarate group increased significantly. This indicates that the GSDME inhibitor dimethyl fumarate can reduce the mechanical pain sensitivity of mice caused by oxaliplatin.

[0083] (3) Effects on cold pain perception in mice: Before the experiment, mice in each group were insensitive to cold pain perception. Figure 6 As shown in (C), after 6 days of the experiment, the cold pain scores of mice in the model group increased significantly, showing obvious cold pain sensitivity. From 11 days to 21 days, the cold pain scores of mice in the dimethyl fumarate group were lower than those in the model group, and there was a significant difference. This shows that the GSDME inhibitor dimethyl fumarate can reduce the cold pain sensitivity of mice caused by oxaliplatin.

[0084] In summary, in the peripheral nerve injury model caused by oxaliplatin, mice were sensitive to mechanical pain and cold pain, while the GSDME inhibitor dimethyl fumarate reduced the sensitivity of mechanical pain and cold pain in mice, and dimethyl fumarate had a protective effect on peripheral nerve injury caused by oxaliplatin in mice. Two-way ANOVA was used, followed by Tukey's post hoc comparison. *P<0.05, **P<0.01 compared with the model group and the normal group; *P<0.05, **P<0.01 compared with the dimethyl fumarate group and the model group.

[0085] Example 6: The GSDME inhibitor dimethyl fumarate has a protective effect on paclitaxel- and bortezomib-induced peripheral nerve injury in mice.

[0086] 1. Experimental materials: Experimental drugs: Oxaliplatin (CAS No.: HY-17371) and dimethyl fumarate (CAS No.: HY-17363) were purchased from MCE, paclitaxel (CAS No.: HWG53914) was purchased from Beijing Huawei Ruike Chemical Co., Ltd., and bortezomib (CAS No.: 179324-69-7) was purchased from Taoshu Biological. Paclitaxel was prepared with polyoxyethylene castor oil and anhydrous ethanol, bortezomib was prepared with normal saline, and dimethyl fumarate was prepared with 50% PEG300 plus 50% normal saline.

[0087] Instruments: Electronic von Frey analgesia meter, Plantar Test (Hargreaves method) and Glass stands were purchased from IITC (IITC Inc. Life Science, Woodland Hills, CA, USA).

[0088] Experimental animals: Male C57BL / 6 mice, weighing 20-24g, clean grade, purchased from Shanghai Slake Experimental Animal Center, and kept in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine. The experimental temperature was controlled at 25±1℃, and free access to drinking water and animal feed was provided. The experiment began after one week of feeding.

[0089] 2. Experimental methods: (1) The experimental animals were randomly divided into the following three groups: normal group, PIPN model group and dimethyl fumarate-PIPN group; normal group, BIPN model group and dimethyl fumarate-BIPN group.

[0090] Normal group: intraperitoneal injection of solvent; PIPN model group: intraperitoneal injection of paclitaxel solution 10 mg / kg (once on the 1st, 3rd, 5th, and 7th day, 4 injections in total for 4 days). The same dose of corn oil solvent was gavaged every day.

[0091] Dimethyl fumarate-PIPN group: dimethyl fumarate solution was administered intragastrically at a dose of 50 mg / kg for 2 hours in advance. Dimethyl fumarate was administered every day. Then paclitaxel solution was injected intraperitoneally at a dose of 10 mg / kg (once on the 1st, 3rd, 5th, and 7th day, for a total of 4 injections over 4 days).

[0092] BIPN model group: Bortezomib 1 mg / kg was injected intraperitoneally (once on the 1st, 3rd, 5th, and 7th day, 4 times in total for 4 days). Corn oil solvent was administered intragastrically at the same dose for consecutive days.

[0093] Dimethyl fumarate-BIPN group: The mice were gavaged with dimethyl fumarate solution at a dose of 50 mg / kg for 2 hours in advance. Dimethyl fumarate was administered every day. Then bortezomib 1 mg / kg was injected intraperitoneally (once on the 1st, 3rd, 5th and 7th day, for a total of 4 injections over 4 days).

[0094] (2) The first day of drug treatment was designated as Day 1, and the mechanical pain threshold of mice was measured on Days 0 and 8, respectively (see Example 2 for the specific experimental method).

[0095] 3. Experimental results: The results are as follows Figure 7 As shown, according to the diagram: (1) Effects on mechanical pain perception in mice: Figure 7 As shown in the figure, during the measurement period, the mechanical paw withdrawal threshold of mice in the normal group tended to be stable. The mechanical paw withdrawal threshold of mice in the PIPN and BIPN model groups decreased significantly; compared with the PIPN and BIPN model groups, the mechanical pain threshold of mice in the dimethyl fumarate group increased significantly. This indicates that the GSDME inhibitor dimethyl fumarate can reduce the mechanical pain sensitivity of mice caused by paclitaxel and bortezomib.

[0096] In summary, in the peripheral nerve injury model caused by paclitaxel and bortezomib, mice were sensitive to mechanical pain and cold pain, while the GSDME inhibitor dimethyl fumarate reduced the sensitivity of mechanical pain and cold pain in mice. Dimethyl fumarate had a protective effect on peripheral nerve injury caused by paclitaxel and bortezomib in mice. Two-way ANOVA was used, followed by Tukey's post hoc comparison. *P<0.05, **P<0.01 compared with the normal group in the PIPN and BIPN model groups; *P<0.05, **P<0.01 compared with the PIPN and BIPN model groups in the dimethyl fumarate group.

[0097] It should be understood that although the embodiments of the present invention use dimethyl fumarate as an example of a GSDME inhibitor to illustrate the use of GSDME inhibitors for preventing and treating peripheral nerve damage caused by chemotherapy drugs, it should be understood that the above embodiments are for illustrative purposes only. After the technical personnel in the industry read the documents of the present invention and understand the principles of the present invention, they can easily understand that other GSDME inhibitors can also be used for the above purposes. Therefore, other GSDME inhibitors should also be included in the scope of protection of the present invention.

[0098] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0099] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified and replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Use of a GSDME inhibitor for the manufacture of a drug for preventing or treating peripheral nerve damage during chemotherapy.

2. The use according to claim 1, characterized in that The peripheral nerve damage is damage to peripheral nerve cells caused by chemotherapy drugs.

3. The use according to claim 2, characterized in that: The chemotherapy drug is a cancer chemotherapy drug.

4. The use according to claim 3, characterized in that The cancer chemotherapy drug is selected from one or more of platinum chemotherapy drugs, taxane chemotherapy drugs, vincristine chemotherapy drugs, proteasome inhibitor chemotherapy drugs and their derivatives.

5. The use according to claim 4, characterized in that The platinum chemotherapy drug is one or more of cisplatin, carboplatin, cyclothioplatin, nedaplatin, oxaliplatin, and lobaplatin.

6. The use according to claim 4, characterized in that The taxane chemotherapy drug is one or more of paclitaxel and docetaxel.

7. The use according to claim 4, characterized in that The vincristine chemotherapy drug is one or more of vinblastine and vincristine.

8. The use according to claim 2, characterized in that: The peripheral nerve cells are ND7 / 23 cells.

9. The use according to claim 1, characterized in that The symptoms of peripheral nerve injury include one or more of the following: numbness and pain in the skin of the extremities, glove or stocking-like sensation, cold skin, paresthesia, a sense of ants, weakness in holding objects, a feeling of walking on cotton, burning pain, shooting pain, and knife-like pain.

10. The use according to claim 1, characterized in that The GSDME inhibitor is one or more of dimethyl fumarate (DMF), SP600125, Ac-DMPD-CMK, and Ac-DMLD-CMK.

11. A drug for preventing or treating peripheral nerve damage, characterized in that: The medicament comprises a GSDME inhibitor and a pharmaceutically acceptable carrier.

12. A method for preparing a drug for preventing or treating peripheral nerve damage during chemotherapy, characterized in that: The method comprises mixing a GSDME inhibitor and a pharmaceutically acceptable carrier in proportions.

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