Use of piperidine derivative in preparation of drug for preventing and treating sepsis and sepsis-induced organ damage

Abstract

A use of a piperidine derivative or a pharmaceutically acceptable salt thereof in preparation of a drug for preventing and treating sepsis and sepsis-induced organ damage. Pharmacodynamic experimental research shows that the piperidine derivative significantly reduces the level of inflammatory factors of the serum of sepsis models, effectively prevents and treats sepsis and sepsis-induced organ damage, protects the liver, kidneys and heart, and improves cognitive function, reduces brain index and relieves brain tissue damage in patients with sepsis-related encephalopathy, and thus has relatively high clinical application prospects.

Description

Use of a piperidine derivative in preparing a drug for preventing and treating sepsis and septic organ damage Technical Field

[0001] The present invention belongs to the field of medical technology and relates to the medical use of a piperidine derivative, and specifically relates to the use of N-(3-chlorophenyl)-1-(1-methyl-1H-indole-2-carbonyl)piperidine-4-carboxamide in the preparation of a medicament for preventing and treating sepsis and septic organ damage. Background Art

[0002] Sepsis is a potentially fatal systemic disease caused by pathogens and / or a systemic inflammatory response. It often accompanies trauma, burns, and surgical procedures. It is a serious condition with high morbidity and mortality. Globally, over 18 million patients develop severe sepsis annually, and this number is increasing at an annual rate of 1.5% to 8.0%. According to international epidemiological surveys, the mortality rate from sepsis has surpassed that of myocardial infarction, becoming the leading cause of death among non-cardiac patients in intensive care units.

[0003] Sepsis, caused by a persistent and uncontrolled inflammatory response, leads to the release of large amounts of inflammatory and anti-inflammatory mediators into the bloodstream, resulting in multiple organ damage, lymphocyte apoptosis, and immune paralysis. Sepsis-induced organ damage is the primary cause of poor prognosis in sepsis. Reducing the extent of sepsis-induced organ damage is also crucial for its treatment. Sepsis complications are essentially clinical manifestations of various stages of the pathophysiology of sepsis. Common complications include shock, acute lung injury, and acute respiratory distress syndrome. Sepsis is a systemic inflammatory response with profound effects on various organ systems. In addition to the liver and kidneys, the central nervous system is also commonly affected, often in the early stages of the disease, before other organs are affected. In the absence of central nervous system infection or other potential causes of brain dysfunction, diffuse brain dysfunction caused by infection is termed sepsis-associated encephalopathy (SAE). Studies have shown that up to 70% of patients with sepsis develop SAE. Clinical manifestations include varying degrees of brain dysfunction, ranging from mild delirium to coma and even death. SAE manifests in the acute phase with changes in consciousness, while the chronic phase presents with cognitive impairment and even dementia.

[0004] Currently, there is a lack of specific medications for the clinical treatment of sepsis. Treatment options for sepsis primarily focus on symptomatic and supportive care, including antibiotic therapy, surgical or interventional elimination of the source of infection, intravenous fluids to maintain adequate blood volume, administration of inotropic and / or inotropic drugs (such as vasopressin and norepinephrine), and supportive care such as mechanical ventilation, hemodialysis, and extracorporeal oxygenation. Antimicrobial agents are key to the treatment of sepsis, but their overuse and increasing severity of nosocomial infections have led to the emergence of multiple drug-resistant strains, significantly reducing their effectiveness. Despite significant advances in anti-infective therapy and organ function support, the mortality rate of sepsis remains as high as 30%-70%. Recommended treatment for sepsis-associated encephalopathy focuses on systemic infection management, but few therapies are effective in alleviating cognitive impairment. Most established inhibitors of peripheral inflammation are not developed for the brain and lack the ability to penetrate the blood-brain barrier. Clinical treatment of sepsis and related diseases mainly relies on infection control or non-specific treatments such as glucocorticoids, as well as symptomatic treatments such as preventing and treating organ failure and shock. Although these treatments can alleviate sepsis symptoms and prolong life in some patients, the results are limited.

[0005] Summary of the Invention

[0006] To identify new and effective drugs for preventing and treating sepsis and septic organ damage, the inventors creatively used N-(3-chlorophenyl)-1-(1-methyl-1H-indole-2-carbonyl)piperidine-4-carboxamide (Compound I) for the treatment of sepsis. They unexpectedly discovered that the compound exhibited significant therapeutic effects, including significant effects on sepsis-induced organ damage. Therefore, the present invention provides the use of Compound I or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating sepsis and sepsis-induced organ damage.

[0007] The piperidine derivative of the present invention is N-(3-chlorophenyl)-1-(1-methyl-1H-indole-2-carbonyl)piperidine-4-carboxamide, and its specific structural formula is:

[0008] The compounds I mentioned in the present invention are all N-(3-chlorophenyl)-1-(1-methyl-1H-indole-2-carbonyl)piperidine-4-carboxamide.

[0009] In the use of the present invention, sepsis includes early sepsis, severe sepsis and septic shock.

[0010] In the use of the present invention, the sepsis is the sepsis caused by the stress of severe burns, combat injuries, infection, major surgery, severe shock, radiotherapy, chemotherapy, organ transplantation, or severe hepatitis, cirrhosis, gastrointestinal diseases, malnutrition, biliary or intestinal obstruction.

[0011] In the use of the present invention, the sepsis is caused by microbial infection.

[0012] Furthermore, the microorganisms include but are not limited to Gram-negative bacteria or Gram-positive bacteria.

[0013] In the use of the present invention, the sepsis-induced organ damage is sepsis-induced liver damage and / or sepsis-induced kidney damage.

[0014] In the use of the present invention, the sepsis-induced organ damage may also be sepsis-related encephalopathy.

[0015] In the use of the present invention, the drug is a drug for improving serum biochemical indicators.

[0016] Furthermore, the drug is a drug that reduces serum AST, ALT, UREA, and CREA-S levels.

[0017] In the use of the present invention, the medicine is a preparation prepared by using compound I or its salt as the active ingredient and adding conventional pharmaceutically acceptable excipients.

[0018] Furthermore, the pharmaceutically acceptable excipient may be an excipient, a filler, a diluent, a binder, a disintegrant, a solubilizer, an adsorbent, a pH buffer, a preservative, a flavoring agent, or a combination thereof.

[0019] Furthermore, the preparation is an oral preparation, an injection or an external preparation.

[0020] Furthermore, the oral preparations include tablets, pills, powders, granules, capsules, and syrups; the injections include intravenous injections, intraperitoneal injections, intracavitary injections, lavages, or irrigations; and the external preparations include patches, ointments, or gels.

[0021] In order to prepare the composition of the present invention into tablets, various excipients known in the relevant field can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropyl alcohol, etc.; binders can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0022] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0023] The present invention also provides a method for treating sepsis and its complications, comprising providing an effective amount of Compound I of the present invention to a patient in need thereof.

[0024] In the use of the present invention, the content of compound I in each unit of the pharmaceutical preparation is 0.1-99wt%

[0025] In some examples, the content of Compound I in each unit of the pharmaceutical preparation is 0.1-80 wt %.

[0026] In some examples, the content of Compound I in each unit of the pharmaceutical preparation is 0.1-60 wt %.

[0027] In some examples, the content of Compound I in each unit of the pharmaceutical preparation is 0.1-40 wt %.

[0028] In some examples, the content of Compound I per unit of the pharmaceutical preparation is 0.1-20 wt %.

[0029] In some embodiments of the present invention, Compound I can reduce the levels of serum inflammatory factors TNF-α and IL-1β in patients with sepsis.

[0030] In some embodiments of the present invention, Compound I can reduce the serum AST, ALT, UREA, and CREA-S levels in patients with sepsis.

[0031] In some embodiments of the present invention, Compound I can prevent and treat organ damage caused by sepsis, such as liver damage caused by sepsis and / or kidney damage caused by sepsis.

[0032] In some embodiments of the present invention, Compound I can improve the survival rate of sepsis patients.

[0033] In some embodiments of the present invention, Compound I can delay or halt the progression of sepsis

[0034] In some embodiments of the present invention, Compound I can reduce the severity of sepsis and / or improve the prognosis of sepsis.

[0035] In some embodiments of the present invention, Compound I can reduce brain index in patients with sepsis-related encephalopathy.

[0036] In some embodiments of the present invention, Compound I can improve cognitive dysfunction in patients with sepsis-related encephalopathy.

[0037] In some embodiments of the present invention, Compound I can reduce brain tissue damage in patients with sepsis-related encephalopathy.

[0038] As used herein, the term "pharmaceutically acceptable" means that the compound, composition or carrier is suitable for administration to a subject for the purposes or methods described herein without undue adverse side effects.

[0039] As used herein, the term "treat," "treat," "treat," and other similar synonyms include alleviating, relieving, or ameliorating the symptoms of a disease or condition, inhibiting a disease or condition, such as preventing the development of the disease or condition, relieving the disease or condition, improving the disease or condition, alleviating the symptoms caused by the disease or condition, or stopping the symptoms of the disease or condition, preventing other symptoms, improving or preventing the underlying metabolic causes of the symptoms, and further, the term encompasses the purpose of prevention. The term also encompasses obtaining a therapeutic effect and / or a prophylactic effect. The therapeutic effect refers to curing or improving the underlying disease being treated. In addition, curing or improving one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, for example, although the patient may still be affected by the underlying disease, an improvement in the patient's condition is observed. With respect to a prophylactic effect, the composition can be administered to a patient at risk for a particular disease, or even if a diagnosis of the disease has not yet been made, the composition can be administered to a patient who exhibits one or more physiological symptoms of the disease.

[0040] As used herein, the terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" refer to an amount of at least one active substance (such as a compound of the present application) sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide a clinically significant symptom alleviation effect. Techniques such as dose escalation trials can be used to determine the effective amount appropriate for any individual case.

[0041] As used herein, the terms "subject" and "patient" include humans and mammals in this application. Mammals refer to any animal species of the class mammals. Examples of mammals include humans; non-human primates such as monkeys; laboratory animals such as rats, mice, and guinea pigs; domestic animals such as cats, dogs, rabbits, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions and tigers.

[0042] Terms not defined herein have their ordinary meanings in the art.

[0043] The beneficial effects of the present invention are:

[0044] This study induced a sepsis model in mice by intraperitoneal injection of lipopolysaccharide (LPS). Compound I was then administered intraperitoneally. The therapeutic efficacy of Compound I on sepsis and septic organ damage was evaluated based on changes in serum inflammatory factors, serum biochemical profiles, and organ tissue pathology. Animal experiments demonstrated that Compound I significantly reduced serum levels of the major inflammatory factors TNF-α and IL-1β in septic mice, as well as serum levels of AST, ALT, UREA, and CREA-S, effectively protecting organs such as the liver and kidneys.

[0045] Animal experiments observed the effects of the compounds of the present invention on individual survival rates, and the results showed that the compounds of the present invention significantly increased the survival rates of the individuals, delayed or halted the progression of sepsis, reduced the severity of sepsis, and / or improved the prognosis of sepsis.

[0046] This study investigated the pharmacological effects of Compound I on sepsis-related encephalopathy (SERE). A SERE model was established in mice by intraperitoneal injection of lipopolysaccharide (LPS). The behavioral changes and brain pathological changes following intraperitoneal injection of Compound I were observed. Animal studies demonstrated that Compound I improved cognitive impairment, reduced brain function index, and alleviated brain damage in SERE-induced mice.

[0047] The piperidine derivatives of the present invention have good application prospects in preventing and treating sepsis and organ damage caused by sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is the HE staining images of the kidneys of the normal group, model group and compound I group after autopsy at 100 times magnification under a microscope.

[0049] FIG2 is a graph showing the effects of Compound I, positive drugs and control group on the survival rate of mice with LPS-induced sepsis within 48 hours.

[0050] FIG3 shows the brain index of mice in the normal group, model group, and compound I-administered groups.

[0051] Figure 4 is the Nissl staining images of the hippocampus of the brain tissue of rats in the normal group, model group and compound I treatment groups after 100 times magnification under a microscope.

[0052] FIG5 shows the results of the eight-arm maze test of mice in the normal group, model group, and compound I-administered groups.

[0053] FIG6 shows the results of the novel object recognition experiment of mice in the normal group, model group, and compound I-administered groups.

[0054] Note: Compared with the normal group, #p<0.05, ##p<0.01; compared with the model group, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0055] After extensive and in-depth research, the inventors of the present invention have provided the use of compound I in the preparation of a drug for preventing and treating sepsis and sepsis-induced organ damage.

[0056] The present invention will be further described in detail below through specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following examples. The following examples are intended to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Modifications or equivalent substitutions of the technical solutions of the present invention by persons of ordinary skill in the art without departing from the essence and scope of the technical solutions of the present invention are still within the scope of protection of the present invention.

[0057] Pharmacological Effect Example 1 Pharmacological Effect of Compound I on LPS-Induced Sepsis in Mice

[0058] 1. Drugs and reagents

[0059] Compound I was prepared by the method disclosed in CN111518020A and provided by Shandong Xinshidai Pharmaceutical Co., Ltd.

[0060] The specific structure of the control compound 1 is shown in the following formula: Prepared by the method disclosed in CN111518020A and provided by Shandong New Era Pharmaceutical Co., Ltd.

[0061] The specific structure of the control compound 2 is shown in the following formula: It was prepared by the method disclosed in CN111518020A and provided by Shandong New Era Pharmaceutical Co., Ltd.

[0062] Endotoxin (LPS), produced by Sigma;

[0063] TNF-α, IL-6, and IL-1β kits were produced by Shanghai ELISA Biotechnology Co., Ltd.;

[0064] ALT, AST, UREA, and CREA-S test kits were produced by Shenzhen Mindrary Company;

[0065] Hematoxylin and eosin staining kit was produced by Biyuntian Biotechnology Co., Ltd.

[0066] 2. Experimental Animals

[0067] One hundred and sixty C57BL / 6J male mice (6-8 weeks old, 18-22 g) were purchased from Beijing Life River Laboratory Animal Technology Co., Ltd. All animals were housed in an animal room at room temperature (22 ± 2°C), 60 ± 5% relative humidity, and a 12-h light:12-h dark cycle with free access to water and food.

[0068] 3. Experimental Methods

[0069] After one week of acclimation, mice were randomly divided into eight groups: normal group, model group, positive drug group, control group 1, control group 2, and low-, medium-, and high-dose Compound I groups. Each group consisted of 20 mice. Ten mice were autopsied 24 hours later, and the remaining ten mice were observed for survival for 48 hours. At the start of the experiment, normal and model groups were intraperitoneally injected with normal saline according to body weight. The positive drug group was intraperitoneally injected with 1 mg / kg dexamethasone. The low-, medium-, and high-dose Compound I groups were intraperitoneally injected with 1 mg / kg, 2.5 mg / kg, and 5 mg / kg of Compound I according to body weight. The control group was intraperitoneally injected with 5 mg / kg of control Compound 1 according to body weight, and the control group was intraperitoneally injected with 5 mg / kg of control Compound 2 according to body weight. One hour later, normal saline was injected intraperitoneally according to body weight in the normal group, and 20 mg / kg of LPS was injected in the model group and each drug-treated group. Autopsies were performed 24 hours later.

[0070] 4. Detection indicators

[0071] 4.1 Serum inflammatory factors

[0072] After autopsy, blood was collected from the paracardial vein of the mouse. After standing at room temperature for 1 hour, the blood was centrifuged at 3000 rpm to obtain serum, which was divided into two aliquots. One aliquot was assayed according to the instructions for the TNF-α and IL-1β ELISA kits. Each well was analyzed on a microplate reader at 450 nm. The OD value of each sample was calculated by subtracting the value of the blank control well from the value obtained. A standard curve was constructed based on the concentration of the standard and the OD values ​​to calculate the sample concentration.

[0073] 4.2 Serum biochemical indicators

[0074] The serum was subjected to a series of operations according to the requirements of the kit instructions to detect the concentrations of ALT, AST, UREA, and CREA-S.

[0075] 4.3 Kidney Pathology Section

[0076] Mouse kidney tissue sections were stained with hematoxylin-eosin (HE). First, the baked sections were dewaxed with xylene until transparent. Rinse with ethanol to remove the xylene, then rinse with distilled water. After drying, stain with hematoxylin solution for 5-10 minutes, then soak in hydrochloric acid alcohol for 10 seconds. After washing the sections, stain with eosin for 1-2 minutes, wash with gradient ethanol, and finally soak in xylene twice for 5 minutes each time to make it transparent. After drying, seal the sections with neutral resin and observe under an optical microscope.

[0077] 4. After 448 hours, the survival rate of the remaining 10 mice in each group was observed.

[0078] 5. Statistical processing

[0079] SPSS 22.0 software was used for statistical analysis of the data. Measurement data were expressed as (x ± s). One-way analysis of variance was used for comparisons between groups, and t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0080] 6. Experimental Results and Analysis

[0081] 6.1 Effects of Compound I on Serum Inflammatory Factors in LPS-Induced Sepsis Mice

[0082] Table 1 Serum inflammatory factor levels of mice in each group ( n=6)

[0083] Note: Compared with the normal group, #p<0.05, ##p<0.01; compared with the model group, *p<0.05, **p<0.01.

[0084] As shown in Table 1, compared with the normal group, the serum TNF-α level of mice in the model group was significantly increased (p < 0.01). Compared with the model group, the serum TNF-α level of mice in the positive drug group was significantly decreased (p < 0.01). The serum TNF-α level of mice in the low-, medium-, and high-dose groups of Compound I all decreased significantly (p < 0.01), and this was dose-dependent. Compared with the normal group, the serum IL-6 level of mice in the model group was significantly increased (p < 0.01). Compared with the model group, the serum IL-6 level of mice in the positive drug group was significantly decreased (p < 0.01). The serum IL-6 level of mice in all Compound I-treated groups decreased significantly (p < 0.05, p < 0.01), with the medium- and high-dose groups of Compound I showing the most significant effect (p < 0.01). No significant improvement in serum cytokine levels was observed in the control groups 1 and 2 compared with the model group. These experimental results indicate that Compound I can reduce the levels of inflammatory factors in mice with LPS-induced sepsis, thereby improving inflammatory symptoms.

[0085] 6.2 Effects of Compound I on Serum Biochemical Parameters in LPS-Induced Sepsis Mice

[0086] Table 2 Serum biochemical parameters of mice in each group ( n=7)

[0087] Note: Compared with the normal group, #p<0.05, ##p<0.01; compared with the model group, *p<0.05, **p<0.01.

[0088] As shown in Table 2, compared with the normal group, serum ALT, AST, UREA, and CREA-S levels in the model group were significantly increased (p < 0.01). Compared with the model group, serum ALT, AST, UREA, and CREA-S levels in the positive drug group were significantly decreased (p < 0.01). Compared with the model group, serum ALT levels in the low, medium, and high-dose Compound I groups were significantly decreased (p < 0.05, p < 0.01), with the high-dose Compound I group showing a particularly significant effect (p < 0.01). Compared with the model group, serum AST levels in the low, medium, and high-dose Compound I groups were significantly decreased (p < 0.05, p < 0.01), with the medium and high-dose Compound I groups showing a particularly significant effect (p < 0.01). Compared with the model group, serum UREA levels in the low, medium, and high-dose Compound I groups were significantly decreased (p < 0.01), with the medium and high-dose Compound I groups showing a particularly significant effect (p < 0.01). Compared with the model group, the serum levels of CREA-S in mice treated with low, medium, and high doses of Compound I were significantly reduced (p<0.01). There was no significant improvement in serum ALT, AST, UREA, and CREA-S levels in control groups 1 and 2 compared with the model group. ALT and AST are clinically used to identify liver damage, while elevated UREA and CREA-S are a sign of kidney disease. Therefore, these experimental results demonstrate that Compound I of the present invention can protect the liver and kidneys of mice with LPS-induced sepsis. It demonstrates a robust protective effect against liver and kidney damage caused by sepsis.

[0089] 6.3 HE staining and scoring of mouse kidney tissue

[0090] As can be seen from Figure 1, 24 hours after intraperitoneal injection of LPS, the regular shape of the renal tubules in the model group mice changed significantly, specifically manifested as abnormal tubular dilation, atrophy of epithelial cells, and lighter cytoplasm color. The condition showed significant improvement after the administration of compound I.

[0091] 6.4 After 48 hours, the survival rate of the remaining 10 mice in each group was observed, as shown in Figure 2.

[0092] Animal experimental results demonstrate that Compound I significantly reduces serum levels of the major inflammatory factors TNF-α and IL-1β in mice with sepsis, lowering serum levels of AST, ALT, UREA, and CREA-S, effectively protecting organs such as the liver and kidneys. The compound significantly improves survival rates, slows or halts the progression of sepsis, reduces the severity of sepsis, and / or improves the prognosis of sepsis.

[0093] Effect of Compound I on the Brain Tissue of Mice with LPS-Induced Sepsis-Related Encephalopathy

[0094] 1. Drugs and reagents

[0095] Compound I was prepared by the method disclosed in CN111518020A and provided by Shandong Xinshidai Pharmaceutical Co., Ltd.

[0096] Endotoxin (LPS), produced by Sigma;

[0097] Nissl staining solution, produced by Biyuntian Company.

[0098] 2. Experimental Animals

[0099] Sixty male C57BL / 6J mice weighing 18–22 g were obtained from Beijing Huafukang Biotechnology Co., Ltd. (license number: SYXK(Lu)20230023). Mice were housed in an SPF-grade animal housing facility under strictly controlled environmental conditions: a temperature of 20–26°C, a humidity of 40–70%, a 12:12 h light–dark cycle, and a growth and breeding diet with free access to food.

[0100] 3. Experimental Methods

[0101] After one week of adaptive feeding, 60 C57BL / 6J male mice were randomly divided into five groups, namely, normal group, model group, low-dose compound I group, medium-dose compound I group, and high-dose compound I group, with 12 mice in each group.

[0102] At the start of the experiment, normal and model groups were intraperitoneally injected with saline according to body weight. The low-, medium-, and high-dose groups of Compound I were intraperitoneally injected with 1 mg / kg, 2.5 mg / kg, and 5 mg / kg of Compound I according to body weight. One hour later, the normal group was intraperitoneally injected with the corresponding volume of saline according to mouse body weight. The model group and each treatment group were injected with 25 mg / kg LPS, with a dosing volume of 10 ml / kg. 24 hours later, the mice were anesthetized with sodium pentobarbital, the brains were removed from the skull, rinsed with saline, blotted dry with filter paper, and weighed. The brain index was calculated as follows:

[0103] Brain index = brain mass / body mass*100%.

[0104] Prepared sections were dried and dewaxed, then stained in Nissl stain for 90 minutes. The stain was rinsed with running water and then separated with 0.1% glacial acetic acid until a clear background was visible under the microscope. After rinsing with running water, the sections were placed in a stainer to counterstain nuclei with hematoxylin. Dehydrated three times with anhydrous alcohol, the sections were cleared twice with xylene, each for 5 minutes. After completion, the sections were left in a well-ventilated place overnight, mounted with neutral gum, and observed under a microscope for changes in the structure and number of neurons in different groups.

[0105] 4. Experimental Results and Analysis

[0106] 4.1 Nissl staining results

[0107] Nissl bodies, as one of the specific structures in the cytoplasm of neurons, are an important site for protein synthesis. They have a certain basophilicity and can therefore be stained blue-purple by alkaline stains. Nissl staining is often used to show changes in the structure and number of neurons in nervous system diseases such as the brain and spinal cord.

[0108] As shown in the Nissl staining results in Figure 3, compared with the normal group, the model group showed damage in the hippocampus region of the brain tissue, with pyknosis of the nerve cell nuclei, shrinkage and deformation of the cell body, and disappearance of the cytoplasmic Nissl bodies, indicating that cell metabolism in the hippocampus region of the brain tissue was impaired; compared with the model group, the pyknosis of the nerve cell nuclei in the hippocampus region of the brain tissue of mice in the low, medium and high dose groups of compound I was reduced, the cell body morphology was restored, and the Nissl bodies were normal. The above results indicate that compound I of the present invention can significantly reduce the brain tissue damage of mice with LPS-induced sepsis-related encephalopathy.

[0109] 4.2 Brain Index

[0110] As shown in Figure 4, compared with the normal group, the brain index of the model group was significantly increased; compared with the model group, the brain index of mice in the low, medium and high groups of compound I were significantly decreased (p<0.01).

[0111] Efficacy Example 3 Behavioral Effects of Compound I on LPS-Induced Sepsis-Related Encephalopathy Mice (Eight-Arm Maze Test)

[0112] 1. Drugs and reagents

[0113] Compound I was prepared by the method disclosed in CN111518020A and provided by Shandong Xinshidai Pharmaceutical Co., Ltd.

[0114] Endotoxin (LPS) was produced by Sigma.

[0115] 2. Experimental Animals

[0116] Sixty male C57BL / 6J mice weighing 18–22 g were obtained from Beijing Huafukang Biotechnology Co., Ltd. (license number: SYXK(Lu)20230023). Mice were housed in an SPF-grade animal housing facility under strictly controlled environmental conditions: a temperature of 20–26°C, a humidity of 40–70%, a 12:12 h light–dark cycle, and a growth and breeding diet with free access to food.

[0117] 3. Experimental Methods

[0118] After one week of adaptive feeding, 60 C57BL / 6J male mice were randomly divided into five groups, namely, normal group, model group, low-dose compound I group, medium-dose compound I group, and high-dose compound I group, with 12 mice in each group.

[0119] After the experiment started, the normal group and the model group were intraperitoneally injected with normal saline according to body weight, and the low-, medium-, and high-dose groups of compound I were intraperitoneally injected with 1 mg / kg, 2.5 mg / kg, and 5 mg / kg of compound I according to body weight. One hour later, the normal group was intraperitoneally injected with the corresponding volume of normal saline according to the body weight of the mice, and the model group and each drug-treated group were injected with 10 mg / kg LPS, with a dosage volume of 10 ml / kg.

[0120] Behavioral testing was conducted in an eight-arm maze with automatic doors. Before the start of the experiment, all mice had to undergo an adaptation test. The diet of the mice was controlled every day to promote the rapid discovery and memory of food pellets placed in the first, second, fourth and seventh arms during the test. On the first day, an equal amount of food pellets was placed in each arm. After the mice were placed in, they were allowed to explore freely for 300 seconds before the adaptation ended. Starting from the second day, the automatic door was first closed, and the mice were placed in the center of the maze for 15 seconds. Then the automatic door was opened and the mice were released for testing. The training ended when the time reached 300 seconds or the mice found the pellets. The training lasted for a total of five days. The mice repeatedly entered the arms where the pellets had been found and eaten. The mice entered the arms without food pellets. It was a reference memory error. The total number of times the mice entered the arms was the total number of arm entries. The time required for the mice to find all the food pellets was the test time. When the seventh day of the experiment, the formal test began and the steps remained unchanged. The working memory error frequency and reference memory error frequency were calculated based on the recorded working memory errors, reference memory errors and total arm entries.

[0121] 4. Statistical Processing

[0122] SPSS 22.0 software was used for statistical analysis of the data. Measurement data were expressed as (x ± s). One-way analysis of variance was used for comparisons between groups, and t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0123] 5. Experimental Results and Analysis

[0124] Working memory is the ability to temporarily store and process information in a short period of time. Its error frequency can reflect short-term memory function and is mainly related to the prefrontal cortex; reference memory refers to information stored for a long time, and its error frequency can reflect long-term memory ability. The storage and retrieval of long-term memory mainly involve the hippocampus and cerebral cortex.

[0125] As shown in Figure 5, compared with the normal group, the working memory error frequency and reference memory error frequency of the model group were increased; compared with the model group, the working memory error frequency and reference memory error frequency of each compound I treatment group were decreased, among which the medium dose compound I group had the best effect.

[0126] Compared with the model group, the test time of the medium-dose and high-dose groups of Compound I decreased; compared with the model group, the total number of arm entries in the medium-dose and high-dose groups of Compound I decreased, among which the decrease in the medium-dose group of Compound I was the most obvious.

[0127] Based on the above experimental results, it can be seen that compared with the normal group, the model group had slightly worse cognitive function and a higher frequency of reference memory errors, indicating damage to the hippocampus or cerebral cortex; compared with the model group, the cognitive function of each compound I-treated group was significantly improved, among which the cognitive function improvement in the medium-dose and high-dose groups was the most obvious.

[0128] Efficacy Example 4: Behavioral Effects of Compound I on LPS-Induced Sepsis-Related Encephalopathy in Mice (Novel Object Recognition)

[0129] 1. Drugs and reagents

[0130] Compound I was prepared by the method disclosed in CN111518020A and provided by Shandong Xinshidai Pharmaceutical Co., Ltd.

[0131] Endotoxin (LPS) was produced by Sigma.

[0132] 2. Experimental Animals

[0133] Sixty male C57BL / 6J mice weighing 18–22 g were obtained from Beijing Huafukang Biotechnology Co., Ltd. (license number: SYXK(Lu)20230023). Mice were housed in an SPF-grade animal housing facility under strictly controlled environmental conditions: a temperature of 20–26°C, a humidity of 40–70%, a 12:12 h light–dark cycle, and a growth and breeding diet with free access to food.

[0134] 3. Experimental Methods

[0135] After one week of adaptive feeding, 60 C57BL / 6J male mice were randomly divided into five groups, namely, normal group, model group, low-dose compound I group, medium-dose compound I group, and high-dose compound I group, with 12 mice in each group.

[0136] On the first day of the experiment, the normal group and the model group were intraperitoneally injected with normal saline according to body weight, and the low-, medium-, and high-dose groups of compound I were intraperitoneally injected with 1 mg / kg, 2.5 mg / kg, and 5 mg / kg of compound I according to body weight. One hour later, the normal group was intraperitoneally injected with the corresponding volume of normal saline according to the body weight of the mice, and the model group and each drug-treated group were injected with 7.5 mg / kg LPS, with a dosage volume of 10 ml / kg.

[0137] The same procedures as on the first day were repeated on the second day. On the third day, the normal and model groups received intraperitoneal injections of saline corresponding to the mice's body weight. The low-, medium-, and high-dose groups received intraperitoneal injections of 10 ml / kg of Compound I at 1 mg / kg, 2.5 mg / kg, and 5 mg / kg, respectively. Seven days later, a novel object recognition test was performed to analyze the mice's cognitive function.

[0138] Mice were individually placed in a 40 × 40 × 40 cm square arena and allowed to move freely for 10 minutes. The next day, two identical objects, A1 and A2, were placed at opposite corners of the arena, and the mice were allowed to move freely for 5 minutes. 120 minutes later, the familiar object A1 and the novel object A3 were placed in the arena, and the mice were allowed to move freely for 5 minutes. The time and number of times the mice touched the objects or explored the objects within 2 cm of the objects (such as placing their forepaws on the objects, sniffing the objects, or licking the objects) were recorded, and the preference index (PI) and discrimination index were calculated.

[0139] Preference index (PI) = [time observing the novel object (s) (A3) / time observing the two objects (s) (A1 and A3)] × 100%;

[0140] Discrimination index = (time (s) of observing the novel object (A3) - time (s) of observing the familiar object (A1)) / (time (s) of observing the novel object (A3) + time (s) of observing the familiar object (A1)).

[0141] 4. Statistical Processing

[0142] SPSS 22.0 software was used for statistical analysis of the data. Measurement data were expressed as (x ± s). One-way analysis of variance was used for comparisons between groups, and t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0143] 5. Experimental Results and Analysis

[0144] The closer the preference index is to 1, the more it likes new objects; the closer it is to 0, the more it likes old objects, reflecting the animal's exploratory ability. As shown in Figure 6, compared with the normal group, the preference index of the model group was reduced, indicating that the exploratory ability of the model group mice was reduced; compared with the model group, the preference index of each compound I administration group was improved. Compared with the normal group, the discrimination index of the model group was slightly reduced, indicating that its cognitive ability for familiar objects was poor; compared with the model group, the discrimination index of each compound I administration group was improved, indicating that its cognitive ability for familiar objects was better. All publications and patent applications cited in this application indicate the technical level of the field to which the present disclosure belongs. They are all incorporated herein by reference in their entirety.

[0145] The above embodiments do not limit the present invention in any form. For those skilled in the art, any modifications, replacements, etc. made within the scope of the technical solution of the present invention based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Use of a piperidine derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and treating sepsis and sepsis-induced organ damage, wherein the piperidine derivative is N-(3-chlorophenyl)-1-(1-methyl-1H-indole-2-carbonyl)piperidine-4-carboxamide.

2. The use according to claim 1, characterized in that The sepsis includes early sepsis, severe sepsis and septic shock.

3. The use according to claim 1, characterized in that The sepsis is sepsis caused by stress of severe burns, combat injuries, infection, major surgery, severe shock, radiotherapy, chemotherapy, organ transplantation, or sepsis complicated by severe hepatitis, cirrhosis, gastrointestinal disease, malnutrition, bile duct or intestinal obstruction.

4. The use according to claim 1, characterized in that The sepsis is caused by infection with microorganisms.

5. The use according to claim 4, characterized in that The microorganisms include, but are not limited to, Gram-negative bacteria or Gram-positive bacteria.

6. The use according to claim 1, characterized in that The sepsis-induced organ damage is sepsis liver damage, sepsis kidney damage and / or sepsis heart damage.

7. The use according to claim 1, characterized in that The sepsis-induced organ damage is sepsis-related encephalopathy.

8. The use according to claim 1, characterized in that The medicine is a medicine for improving serum biochemical indicators.

9. The use according to claim 8, characterized in that The drug is a drug for reducing serum AST, ALT, UREA, and CREA-S levels.

10. The use according to claim 1, characterized in that The drug is a preparation prepared by using N-(3-chlorophenyl)-1-(1-methyl-1H-indole-2-carbonyl)piperidine-4-carboxamide or its salt as an active ingredient and adding conventional pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients may be excipients, fillers, diluents, adhesives, disintegrants, solubilizers, adsorbents, pH buffers, preservatives, flavoring agents or a combination thereof.

11. The use according to claim 10, characterized in that The preparation is an oral preparation, an injection or an external preparation; the oral preparation is a tablet, a pill, a powder, a granule, a capsule, or a syrup; the injection is an intravenous injection, an intraperitoneal injection, an intracavitary injection, an lavage or an irrigation; the external preparation includes a patch, an ointment or a gel.

Images