Application of dehydrounionine in the preparation of drugs that reduce polymyxin toxicity

Abstract

This invention provides the application of dehydrouncitonin in the preparation of drugs that reduce polymyxin toxicity, belonging to the field of pharmaceutical technology. This invention uses dehydrouncitonin in combination with polymyxin. The combined use of dehydrouncitonin and polymyxin E increased the viability of HEK293T and RAW264.7 cells to 87.7% and 94.3%, respectively, with highly significant differences compared to the control, indicating that treatment with dehydrouncitonin in combination with polymyxin E can significantly inhibit the cytotoxic effect of polymyxin E. This invention uses a mouse model to demonstrate that treatment with dehydrouncitonin in combination with polymyxin E can significantly reduce the neurotoxicity and nephrotoxicity of polymyxin E alone in mice, specifically manifested in improved neurobehavioral effects and improved histopathological effects on brain and kidney tissues. This invention uses dehydrouncitonin in combination with polymyxin E to significantly inhibit the cytotoxic, neurotoxic, and nephrotoxic effects of polymyxin E.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of dehydrouncitonin in the preparation of drugs that reduce polymyxin toxicity. Background Technology

[0002] Polymyxins are antimicrobial polypeptides found in cultures of *Bacillus polymyxa*, and there are five types: A, B, C, D, and E. They have similar but broad antimicrobial spectra, and are particularly effective against Gram-negative bacteria such as *Escherichia coli*, *Klebsiella pneumoniae*, and *Pseudomonas aeruginosa*. Polymyxin E (also known as polymyxin E, CAS number: 1066-17-7, English name: Colistin, trade names: Colistin, Colistin E) is mainly used clinically in sulfate or mesylate forms, namely polymyxin E sulfate and polymyxin E mesylate.

[0003] However, polymyxin injections currently have significant toxic effects in clinical practice. The main toxic side effects of polymyxin are: (1) nephrotoxicity, which is a common and serious side effect that may cause a decline in renal function, such as proteinuria, casts in urine, azotemia, and an increase in creatinine levels. This is its main damage to renal function; (2) neurotoxicity, which may cause dizziness, ataxia, drowsiness, and peripheral sensory abnormalities during the use of polymyxin. These are all neurological side effects caused by polymyxin. Nephrotoxicity and neurotoxicity are the most common toxic side effects of polymyxin E in clinical application. Among them, the incidence of clinical nephrotoxicity is as high as 60%, and the incidence of neurotoxicity is about 7%. Mild peripheral neurotoxicity symptoms are more obvious, with an incidence rate as high as 100%. In addition, polymyxin also has clinical application problems such as a narrow therapeutic window and the spread of drug resistance.

[0004] In order to improve the therapeutic efficacy of polymyxins and reduce their cytotoxic effects, there is an urgent need in the art to provide agents that reduce the cytotoxicity, neurotoxicity and nephrotoxicity of polymyxins. Summary of the Invention

[0005] The purpose of this invention is to provide the use of dehydrouncitonin in the preparation of drugs that reduce the cytotoxicity and / or neurotoxicity and nephrotoxicity of polymyxins, pharmaceutical compositions and applications, to solve the problems of cytotoxicity and organ toxicity caused by the use of polymyxins for treatment.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the use of dehydrouncitonin in the preparation of drugs that reduce polymyxin cytotoxicity and / or organ toxicity, wherein the organ toxicity includes neurotoxicity and nephrotoxicity.

[0008] Preferably, the polymyxin is polymyxin E or polymyxin B.

[0009] This invention provides a pharmaceutical composition for reducing polymyxin toxicity, comprising dehydrolysine and polymyxin.

[0010] Preferably, the polymyxin is polymyxin E sulfate.

[0011] Preferably, the polymyxin toxicity includes cytotoxicity or organ toxicity; the organ toxicity includes neurotoxicity and nephrotoxicity.

[0012] Preferably, when the pharmaceutical composition is used to reduce cytotoxicity, the mass ratio of dehydrouncitonin to polymyxin E sulfate is (5-20):840 or (5-20):210.

[0013] Preferably, when the pharmaceutical composition is used to reduce organ toxicity, the mass ratio of dehydrouncitonin to polymyxin sulfate is (0.25-1):1.

[0014] Preferably, the dosage form of the pharmaceutical composition is a tablet, capsule, sustained-release tablet, controlled-release tablet, oral liquid, syrup, injection, drop pill, or lyophilized powder injection.

[0015] The present invention provides the use of the pharmaceutical composition for reducing polymyxin toxicity described above in the preparation of drugs for reducing polymyxin cytotoxicity and / or organ toxicity, wherein the organ toxicity includes neurotoxicity and nephrotoxicity.

[0016] This invention employs dehydrorheumatine in combination with polymyxin E, which significantly increases cell viability compared to polymyxin E alone. Specifically, when the final concentration of dehydrorheumatine was 5, 10, and 20 μg / mL, the cell viability of HEK293T increased from 62.9% to 75.63%, 87.7%, and 81.1%, respectively, showing significant differences compared to polymyxin E alone. Similarly, when the final concentration of dehydrorheumatine was 5, 10, and 20 μg / mL, the cell viability of RAW264.7 increased from 65.5% to 79.5%, 83.75%, and 94.2%, respectively, also showing significant differences compared to polymyxin E alone. Therefore, this invention, using dehydrorheumatine in combination with polymyxin E, can significantly inhibit the cytotoxic effects of polymyxin E.

[0017] This invention demonstrates, through the establishment of a mouse model of polymyxin E-induced kidney injury and neurotoxicity, that treatment with dehydrouncitonin combined with polymyxin E can significantly reduce the neurotoxicity and nephrotoxicity induced by polymyxin E alone in mice. The protective effect against neurotoxicity is mainly manifested in changes in mouse neurobehavior, with the main phenotype being a significant restoration of motor function in mice treated with dehydrouncitonin. The protective effect against nephrotoxicity is mainly manifested in the improvement of kidney histopathology and renal function, specifically, the significant reduction in colistin sulfate-induced tubular necrosis, epithelial cell shedding, and cast formation in dehydrouncitonin combined with treatment, and a significant inhibition of the cytotoxic, neurotoxic, and nephrotoxic effects of polymyxin E.

[0018] This invention provides a solution to the problem of high toxicity and side effects in the clinical application of polymyxins, and can provide a basis for clinical combination therapy, reduce the clinical toxicity and side effects of polymyxins, thereby improving clinical output and therapeutic index, and can solve the problems of low clinical therapeutic index and insufficient efficacy of polymyxins. Attached Figure Description

[0019] Figure 1 The results show the protective effect of dehydrouncitonin against polymyxin E-induced HEK293T cell toxicity; ** represents highly significant difference compared with the control group; ## represents highly significant difference compared with the polymyxin E-only treatment group.

[0020] Figure 2 The results show the protective effect of dehydrouncitonin against polymyxin E-induced cytotoxicity in RAW264.7 cells; ** represents highly significant difference compared with the control group; ## represents highly significant difference compared with the polymyxin E-only treatment group.

[0021] Figure 3 Serum urea nitrogen (A) and creatinine (B) levels were measured in mice in the control group, polymyxin E sulfate model group, high-dose dehydrorheumatoidin protection group, and low-dose dehydrorheumatoidin protection group. Compared with the control group, ** indicates extremely significant difference; compared with the polymyxin E sulfate treatment group alone, ## indicates extremely significant difference.

[0022] Figure 4 The images show the histopathological sections (A) and semi-quantitative damage scores (B) of the kidney tissues in the control group, polymyxin E sulfate model group, high-dose dehydrorheumatoidin protection group, and low-dose dehydrorheumatoidin protection group. Compared with the control group, ** represents extremely significant differences; compared with the polymyxin E sulfate treatment group alone, ## represents extremely significant differences.

[0023] Figure 5The results of open field tests on the neurobehavioral characteristics of mice in the control group, polymyxin E sulfate model group, high-dose dehydrorheumatoidin protection group, and low-dose dehydrorheumatoidin protection group are shown. Compared with the control group, ** represents extremely significant difference; compared with the polymyxin E sulfate treatment group alone, ## represents extremely significant difference.

[0024] Figure 6 Results of the binding modes of dehydrouncitonin to protein molecules such as CYP1A1, ERK1, JNK, Nrf2 and NF-κB. Detailed Implementation

[0025] This invention provides the use of dehydrouncitonin in the preparation of drugs that reduce polymyxin cytotoxicity and / or organ toxicity, wherein the organ toxicity includes neurotoxicity and nephrotoxicity.

[0026] The present invention does not have any special limitation on the source of the dehydrouncitonine (CAS: 630-94-4), and any commercially available product well known in the art is acceptable.

[0027] In this invention, the dehydrounionine is used to prepare drugs that reduce polymyxin cytotoxicity, or to prepare drugs that reduce polymyxin organ toxicity; or to prepare both drugs that reduce polymyxin cytotoxicity and drugs that reduce organ toxicity.

[0028] In this invention, the polymyxin is preferably polymyxin E or polymyxin B. This invention does not have a specific limitation on the source of the polymyxin; commercially available products well-known in the art are acceptable.

[0029] The present invention provides a pharmaceutical composition for reducing polymyxin toxicity, comprising dehydrouncitonin and polymyxin.

[0030] In this invention, the polymyxin is preferably polymyxin E sulfate.

[0031] In this invention, the polymyxin toxicity includes cytotoxicity or organ toxicity; the organ toxicity includes neurotoxicity and nephrotoxicity.

[0032] In this invention, when the pharmaceutical composition is used to reduce cytotoxicity, the mass ratio of dehydrouncitonin to polymyxin E sulfate is (5-20):840 or (5-20):210, more preferably 1:84 or 2:21.

[0033] In this invention, when the pharmaceutical composition is used to reduce organ toxicity, the mass ratio of dehydrouncitonin to polymyxin sulfate is preferably (0.25-1):1, more preferably 1:1.

[0034] In this invention, the dosage form of the pharmaceutical composition is tablets, capsules, sustained-release tablets, controlled-release tablets, oral liquids, syrups, injections, pellets, or lyophilized powder for injection. This invention does not impose any particular limitation on the preparation method for different dosage forms; any dosage form meeting the above-mentioned dosage requirements can be obtained by following methods well-known in the art.

[0035] The present invention provides the use of the pharmaceutical composition for reducing polymyxin toxicity described above in the preparation of drugs for reducing polymyxin cytotoxicity and / or organ toxicity, wherein the organ toxicity includes neurotoxicity and nephrotoxicity.

[0036] When the pharmaceutical composition is used to prepare drugs that reduce polymyxin cytotoxicity and / or organ toxicity, the present invention does not impose any special limitation on the concentrations of polymyxin and dehydrorheumatine in the pharmaceutical composition; the concentrations can be adjusted according to actual needs. In the experimental model of the present invention, when the pharmaceutical composition is used to prepare drugs that reduce polymyxin cytotoxicity, the effective concentration of polymyxin sulfate E is preferably 600 or 150 μM (equivalent to a mass concentration of polymyxin sulfate E of 840 μg / mL or 210 μg / mL, respectively), and the effective concentration of dehydrorheumatine is preferably 5–20 μg / mL, more preferably 10 or 20 μg / mL; the effective concentration is the concentration at which the efficacy is achieved.

[0037] In the experimental model of the present invention, when the pharmaceutical composition is used to prepare a drug that reduces the organ toxicity of polymyxins, the preferred dosage of the dehydrouncitonin is 5 to 20 mg / kg body weight per day, and the preferred dosage of the polymyxin E sulfate is 20 mg / kg body weight per day.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] The drugs used in this invention embodiment are: Dehydrouncitonin, purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity ≥98%; and Polymyxin E sulfate, purchased from Hebei Shengxue Dacheng Tangshan Pharmaceutical Co., Ltd., with a potency ≥23000 U / mg.

[0040] Weigh a certain amount of polymyxin E sulfate to prepare a polymyxin E sulfate stock solution with a concentration of 28 mg / mL; prepare a dehydrorheumatoidin stock solution with a concentration of 20 mg / kg using DMSO.

[0041] All prepared mother liquors were stored in a -20°C freezer.

[0042] Example 1: Protective effect of dehydrouncitonin against polymyxin cytotoxicity

[0043] 1.1 Test cell lines

[0044] Human embryonic kidney cells (HEK293T) (Cat. No: BC-C-HU-044) were purchased from Nanjing Shenghang Biotechnology Co., Ltd.; mouse mononuclear macrophages (RAW264.7) were preserved by the National Veterinary Drug Safety Evaluation Center of China Agricultural University.

[0045] 1.2 Cell viability assay

[0046] Dehydrounerectin pretreatment: HEK293T cells were pretreated with 1×10⁻⁶ molluscine. 4 HEK293T cells were seeded per well in 96-well plates. When the cell density reached 50%, the original culture medium was discarded. 100 μL of culture medium was added to each well of the 96-well plate, followed by an appropriate volume of dehydrorheumatine stock solution to achieve final concentrations of 5, 10, and 20 μg / mL (two groups of 96-well plate experiments). HEK293T cells were pretreated for 2 hours, and then an appropriate amount of polymyxin E sulfate stock solution was added to achieve a final concentration of 600 μM (equivalent to 840 μg / mL). Corresponding solvent control groups (containing 0.1 wt% DMSO), dehydrorheumatine monotherapy groups (with a final dehydrorheumatine concentration of 20 μg / mL), and polymyxin E sulfate monotherapy groups (with a final polymyxin E concentration of 600 μM) were set up. All cells were cultured for 24 hours.

[0047] Following the above-described treatment steps for HEK293T cells with dehydrouncitonin, RAW264.7 cells were pretreated with dehydrouncitonin at final concentrations of 5, 10, and 20 μg / mL. After 2 hours of pretreatment, polymyxin E sulfate stock solution was added to bring the final concentration of polymyxin E sulfate to 150 μM (equivalent to 210 μg / mL). Corresponding solvent control groups (containing 0.1% DMSO), dehydrouncitonin monotherapy groups (with corresponding final concentrations of 20 μg / mL), and polymyxin E sulfate groups (with a final concentration of 150 μM, equivalent to 210 μg / mL) were set up, and the cells were cultured together for 24 hours.

[0048] After drug treatment, the culture medium was discarded, and 100 μL of CCK-8 working solution (containing 10 μL of CCK-8 reagent and 90 μL of complete culture medium) was added to each group of cells in the dark. Then, the cells were transferred to a 37°C cell culture incubator and incubated for 1 h. After the incubation, the OD value was measured at a wavelength of 450 nm.

[0049] Cell viability = [(As-Ab) / (Ac-Ab)]×100%; where As is the OD value of the drug-treated group; Ab is the OD value of the background blank control group; and Ac is the OD value of the control group.

[0050] 1.3 Test Results

[0051] The results of dehydrouncitonin reducing the cytotoxicity of polymyxin E sulfate to HEK293T cells are shown in the figure. Figure 1 The results of dehydrouncitonin reducing the cytotoxicity of polymyxin E sulfate on RAW264.7 cells are shown in [the original text]. Figure 2 Compared with the control group, ** indicates a highly significant difference; compared with the polymyxin E sulfate treatment group alone, ## indicates a highly significant difference; where "-" represents no treatment and "+" represents the corresponding drug treatment;

[0052] Figures 1-2 The specific results are analyzed as follows:

[0053] like Figure 1 In HEK293T cells, compared with the control group, treatment with polymyxin E at 600 μM (equivalent to 840 μg / mL) for 24 h significantly reduced the cell viability to 62.9% (**). When the final concentrations of dehydrouncitonin were 5, 10, and 20 μg / mL, the cell viability of HEK293T cells increased from 62.9% to 75.63%, 87.7%, and 81.1%, respectively, all significantly different from polymyxin E treatment alone (##). Figure 2 In RAW264.7 cells, compared with the control group, treatment with polymyxin E at 150 μM (equivalent to 210 μg / mL) for 24 h significantly reduced cell viability to 65.5% (**). When the final concentrations of dehydrouncitonin were 5, 10, and 20 μg / mL, the cell viability of RAW264.7 cells increased from 65.5% to 79.5%, 83.75%, and 94.2%, respectively, all significantly different from polymyxin E treatment alone (##). These data indicate that treatment with dehydrouncitonin in combination with polymyxin E significantly inhibits the cytotoxic effects of polymyxin E.

[0054] Example 2: Protective effect of dehydrouncitonin combined treatment on neurological and renal damage induced by polymyxin E sulfate in mice.

[0055] 2.1 Laboratory Animals

[0056] C57BL / 6, female, 8 weeks old, weighing 18-22g, purchased from Beijing Sibeifu Technology Co., Ltd.

[0057] 2.2 Feeding and handling of laboratory animals

[0058] Mice were housed at the National Veterinary Drug Safety Evaluation Center of the College of Veterinary Medicine, China Agricultural University, fed with routine pelleted feed and free access to water. The temperature was controlled at (22±2)℃ and the humidity at (50±10)%. Mice underwent a one-week acclimatization period before the experiment. Forty animals were randomly divided into five groups of eight each, as follows:

[0059] Control group: Mice were intraperitoneally injected with an equal volume of physiological saline containing 1 wt% DMSO at a dose of 0.1 mL / 10 g body weight.

[0060] Polymyxin E sulfate model group: Mice were intraperitoneally injected with polymyxin E at a single dose of 10 mg / kg body weight, twice a day with an 8-hour interval between injections; that is, mice were given 20 mg / kg body weight of polymyxin E daily. At the same time, mice were intraperitoneally injected with physiological saline containing 1 wt% DMSO at a dose of 0.1 mL / 10 g body weight for 7 consecutive days.

[0061] Dehydrorheumatine control group: An appropriate amount of dehydrorheumatine was dissolved in DMSO, and then dissolved in physiological saline to prepare a suspension with a dehydrorheumatine concentration of 2 mg / mL. The concentration of DMSO in the suspension was 1 wt%. Mice were given intraperitoneal injection at a dose of 20 mg / kg body weight; that is, mice were given 20 mg / kg body weight of dehydrorheumatine daily for 7 consecutive days.

[0062] High-dose protection group of dehydrorheumatoidin: An appropriate amount of dehydrorheumatoidin was dissolved in DMSO, and then dissolved in physiological saline to prepare a suspension with a dehydrorheumatoidin concentration of 2 mg / mL. The DMSO concentration in this suspension was 1 wt%. Mice were given intraperitoneal injection at a dose of 20 mg / kg body weight for 7 consecutive days. At the same time, polymyxin E was injected twice, with a single injection dose of 10 mg / kg body weight and an interval of 8 hours between each injection. That is, mice were given 20 mg / kg body weight of dehydrorheumatoidin and 20 mg / kg body weight of polymyxin E daily for 7 consecutive days.

[0063] Low-dose protection group of dehydrorheumatoidin: An appropriate amount of dehydrorheumatoidin was dissolved in DMSO, and then dissolved in physiological saline to prepare a suspension with a dehydrorheumatoidin concentration of 2 mg / mL. The DMSO concentration in this suspension was 1 wt%. Mice were given an intraperitoneal injection of 5 mg / kg body weight for 7 consecutive days. At the same time, polymyxin E was injected twice, with a single injection dose of 10 mg / kg body weight and an interval of 8 hours between each injection. That is, mice were given 5 mg / kg body weight of dehydrorheumatoidin and 20 mg / kg body weight of polymyxin E daily for 7 consecutive days.

[0064] The establishment of the polymyxin E nephrotoxicity mouse model was carried out in accordance with the method described in the existing technology (Dai C, Tang S, Wang Y, Velkov T, Xiao X. Baicalein acts as a nephroprotectant that amelioratescolistin-induced nephrotoxicity by activating the antioxidant defense mechanism of the kidneys and down-regulating the inflammatory response. J Antimicrob Chemother. 2017 Sep 1; 72(9):2562-2569).

[0065] All mice were euthanized 24 hours after the last administration of the drug, following 7 consecutive days of injection. The mice were then injected intraperitoneally with an excessive amount of sodium pentobarbital (80 mg / kg body weight). Blood, kidney, and nerve tissue were collected rapidly. Serum and tissue were processed as follows.

[0066] 2.3 Determination of Biochemical Indicators

[0067] Collected mouse blood samples were centrifuged at 3000 rpm for 10 min, and the supernatant was collected. Changes in serum urea nitrogen and serum creatinine were detected using an automated biochemical analyzer.

[0068] 2.4 Open Field Test

[0069] The open field test was conducted according to the literature (Wang Qiuzhen, Deng Ziteng, Lan Jing, Liu Xuelian, Fan Xiangwei, Xue Yi, Ma Yunfei. Effects of Lactobacillus casei on anxiety-depression-like behavior in DSS-induced colitis mice and its protective effect on the colon, Journal of China Agricultural University, 2023, 28(4):139-146). The specific steps are as follows: On the 7th day of the experiment, the open field test was conducted. The specifications of the open field box were length x width x height = 45cm x 45cm x 45cm. The box was white and divided into 25 small squares. The 16 squares close to the perimeter were the outer area, and the 9 small squares in the middle were the central area. On the 14th day of the experiment, the mice were placed in the open field box and timed for 5 minutes using ANY-maze software (Stoelting, USA). The path of the mice walking in the open field box was completely captured. One mouse was tested each time, and the side walls and bottom of the open field box were cleaned with alcohol after each test. The total distance each mouse traveled in the open field box and the time spent in the central area within 5 minutes were recorded to assess the mice's behavior.

[0070] 2.5 Pathological examination and semi-quantitative scoring of kidney and brain tissue

[0071] Freshly isolated kidney tissue was washed with physiological saline, then sampled and fixed in 10% neutral formalin. After 48 hours, it was rinsed with water and then subjected to dehydration, clearing, paraffin embedding, sectioning, and staining. The histopathological changes of the kidney tissue were observed and photographed under a microscope. HE staining: Paraffin sections were removed and placed in a 60℃ constant temperature oven for 30 minutes; then dewaxed and dehydrated with ethanol, and immersed in double-distilled water; hematoxylin staining for 10 minutes, then rinsed with tap water, and differentiated with hydrochloric acid and ethanol; rinsed with running water until the sections turned blue; stained with 95% ethanol for 30 seconds, alcoholic eosin for 30 seconds, 95% ethanol for 30 seconds, and 100% ethanol for 30 seconds; cleared with xylene, and mounted with neutral resin.

[0072] After HE staining, a semi-quantitative scoring system (SQS) was used to evaluate the extent of kidney tissue damage. The SQS scoring was based on publicly available literature (Antimicrob Agents Chemother, 2014 Jul; 58(7):4075-85.), and the specific criteria are as follows: SQS = grade score × percentage of damage score; where, grade 1 (1 point): mild tubular damage, i.e., tubular dilation, protruding cell nuclei, and a small number of tubular casts appear; grade 2 (4 points): severe tubular damage, i.e., necrosis of tubular epithelial cells and a large number of tubular casts appear; grade 3 (10 points): acute necrosis or infarction of the renal cortex or medulla. Kidney tissue section injury percentage scoring: <1% = 0 points, 1%–<5% = 1 point, 5%–<10% = 2 points, 10%–<20% = 3 points, 20%–<30% = 4 points, 30%–<40% = 5 points, ≥40% = 6 points. Finally, the SQS evaluation results are as follows: SQS+0 = No significant change (overall score, <1 point); SQS+1 = Mild injury (overall score, 1–<15 points); SQS+2 = Mild to moderate injury (overall score, 15–<30 points); SQS+3 = Moderate injury (overall score, 30–<45 points); SQS+1 = Mild to moderate injury (overall score, 45–<60 points); SQS+1 = Severe injury (overall score, ≥60 points).

[0073] 2.5 Statistical Analysis

[0074] All results are expressed as mean ± standard deviation. One-way ANOVA was performed using GraphPad 9.0 software. ** indicates P < 0.01 compared with the control group; ## indicates P < 0.01 compared with the polymyxin E sulfate model group.

[0075] The results are as follows Figures 3-5 As shown:

[0076] Compared with the control group, polymyxin E injection at 20 mg / kg daily for 7 consecutive days significantly increased serum urea nitrogen and creatinine levels in mice. Compared with the polymyxin E model group, serum urea nitrogen levels in mice in the low-dose and high-dose dehydrorutin protection groups were significantly higher (see the serum urea nitrogen test results for the control group, polymyxin E sulfate model group, dehydrorutin control group, low-dose dehydrorutin protection group, and high-dose dehydrorutin protection group as shown in the figure). Figure 3 The serum creatinine levels of mice in the control group, polymyxin E sulfate model group, dehydrorheumatine control group, low-dose dehydrorheumatine protection group, and high-dose dehydrorheumatine protection group (as shown in Figure A) are as follows: Figure 3 (As shown in B) were all significantly reduced.

[0077] The histopathological sections and semi-quantitative scores of pathological changes in the kidney tissues of mice in the control group, polymyxin E sulfate model group, dehydrorheumatine control group, low-dose dehydrorheumatine protection group, and high-dose dehydrorheumatine protection group of this invention are as follows: Figure 4 As shown in Figures A and B, compared with the control group, the mice in the polymyxin E sulfate model group showed significant pathological damage to the kidneys, including shedding and necrosis of renal tubular epithelial cells and formation of casts. The corresponding pathological damage scores increased to 2.45, with highly significant differences (**). Compared with the polymyxin E sulfate model group, the pathological damage to the kidneys of mice in the low-dose and high-dose dehydrorumengin protection groups was significantly improved, with the corresponding semi-quantitative scores decreasing to 1 and 0.6, respectively, both with highly significant differences (##). This indicates that treatment with dehydrorumengin in combination with polymyxin E can significantly inhibit the nephrotoxicity of polymyxin E.

[0078] like Figure 5 As shown in the figure, the mouse movement distance results in the open field experiment of the control group, polymyxin E sulfate model group, dehydrorheumatine control group, low-dose dehydrorheumatine protection group, and high-dose dehydrorheumatine protection group are as follows. Figure 5 As shown; compared with the control group, the total movement distance of mice in the polymyxin E sulfate model group was significantly reduced, with a highly significant difference (**); compared with the polymyxin E sulfate model group, the movement distance of mice in the low-dose dehydrorutin protection group and the high-dose dehydrorutin protection group was significantly increased in the open field, with a highly significant difference (##).

[0079] Example 3: Investigation into the protective mechanism of dehydrouncitonin against the toxicity of polymyxin E sulfate

[0080] The binding affinity and interaction patterns between dehydrouncitonin and Nrf2 and CYP1A1, ERK1, JNK, and NF-κB proteins were analyzed using AutoDockVina 1.2.2 software. Specifically, the 3D coordinates of mouse Nrf2 (PDB: 1X2R), CYP1A1 (PDB: AF-P00184-F1), ERK1 (PDB: AF-P63085-F1), JNK (PDB: AF-Q61831-F1), and NF-κB (PDB: 1IKN) proteins were downloaded from the PDB website, and molecular docking studies were performed using AutoDockVina 1.2.2 software (NIH, MD, USA).

[0081] The results are as follows Figure 6 As shown, the binding energies of dehydrorheumatoidin to proteins CYP1A1, Nrf2, ERK1, JNK, and NF-κB are -8.3, -13.5, -12.2, -12.5, and -12.2 kcal / mol, respectively. This indicates that dehydrorheumatoidin can exert a protective effect by comprehensively regulating these signaling pathways.

[0082] As can be seen from the above embodiments, the present invention uses dehydrouncitonin in combination with polymyxin E sulfate. Compared with the polymyxin E sulfate treatment group alone, the combination of dehydrouncitonin and polymyxin E sulfate can significantly improve the polymyxin E sulfate-induced cytotoxicity. Furthermore, by establishing a mouse model of neurotoxicity and nephrotoxicity induced by polymyxin E sulfate, treatment with polymyxin E sulfate significantly reduced renal function in mice, specifically manifested as a significant increase in serum urea nitrogen and creatinine levels, while also leading to significant renal tubular necrosis. Treatment with dehydrouncitonin significantly reduced serum urea nitrogen and creatinine levels, and significantly improved renal tubular pathological changes. This indicates that treatment with dehydrouncitonin in combination with polymyxin E sulfate can significantly reduce the nephrotoxicity induced by polymyxin E sulfate. Simultaneously, after 7 consecutive days of injection of polymyxin E sulfate, mice exhibited significant neurotoxic symptoms, specifically depressive-like symptoms, which were confirmed by the open field test, demonstrating that polymyxin E sulfate can induce a decline in the motor ability of mice. Supplementation with dehydrouncitonin significantly increased the movement distance of mice, indicating that dehydrouncitonin can reduce the neurotoxicity induced by polymyxin E sulfate. Mechanistic studies have revealed that, unlike previous protective agents, dehydrouncitonin exerts its protective effect by comprehensively regulating the expression of related proteins such as CYP1A1, Nrf2, ERK1, JNK, and NF-κB.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a pharmaceutical composition in the preparation of drugs that reduce polymyxin cytotoxicity and / or organ toxicity, characterized in that, The organ toxicity is nephrotoxicity; the cytotoxicity is mononuclear macrophage cytotoxicity; the pharmaceutical composition includes dehydrouncitonin and polymyxin; the polymyxin is polymyxin E sulfate.

2. The application according to claim 1, characterized in that, When the pharmaceutical composition is used to reduce cytotoxicity, the mass ratio of dehydrouncitonin to polymyxin E sulfate is (5~20):840 or (5~20):

210.

3. The application according to claim 1, characterized in that, When the pharmaceutical composition is used to reduce organ toxicity, the mass ratio of dehydrouncitonin to polymyxin E sulfate is (0.25~1):

1.

4. The application according to claim 1, characterized in that, The dosage form of the pharmaceutical composition is tablet, capsule, oral liquid, syrup, injection, pellet, or lyophilized powder for injection.

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