CN119326894A - Use of GSDME inhibitors for manufacturing drugs for preventing and treating peripheral nerve damage during chemotherapy - Google Patents

Abstract

The present invention provides the use of a GSDME inhibitor for manufacturing a drug for preventing and treating peripheral nerve damage during chemotherapy. The present invention discloses the role of GSDME in peripheral nerve damage induced by chemotherapy, and proves that the GSDME inhibitor can be used as an adjuvant of chemotherapy drugs for cancer treatment, and has a mitigating effect on neurotoxicity caused by chemotherapy, thereby providing a treatment idea and theoretical basis for peripheral nerve damage induced by various chemotherapy drugs, and can also provide molecular targets and preparation ideas for screening and preparing drugs for treating peripheral nerve damage induced by chemotherapy drugs.

Description

Use of GSDME inhibitors for the manufacture of a medicament for the prevention and treatment of peripheral nerve damage during chemotherapy

Technical Field

The invention relates to the technical field of biological medicines, in particular to an application of GSDME inhibitor in preparing medicines for preventing and treating peripheral nerve injury during chemotherapy.

Background

Methods of comprehensive treatment of tumors are increasingly different, but for most tumors, particularly for patients with advanced tumors, chemotherapy is still one of the main methods of treating malignant tumors. The adverse reaction of the chemotherapeutic medicine is aggravated and even irreversible damage to tissues is caused after repeated chemotherapy for many times. CIPN (chemotherapy-induced peripheral neuropathy, CIPN) is a common dose-limiting adverse reaction caused by motor, sensory and autonomic nerve injury caused by anti-tumor drugs, wherein sensory dysfunction is most prominent, and typical symptoms include limb numbness, proprioception, obvious needle-punching sensation, hyperalgesia or allodynia, and the like [1]. The american society of clinical oncology (American society of clinical oncology, ASCO) in 2014 purposely promulgated guidelines for the control of chemotherapy-induced peripheral neuropathy CIPN [2,3], indicating that "CIPN is a very important adverse event that needs to be addressed, however no effective treatment is currently available against CIPN" [4]. CIPN-related antitumor drugs include platinum drugs (such as carboplatin, cisplatin and oxaliplatin), taxane drugs (such as paclitaxel and docetaxel), epothilone drugs (such as ib Sha Baitong), vincristine alkaloids (such as vincristine and vinblastine), bortezomib and thalidomide, etc., CIPN limits the increase of the dosage and efficacy of chemotherapeutic drugs to some extent, and is the main dose-limiting toxicity of chemotherapeutic drugs [5-8].

In recent years, how to prevent and alleviate the neurotoxicity induced by chemotherapy drugs has become a common problem in the field of cancer drug treatment. Based on the pathogenesis of CIPN, a number of compounds have been developed to prevent or treat CIPN by blocking ion channels, targeting inflammatory cytokines, and antioxidant stress. Glutathione and manganese fossilide are promising for preventing CIPN, duloxetine is expected to be effective for CIPN intervention, but the mechanism of preventing and intervening CIPN by these drugs has not been elucidated [9-12], whereas drugs such as calcium magnesium injection, venlafaxine, minocycline hydrochloride and gabapentin have limited efficacy in preventing and alleviating CIPN [13-15], and erythropoietin, menthol and amifostine are accompanied with various adverse reactions in use [16]. Because the mechanism of peripheral neurotoxicity caused by chemotherapeutic drugs is not clear, no target point exists in the drug treatment, and the expected curative effect of the drug with proved application prospect in large-scale clinical experiments is not obtained in animal experiments [17], no specific drug of CIPN is found so far, the clinical control is mainly performed, and the drug treatment is still controversial.

Cell apoptosis is a pro-inflammatory cell death mainly characterized by cell swelling, cell membrane cleavage, and the concomitant release of inflammatory factors such as HMGB1, IL-1 beta, IL-18, and LDH, and is also an important natural immune defense mechanism of a host against intracellular pathogen infection. When cells are stimulated by exogenous pathogenic related molecular patterns such as bacteria and viruses, receptor molecules in cytoplasm (pattern recognition receptors such as NLRP1, NLRP2, NLRP3, NLRC4, AIM2 and the like), linker proteins (apoptosis-related plaque spotting proteins (apoptosis associated speck-like proteincontaining a CARD, ASC)) and effector molecules (Caspase-1) form inflammatory bodies, activate Caspase-1/GSDME, mediate cell apoptosis, and release HMGB1, IL-1 beta and IL-18 inflammatory factors and LDH. There is a need in the art to address whether polypeptide inhibitors directed against GSDME can be utilized as potential drugs for the prevention and treatment of chemotherapeutic neurotoxicity. The difference of GSDME expression in tumor cells and normal cells is the key of adverse reaction of tissue injury caused by chemotherapeutics. The compound can also be used as a treatment target to improve side effects such as peripheral nerve injury, digestive tract injury and the like of chemotherapy without affecting the anti-tumor effect of the chemotherapy.

In summary, cell apoptosis plays an important role in peripheral nerve injury induced by tumor chemotherapy, and GSDME is a key molecule mediating chemotherapy drug induced cell apoptosis. Inhibitors against GSDME as potential drugs for tumor chemotherapy-induced peripheral nerve injury prevention and treatment are a major concern for those skilled in the art.

Disclosure of Invention

The invention aims to provide the application of GSDME inhibitor in preparing medicines for preventing and treating peripheral nerve injury, so as to solve the technical problem of preventing or treating peripheral nerve injury caused by chemotherapeutic medicines during chemotherapy.

In order to achieve the above purpose, the present invention provides the following technical solutions:

In a first aspect, the present invention discloses the use of GSDME inhibitors for the manufacture of a medicament for the prevention or treatment of peripheral nerve damage during chemotherapy.

Preferably, the peripheral nerve injury is injury to peripheral nerve cells caused by a chemotherapeutic agent.

Further, the chemotherapeutic agent is a cancer chemotherapeutic agent.

More preferably, the cancer chemotherapeutic is one or more selected from platinum chemotherapeutic, taxane chemotherapeutic, vincristine chemotherapeutic, bortezomib and derivatives thereof.

Further, the platinum chemotherapeutic medicine is one or more of cisplatin, carboplatin, cyclothioplatin, nedaplatin, oxaliplatin and lobaplatin.

In addition, the taxane chemotherapeutic drug is paclitaxel.

In addition, the vincristine chemotherapeutic medicine is one or more of vincristine and vincristine.

Preferably, the peripheral nerve cell is an ND7/23 cell.

Preferably, the symptoms of peripheral nerve injury include one or more of limb end skin tingling, pain, glove, sock-like feel, skin coldness, paresthesia, formicary, weakness in hand, feeling of stepping on cotton, burn-like pain, flash pain, and knife-like pain.

Preferably, the GSDME inhibitor is dimethyl fumarate (Dimethyl Fumarate, DMF).

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

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

Compared with the prior art, the invention has the beneficial effects that:

GSDME is used as an action target of chemotherapy-induced cell apoptosis, and the inventor determines the action of GSDME in chemotherapy-induced peripheral nerve injury, so that a treatment thought and a theoretical basis are provided for peripheral nerve injury induced by various chemotherapeutic drugs, and a molecular target and a preparation thought can be provided for screening and preparing drugs for treating the peripheral nerve injury induced by the chemotherapeutic drugs.

The invention screens the inhibitor dimethyl fumarate aiming at GSDME, further reveals the application of the inhibitor in preparing medicines for preventing and treating peripheral nerve injury caused by chemotherapeutic medicines, further discovers that the dimethyl fumarate has a protective effect on the neurotoxicity of ND7/23 cells induced by oxaliplatin, paclitaxel and bortezomib through experiments, and has a protective effect on the peripheral neurotoxicity of mice induced by oxaliplatin, paclitaxel and bortezomib on an animal model. The medicine dimethyl fumarate can be used as an adjuvant of a chemotherapeutic medicine for treating cancers, and has a relieving effect on neurotoxicity caused by chemotherapy.

Drawings

FIG. 1 shows the Western detection of mouse GSDME protein activation in oxaliplatin-induced peripheral nerve injury model;

FIG. 2 is a graph showing the protective effect of GSDME knockdown on oxaliplatin-induced peripheral nerve injury model mice;

FIG. 3 is a diagram of the structure of GSDME inhibitor dimethyl fumarate;

FIG. 4 is a graph showing the effect of dimethyl fumarate and oxaliplatin, paclitaxel, bortezomib on neuronal cell viability;

FIG. 5 is a graph showing the effect of GSDME inhibitor dimethyl fumarate on GSDME activation levels in mice DRG of oxaliplatin-induced peripheral nerve injury model;

FIG. 6 is a graph showing the effect of GSDME inhibitor dimethyl fumarate on oxaliplatin-induced peripheral nerve injury model mice;

FIG. 7 is a graph showing the effect of GSDME inhibitor dimethyl fumarate on mechanical pain threshold in paclitaxel, bortezomib-induced peripheral nerve injury model mice.

Note that the data are expressed as mean ± standard deviation (n=3). FIGS. 1-7 employ Two-way ANOVA followed by a Tukey post-hoc comparison. Treatment group compared to control group P <0.05, P <0.01.

Detailed Description

The invention provides an application of GSDME inhibitor in preventing peripheral nerve injury during chemotherapy, belonging to the technical field of biological medicine. The invention provides GSDME inhibitor which can reduce GSDME oligomerization and inhibit the formation of holes on plasma membranes, so that GSDME mediated cell apoptosis can be inhibited in vivo and used for preventing and treating peripheral nerve injury.

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the description of the present invention, it should be noted that the terms "upper," "lower," "inner," "outer," "front," "rear," "both ends," "one end," "the other end," and the like indicate or are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

Reference in the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or attribute is incorporated into the embodiment. Wherein these features and structures are included in at least one embodiment. Thus, the expression "in one embodiment" or "in an embodiment" does not mean necessarily equivalent to the same embodiment. Furthermore, the particular features, structures, and attributes may be combined and utilized in any manner as desired in one or more embodiments. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Finally, the term "or" is generally employed in its sense including "and/or" unless the context clearly dictates otherwise.

As used herein, the term "peripheral nerves" referred to in the article generally refers to those types of nerves that branch from the spinal cord and extend to various sites throughout the body. The phenomenon of nerve repair in the peripheral nervous system is more common than in the central nervous system.

As used herein, chemotherapy-induced peripheral nerve injury (Chemotherapy-Induced Peripheral Neuropathy, CIPN) is a type of nerve injury caused by chemotherapeutic agents, often manifested as symptoms of paresthesia, pain, numbness, tingling, and the like. Chemotherapy drugs cause impaired nerve conduction by affecting the normal function of nerve cells, thereby causing peripheral nerve damage. Common chemotherapeutic agents include paclitaxel (e.g., paclitaxel), platinum (e.g., oxaliplatin), enzyme inhibitors (e.g., bortezomib), and the like. Peripheral nerve damage can affect the quality of life of patients, and thus the occurrence and progression of CIPN needs to be closely monitored and managed during chemotherapy.

Terms such as "therapeutic", "treatment" or "palliative", "alleviating" in the present invention refer not only to medical methods for treating a disease (e.g., slowing or curing symptoms), halting (or reducing) the progression of a diagnosed condition, and preventing peripheral nerves from being damaged, but also to measures for preventing the peripheral nerve regeneration process from slowing. Thus, the patients to be treated include those suffering from peripheral nerve injury and those susceptible to peripheral nerve injury. Desirable medical effects may include, but are not limited to, alleviation of symptoms, diminishment of extent of nerve tissue damage, maintaining a stable (i.e., non-worsening) state of nerve damage, slowing or arresting the development of damage, restoration (whether partial or complete) of nerve tissue, and even regeneration. The treated persons include those already suffering from peripheral nerve injury or suspected of having the condition, as well as those who may be susceptible to such injury, who may for various reasons need to be protected from such conditions or from greater injury. In addition, the population in need of special treatment also includes animals with a deterioration of their ability to re-live with age.

In the present invention, the term "therapeutically effective amount" refers to an amount of a small organic molecule or other drug that is effective for "treating" a disease or disorder in a subject or mammal. In the case of peripheral nerve injury or trauma (e.g., sciatic nerve trauma), a therapeutically effective amount of the agent may promote axonal regeneration, epidermal innervation, and functional recovery of neurons following nerve injury in a subject with decline in nerve regeneration by increasing, e.g., proliferation, differentiation, migration, and/or survival of neural stem cells/precursor cells, reducing, delaying, or stopping the reduction of nerve cells, inhibiting, e.g., suppressing, delaying, preventing, stopping, or reversing the reduction of nerve cells, increasing the number, density, and/or concentration of nerve cells, alterations in nerve cell morphology or function, or alterations in interactions between nerve cells, alleviating to some extent one or more symptoms associated with nerve injury, e.g., pain, improving quality of life, or a combination of these effects.

The subject of the present invention includes individuals, animals and mammals. Such animals include, but are not limited to, humans, domestic pets, livestock, animals in wild zoos, athletic animals, and pets such as dogs, cats, hamsters, rabbits, rats, mice, horses, cows, and bears, among others.

The mode of administration of the compounds of the invention may be by oral administration, but as an alternative to oral administration, other routes of administration may be applied to the techniques and application scenarios of the present description, such as intravenous administration, in particular contexts. There are various routes of intravenous administration, such as subcutaneous, intravenous, intramuscular, articular, synovial, sternal, intrathecal, hepatic, focal and intracranial. For example, the compounds may also be administered by intravenous, intramuscular, intrathecal or subcutaneous injection. In certain applications, for the purpose of oral administration, the dosage of the compound may be reduced to a level suitable for intravenous administration, and further by injection.

Depending on the use and function described herein, there are other suitable scenarios in which the compound may be administered, such as sublingual, intra-cheek (such as by applying a film or other mixture under the tongue or inside the cheek that can dissolve in the mouth), eye (such as an eye drop), ear (such as by ear drops), oral inhalation (such as by blowing or nebulizing), skin or topical area (such as by applying an emulsion or lotion), or directly to the skin (such as by a patch). In addition to oral administration, delivery may be by other enteral routes such as vaginal and rectal administration, such as by ointments, suppositories, or enemas.

In practice, the procedures and purposes involved in this study are generally achieved by adding an effective compound (e.g., orally administered) to achieve the goal of alleviating symptoms. Effective dosages for specific use are in the range of 0.05mg/kg to 20mg/kg, including 0.05mg/kg to 10mg/kg,0.1mg/kg to 20mg/kg,0.1mg/kg to 10mg/kg,0.05mg/kg to 5mg/kg,0.1mg/kg to 5mg/kg,0.05mg/kg to 2mg/kg,0.1mg/kg to 2mg/kg, and any dosage ranges by terminating at any interval set at 0.05mg/kg and 20mg/kg or at other intermediate values. In some cases, this strategy may require the use of a compound in a dosage range of 0.1-1mg/kg, or the use of a compound in a dosage range of 0.2-0.5 mg/kg. Likewise, the method or use may involve the use of a compound in a dosage range of 0.05-20mg/kg in some cases, or in a dosage range of 1-10mg/kg in some cases.

In some embodiments, the methods and uses described herein, e.g., methods or uses for treating pain in a subject (e.g., human) in need thereof, are achieved by administering (e.g., orally) a therapeutically effective amount of a compound per day, e.g., 5 to 1000mg of a compound per day, e.g., 5 to 500mg or 5 to 250mg of a compound per day. For example, the method or use may comprise daily administration (e.g., orally) of about 5mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 105mg, about 110mg, about 115mg, about 120mg, about 125mg, about 130mg, about 135mg, about 140mg, about 145mg, about, 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, and about, 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, 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 the compound, or any range of amounts created by using two of the above amounts as endpoints, administered daily (e.g., orally). In some aspects, the method or use comprises orally administering 10 to 200mg of the compound to a subject (e.g., a human) per day, e.g., 10, 15, 20, 25, 30, 35, or 40mg to 75, 100, 125, 150, 175, or 200mg of the 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 125mg per day, e.g., 100mg per day. In some cases, the daily dosage of the compound may be administered by multiple administrations (e.g., orally), such as 5 times 20mg,4 times 25mg,3 times 33.3mg, or 2 times 50mg, etc. for each administration. For example, a daily dose of 100mg may be administered in different dosage regimens of 5 times 20mg, 4 times 25mg, 3 times 33.3mg, or 2 times 50mg per day. In certain methods, the daily dose of a compound may also be considered a single dose for use (e.g., orally). For example, a daily dose of one compound may be allocated to take 5mg, 10mg, 15mg, 20mg, 25mg or 30mg alone, or one of 5mg, 10mg, 15mg, 20mg, 25mg or 30mg, such as 5mg once a day or 10mg once a day, etc. Also, any of the dosages of the compounds discussed in the preceding section may be dispensed into single-dose unit or multi-dose unit dosage forms, such as two, three or four unit dosage forms.

In some embodiments, methods and uses of treatment by administration (e.g., oral) of a compound described herein, including treatment regimens in which the compound is administered concurrently with, or before, or after, the administration of the chemotherapy, e.g., 0.5h, 1h, 1.5h, 2h, or 0.5h, 1h, 1.5h, 2h after the administration of the chemotherapy, and also in the first 1 day, 2 days, 3 days, etc. of the administration of the chemotherapy.

The pharmaceutical compositions of the present invention encompass various forms (without limitation) injection solutions, tablets, capsules, gas releasing agents, patches, films, drops, external application, controlled release or slow release or nanoparticles. The pharmaceutical compositions comprise a primary compound or a pharmaceutically acceptable salt thereof which is safe and pharmaceutically acceptable within the effective dosage range thereof, and a pharmaceutically acceptable carrier. By "effective dose" it is meant that the amount of the drug is such that the condition is significantly ameliorated without causing serious side effects. Generally, the pharmaceutical compositions contain 1-2000mg of the primary compound per dose, and even more preferably, 10-1000mg of the primary compound per dose. Preferably, the "unit" is a capsule or tablet. By "acceptable carrier" is meant one or more compatible solid or liquid filling materials suitable for use in the human body, which must be of sufficient purity and of sufficiently low toxicity. "compatible" as used herein means that the individual components of the pharmaceutical composition are capable of being mixed with each other in the primary compound and between them without significantly reducing the efficacy of the compound. For example, some of the raw materials of acceptable carriers are cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween 80), and colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

Among the solid formulations for oral administration are various types of formulations, such as capsules, tablets, pills, powders and granules. The active ingredients of these formulations are mixed with some common inactive adjuvants, such as sodium citrate or dicalcium phosphate, or with other specific ingredients (one) fillers or thickeners, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (two) binders, such as, for example, hydroxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (three) humectants, such as glycerin, (four) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (five) slow-release agents, such as, for example, paraffin, (six) absorption accelerators, such as quaternary ammonium compounds, (seven) wetting agents, such as cetyl alcohol and glycerol monostearate, (eight) adsorbents, such as kaolin, (nine) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. Buffers may also be added to the three formulations, capsules, tablets and pills. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be obtained through wrapping and overcladding processes such as enteric coatings and other common materials. These formulations may also contain opaque fillers and in the formulations the active compound or its release pattern may be released in specific sites of the digestive tract. Buffers may also be added to capsules, tablets and pills. In oral liquid preparations, emulsions, solutions, suspensions, syrups or tinctures which are accepted in the pharmaceutical field are included. In addition, the liquid formulations may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like. In addition, the compositions may contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents, in addition to such inactive diluents. Suspending agents may also be included in the suspension, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances, and the like. In parenteral injection compositions, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions may be included. Suitable as water, ethanol, polyols and suitable mixtures thereof. The topical pharmaceutical formulations include ointments, powders, patches, sprays, inhalants and the like. Under sterile conditions, the active ingredient will be mixed with a physiologically acceptable carrier, any preservatives, buffers, or propellants which may be required. The treatment method can be used independently or in combination with other treatment methods or drugs.

Example 1 intravenous injection of AAV-U6-GSDME SHRNA adeno-associated virus into the tail of mice the level of GSDME expression in the dorsal root ganglion of the mice was knocked down.

1. Experimental materials:

AAV-U6-GSDME SHRNA adeno-associated virus was purchased from Nanjing next family Biotechnology Inc., oxaliplatin (CAS No. HY-17371) was purchased from MCE, and oxaliplatin was formulated in a 5% dextrose solution at a dosing rate of 3mg/kg.

The experimental animals are male C57BL/6 mice, have the weight of 20-24g, are clean, are purchased from Shanghai Laike experimental animal center, and are fed into SPF-grade experimental animal houses of Chinese medical institute of Jiangsu province. The experimental temperature is controlled at 25+/-1 ℃, and the animal feed is freely drunk and eaten. Experiments were started one week after feeding.

2. The experimental method comprises the following steps:

The experimental animals were randomly divided into two groups, a control group and AAV-GSDME SHRNA group.

The control group is that 3mg/kg oxaliplatin 5% glucose solution is injected into the abdominal cavity at the dosage of 5ml/kg, and the drug is stopped for 5 days after the drug administration, and the two circulation are carried out for 20 days;

AAV-GSDME SHRNA groups, i.e. 3mg/kg oxaliplatin 5% glucose solution is injected into abdominal cavity at 5ml/kg dose, the administration is stopped for 5 days, and two cycles are carried out for 20 days, and AAV-GSDME SHRNA virus is injected into tail vein two weeks before oxaliplatin administration.

Mice were sacrificed on day 21, the dorsal root ganglion of the mice collected, and recorded by weighing. Protein lysates were added and protein was collected for Western detection.

3. Experimental results:

The activation condition of GSDME is detected by Western analysis of the 2 part protein lysate by using an anti-mouse GSDME antibody and an anti-mouse GAPDH antibody, and Western results show that the expression and activation condition of GSDME are obviously reduced after the tail vein is injected with AAV-GSDME SHRNA virus. See FIG. 1 (lanes 1-3 are control and lanes 4-6 are AAV-GSDME SHRNA virus).

Example 2 protection against oxaliplatin-induced peripheral nerve injury in mice following GSDME knockdown.

1. Experimental materials:

experimental drugs oxaliplatin (CAS No.: HY-17371), AAV-U6-GSDME SHRNA adeno-associated virus was purchased from Nanjing next family Biotechnology Co., ltd. Oxaliplatin was formulated with a 5% dextrose solution at a dose of 3mg/kg.

Instruments electronic von Frey pain measuring instruments, PLANTAR TEST (HARGREAVES METHOD) GLASS STANDS, hot Cold PLATE ANALGESIA METER for MICE AND RATS are all available from IITC (IITC Inc. Life science, woodland Hills, calif., USA).

The experimental animals are male C57BL/6 mice, have the weight of 20-24g, are clean, are purchased from Shanghai Laike experimental animal center, and are fed into SPF-grade experimental animal houses of Chinese medical institute of Jiangsu province. The experimental temperature is controlled at 25+/-1 ℃, and the animal feed is freely drunk and eaten. Experiments were started one week after feeding.

2. The experimental method comprises the following steps:

(1) The experimental animals were randomly divided into two groups, a control group and AAV-GSDME SHRNA group.

The control group is that 3mg/kg oxaliplatin 5% glucose solution is injected into the abdominal cavity at the dosage of 5ml/kg, and the drug is stopped for 5 days after the drug administration, and the two circulation are carried out for 20 days;

AAV-GSDME SHRNA group, wherein 3mg/kg oxaliplatin 5% glucose solution is injected into abdominal cavity at 5ml/kg dose, and the administration is stopped for 5 days, and the two circulation is completed for 20 days;

(2) The first Day of drug treatment was designated Day1, and mice were tested for body weight and behavioural (mechanical mice pain test and cold mice pain test) on days 0, 6, 11, 16, and 21, respectively.

(A) Body weight mice were measured daily at the same time point.

(B) Mechanical pain test determination in mice mechanical hyperalgesia in mice was assessed at a mechanical foot-reduction threshold (von Frey test) by reference to method (K.I.Meng Zhao,SakiNakamura,TakayuK1 nakagawa,Shuji Kaneko,Acute cold hypersensitivitycharacteristically induced by oxaliplatin is caused by the enhancedresponsiveness of TRPA1 in mice,Molecular Pain.8(2012)55), of Meng Zhao et al. Mice were placed in (20 x 17 x 13 cm) plexiglas square boxes with a metal screen on the bottom mouse toe interface. When the mice were stationary after 15 minutes of adaptation to the measuring square, the heart of the mice were stimulated vertically with von Frey fiber filaments, and after the mice had developed a paw withdrawal response, electronic display stress values were recorded. Each mouse was assayed 10 times.

(C) Mice cold pain experiments were assayed by placing the mice in the measurement environment for 30 minutes prior to measurement, and the mice were placed on a cold plate at 5 ℃ with a clear plexiglas square box. The measurement time was 60s, and the mice were scored for escape behavior during the measurement time, 0min = no response, 1 min = slight cold escape response, such as lifting hindfoot or rewinding, 2 min = strong cold escape response, such as jumping, etc. The sum of scores over the measurement time period of 60s was recorded, and each mouse was measured 3 times, and the average was taken.

3. Experimental results:

the results are shown in fig. 2, and can be seen from the graph:

(1) The effect on the body weight of mice in the control group showed a remarkable body weight decrease phenomenon due to gastrointestinal adverse reaction induced by oxaliplatin stimulation, and the body weight of mice in the AAV-GSDME SHRNA group showed a slow increase trend during the period of oxaliplatin administration, and the body weight of mice in the AAV-GSDME SHRNA group showed a slow decrease trend during the period of oxaliplatin administration, and the body weight of mice in the control group showed a remarkable body weight increase after oxaliplatin administration, compared with the control group. AAV-GSDME SHRNA mice had a significant change in body weight compared to the control mice.

(2) Effect on mechanical pain in mice as shown in fig. 2 (B), the mechanical paw withdrawal threshold was significantly reduced in mice in the control group and significantly increased in AAV-GSDME SHRNA group compared to the control group during the measurement. It is shown that GSDME knockdown can reduce mechanical pain sensitivity of mice caused by oxaliplatin.

(3) Effect on cold pain in mice each group of mice was insensitive to cold pain prior to the experiment. As shown in fig. 2 (C), after the experiment was performed for 6 days, the cold pain score of the control mice was significantly increased, showing a significant cold pain sensitivity. The cold pain scores were lower for the AAV-GSDME SHRNA mice than for the control group, with significant differences, measured from 11 days to 21 days. It is shown that GSDME knockdown can reduce mice cold pain sensitivity caused by oxaliplatin.

In summary, in the peripheral nerve injury model caused by oxaliplatin, mice are sensitive to mechanical pain and cold pain, and the mechanical pain and cold pain of the mice are relieved after GSDME is knocked down, and the peripheral nerve injury of the mice caused by oxaliplatin is protected after GSDME is knockdown. A Two-way ANOVA was used followed by a Tukey post-hoc comparison. AAV-GSDME SHRNA groups compared to control groups P <0.05, < P <0.01.

EXAMPLE 3 dimethyl fumarate, a GSDME inhibitor, has the structural formula.

EXAMPLE 4 the GSDME inhibitor dimethyl fumarate has protective effects on oxaliplatin, paclitaxel and bortezomib-induced ND7/23 cell damage.

1. Experimental materials:

Oxaliplatin (CAS No.: HY-17371), dimethyl fumarate (CAS No.: HY-17363) were all purchased from MCE corporation, paclitaxel (CAS No.: HWG 53914) was purchased from Beijing Wallich chemical Co., ltd, and bortezomib (CAS No.: 179324-69-7) was purchased from a ceramic organism. The stock solutions were all 10mM (dissolved in DMSO) and directly diluted in culture medium for use.

ND7/23 cells are rat/mouse neuronal fusion cells purchased from Shanghai cell bank of China academy of sciences, and the cells are cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) and placed in a 37 ℃ and 5 ℃ constant temperature incubator for culture until the cells grow to 70% -80% confluence degree for experiments.

2. The experimental method comprises the following steps:

ND7/23 cells were seeded on 96-well plates with 100uL of cell suspension added to each well and incubated overnight in an incubator. ND7/23 cells were treated with different concentrations (1. Mu.M, 10. Mu.M) of dimethyl fumarate for 2 hours the next day followed by 2.5. Mu.M oxaliplatin, 0.1. Mu.M paclitaxel, 100nM bortezomib, respectively. After 24 hours of co-treatment of cells with dimethyl fumarate and chemotherapeutic drugs, 10. Mu.L of CCK-8 (Cat. No.: HY-K0301, available from China MCE) was added to each well, and the absorbance was measured using a full-wave multifunctional microplate reader (model: varioskan flash, available from U.S. Thermo Scientific) after incubation in an incubator for 2 hours.

3. Experimental results:

The results are shown in fig. 4, and it can be seen from the graph that the administration of oxaliplatin, paclitaxel and bortezomib alone resulted in a significant decrease in cell viability, and that the cell viability was increased when oxaliplatin, paclitaxel and bortezomib were co-treated with 10 μm dimethyl fumarate, respectively, which indicates that dimethyl fumarate has a protective effect on the decrease in cell viability caused by oxaliplatin, paclitaxel and bortezomib. One-way ANOVA was used followed by a Tukey post-hoc comparison. P <0.05, # P <0.01 compared to the control group and #p <0.05, # P <0.01 compared to the model group.

Example 4 the GSDME inhibitor dimethyl fumarate reduced GSDME activation levels in the dorsal root ganglion in mice.

1. Experimental materials:

Oxaliplatin (CAS No.: HY-17371), dimethyl fumarate (CAS No.: HY-17363) were all purchased from MCE company. Oxaliplatin was prepared with a 5% dextrose solution, and dimethyl fumarate was prepared with 50% PEG300 plus 50% physiological saline.

The experimental animals are male C57BL/6 mice, have the weight of 20-24g, are clean, are purchased from Shanghai Laike experimental animal center, and are fed into SPF-grade experimental animal houses of Chinese medical institute of Jiangsu province. The experimental temperature is controlled at 25+/-1 ℃, and the animal feed is freely drunk and eaten. Experiments were started one week after feeding.

2. The experimental method comprises the following steps:

The experimental animals were randomly divided into two groups, a normal group, a model group and a dimethyl fumarate group.

Normal group, i.e. intraperitoneal injection of solvent;

The model group comprises injecting 3mg/kg oxaliplatin 5% glucose solution into abdominal cavity at 5ml/kg dose, stopping administration for 5 days, and repeating the two steps for 20 days, and administering corn oil solvent at equal dose for continuous daily administration.

Dimethyl fumarate group A dimethyl fumarate solution was infused in advance at a dose of 50mg/kg for 2 hours, and 3mg/kg oxaliplatin 5% dextrose solution was injected intraperitoneally at a dose of 5 ml/kg. Dimethyl fumarate is required to be administered daily, oxaliplatin is administered for 5 days and 5 days, and two cycles are performed for 20 days.

Mice were sacrificed on day 21, the dorsal root ganglion of the mice collected, and recorded by weighing. Protein lysates were added and protein was collected for Western detection.

3. Experimental results:

Western detection of GSDME activation of part 5 protein lysates with anti-murine GSDME antibody, anti-murine GAPDH antibody revealed that GSDME was significantly activated in the dorsal root ganglion of mice in the oxaliplatin group compared to the control group, whereas the GSDME inhibitor dimethyl fumarate significantly reduced the level of GSDME activation in the dorsal root ganglion compared to the oxaliplatin group. It was demonstrated that the GSDME inhibitor dimethyl fumarate significantly reduced the level of activation of GSDME in the dorsal root ganglion of mice model of oxaliplatin-induced peripheral nerve injury. See FIG. 5 (lanes 1-3 for model group, lanes 4-6 for dimethyl fumarate group).

Example 5 the GSDME inhibitor dimethyl fumarate has a protective effect on oxaliplatin-induced peripheral nerve injury in mice.

1. Experimental materials:

Experimental drugs oxaliplatin (CAS No.: HY-17371), dimethyl fumarate (CAS No.: HY-17363) were all purchased from MCE company. Oxaliplatin was prepared with a 5% dextrose solution, and dimethyl fumarate was prepared with 50% PEG300 plus 50% physiological saline.

Instruments electronic von Frey pain measuring instruments, PLANTAR TEST (HARGREAVES METHOD) GLASS STANDS, hot Cold PLATE ANALGESIA METER for MICE AND RATS are all available from IITC (IITC Inc. Life science, woodland Hills, calif., USA).

The experimental animals are male C57BL/6 mice, have the weight of 20-24g, are clean, are purchased from Shanghai Laike experimental animal center, and are fed into SPF-grade experimental animal houses of Chinese medical institute of Jiangsu province. The experimental temperature is controlled at 25+/-1 ℃, and the animal feed is freely drunk and eaten. Experiments were started one week after feeding.

2. The experimental method comprises the following steps:

(1) The experimental animals were randomly divided into three groups, a normal group, a model group and a dimethyl fumarate group.

Normal group, i.e. intraperitoneal injection of solvent;

The model group comprises injecting 3mg/kg oxaliplatin 5% glucose solution into abdominal cavity at 5ml/kg dose, stopping administration for 5 days, and repeating the two steps for 20 days, and administering corn oil solvent at equal dose for continuous daily administration.

Dimethyl fumarate group A dimethyl fumarate solution was infused in advance at a dose of 50mg/kg for 2 hours, and 3mg/kg oxaliplatin 5% dextrose solution was injected intraperitoneally at a dose of 5 ml/kg. Dimethyl fumarate is required to be administered daily, oxaliplatin is administered for 5 days and 5 days, and two cycles are performed for 20 days.

(2) The first Day of drug treatment was designated Day1 and mice were tested for body weight and behavioural on days 0, 6, 11, 16, 21, respectively (mice were tested for mechanical pain experiments and mice were tested for cold pain experiments, see example 2 for specific experimental methods).

3. Experimental results:

the results are shown in fig. 6, and can be seen from the graph:

(1) The effect on the body weight of mice in the normal group showed a trend of increasing body weight with time as shown in FIG. 6 (A), the mice in the model group showed a remarkable body weight decrease due to the stimulation of oxaliplatin, and the mice showed a trend of slowly increasing body weight during the period of withdrawal of oxaliplatin, and the mice in the dimethyl fumarate group showed a trend of decreasing body weight during the period of administration of oxaliplatin, and the mice showed a remarkable increase in body weight after withdrawal of oxaliplatin, and the body weight was generally decreased as compared with the control group. The body weight of the mice in the dimethyl fumarate group was significantly changed from that of the mice in the control group.

(2) Effect on mechanical pain sensation in mice as shown in fig. 6 (B), the mechanical paw withdrawal threshold of normal mice tended to stabilize during the measurement. The mechanical foot contraction threshold of the mice in the model group is obviously reduced, and compared with the mice in the model group, the mechanical pain threshold of the mice in the dimethyl fumarate group is obviously increased. The GSDME inhibitor dimethyl fumarate was shown to reduce mechanical pain sensitivity in mice caused by oxaliplatin.

(3) Effect on cold pain in mice each group of mice was insensitive to cold pain prior to the experiment. As shown in fig. 6 (C), after the experiment was performed for 6 days, the model group mice showed a significant increase in cold pain score, indicating a significant cold pain sensitivity. Mice from the dimethyl fumarate group had lower cold pain scores than the model group, with significant differences, measured from 11 days to 21 days. It is shown that GSDME the inhibitor dimethyl fumarate reduces the cold pain sensitivity in mice caused by oxaliplatin.

In summary, in the model of peripheral nerve injury caused by oxaliplatin, mice are sensitive to mechanical pain and cold pain, while GSDME inhibitor dimethyl fumarate reduces the mechanical pain and cold pain sensitivity of mice, and dimethyl fumarate has a protective effect on the peripheral nerve injury of mice caused by oxaliplatin. A Two-way ANOVA was used followed by a Tukey post-hoc comparison. Comparison of model group with normal group P <0.05, < P <0.01, comparison of dimethyl fumarate group with model group P <0.05, < P <0.01.

EXAMPLE 6 the GSDME inhibitor dimethyl fumarate has protective effect on paclitaxel-induced peripheral nerve injury in mice.

1. Experimental materials:

Experimental drugs oxaliplatin (CAS No.: HY-17371) and dimethyl fumarate (CAS No.: HY-17363) were both purchased from MCE corporation, paclitaxel (CAS No.: HWG 53914) was purchased from North Beijing Wallich chemical Co., ltd, and bortezomib (CAS No.: 179324-69-7) was purchased from a ceramic organism. Paclitaxel is prepared from polyoxyethylated castor oil and absolute ethanol, bortezomib is prepared from physiological saline, and dimethyl fumarate is prepared from 50% PEG300 and 50% physiological saline. .

Instruments electronic von Frey pain measuring instruments PLANTAR TEST (HARGREAVES METHOD) GLASS STANDS were purchased from IITC (IITC Inc. Life science, woodland Hills, calif., USA).

The experimental animals are male C57BL/6 mice, have the weight of 20-24g, are clean, are purchased from Shanghai Laike experimental animal center, and are fed into SPF-grade experimental animal houses of Chinese medical institute of Jiangsu province. The experimental temperature is controlled at 25+/-1 ℃, and the animal feed is freely drunk and eaten. Experiments were started one week after feeding.

2. The experimental method comprises the following steps:

(1) The experimental animals were randomly divided into three groups, a normal group, a PIPN model group and a dimethyl fumarate-PIPN group, and a normal group, a BIPN model group and a dimethyl fumarate-BIPN group.

Normal group, i.e. intraperitoneal injection of solvent;

PIPN model group, paclitaxel solution 10mg/kg was injected intraperitoneally (1 injection, 4 injections total 4d injections 1, 3,5, 7 d). The corn oil solvent is infused in equal doses for continuous daily administration.

Dimethyl fumarate-PIPN, which was previously administered by gavage of dimethyl fumarate solution at a dose of 50mg/kg for 2 hours, dimethyl fumarate was administered daily, and 10mg/kg of paclitaxel solution was injected intraperitoneally (1 st, 3 rd, 5 th, 7d each, 4 th total injections of 4 d).

BIPN model group bortezomib 1 mg/kg was intraperitoneally injected (1 injection for each of 1 st, 3 rd, 5 th, and 7 d) for a total of 4d injections for a total of 4 times. The corn oil solvent is infused in equal doses for continuous daily administration.

Dimethyl fumarate-BIPN, in which a 50mg/kg dose of dimethyl fumarate solution was administered for 2 hours in advance, dimethyl fumarate was administered daily, and bortezomib 1 mg/kg was injected intraperitoneally (1 injection for 1,3, 5, and 7d, and 4 injections for a total of d injections).

(2) The first Day of drug treatment was designated Day1 and mechanical pain threshold test measurements were performed on Day 0 and Day 8, respectively (see example 2 for specific experimental methods).

3. Experimental results:

the results are shown in fig. 7, and can be seen from the graph:

(1) Effect on mechanical pain sensation in mice as shown in figure 7, the mechanical paw withdrawal threshold of normal mice tended to stabilize during the measurement. Compared with the PIPN and BIPN model groups, the mechanical foot contraction threshold of the mice in the PIPN and BIPN model groups is obviously reduced, and the mechanical pain threshold of the mice in the dimethyl fumarate group is obviously increased. It is shown that GSDME inhibitor dimethyl fumarate can reduce mechanical pain sensitivity of mice caused by paclitaxel and bortezomib.

In summary, in the peripheral nerve injury model caused by paclitaxel and bortezomib, mice are sensitive to mechanical pain and cold pain, and GSDME inhibitor dimethyl fumarate reduces mechanical pain and cold pain sensitivity of mice, and dimethyl fumarate has a protective effect on peripheral nerve injury of mice caused by paclitaxel and bortezomib. A Two-way ANOVA was used followed by a Tukey post-hoc comparison. Comparison of PIPN, BIPN model group with normal group with P <0.05, P <0.01, comparison of dimethyl fumarate group with PIPN, BIPN model group with P <0.05, P <0.01.

It should be appreciated that while the present embodiment describes the use of the GSDME inhibitor for the prevention and treatment of chemotherapy-induced peripheral nerve damage using dimethyl fumarate as an example of the GSDME inhibitor, it should be understood that the above examples are for illustrative purposes only and that other GSDME inhibitors may be readily used for the above purposes as would be understood by one of ordinary skill in the art upon reading the present disclosure, and that other GSDME inhibitors are intended to be within the scope of the present disclosure.

The present invention is not described in detail in the present application, and is well known to those skilled in the art.

It is finally intended that the foregoing detailed description be regarded as illustrative rather than limiting, and that it be understood that it is understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (12)

1. Use of GSDME inhibitors for the manufacture of a medicament for the prevention or treatment of peripheral nerve damage during chemotherapy.

2. The use according to claim 1, wherein the peripheral nerve injury is injury to peripheral nerve cells induced by a chemotherapeutic agent.

3. The use according to claim 2, wherein the chemotherapeutic agent is a cancer chemotherapeutic agent.

4. The use according to claim 3, wherein the cancer chemotherapeutic is one or more selected from the group consisting of platinum chemotherapeutics, taxane chemotherapeutics, vincristine chemotherapeutics, proteasome inhibitor chemotherapeutics and derivatives thereof.

5. The use according to claim 4, wherein the platinum-based chemotherapeutic agent is one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin.

6. The use according to claim 4, wherein the taxane chemotherapeutic agent is one or more of paclitaxel and docetaxel.

7. The use according to claim 4, wherein the vincristine-based chemotherapeutic agent is one or more of vinblastine and vincristine.

8. The use according to claim 2, wherein the peripheral nerve cells are ND7/23 cells.

9. The use according to claim 1, wherein the symptoms of peripheral nerve injury include one or more of limb end skin tingling, pain, glove, sock-like feel, skin coldness, paresthesia, formicary, weakness in hand, cotton feeling on walking, burn-like pain, flash pain, and knife-like pain.

10. The use according to claim 1, wherein the GSDME inhibitor is one or more of dimethyl fumarate (Dimethyl Fumarate, DMF), SP600125, ac-DMPD-CMK, ac-DMLD-CMK.

11. A medicament for preventing or treating peripheral nerve injury, comprising a GSDME inhibitor and a pharmaceutically acceptable carrier.

12. A method of preparing a medicament for preventing or treating peripheral nerve damage during chemotherapy, the method comprising mixing GSDME inhibitor in proportion with a pharmaceutically acceptable carrier.