Application of spiropyrone in the preparation of drugs for preventing and treating sepsis

Abstract

The present invention belongs to the field of pharmaceutical technology and relates to the use of spirocycline in the preparation of drugs for the prevention and treatment of sepsis. Through extensive animal experiments, the present invention has discovered that spirocycline can also be used to prevent sepsis in mice. In a cecum ligation and puncture (CLP)-induced sepsis mouse model, it was confirmed that spirocycline can significantly reduce the mortality rate and improve the survival rate of septic mice. Spirocycline can also significantly alleviate sepsis-induced liver and kidney damage and improve liver and kidney function indicators. This provides new ideas for the prevention and treatment of sepsis and a new use of spirocycline in drugs for the prevention or treatment of sepsis.

Description

Technical field:

[0001] The invention belongs to the technical field of medicine and relates to the application of spironolactone in the preparation of medicines for preventing and treating sepsis. Background technology:

[0002] Sepsis, a systemic inflammatory response syndrome caused by infection, is one of the leading causes of childhood mortality worldwide. Its treatment often places a significant financial burden on patients' families, making its prevention and early treatment crucial for their families. Despite significant advances in anti-infective therapy (antibiotics), intensive care, and organ support technologies, the in-hospital mortality rate remains as high as 15% to 50%. In sepsis, a large number of inflammatory and anti-inflammatory mediators are simultaneously released into the bloodstream, leading to multi-organ damage, lymphocyte apoptosis, and immune paralysis. An imbalance between inflammatory and anti-inflammatory responses is considered the primary mechanism of sepsis. Currently, no effective, targeted treatment for sepsis has been successfully developed, posing a significant challenge for critical care medicine. Current clinical treatments for sepsis are limited in effectiveness and can easily worsen the course of the disease, placing a significant financial burden on patients' families and causing significant psychological distress, hindering further treatment. Therefore, the search for new preventive and therapeutic agents is of great scientific and clinical significance.

[0003] Spiperone, also known as spiroperidone and spiroperone, has a molecular formula of C23H26FN3O2, a molecular weight of 395.47, and a CAS number of 749-02-0. It is a white powder at room temperature, poorly soluble in water (50.1 μg / ml) but readily soluble in dimethyl sulfoxide. Due to its strong affinity for dopamine and serotonin receptors, it is clinically recommended as an antipsychotic. Positron-labeled spiroperidone can also be used for dopamine type II receptor imaging using positron emission tomography.

[0004] There are currently no reports on the use of spironolactone in the treatment or prevention of sepsis. Summary of the invention:

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a use of spironol in the preparation of a medicament for preventing and treating sepsis.

[0006] In order to achieve the above object, the present invention provides a use of spiropyrone in the preparation of a drug for preventing and treating sepsis. Spiropyrone can inhibit the increase in serum immune factor expression levels caused by sepsis and alleviate vasodilation disorders.

[0007] Through extensive animal experiments, the applicant has confirmed that spirocycline can significantly reduce mortality and increase survival rates in septic mice induced by cecum ligation and puncture (CLP). Spirocycline can be used to prevent sepsis in mice.

[0008] The present invention also provides the use of spiropyridine in preparing a drug for preventing and treating target organ damage caused by sepsis, wherein the target organ is the liver or the kidney.

[0009] The applicant found through a large number of animal experiments that spirocycline can significantly alleviate the liver and kidney function damage caused by sepsis in a mouse model induced by cecum ligation and puncture (CLP), and alleviate liver and kidney function indicators.

[0010] The structural formula of spiropyridone is shown below.

[0011]

[0012] The source of the spirocycline piperidone of the present invention is not limited.

[0013] Compared with the existing technology, the present invention demonstrates through animal experiments that spirocycline can reduce the increase in serum immune factors caused by sepsis, alleviate vasodilation disorders, and improve damaged liver and kidney function in a cecum ligation and puncture (CLP)-induced sepsis mouse model, thereby increasing the survival rate of sepsis model mice. This provides a new approach to the prevention and treatment of sepsis and a new use of spirocycline in drugs for the prevention or treatment of sepsis. Description of the drawings:

[0014] Figure 1 This is a schematic diagram of experimental results on the preventive effect of spiropyrone on sepsis in mice.

[0015] Figure 2 This is a schematic diagram of the experimental results of Example 2 of the present invention regarding the effect of spiropyridine on the microvascular dilation ability of mice when used to prevent sepsis in mice, wherein A is a curve diagram showing the change of microvascular dilation ability with acetylcholine concentration; B is the maximum dilation diagram; and C is the area under the curve.

[0016] Figure 3 This is a schematic diagram of the experimental results of Example 3 of the present invention regarding the effect of spiropyridine on mouse serum inflammatory factors when used to prevent sepsis in mice, wherein A is IL-1β, B is IL-6, and C is TNF-α.

[0017] Figure 4 This is a schematic diagram of the results of Example 4 of the present invention showing the effect of spironolactone on the survival period of mice with CLP-induced sepsis.

[0018] Figure 5 This is a schematic diagram of the experimental results of Example 5 of the present invention showing the effect of spiropyrone on the microvascular dilation ability of mice with CLP-induced sepsis, wherein A, B, and C are respectively the percentage of microvascular dilation, maximum dilation percentage, and area under the curve of mice 3 hours after administration; D, E, and F are respectively the percentage of microvascular dilation, maximum dilation percentage, and area under the curve of mice 6 hours after administration; and G, H, and I are respectively the percentage of microvascular dilation, maximum dilation percentage, and area under the curve of mice 12 hours after administration.

[0019] Figure 6 This is a schematic diagram of the experimental results of Example 6 of the present invention on the effect of spiropyrone on the expression levels of serum inflammatory factors in CLP-induced septic mice, wherein A is IL-1β; B is IL-6; and C is TNF-α.

[0020] Figure 7 This is a schematic diagram of the experimental results of Example 7 of the present invention on the effects of spiropyridine on liver and kidney function indicators in CLP-induced septic mice, wherein A is urea nitrogen; B is creatinine; C is aspartate aminotransferase; and D is alanine aminotransferase. Specific implementation method:

[0021] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0022] In the following examples, spironolactone was purchased from MCE (item number: HY-B1371); the Elisa kit for detecting inflammatory factors was purchased from R&D Company in the United States; the urea nitrogen (BUN) test kit was purchased from Nanjing Jiancheng (item number: C013-2-1); the creatinine (Cr) detection kit was purchased from Leagene (item number: TC1191); the aspartate aminotransferase test kit and the alanine aminotransferase test kit were both purchased from Nanjing Jiancheng (item numbers: C010-2-1, C009-2-1); and acetylcholine was purchased from MCE (item number: HY-B0282).

[0023] The reagents involved in the following examples were prepared as follows:

[0024] Phosphate buffer: Dissolve 0.818 g NaCl, 0.037 g KCl, 0.022 g CaCl2, 0.238 g HEPES, and 0.18 g glucose in 90 mL of double-distilled water, adjust the pH to 7.4, and make up to 100 mL.

[0025] Example 1:

[0026] This example relates to an experiment on the preventive effect of spironolactone on sepsis in mice, and the specific steps are as follows:

[0027] (1) Sixty C57 mice weighing 21-25 g were purchased and randomly divided into sham operation group, model group, and drug pretreatment group, with 20 mice in each group;

[0028] (2) The drug pretreatment group was given spironolactone by gavage every day at a dose of 0.585 mg / kg; the model group and the sham operation group were given an equal volume of normal saline by gavage once a day for five consecutive days;

[0029] (3) Cecum ligation and puncture (CLP) was performed on the model group and the drug pretreatment group to establish a sepsis model. The specific process is as follows:

[0030] a. Mice were fasted for 12 hours before the experiment;

[0031] b. After anesthesia, the animal was placed supine on a surgical board. The abdominal surgical area was routinely disinfected and hair removed. Under sterile conditions, a 2-cm incision was made in the abdominal wall using a scalpel. The cecum was dissected distal to the ileocecal valve and ligated at 1 / 3 of the cecum with No. 3 silk suture.

[0032] c. Use an 18-gauge needle to pierce the ligature and squeeze out a small amount of feces, minimizing damage to blood vessels. Then, use 4-gauge silk sutures to suture the peritoneum and skin intermittently. Immediately inject 50 ml / kg of body weight of normal saline subcutaneously to prevent shock.

[0033] In the sham operation group, the abdominal cavity was opened but the cecum was not ligated or punctured.

[0034] (4) Observe the death of mice at 12h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after surgery, and draw a survival curve. Figure 1 As shown, * indicates significance at p < 0.05; ** indicates significance at p < 0.01; *** indicates significance at p < 0.001; **** indicates significance at p < 0.0001.

[0035] from Figure 1 As can be seen, no mice in the sham-operated group died within 7 days; however, mice in the model group died 12 hours after modeling, and the 7-day survival rate was only 20%. Compared with the model group, the 7-day survival rate in the drug pretreatment group was significantly improved. This suggests that spironolactone can prevent sepsis in mice and improve the survival rate of septic mice.

[0036] Example 2:

[0037] This embodiment relates to the detection of microvascular dilation ability of mice when spirocycline is used to prevent sepsis in mice. The specific method is as follows:

[0038] (1) The mouse grouping, drug administration, and CLP modeling procedures were the same as in Example 1;

[0039] (2) 12 hours after CLP modeling, the dilation capacity of the mesenteric artery microvessels of mice was measured:

[0040] a. Prepare phosphate buffer;

[0041] b. The mouse was killed by cervical dislocation and placed on the operating table with the abdomen facing upwards. The abdominal wall was opened and the mesenteric artery was isolated.

[0042] c. Slowly insert the first silver wire into the artery under a microscope and place it, along with the blood vessel, in another dish for later use;

[0043] d. Use microtweezers and a small screwdriver to secure the two ends of the silver wire to one end of the test clamp. Bring the two measuring clamps together, and use fiber tweezers to hold the second silver wire in place. Insert it parallel to and close to the first silver wire. Once the second silver wire is fully inserted, bring it closer to the measuring clamps, secure the silver wire to the other end of the measuring clamps, and slightly separate the clamps.

[0044] e. After 20 minutes of heating and ventilation equilibrium, norepinephrine was added to constrict the blood vessels;

[0045] f. Add acetylcholine / phosphate buffer solution in descending order (the concentration gradient of acetylcholine in the solution is 10 -8 -10 -3 mol / L), to detect the tension after vasodilation;

[0046] (3) Analyze the data and obtain the statistical results. Figure 2 As shown in the table, * indicates significance at p < 0.05; ** indicates significance at p < 0.01; *** indicates significance at p < 0.001; **** indicates significance at p < 0.0001.

[0047] from Figure 2 A As can be seen from the results, as the concentration of acetylcholine increases, the vasodilation percentage of the model group can only reach about 20%, indicating that the vasodilation ability is severely impaired, while the vasodilation ability of the drug pretreatment group is significantly improved compared with the model group. The maximum vasodilation percentages of the sham operation group, model group and drug pretreatment group are 82%, 27% and 76% respectively. Figure 2 B); Figure 2 The area under the curve of C can also be used to evaluate the vasodilation effect of acetylcholine. In the figure, the areas under the curve of the sham operation group, model group and drug pretreatment group are 0.081, 0.022 and 0.066, respectively, indicating that the vasodilation ability of the model group is severely impaired, while the vasodilation ability of the drug pretreatment group is significantly improved compared with the model group.

[0048] Example 3:

[0049] This embodiment relates to the detection of the expression level of inflammatory factors in mice when spironolone is used to prevent sepsis in mice. The specific method is as follows:

[0050] (1) The mouse grouping, drug administration, and CLP modeling procedures were the same as in Example 1;

[0051] (2) Twelve hours after CLP modeling, mice were killed by cervical dislocation, and their eyeballs were removed to collect blood. After blood coagulation, serum was collected.

[0052] (3) Detect the serum inflammatory factor levels of each group of mice according to the instructions of the Elisa kit;

[0053] (4) Analyze the data and obtain the statistical results. Figure 3 As shown in the table, * indicates significance at p < 0.05; ** indicates significance at p < 0.01; *** indicates significance at p < 0.001; **** indicates significance at p < 0.0001.

[0054] from Figure 3 It can be seen that the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in the serum of the model group were significantly increased compared with the sham operation group; the increase in the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in the serum of mice in the drug pretreatment group was not as great as that in the model group, indicating that spironolactone can reduce the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in the serum caused by sepsis, and spironolactone can significantly control the inflammatory response caused by sepsis and prevent sepsis.

[0055] Example 4:

[0056] This example relates to an experiment on the effect of spironolactone on the survival of CLP-induced septic mice, and the specific steps are as follows:

[0057] (1) One hundred C57 mice weighing 21-25 g were purchased and randomly divided into a sham operation group, a model group, a 3 h postoperative administration group, a 6 h postoperative administration group, and a 12 h postoperative administration group, with 20 mice in each group.

[0058] (2) CLP was performed on the model group and the groups receiving the drug 3 h, 6 h, and 12 h after surgery to establish a sepsis model. The model construction process was the same as in Example 1. In the sham operation group, only the abdominal cavity was opened, but the cecum was not ligated or punctured.

[0059] (3) The 3-hour, 6-hour, and 12-hour postoperative administration groups were administered spironolactone orally at a dose of 0.585 mg / kg 3, 6, and 12 hours after surgery, respectively;

[0060] (4) The death of mice was observed at 12h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after surgery, and the survival curve was drawn and statistically analyzed using GraphPad software. Figure 4 As shown, * indicates significance at p < 0.05; ** indicates significance at p < 0.01; *** indicates significance at p < 0.001; **** indicates significance at p < 0.0001.

[0061] from Figure 4 As can be seen, no mice in the sham-operated group died within 7 days; however, mice in the model group died 12 hours after modeling, and the 7-day survival rate was only 20%. Compared with the model group, the 7-day survival rates of the groups administered 3 hours and 6 hours after surgery were significantly improved, indicating that spironolone has a good therapeutic effect on sepsis in mice.

[0062] Example 5:

[0063] This example relates to an experiment on the effect of spironolactone on the vasodilation capacity of CLP-induced septic mice, and the specific method is as follows:

[0064] (1) The mouse grouping, drug treatment, and CLP modeling procedures were the same as in Example 4;

[0065] (2) 24 hours after CLP modeling, the dilation capacity of mesenteric artery microvessels was measured. The specific experimental steps were the same as those in steps (2) af of Example 2;

[0066] (3) Analyze the data and obtain the statistical results. Figure 5 As shown in the table, * indicates significance at p < 0.05; ** indicates significance at p < 0.01; *** indicates significance at p < 0.001; **** indicates significance at p < 0.0001.

[0067] from Figure 5 As can be seen, the percentage of vasodilation in the model group was only 26%, indicating that the model group's vasodilation capacity was severely impaired. The percentage of vasodilation in the 3-hour and 6-hour postoperative administration groups increased with increasing acetylcholine concentrations, and the vasodilation capacity of the 3-hour and 6-hour postoperative administration groups was significantly improved compared with the model group. The percentage of vasodilation in the 12-hour postoperative administration group was greater than that of the model group, but less than that of the 3-hour and 6-hour postoperative administration groups. This indicates that administering spironolamine within 6 hours after modeling can effectively improve the vasodilation capacity of septic mice and treat sepsis in mice.

[0068] Example 6:

[0069] This example relates to an experimental test of the effect of spironolactone on the expression levels of inflammatory factors in CLP-induced septic mice. The specific method is as follows:

[0070] (1) The mouse grouping, drug treatment, and CLP modeling procedures were the same as in Example 4;

[0071] (2) 24 hours after CLP modeling, mice were killed by cervical dislocation, eyeballs were removed to collect blood, and serum was collected after blood coagulation;

[0072] (3) Detect the serum inflammatory factor levels of each group of mice according to the instructions of the Elisa kit;

[0073] (4) Analyze the data and obtain the statistical results. Figure 6 As shown in the table, * indicates significance at p < 0.05; ** indicates significance at p < 0.01; *** indicates significance at p < 0.001; **** indicates significance at p < 0.0001.

[0074] from Figure 6 As can be seen, the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in the serum of the model group were significantly higher than those in the sham-operated group. However, the levels of these factors in the serum of mice given the drug 3 and 6 hours after surgery did not increase to the same extent as in the model group. This suggests that spironolactone can significantly reduce the serum levels of the proinflammatory factors IL-1β, IL-6, and TNF-α caused by sepsis. Furthermore, administering spironolactone within 6 hours of modeling can significantly control the inflammatory response caused by sepsis and treat sepsis.

[0075] Example 7:

[0076] This example relates to an experiment on the effect of spironolactone on the liver and kidney function of CLP-induced septic mice, and the specific method is as follows:

[0077] (1) The mouse grouping, drug treatment, and CLP modeling procedures were the same as in Example 4;

[0078] (2) 24 hours after CLP modeling, mice were killed by cervical dislocation, eyeballs were removed to collect blood, and serum was collected after blood coagulation;

[0079] (3) According to the kit instructions, the levels of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea nitrogen in the serum of each group of mice were detected;

[0080] (4) Analyze the data and obtain the statistical results. Figure 7 As shown in the table, * indicates significance at p < 0.05; ** indicates significance at p < 0.01; *** indicates significance at p < 0.001; **** indicates significance at p < 0.0001.

[0081] from Figure 7As can be seen, the serum levels of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea nitrogen in the model group were significantly higher than those in the sham-operated group. The serum levels of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea nitrogen in the group administered 3 hours after surgery were significantly lower than those in the model group, indicating that administration 3 hours after modeling can effectively inhibit the increase in serum levels of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea nitrogen. The serum levels of alanine aminotransferase, aspartate aminotransferase, and urea nitrogen in the group administered 6 hours after surgery were significantly lower than those in the model group, indicating that administration 6 hours after modeling can effectively inhibit the increase in serum levels of alanine aminotransferase, aspartate aminotransferase, and urea nitrogen. The serum level of alanine aminotransferase in the group administered 12 hours after surgery was lower than that in the model group, indicating that administration 12 hours after modeling can effectively inhibit the increase in serum alanine aminotransferase. In summary, administration of spironolamine within 6 hours after modeling can alleviate liver and kidney damage in septic mice and is useful for the treatment of sepsis.

Claims

1. Use of spironolactone as the sole active ingredient in the preparation of a drug for preventing and treating sepsis, characterized in that: Spiroperidinone can inhibit the increase in serum immune factor expression levels caused by sepsis and alleviate vasodilation disorders.

2. The use of spironolactone as the sole active ingredient in the preparation of a drug for preventing and treating sepsis according to claim 1, characterized in that: In the mouse model of sepsis induced by cecal ligation and puncture, spironolactone can significantly reduce the mortality rate of septic mice and increase the survival rate.

3. Use of spironolactone as the sole active ingredient in the preparation of a drug for preventing and treating sepsis according to claim 1, characterized in that: Spiroperidinone can be used to prevent sepsis in mice.

4. Use of spironolactone as the sole active ingredient in the preparation of a drug for preventing and treating target organ damage caused by sepsis, characterized in that: The target organ is the liver or the kidney.

5. Use of the spironolactone as the sole active ingredient in the preparation of a drug for preventing and treating target organ damage caused by sepsis according to claim 4, characterized in that: In the mouse model of sepsis induced by cecal ligation and puncture, spironolactone can significantly alleviate the liver and kidney function damage caused by sepsis and relieve liver and kidney function indicators.

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